THE DIFFERENTIATION AND SPECIFICITY OF CORRESPONDING PROTEINS AND OTHER VITAL SUBSTANCES IN RELATION TO BIOLOGICAL CLASSIFICATION AND ORGANIC EVOLUTION: THE CRYSTALLOGRAPHY OF HEMOGLOBINS. BY EDWAED TYSON EEICHEET, M.D., Professor of Physiology in the University of Pennsylvania, AND AMOS PEASLEE BBOWN, PH.D., Profeesor of Mineralogy and Geology in the University of Pennsylvania, WASHINGTON, D. C. PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON 1909 CARNEGIE INSTITUTION OF WASHINGTON PUBLICATION No. 116 PRESS OF J. B. LJPPINCOTT COMPANY PHILADELPHIA PREFACE. This research was begun by me in October, 1902, and after considerable preliminary laboratory investigation I found that in the solution of my problems the crystallographic method promised at the present time to be the most likely to yield satisfactory results. Not being an authority in the science of crystallography, I associated with me in 1904 one of my colleagues, Professor Amos Peaslee Brown, upon whom has fallen especially that por- tion of the work which demanded the services of an expert crystallographer. The trend of modern biological science seems to be irresistibly toward the explanation of all vital phenomena on a physico-chemical basis, and this movement has already brought about the development of a physico- chemical physiology, a physico-chemical pathology, and a physico-chemical therapeutics. The striking parallelisms that have been shown to exist in the properties and reactions of colloidal and crystalloidal matter in vitro and in the living organism lead to the assumption that protoplasm may be looked upon as consisting essentially of an extremely complex solution of interacting and interdependent colloids and crystalloids, and therefore that the phenomena of life are manifestations of colloidal and crystalloidal inter- actions in a peculiarly organized solution. We imagine this solution to con- sist mainly of proteins with various organic and inorganic substances. The constant presence of protein, fat, carbohydrate, and inorganic salts, together with the existence of protein-fat, protein-carbohydrate, and protein-inorganic salt combinations, justifies the belief that not only such substances, but also such combinations, are absolutely essential to the existence of life. The very important fact that the physical, nutritive, or toxic properties of given substances may be greatly altered by a very slight change in the arrangement of the atoms or groups of molecules may be assumed to be conclusive evidence that a trifling modification in the chemical constitu- tion of a vital substance may give rise to even a profound alteration in its physiological properties. This, coupled with the fact that differences in cen- tesimal composition have proved very inadequate to explain the differences in the phenomena of living matter, implies that a much greater degree of importance is to be attached to peculiarities of chemical constitution than is universally recognized. The possibilities of an inconceivable number of constitutional differences in any given protein are instanced in the fact that the serum albumin mole- cule may, as has been estimated, have as many as 1,000 million stereoisomers. If we assume that serum globulin, myoalbumin, and other of the highest proteins may have a similar number, and that the simpler proteins and the fats and carbohydrates, and perhaps other complex organic substances, may each have only a fraction of this number, it can readily be conceived how, primarily by differences in chemical constitution of vital substances, and secondarily by differences in chemical composition, there might be brought iv PREFACE. about all of those differences which serve to characterize genera, species, and individuals. Furthermore, since the factors which give rise to consti- tutional changes in one vital substance would probably operate at the same time to cause related changes in certain others, the alterations in one may logically be assumed to serve as a common index of all. In accordance with the foregoing statement it can readily be under- stood how environment, for instance, might so affect the individual's meta- bolic processes as to give rise to modifications of the constitutions of certain corresponding proteins and other vital molecules which, even though they be of too subtle a character for the chemist to detect by his present methods, may nevertheless be sufficient to cause not only physiological and morpho- logical differentiations in the individual, but also become manifested physio- logically and morphologically in the offspring. Furthermore, if the corresponding proteins and other complex organic structural units of the different forms of protoplasm are not identical in chemical constitution it would seem to follow, as a corollary, that the homologous organic metabolites should have specific dependent differ- ences. If this be so it is obvious that such differences should constitute a preeminently important means of determining the structural and physio- logical peculiarities of protoplasm. It was such germinal thoughts that led to the present research, which I began upon the hypothesis that if it should be found that corresponding vital substances are not identical, the alterations in one would doubtless be associated with related changes in others, and that if definite relationships could be shown to exist between these differences and peculiarities of the living organism, a fundamental principle of the utmost importance would be established in the explanation of heredity ,'mutations, the influences of food and environment, the differentiation of sex, and other great problems of biology, normal and pathological. To what extent this hypothesis is well founded may be judged from this partial report of the results of our investigations: It has been conclu- sively shown not only that corresponding hemoglobins are not identical, but also that their peculiarities are of positive generic specificity, and even much more sensitive in their differentiations than the " zooprecipitin test. " More- over, it has been found that one can with some certainty predict by these peculiarities, without previous knowledge of the species from which the hemo- globins were derived, whether or not interbreeding is probable or possible, and also certain characteristics of habit, etc., as will be seen by the context. The question of interbreeding has, for instance, seemed perfectly clear in the case of Canute and Muridoe, and no difficulty was experienced in forecasting similarities and dissimilarities of habit in Sciuridce, Muridoe, Felidce, etc., not because hemoglobin is per se the determining factor, but because, according to this hypothesis, it serves as an index (gross though it be, with our present very limited knowledge) of those physico-chemical properties which serve directly or indirectly to differentiate genera, species, and individuals. In other words, vital peculiarities may be resolved to a physico-chemical basis. EDWARD TYSON REICHERT. CONTENTS. PAGE CHAPTER I. THE DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES IN THE ANIMAL KINGDOM . . . 1-27 Statement of the Distribution 2 Histohematins and Myohematins 3 Echinochrome, Hemerythrin, and Chlorocruorin, etc 4 Respiratory Metal-free Colorless Proteins 6 The Distribution of Hemocyanin 7 The Distribution of Hemoglobin in the Invertebrates 15 Causes of the Peculiarities in the Distribution of Respiratory Pigments 18 The Source of Hemoglobin Probably in Chlorophyl 19 Chemical Nature of Typical Respiratory Substances, etc 20 The Non-identity of Hemocyanins 27 The Identity or Non-identity of Corresponding Respiratory Substances 27 CHAPTER II. SPECIFICITY OF THE BLOOD OF VERTEBRATES IN RELATION TO ZOOLOGICAL DISTINCTION . . 29-66 The Quantity of Blood in Relation to Body-weight in Reference to Genera 29 The Specific Gravity of the Blood in Relation to Genera 35 The Alkalinity of the Blood in Relation to Genera 36 The Proportions of Sodium and Potassium in the Blood, Serum, and Corpuscles in Relation to Genera 39 The Phosphoric Acid of the Ash of the Corpuscles in Relation to Genera 41 The Proportions of Cholesterin in the Serum and Corpuscles in Relation to Genera 42 The Proteins of the Serum in Relation to Genera 43 The Proteins of Muscle Plasma and Seeds in Relation to Genera 45 The Zooprecipitins and Phytoprecipitins and Immune Sera in Relation to Genera 47 The Specificity of the Blood in Zoological Differentiation as Shown by the Phenomena of Coagulation 47 The Leucocytes of the Blood in Relation to Genera 48 The Proportion of Corpuscles to Serum in Relation to Genera 50 The Blood Platelets in Relation to Genera 51 The Form of the Erythrocytes in Relation to Genera 51 The Number of Erythrocytes in Relation to Genera 52 Table 20. The Number of Erythrocytes per Cubic Millimeter in Relation to Genera 53 The Size of the Erythrocytes in Relation to Genera 54 Table 21. The Sizes of the Erythrocytes in Different Genera according to the Measurements of Gulliver, Wormley, Treadwell, Formad, Welcker, and Malassez 55 Gulliver's Micrometry of the Red Blood Corpuscles (Plate A) 58-59 Certain Properties of the Erythrocyte in Relation to Genera 59 The Percentages of Hemoglobin in the Dry Erythrocytes in Relation to Genera 60 The Percentage of Hemoglobin in the Moist Erythrocytes in Relation to Genera 61 The Percentage of Hemoglobin in the Whole Blood in Relation to Genera 61 General Consideration of the Zoological Specificities of the Blood 63 CHAPTER III. HEMOGLOBIN; ITS GENERAL CHEMICAL AND PHYSICAL CHARACTERS, AND ITS SPECIFICI- TIES 67-82 Constituents and Relations to the Other Constituents of the Erythrocytes 67 The Elementary Composition of Hemoglobin 70 The Molecular Formula and Weight of Hemoglobin 74 The Solubility of Hemoglobin _ 75 The Quantity of Water of Crystallization 76 The Extinction Coefficients and Quotients 77 The Differences in the Decomposability of the Hemoglobin of Different Species. 80 Is the Oxyhemoglobin of the Blood of any Individual a Single Substance.? 82 CHAPTER IV. THE PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS PREVIOUS TO THE INVESTI- GATIONS OF PREYER 83-92 VJ CONTENTS. CHAPTER V THE INVESTIGATIONS OF PREYER ON THE CRYSTALLOGRAPHY or HEMOGLOBIN 93-106 Processes Used by Preycr for Obtaining Crystals in Large Quantities 94 Processes Given by Preyer for Obtaining Crystals in Small Quantities 98 The Forms and Systems of Crystallization of Hemoglobins. ... . . . •••••••• • • .......... Table 31. Preyer's Table Showing the Source of Hemoglobin Crystals, Crystalline Form, Crystalline System, etc 103-106 CHAPTER VI. THE PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS SINCE PREYER'S INVES- TIGATIONS 107-130 CHAPTER VII. CRYSTALLOGRAPHY OF HEMOGLOBIN IN RELATION TO SPECIES, ACCORDING TO PRE- VIOUS INVESTIGATORS, WITH EXPLANATIONS OP VARIOUS CONTRADICTORY STATE- MENTS, ETC 131-140 CHAPTER VIII. METHODS FOR PREPARING, EXAMINING, AND MEASURING CRYSTALS OF THE HEMO- GLOBINS EMPLOYED IN THIS RESEARCH 141-147 Methods for Preparing Crystals of Hemoglobin 141 The Value of the Crystallographic Method of Investigation 144 The Petrographical Microscope and its Use 145 CHAPTER IX. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF PISCES, BATRACHIA, AND REPTILIA . . 149-160 Pisces 149 Oxyhemoglobin of Raia Isevis (Barndoor Skate) 149 Oxyhemoglobin of Acipenser sturio (Sturgeon) 150 Oxyhemoglobin of Alosa sapidissima (Shad) 152 Metoxyhemoglobin of Alosa sapidissima (Shad) 153 Reduced Hemoglobin of Alosa sapidissima (Shad) 154 Methemoglobin of Alosa sapidissima (Shad) 155 Metoxyhemoglobin of Cyprinus carpio (Carp) 156 Reduced Hemoglobin of Cyprinus carpio (Carp) 156 Batrachia 157 Oxyhemoglobin of Necturus maculatus (Necturus) 157 Reduced Hemoglobin of Necturus maculatus (Necturus) 158 Reptilia 158 a-Oxyhemoglobin of Python molurus (Indian Python) 159 ^-Oxyhemoglobin of Python molurus (Indian Python) 160 CHAPTER X. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES 161-171 Aves 161 Methemoglobin (?) of Struthio camelus (African Ostrich) 162 Oxyhemoglobin of Casuarius galeatus (Cassowary) 162 Oxyhemoglobin of Anser anser (Goose) 163 Oxyhemoglobin of Olor buccinator (Trumpeter Swan) 164 Oxyhemoglobin of Olor columbianus (Whistling Swan) 164 Oxyhemoglobin of Gallus domestica (Chicken) 165 Oxyhemoglobin of Colinus virginianus (Quail) 166 Oxyhemoglobin of Numida meleagris (Guinea-fowl) 167 "-Oxyhemoglobin of Columba livia var. (Carrier Pigeon) 168 Metoxyhemoglobin of Columba livia var. (Carrier Pigeon) 168 Reduced Hemoglobin of Columba livia var. (Carrier Pigeon) 169 /'-Oxyhemoglobin of Columba livia var. (Carrier Pigeon) 169 Oxyhemoglobin of Corvus americanus (Crow) 170 Reduced Hemoglobin of Corvus americanus (Crow) 171 CHAPTER XI. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE MARSUPIALIA, EDENTATA, AND SIRENIA 173-188 Marsupialia 174 n-Oxy hemoglobin of Didelphis virginiana (Opossum) 175 /'-Oxyhemoglobin of Didelphis virginiana (Opossum) 176 Reduced Hemoglobin of Didelphis virginiana (Opossum) 177 n-CO-hemoglobin of Didelphis virginiana (Opossum) 178 ^-CO-hemoglobin of Didelphis virginiana (Opossum) 179 Oxyhemoglobin of Sarcophilus ureinus (Tasmanian Devil) 180 Reduced Hemoglobin of Dasyurus maculatus (Spotted Dasyure) 181 Oxyhemoglobin of Dasyurus viverrinus (Australian Cat) 182 "-Oxyhemoglobin of Thylacynus cynocephalus (Tasmanian Wolf) 182 '-Oxyhemoglobin of Thylacynus cynocephalus (Tasmanian Wolf) 183 Oxyhemoglobin of Trichosurus vulpecula (Vulpine Phalanger or Cuscus) 183 CONTENTS. Vll CHAPTER XI. CRYSTALLOGRAPHY OP THE HEMOGLOBINS OF THE MARSUPIALIA, EDENTATA, AND SIRENIA (continued) 173-188 Marsupialia (continued) 174 Oxyhemoglobin of .lEpyprymnus rufescens (Rat-kangaroo) 185 Oxyhemoglobin of Macropus giganteus (Kangaroo) 186 Oxyhemoglobin of Petrogale sp. (Rock-kangaroo) 187 Edentata 187 Oxyhemoglobin of Myrmecophaga (?) (Ant-eater) 187 Sirenia Oxyhemoglobin of Manatus americanus (Manatee) 188 CHAPTER XII. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE UNGULATES 189-216 Ungulata 190 n-Oxyhemoglobin of Equus caballus (Horse) 190 /3-Oxyhemoglobin of Equus caballus (Horse) 191 «-CO-hemoglobin of Equus caballus (Horse) 193 /3-CO-hemoglobin of Equus caballus (Horse) 194 a-Oxyhemoglobin of Mule 195 /3-Oxyhemoglobin of Mule 196 Oxyhemoglobin of Hippopotamus amphibius (Hippopotamus) 197 Oxyhemoglobin of Dicotyles labiatus (Peccary) 198 Oxyhemoglobin of Dicotyles tajacu (Collared Peccary) 199 Oxyhemoglobin of Domesticated Variety of Sus scrofa (Pig) 200 Reduced Hemoglobin of Sus scrofa, Domesticated Variety (Pig) 200 Oxyhemoglobin of Tragulus meminna (Muis Deer or Chevrotain) 201 Reduced Hemoglobin of Tragulus meminna (Muis Deer or Chevrotain) 202 Oxyhemoglobin of Cervus canadensis (Elk or Wapiti) 202 Reduced Hemoglobin of Cariacus rufus (Red-backed Deer) 203 Reduced Hemoglobin of Mazama americana savannarum (Venezuela Deer) 204 Oxyhemoglobin of Cervus darna (Fallow Deer) 205 Reduced Hemoglobin of Cervus dama (Fallow Deer) 205 Oxyhemoglobin of Cervulus muntjak (Muntjak) 206 Reduced Hemoglobin of Cervulus muntjak (Muntjak). 206 Oxyhemoglobin of Antilope cervicapra (Indian Antelope) 207 Oxyhemoglobin of Cervicapra redunca (Redunca Antelope, Nagor) 208 Oxyhemoglobin of Gazella dorcas (Dorcas Gazelle) 209 «-Oxyhemoglobin of Cephalophus grimmi (Duickerbok) 210 /3-Oxyhemoglobin of Cephalophus grimmi (Duiokerbok) 211 Oxyhemoglobin of Ovis aries (Sheep) 211 Reduced Hemoglobin of Ovis aries (Sheep) 212 Oxyhemoglobin of Ovis nahura (Burrell or Bharal) 213 Reduced Hemoglobin of Ovis nahura (Burrell or Bharal) 214 Oxyhemoglobin of Bos taurus (Bullock or Ox) 214 Oxyhemoglobin of Bos bison (Buffalo) 215 Table 41. Crystallographic Characters of the Hemoglobins of the Ungulata 216 CHAPTER XIII. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE RODENTIA 217-246 Rodentia 218 a-Oxyhemoglobin of Sciurus vulgaris (European Red Squirrel), from Fresh Blood 219 /3-Oxyhemoglobin of Sciurus vulgaris (European Red Squirrel), from Putrid Blood 219 Oxyhemoglobin of Sciurus rufiventer neglectus (Fox-squirrel) 220 Oxyhemoglobin of Sciurus carolinensis (Gray Squirrel) 221 Oxyhemoglobin of Sciuropterus volans (Flying-squirrel) 222 Oxyhemoglobin of Tamias striatus (Ground-squirrel or Hackee) 223 Oxyhemoglobin of Cynomys ludovicianus (Prairie-dog) 223 a-Oxyhemoglobin of Marmota monax (Ground-hog or Woodchuck) 225 /3-Oxyhemoglobin of Marmota monax (Ground-hog or Woodchuck) 225 y-Oxyhemoglobin of Marmota monax (Ground-hog or Woodchuck) 226 Reduced Hemoglobin and Metoxyhemoglobin of Marmota monax (Ground-hog or Wood- chuck) Oxyhemoglobin of Castor canadensis (Beaver) Oxyhemoglobin of Fiber zibethicus (Muskrat) 229 Oxyhemoglobin of Albino of Mus norvegicus (White Rat) 230 a-Oxyhemoglobin of Mus norvegicus (Norway or Brown Rat) /3-Oxyhemoglobin of Mus norvegicus (Norway or Brown Rat) 233 Oxyhemoglobin of Mus rattus (Black Rat) 234 Oxyhemoglobin of Mus alexandrinus (Alexandrine Rat) 236 n-Oxyhemoglobin of Erethizon dorsatus (Canadian Porcupine) 238 CONTENTS. CHAPTER XIII. CRTBTALLOGRAPHT or THE HEMOGLOBINS OF THE RODENTIA (continued) .... 217-246 Rodentia (continued) ; • • • • • • • • • • : • • • ^18 /?-Oxyhemoglobin of Erethizon dorsatus (Canadian Porcupine) 239 Oxyhemoglobin of Domesticated Variety of Cavia cutleri (Guinea-pig) 240 a-Oxyhemoglobin of Hydrochoerus capyvara (Capybara) 242 £-Oxyhemog]obin of Hydrochcerus capyvara (Capybara) 243 n-Oxyhemoglobin of Lepus caniculus (Domestic Rabbit) 243 /3-Oxyhemoglobin of Lepus caniculus (Domestic Rabbit) 244 a-Oxyhemoglobin of Lepus europaeus (Belgian Hare) 245 /3-Oxyhemoglobin of Lepus europaeus (Belgian Hare) 246 Table 42. Crystallographic Characters of the Hemoglobins of the Rodentia 246 CHAPTER XIV. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE OTARIID.E, PHOCID^E, MUSTEL- ID.S:, PROCYONID-E, AND URSINE 247-263 Phocidae 248 Oxyhemoglobin of Phoca vitulina (Harbor Seal) 248 Oxyhemoglobin of Otaria gillespii (California Sea-h'on) 250 CO-hemoglobin of Otaria gillespii (California Sea-lion) 250 Mustelidae 252 Oxyhemoglobin of Domesticated Variety of Mustela putorius (Ferret) 252 Oxyhemoglobin of Mephitis mephitica putida (Skunk) 253 Oxyhemoglobin of Taxidea americana (Badger) 254 Oxyhemoglobin of Lutra canadensis (Otter) 256 Procyonidae 256 Oxyhemoglobin of Cercoleptes caudivolvulus (Kinkajou) 257 Reduced Hemoglobin of Cercoleptes caudivolvulus (Kinkajou) 258 Oxyhemoglobin of Bassariscus astuta (Cacomvxl or Cacomistle) 258 Ursidss 258 Oxyhemoglobin of TJrsus americanus (Black Bear) 259 Metoxyhemoglobin of Ursus americana (Black Bear) 260 Oxyhemoglobin of Ursus maritimus (Polar Bear) 261 Oxyhemoglobin of Melursus ursinus (Sloth Bear) 262 Table 43. Crystallographic Characters of the Hemoglobins of the Phocidas, Otariida, Mus- telidjB, Procyonidae, and Ursidae 263 CHAPTER XV. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE CANID^E — DOGS, WOLVES, AND FOXES 265-279 Canidie 266 a-Oxyhemoglobin of Canis familiaris (Dog) 266 /3-Oxyhemoglobin of Canis familiaris (Dog) 267 Oxyhemoglobin of Canis familiaris var. (Chow Dog) 268 Oxyhemoglobin of Cross between C. familiaris (Collie Dog) and C. latrans (Coyote) 269 Metoxyhemoglobin of Canis lupus mexicanus (Gray Wolf) 270 Oxyhemoglobin of Canis latrans (Coyote or Prairie Wolf) 271 Oxyhemoglobin of Canis aureus (Jackal) 271 Oxyhemoglobin of Canis dingo (Dingo or Australian Wild Dog) 272 Oxyhemoglobin of Canis azarae (Azara's Wild Dog) 274 Oxyhemoglobin of Vulpes vulpes (Swiss Fox) 275 Oxyhemoglobin of Vulpes fulvus (Red Fox) 276 Oxyhemoglobin of Vulpes lagopus (Blue or Arctic Fox) 277 Oxyhemoglobin of Urocyon cinereoargenteus (Gray Fox) 278 Table 44. Crystallographic Characters of the Hemoglobins of the Canidae 279 CHAPTER XVI. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE FELID.B AND VIVERRID.E — CATS AND CIVETS 281-298 Table 45. Crystallographic Characters of the Reduced Hemoglobins of the Felida: 282 Felidse 283 Oxyhemoglobin of Felis leo (Lion) 283 Metoxyhemoglobin of Felis leo (Lion) 284 Reduced Hemoglobin of Felis leo (Lion) 284 Reduced Hemoglobin of Felis tigris (Bengal Tiger) 285 n-Oxyhemoglobin of Felis onca (Jaguar) 286 •''-Oxyhemoglobin of Felis onca (Jaguar) 287 Reduced Hemoglobin of Felis onca (Jaguar) 287 «-Oxyhemoglobin of Felis concolor (Mountain-lion or Puma) 288 /'-Oxyhemoglobin of Felis concolor (Mountain-lion or Puma) 289 Reduced Hemoglobin of Felis concolor (Mountain-lion or Puma) 289 Reduced Hemoglobin of Felis bengalensis (Leopard-cat) 290 CONTENTS. IX CHAPTER XVI CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE FELIDJE AND VIVERRID^H (continued) 281-298 Felidae (continued) 282 Oxyhemoglobin of Felis bengalensis (Leopard-cat) 290 Reduced Hemoglobin of Felis pardalis (Ocelot) 291 Reduced Hemoglobin of Felis domestica (Domestic Cat) 292 Oxyhemoglobin of Lynx ruf us (Wild Cat or Bay Lynx) 294 Reduced Hemoglobin of Lynx rufus (Wild Cat or Bay Lynx) 295 "-Reduced Hemoglobin of Lynx canadensis var. (Florida Lynx) 296 /5-Reduced Hemoglobin of Lynx canadensis var. (Florida Lynx) 297 Oxyhemoglobin of Lynx canadensis var. (Florida Lynx) 297 Viverridte 298 Oxyhemoglobin of Arctictis binturong (Binturong) 298 Table 46. Crystallographic Characters of the Hemoglobins of the Felidse and Viverridse. . . . 298 CHAPTER XVII. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE INSECTTVOBA AND CHIROPTERA. 299-303 Insectivora 300 Oxyhemoglobin of Scalops aquaticus (Mole) 300 Hemoglobin of Scalops aquaticus (Mole) 301 Chiroptera 301 Oxyhemoglobin of Pteropus medius (Fox-bat or Flying-fox) 301 Oxyhemoglobin of Vespertilio fuscus (Brown Bat) 302 Table 47. Crystallographic Characters of the Hemoglobins of the Insectivora and of the Chi- roptera examined 303 CHAPTER XVIII. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE PRIMATES — LEMURS, BA- BOONS, AND MAN 305-319 Table 48. The Three Kinds of Oxyhemoglobin Observed in Baboons and in Man, with their Optical Characters 305 Primates 306 Oxyhemoglobin of Lemur catta (Ring-tailed Lemur) 306 n-Oxyhemoglobin of Papio babuin (Yellow Baboon) 308 /3-Oxyhemoglobin of Papio babuin (Yellow Baboon) 309 y-Oxyhemoglobin of Papio babuin (Yellow Baboon) 309 Reduced Hemoglobin of Papio babuin (Yellow Baboon) 310 a-Oxyhemoglobin of Papio Ieucopha3us (Drill) 310 a-Oxyhemoglobin of Papio sphinx (Guinea Baboon) 311 /3-Oxyhemoglobin of Papio sphinx (Guinea Baboon) 311 } -Oxyhemoglobin of Papio sphinx (Guinea Baboon) 312 o-Oxyhemoglobin of Papio langheldi (Long-armed Baboon) 313 /3-Oxyhemoglobin of Papio langheldi (Long-armed Baboon) 313 a-Oxyhemoglobin of Papio porcarius (Chacma) 314 /3-Oxyhemoglobin of Papio porcarius (Chacma) 315 n-Methemoglobin of Papio porcarius (Chacma) 315 /3-Methemoglobin of Papio porcarius (Chacma) 316 /3-Oxyhemoglobin of Papio anubis (Anubis Baboon) 316 X-Oxyhemoglobin of Papio anubis (Anubis Baboon) 317 Table 49. /3-Oxyhemoglobins of the Baboons, genus Papio; monoclinic 317 «-Oxyhemoglobin of Homo sapiens africanus (Man) 318 Reduced Hemoglobin of Homo sapiens africanus (Man) 319 Table 50. Crystallographic Characters of the Hemoglobins of the Primates 319 CHAPTER XIX. SUMMARY AND CONCLUSIONS 321-338 Mode of Preparation of Hemoglobins 321 The Different Kinds of Hemoglobins Found in the Bloods 322 Specificity in Generic and Specific Characters 325 Constancy of Generic Characters 325 Constancy and Specificity of the Crystallographic Characters of Individual Species 326 General Crystallographic Characters of the Hemoglobin Crystals 328 The Constant Recurrence of Certain Angles, etc 328 Table 51. Simple Ratios and Mean Angles Computed from them for Ratio of 1:1 = 88° 63' 328 Mimesie, and the Angles of 60° and 90° in the Crystals 329 The Zoological Applications of this Method of Research 332 Table 52. Comparison of Axial Ratios of Species of Canidse 333 The Influence of Certain Physiological Conditions upon the Composition and Coloring Matter of the Blood 336 The Condition of the Hemoglobins in the Corpuscle 337 INTRODUCTION. The extraordinarily large number of researches suggested by the hy- pothesis stated in the Preface are so vast in their scope as to enter every domain of the science of living matter, normal and abnormal ; and they are so diversified and exacting in their technical requirements that at the out- set of our work we realized the necessity, for the time being, of limiting our studies by means of practically a single method of investigation and to a single substance. The crystallographic method was selected, for the reasons stated; and one of the proteins was selected because these sub- stances are universally recognized as constituting the most important class of the body constituents, and also because it would seem that modifications in their molecules would be more likely to occur and to be of a more far- reaching influence and importance than in those of other vital substances. Unfortunately, however, the study of the chemistry of proteins has proven so extraordinarily abstruse that our knowledge is still in an early formative stage. It has only been within very recent years that any really important progress has been made; and notwithstanding a large amount of laboratory investigation and the accumulation of a voluminous literature, our informa- tion is still largely of a rudimentary and fragmentary nature. The undeveloped state of the science of proteins is perhaps nowhere more evident than in the absence of any classification that seems to be other than of a purely tentative character, in the absence of satisfactory knowledge of such fundamental subjects as the molecular constitution of even the best-known proteins, and in our very incomplete data of the primary dissociation products. The methods of analysis of proteins into the primary dissociation products are not only not strictly quantitative, but also very imperfect qualitatively. The figures of different investigators for a given protein often differ quite as much as for different substances, and there are yet large percentages to be accounted for, such as 30 per cent of globin, 74 per cent of egg albumin, 57 per cent of serum albumin, 55 per cent of serum globulin and of fibrin, 59 per cent of lactalbumin, 83 to 90 per cent of proteoses, 39 to 54 per cent of plant globulins, 40 to 65 per cent of glute- lins, 25 to 50 per cent of gliadins, etc. In fact, only one of the proteins, salmine, which is one of the simplest, has been fully accounted for, but even here the data are unsatisfactory, chiefly because a larger per cent (110.5) has been recorded than can exist; Abderhalden found leucin and alanin, which is not admitted by Kossel and Dakin; and there is doubt as to the purity of the substance analyzed, there being several similar bodies in the sper- matozoa of the same species. The data in regard to the other protamines are very incomplete, almost wholly qualitative, and by no means conclusive. Whether or not the corresponding proteins of different species of animals or of plants are chemically identical had not, up to the inception XJj INTRODUCTION. of this research, in any instance been conclusively determined. It is true that in certain instances differences have been noted, but these, as a rule, could readily be accounted for without the assumption of chemical differ- ences inherent in the molecules; and in the exceptional cases it is not certain whether we are dealing with normal individuals or with several individuals or with contaminated individuals, etc. For instance, the fact that the egg- white of the eggs of certain species remains perfectly clear upon boiling, while that of other species becomes opaque, might be taken as meaning a difference in chemical composition, but the difference has been shown to lie in the different amounts of alkali and saline present. That the egg- albumin of the chicken, pigeon, and seed-crow has been crystallized, while the experimenter has not been successful with the egg-albumin of other species, may imply nothing more than different conditions extrinsic to the molecules. The caseinogens of woman's and cow's milk are looked upon as being not identical, yet the primary dissociation products show a great similarity and the elementary analyses of the caseinogen of the cow, goat, and rabbit are identical. The centesimal analyses of corresponding albu- mins and globulins have failed to show any positive differences. Oppen- heimer states, from the results of a recent study of the serum albumins of man, the horse, and ox, that serum albumin is a uniform and specific sub- stance, and that the elementary analyses point to one serum albumin. In the case of the hemoglobins the differences in the analyses of the hemoglo- bins of different species are not, with rare exceptions, greater than those of the hemoglobins from different individuals of the same species. Osborne's assertion that the glutenins of wheat and rye are not chemically alike is founded on the fact that "wheat flour readily yields gluten consisting of glia- din and glutenin, whereas rye flour does not. " (Extract from a letter from Professor Osborne in response to an inquiry for data justifying his statement.) Crystallographic differences believed by Giirber to be shown by the serum albumins of the horse and rabbit were disproved by his pupils; the crystals of lactalbumin closely resemble or are even identical with those of serum albumin. The statements made from time to time that the hemo- globins of different species can be distinguished by differences in crystalline form have been contradicted, and have been based upon manifestly inade- quate investigation. Positive differences have been noted, but the differ- ences between the forms of the crystals of different species have not been greater than the differences in the forms obtained from specimens of blood from individuals of the same species that have been recorded by the same or by different investigators. Preyer, in his well-known monograph Die Blutkrystalle, Jena, 1871, refers in a table (see Chapter V) to the crystalline forms of the hemoglobin from 44 species, and with rare exceptions the crystals are described as prisms, rods, plates, and needles, and occasionally as six-sided rods or plates. All of the crystals, when classified according to system, have been assigned to the rhombic system, except one, and possibly three other instances where they are classified as hexagonal. Indeed, in those exceptional instances we have proven that the crystals do not belong to the hexagonal system. While seeming differences have been recorded INTRODUCTION. Xlll in the shapes of the crystals of different species, differences quite as marked have been described in the crystals of the same species by different observ- ers. Thus, the crystals from the blood of the guinea-pig have been recorded by one as occurring occasionally as octahedra, by another as tetragonal, by another as tetrahedra and rhombic plates and prisms, and by another as six-sided plates; squirrels' crystals are almost invariably described as hexagonal plates, but they have also been seen as needles and rhombic prisms; crystals of the mouse are variously referred to as fine needles, or six-sided plates, or small prisms; rats' crystals are described as tetrahedra, prisms, rods, plates, needles, or hexagons. Even the processes for preparing protein crystals are, except those of the hemoglobins, very limited in their range of usefulness, as has been shown by the failure to obtain crystals of even corresponding proteins save in a very limited number of instances. Moreover, the processes are so far from perfect that crystalline condition per se is not a guarantee that we have necessarily either a pure or a normal body; nor are such crystals free albumin, free globulin, etc., but acetates or sulphates or other forms of combination, etc. Moreover, when purification of proteins has been sought by repeated recrystallization it is by no means clear that the proc- esses have not given rise to abnormal substances through a stripping off of very unstable or feebly combined radicals which constitute normal com- ponents of the molecules, and which of course must contribute to giving the substance its peculiar properties. In the case of the hemoglobins it has been shown that decomposition products are formed at each step of recrystallization. It has even been found that crystalline habit may be so affected by recrystallization that a hemoglobin which normally appears in the form of hexagons may crystallize only in rhombic needles and tetra- hedra. We are not, however, without certain data which indicate differences of corresponding proteins. The very fact of the breaking down of the serum albumin and the serum globulin of the food during the processes of digestion, when these substances are in a natural state, can not be due to a non-absorbability, because in such form they may be rapidly absorbed under appropriate conditions, and it is at least suggestive that the degra- dation of the protein molecules must be, in part at least, for some important purpose in relation to the synthesis of the proteins of the individual. To what extent this disintegration is carried out we do not know, but from our present knowledge it is probable that the molecules are broken down into essentially the primary dissociation products. In what ways and from what derivatives the proteins of the individual are built up is a matter of speculation, but it seems at least that such analyses and syntheses mean a differentiation of the proteins of the food and the corresponding proteins of the animal. The best instance on record which positively indicates or shows chem- ical differences in homologous proteins was brought to light by Kossel and others several years (1904 et seq.) after this research was begun. The differences in the protamines in elementary composition, in rotatory power, INTRODUCTION. and in the primary dissociation products in both percentage and groups, seem to be conclusive that these substances (assuming their purity) are not identical. If Griffiths's work on the achroglobulins be confirmed, we have another such instance. Other indications of differentiation are shown in the difference in the globin of the horse, bullock, and dog on the one hand and that of the goose on the other; the "precipitin test," by which can readily be shown certain zoological differentiations of the blood, milk, and flesh extracts, is admitted to depend upon specificities of proteins; hemoglobins of different species are recorded as differing in solubility, decomposability, water of crystallization, crystallizability, color intensity, and absorptive power in relation to 0 and C02 ; in contradiction to Hiifner, who describes such a striking identity and constancy of the extinction coefficients and quotients of the oxyhemoglobin of all species, we have sufficient evidence in literature to show that these coefficients do vary in different species. Differ- ences have been noted in several hemocyanins in regard to the degree of dissociability of the 0 and C02, and to the temperature of coagulation; and there are indications of differences in echinochromes and chlorocruorins. If chemical differences exist in corresponding proteins they seem to be of so subtle a nature, except in rare instances, as to be beyond the possi- bilities of the present methods of chemical distinction. It was therefore believed that some other method might bring success where the chemist has failed. As it is recognized that crystalline form may depend upon either chemical composition or constitution, it seems that the method of investigating microscopic crystals as developed by Sorby, and later by Zirkel, Rosenbusch, and others, and the resulting lithological microscope with its various attachments, might afford the means of obtaining satis- factory results. By this method of investigation an entire science, the science of petrography, has been built up. The "optical reactions" thus obtained are often as distinctive, and even as exact, as the chemical re- actions, and this instrument is now used by the petrographer and chemist alike for the study of crystals too small to be examined in the usual way. Thus the crystals may be studied in the solution in which they are formed, and fairly accurate measurements may be obtained of their plane angles and various optical properties. Inasmuch as the optical properties, which are dependent upon the internal tensions of the crystal, are often more distinctive than the exterior form, and since even "isomeric substances possess different crystal structures" (Groth), it will be readily seen that this method of investigation may show differences which at present may be or are too obscure for the chemist. Thus far only a very limited number of the proteins have been obtained in crystalline form. A number of hemoglobins and hemoglobin compounds and derivatives, serum albumin, lactalbumin, casein, vitellin, a number of globulins from seeds and nuts (some of them being recorded as albumins), the albumin and globulin of egg-white, hyalin, two proteins from abnormal urines (one of which is a casein-like body and the other probably a hetero- proteose), ichthulin (probably a lecithoprotein) from the eggs of fish, gluto- kyrin, hemocyanin, and phycoerythrin and phycocyanin of algse, include INTRODUCTION. XV all, as far as we have been able to find, that have been obtained in crystals. Excepting hemoglobin, the crystallographic studies of proteins have been very limited and inconclusive, and in so far as this substance is concerned it is clear, from a study of the literature of the subject, that the inquiries have with rare exceptions been of so superficial a character as to possess little or no intrinsic value in indicating positive chemical differentiation. Among the literature on hemoglobin we have found only very rare instances where an adequate study was reported of the geometric characters of the crystals, and we have failed to find any quantitative data of any value in regard to the optical characters of the crystals in polarized light that might be of service in showing zoological differentiations ; yet these very characters, we believe, will be found to prove the best and most easily applied means of differentiating the crystals of hemoglobin and of showing the identity or non-identity of chemical composition or chemical constitution. The comparative readiness with which hemoglobin can be crystallized, together with the exceptional importance of this protein in animal life, led to its selection as the subject of study. The important problems next demanding our attention were in regard to the methods to be adopted to obtain graphic records of the crystals, to the methods for preparing the crystals, and to the sources of supply of the numerous and diverse kinds of blood required in order to yield the necessary data. As to the first, experience has demonstrated that line- drawings, lithography, perspective drawings, photomicrography, etc., each has its advantages and disadvantages, yet it goes without saying that while line-drawings are absolutely essential in the geometric descriptions of crystals, the only means of reproduction which eliminates the personal factor and gives at the same time a permanent and faithful record for veri- fication and further study lies in the photomicroscope and its accessories. The generally very poor reproductions of photomicrographs of crystals that have appeared in print, together with the usual extreme unstability of hemoglobin crystals, those of certain bloods melting at temperatures scarcely above the freezing-point, seemed to us upon first thought to render this method impracticable, except to a limited degree. Moreover, we feared that, owing to the fact that the crystals and the solution in which they have been formed are generally of so nearly the same color and tint, satisfactory reproductions for printing would be found to be practically impossible; but these difficulties we overcame. All of our photomicrographic negatives were made with ordinary laboratory apparatus. We used a standard Bausch and Lomb microscope, and almost without exception a 2-inch eyepiece and a § objective, which gave us a magnification in our negatives of about 250. Occasionally we used higher powers, giving us a magnification of about 500, 800, and 1,200. Many of our negatives are not up to the standard we sought, because of the great sensitivity of many of the crystals to the slightest increase of temperature during the focusing and exposure in the photomicrographic apparatus, or to the little or no contrast in the color of the crystals and solution, in many instances the crystals being discernible solely by the xvj INTRODUCTION. shadows of their outlines. The difficulties of the last instance were usually successfully met by the selection of a proper quality of gelatin plate and by careful development and printing, which often brought out marked contrasts. We occasionally resorted to the use of color screens with excel- lent results, but in general they were not found necessary or of any par- ticular advantage. The negatives, about 2,500 in number, were made by Dr. Reichert, from which we have selected 600 to illustrate the text. The line-drawings, numbering 411, were made by Charles Travis, Ph.D., instructor in geology and mineralogy in the University of Pennsylvania, to whom we are especially indebted for the great care and accuracy with which the work was done. While a large number of methods for preparing hemoglobin crystals in large or small quantities have been published, it was found that in order to obtain satisfactory results we should have to devise means whereby we could have better control over the rapidity of crystallization, and also to avoid any method which might injuriously affect the hemoglobin molecule. We therefore devised methods for promoting or retarding crystallization. By the former we have obtained crystals from small quantities of blood which had not heretofore been obtained ; and by retarding crystallization we have secured measurable crystals from blood in which, on account of their rapid crystallizability, it has heretofore been impossible or difficult to develop them. Moreover, by modifications of our processes we have in specimens of blood of certain species been enabled to crystallize at will one or another of several forms of oxyhemoglobin normally present in the same blood. The pernicious effects of alcohol and of recrystallization led to the avoidance of these agents. Finally, it was obvious that a successful outcome of our research demanded an examination of specimens not only from a large number of species, but also from species related and unrelated, so as to permit of a critical examination of possible generic, family, and other peculiarities. Such supplies as might be obtained from domesticated animals and such small wild animals as could be secured within the possibilities of our grant from the Carnegie Institution of Washington we realized could not meet our necessities. We therefore sought the cooperation of those in authority at the various zoological gardens of this country for specimens of blood from animals that died. A circular letter was forwarded by President R. S. Woodward to the management of each garden, and from a number of them we obtained assistance. We are also indebted to Dr. S. Weir Mitchell and to Dr. Charles D. Walcott for assistance in securing material. Speci- mens were received from Mr. Stone, Rochester Park, N. Y.; Mr. M. P. Hurlbut, commissioner of parks and boulevards, Detroit; Dr. H. H. Donald- son, Wistar Institute of Anatomy; Mr. Ernest Tretow, Highland Park, Pittsburg, Pa.; Mr. P. P. Randolph, Zoological Gardens, Seattle, Wash- ington; Mr. R. G. Rau, Zoological Park, St. Joseph, Mo.; Dr. Herbert Fox, pathologist of the Zoological Society of Philadelphia; Mr. Charles H. Townsend, superintendent of the New York Aquarium ; Mr. H. A. Surface, State zoologist of Pennsylvania; Dr. Frank Baker, National Zoological INTRODUCTION. XV11 Park, Washington, D. C., and Dr. W. Reid Blair, pathologist of the New York Zoological Park. To Dr. John R. Mohler, chief of the division of pathology, U. S. Department of Agriculture, Washington, D. C., we are especially indebted. We have had at our disposal specimens from about 200 species of mammals, most of them received from the zoological gardens, and usually in various stages of putrefaction. It would have been advantageous in many ways if in every case we had had not only fresh blood, but also blood from healthy animals and in larger quantities. Yet in so far as the speci- ficity of the crystals is concerned we believe that neither the presence of putrefactive processes in the blood nor diseased conditions generally have any important influence. The greatest disadvantage of putrid blood con- sists in a greater unstability of the preparations and in the difficulty of securing some of the more evanescent forms of oxy hemoglobin. Owing to an absence of preliminary knowledge of the peculiarities of the hemoglobins of different bloods as regards the degree of crystallizability, or to the exceed- ingly small quantities we usually had to work with, or to extreme putrefac- tion or other conditions over which we had no control, we occasionally failed absolutely to obtain any evidence of crystallization, and many of our speci- mens were lost owing to the perishability of the crystals of certain species or to the very pressing demands of teaching. In fact, what work we have accomplished has been through the utilization of such scattered hours as could be taken from the exacting requirements of the class-room and of routine work. Moreover, owing to the extreme solubility of many of the crystals, our investigations were largely limited to the cooler months, and much of our work was done at temperatures at or near the freezing-point. It was our expectation to include in this memoir the results of a few preliminary studies of certain other corresponding vital substances, espe- cially of plant proteins. Our data are not, however, more than sufficient at present to justify the announcement that we believe that the zoological distinctions we have found to be shown by hemoglobins will be demon- strated in other primary organic substances. This research has proved of exceptional fertility and importance in crystallography. It has brought to light the most extraordinary isomor- phous series known; and it has yielded not only the crystallographic data we have recorded in this memoir, but also much that has been omitted because chiefly of its essentially technical character. This latter we will include in a separate memoir in the near future. We have not in the present memoir attempted to support Dr. Reich- ert's hypothesis beyond the mere presentation of our discoveries. The problems pertaining to the origin of species, heredity, mutations, sex, and the influence of food and environment are of such extraordinary importance as to have engaged the master minds in biological inquiry, and the task of presenting so important a matter in the form which we believe is neces- sary to be acceptable to the critical student has seemed too formidable for us to undertake at present. After all, perhaps, it is sufficient and better that we merely state the important hypothesis upon which we have worked, Xviii INTRODUCTION. together with the unique facts we have brought to light, with the hope, in thus pausing in our research to make this partial announcement of our results, that their publication may be the means of exciting original thought and investigation along the same or collateral lines, especially in the utili- zation of the extraordinarily rich supply of material that must be available in the great zoological gardens of Europe and in the marine laboratories of this country and abroad. The first grant for this research was made in April, 1904, and the second grant in January, 1908. The tentative title of our research was announced in the Year Book of the Carnegie Institution of Washington, 1904. Preliminary Reports of our investigation appeared in the Year Book of 1908, 218; in the Proceedings of the Society for Experimental Biology and Medicine, 1907-08, v, 66; and in the Proceedings of the American Philosophical Society, 1908, XLVII, 298. In conclusion, we take pleasure in acknowledging our great indebted- ness to President R. S. Woodward, whose interest and assistance have been invaluable; and especially, to Dr. S. Weir Mitchell, who has been a most valuable co-worker with us throughout our investigations. EDWARD TYSON REICHERT. AMOS PEASLEE BROWN. FROM THE S. WEIR MITCHELL LABORATORY OF PHYSIOLOGY, University of Pennsylvania. THE DIFFERENTIATION AND SPECIFICITY OF CORRESPONDING PROTEINS AND OTHER VITAL SUBSTANCES IN RELATION TO BIOLOGICAL CLASSIFICATION AND ORGANIC EVOLUTION: THE CRYSTALLOGRAPHY OF HEMOGLOBINS. BY EDWARD TYSON REICHERT, M.D., Professor of Physiology in the University of Pennsylvania, AND AMOS PEASLEE BROWN, PH.D., Professor of Mineralogy and Geology in the University of Pennsylvania. CHAPTER I. THE DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES IN THE ANIMAL KINGDOM. Specific respiratory substances are universally distributed throughout plant and animal life, except probably in some of the very lowest organ- isms. For the most part they are colored, and of a variety of colors and tints, and they exhibit decided variations in their respiratory capacities and other properties. In plant life chlorophyl is preeminently the pigment concerned in the interchange of O and CO2; while in animal life hemo- globin occupies an analogous place, but they are undoubtedly very different in their manner of functionating. In each kingdom the major pigment may be represented or supplemented by physiologically allied bodies, which may or may not be closely related chemically. The alliance between chlorophyl and hemoglobin that was first sug- gested by Hoppe-Seyler has been convincingly shown by the investigations of Schunk and Marchlewski (Annal. d. Chem. u. Pharm., 1894, No. 278, 349; 1895, No. 284, 81, and No. 288, 209; 1896, No. 290, 306; and March- lewski, Bull, de 1'Acad. des Sciences de Cracovie, etc., 1902; Biochem. Centralbl., 1902-03, i, 215), who obtained from chlorophyl a derivative coloring matter which they termed phylloporphyrin (C16H18ON2 — new formula C34H3802N4), which bears a striking resemblance to hematopor- phyrin, C34H3S06N4, an iron-free derivative of hemoglobin, and differing from it only in 4 atoms of O. Moreover, Marchlewski and Zaleski obtained hemopyrrol by reduction from both chlorophyl and hemoglobin. Nencki and Zaleski (Berichte d. deutsch. chem. Ges., 1901, xxiv, 997) attempted to convert hemoporphyrin into phylloporphyrin by reduction, but suc- ceeded in removing only two atoms of O, producing a crystalline inter- mediate body which they named mesoporphyrin (C16H18O2N — new formula C34H3SO4N4), and which they believe to be identical with hematoidin. Zaleski (Zeit. f. phys. Chemie, 1902, xxvn, 74) found that from meso- porphyrin and hematoporphyrin similar salt and ester compounds can be obtained; and Marchlewski, in examining the spectra of hemoporphyrin, mesoporphyrin, and phylloporphyrin, found them to be very similar, and distinguishable from one another only by a slight displacement of the absorption bands towards the violet end of the spectrum. According to Schunk and Marchlewski, both chlorophyl and hemoglobin are pyrrol derivatives. Chlorophyl in granular form (chloroplastids) has been found in a large number of invertebrates and vertebrates. In certain of these animals, especially in the lowest types, as in the Protozoa, it or an almost identical i 2 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES body is a normal functionating constituent, while in others it is an inci- dental inactive body, being introduced as food, etc. According to March- lewski (Die Cheraie d. Chlorophyll, 1895, 63), chlorophyl isolated from whatever plants is identical, but Montverde (Acta horti Petropolit., 1893, xni, 176) and Etard (Compt. rend. soc. biolog., 1895, cxx, 275) hold that there are various kinds. It is probable that the bodies studied by Mont- verde and Etard were impure. Whichever may be true, there is no doubt that isolated chlorophyl pigment is physiologically inert (page 22) and that the chloroplastids (chlorophyl-protein combinations) differ chemically, physically, and biologically, and that the functionating chlorophyl granule of animal life is not identical with that of plant life. There is evidence of intermediate bodies between chlorophyl and histohematin, as MacMunn has found in Helix pomatia. STATEMENT OF THE DISTRIBUTION. Hemoglobin is entirely absent from Protozoa, Porifera, and Coelen- terata; it is rare in Echinodermata; it is quite common in certain classes of Annelida; and it is comparatively rare in Arthropoda. (See page 63, Chapter II.) It is distributed among the invertebrates in a remarkably sporadic and inexplicable way, appearing in only certain classes of a series, or in certain members of a class, etc., sometimes exclusively as a constitu- ent of blood corpuscles or blood plasma, or in nervous matter, or in parts of the musculature, etc. It may be present in members of a certain class, as for instance the Choetopoda, but in certain of them it is found as a con- stituent of special blood corpuscles, and in others in solution in the blood plasma. It may be present in certain members and absent in others, and in the latter it may be represented by closely allied bodies, chemically and physiologically, such as the chlorocruorins, histohematins, etc. The close chemical relationship of chlorocruorin to hemoglobin is shown in its yielding hematin as a decomposition product, while the histo- hematins are in the nature of modified forms or derivatives of hemoglobin. Chlorocruorin probably exists in several forms or modifications and seems to have a very restricted distribution, limited to the invertebrates, while histo- hematins and myohematins are very numerous among both invertebrates and vertebrates. Closely related to hemoglobin, chiefly physiologically, is hemocyanin, which probably exists in several modified forms. Hemo- cyanin is albuminous and contains copper in the molecule in place of the iron of the hemoglobin molecule; it is distributed solely among the inver- tebrates, but more widely and quite as erratically as hemoglobin. A large number of lipochromes, some closely allied to chlorophyl and hemoglobin, have been found in both invertebrates and vertebrates. In all vertebrates, except the Leptocephalus and probably the Amphi- oxus, hemoglobin is present in the red blood corpuscles, but is never normally in solution in the blood plasma. In addition to hemoglobin, we find modifications, compounds, and derivatives as normal constituents of various body fluids and solids. IN THE ANIMAL KINGDOM. O HISTOHEMATINS AND MYOHEMATINS. MacMunn's investigations (Philosoph. Trans., 1886, i, 235, 267; Jour- nal of Physiology, 1886, vn, 240, and 1888, ix, 1) have demonstrated a wide distribution of the histohematins and allied bodies among both inverte- brates and vertebrates, including porifera, echinoderms, molluscs, arthro- pods, worms, amphibia, fishes, reptiles, birds, and mammals. In his studies of the chromatology of the British sponges he has shown the presence of coloring matters which are closely related to hemoglobin, and which he groups under the term histohematins. Out of 12 specimens examined by him, 7 by Krukenberg, and 1 by Ray Lankester, making 20 in all, 18 con- tained chlorophyl, nearly all contained lipochromes, and 7 contained histohe- matins. In a previous communication on the chromatology of the Actini- idce, MacMunn (Philosoph. Trans., 1885, n, 641) reports a coloring matter in Actinia mesembryanthemum, Bunodes crassicornis, and other Actiniidce, which can be changed into a hemochromogen and a hematoporphyrin, which are indistinguishable from the corresponding bodies obtained from hemoglobin. In the echinoderm Ophiactis viriens, Foettinger (Archiv d. Biologic, 1880, i, 405) states that he found hemoglobin, and although the correctness of this statement was questioned by Krukenberg (loc. cit.) it was subse- quently stated by MacMunn to be justified (Journal of Physiology, 1886, vn, 240). MacMunn found hematoporphyrin in Uraster rubens. All of the star-fish showed the presence of histohematins from which hematopor- phyrin could be obtained. Hematoporphyrin he found in the integument of slugs, Limax flavus, Limax variegatus, and Arion ater. In molluscs he noted enterohematin in the bile and histohematins in various tissues and organs. He also reports histohematins in Littorina littorea, Purpura lapillus, Trochus cinerarius, Patella vulgata, Limnceus stagnates, Paludina vivipara, Mytilus edulis, Ostrcea edulis, Unio, Anodonta, Limax, Arion, Helix aspersa, and Helix pomatia. In arthropods MacMunn (Philosoph. Trans., 1886, i, 235, 267) deter- mined that the histohematins are the same as those of the echinoderms and molluscs, and he records finding myohematins in Hydrophilis piceus, Dytiscus marginalis, Lucanus cervus, Periplanate orientalis, Bombus terratus, Apis mellifica, Cerarnbyx moschatus, Creophilis maxillosus, Carabus violaceus, Coccinella bipunctata, Staphylinus olens, Geotropes stercorarius, Gryllus domesticus, Tipula oleracea, Musca domestica, Musca vomitoria, Musca Mora, Vespa vulgaris, Acrida viridissima, Pieris rapce, Epeira diadema, Tegenaria civilis, and others. In dipterous, hymenopterous, and lepi- dopterous insects he made the interesting observation that those which use their wing muscles actively have the greatest amount of myohematin in these structures. The presence of histohematins was found to be well marked in the Crustacea, of which he examined Homarus vulgaris, Cancer pagurus, Carcinus moenas, Astacus ftuviatilis, and Pagurus bernhardus. Among Vermes, in Lumbricus and Hirudo all organs which in other species show histohematin spectra appear to contain a small amount of hemoglobin, which he believes functionates in a similar manner to the histohematins. 4 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES Mosely (quoted by MacMunn) described polyperythrin, which Mac- Munn regards as being identical with hematoporphyrin, in Ceratotrochus diadema, Flabellum variable, Flabellum sp.?, Fungia symmetrica, Stephano- phyllia farmosissima, Stephana phyllia sp.?, Actinia with a coriaceous test and in Discosoma sp.?, and also in Cassiopea (a rhizostomean acaleph). ECHINOCHROME, HEMERYTHRIN, AND CHLOROCRUORIN, ETC. Echinochrome, hemerythrin, and chlorocruorin are very close to hemo- globin. Delle Chiaje (Memoria sulla storia e notomia degli animali senza vertebre del regno di Napoli, i, 33, 127; MacMunn, Quar. Jour. Microscop. Science, 1885, xxv, 476) noted in Sipunculus balanorphus and Echino- rhynchus that the arterial blood is red and the venous blood brown. Schwalbe (Archiv f. mikros. Anat., 1869, v, 248) describes the body fluid of Phascolosoma elongatum as being a bright rose or grayish-red color which became darker and darker until it was of an intense Burgundy-red. A similar or identical coloring matter was found by Krukenberg (loc. cit.} in the blood of Sipunculus mtdus, who found that the change of color was due to oxidation and that C02 restored the original color. The coloring matter, which he termed hemerythrin, he found was decomposed by H2S, and that the O seemed to be more firmly combined than in oxyhemoglobin. Geddes (Gamgee's Physiological Chemistry, 1880, 134; Proc. Roy. Soc., 1880) also observed the color changes in the body fluid of echinoderms upon exposure to the air. The most important investigations of this coloring matter, or of what are closely identical substances, were made by MacMunn (Proc. Birming- ham Philosoph. Soc., 1883, in, 380; Quar. Jour. Microscop. Science, 1885, xxv, 482), who found in various parts of the body, and in the peri visceral fluid, of Echinus (esculentus?) and Sphcera a brown coloring matter which deepened in color upon exposure to the air and which reacted to reducing agents. In the later article he reports studies of the coloring matter of the perivisceral fluid of Strongylocentrotus lividus, which he found is capable of existing in two states of oxidation, and which therefore was regarded by him as being respiratory. To it he gave the name echinochrome. He states that it differs from the blood pigments of most invertebrates, and that it can be obtained in solution by two methods : (a) The fresh blood clot can be extracted by water or by alcohol in which it is partially soluble, or by glycerin, ether, chloroform, benzine, bisulphide of carbon, or petroleum ether, in which it is more soluble, the extract upon evaporation yielding an amorphous precipitate. (b) The clot can be separated from the serum by filtering, the clot dried at room temperature, and then extracted with one of the solvents in a test-tube in the dark. The latter method gives the better results. MacMunn in other articles (Philosoph. Trans. Royal Soc. London, 1886, i, 267; Journal of Physiology, 1886, vn, 240) records the presence of various pigments in the tissues of echinoderms, certain of which he identified as hematoporphyrin, hemochromogen, or other bodies very close to hemo- globin. IN THE ANIMAL KINGDOM. An elementary analysis of echinochrome was made by Griffiths (Compt. rend. soc. biol, 1892, cxiv, 419, 669, 738). This substance he obtained from Echinus esculentus, Strongylocentrotus lividus, Echinus sphcera, etc. He showed its very close relationship to hemoglobin, and he gives the follow- ing as the molecular formula: Griffiths also analyzed hemerythrin, and gives to it the formula Robert (Archiv f. ges. Physiologic, 1903, xcvin, 411) states that the hemerythrin from the corpuscles of Sipunculus nudus contains the iron, unlike in hemoglobin, in loose combination. He failed to obtain hemin crystals, hemochromogen, or hematoporphyrin by means of the ordinary processes. H202 was decomposed by it, but he did not find any blue color- ation with guaiac. The chlorocruorins, which from the molecular formula of Griffiths are more closely related to hemoglobin than either echinochrome or hemerythrin, have been studied by a number of investigators. The green coloration of the blood of certain annelids was first pointed out by Milne-Edwards (Ann. des Sciences Natur., 1838, x, 190) and later by Quatrefages (quoted by Ray Lankester, loc. cit.) in Siphonostoma. Krukenberg (Vergleich. physiol. Studien, 1 Rh., 3 Abth., 1882, 87) noted this green pigment in Spirographis and Branchiomma. Ray Lankester (loc. cit.} studied the chloro- cruorins of Siphonostomum and Sabella. He found that, like hemoglobin and oxyhemoglobin, there exist chlorocruorin and oxychlorocruorin, which show different absorption spectra; and he states his belief that hemoglobin and chlorocruorin have a common base in a so-called cyansulphaem (an undetermined body), or perhaps in Stokes's reduced hematin. MacMunn (loc. cit.} subsequently studied the optical properties of oxy- cruorin and reduced cruorin. The green fluid of Sabella, he found, had a reddish tinge with reflected gaslight, and in most cases it was green with transmitted daylight and reddish with transmitted gaslight. On dilution with water the solution gave two bands: the first between C and D from a 618 to a 593 ; and second between D and E from 2, 576 to ^ 554.5. On then adding ammonium sulphide the first of these extended from X 625 to X 596, but it and also the second bands were very faint. "If now caustic soda were added to this solution a dark band was seen covering D, which recalls to mind the band of alkaline hematin, and this band extended from % 595 to X 576." He also studied the blood of Serpula contortuplicata, which he found presents some resemblance to that of the Sabella. An aqueous solution obtained from 9 serpulse was of a reddish-yellow color by gaslight and yellow by daylight. The band before D was from /I 620.5 to /t 593, the second about A 583.5 to A 572, the third uncertain (about a 551 to A 532). After adding sulphide of ammonium the only band seen with certainty was that before D, which seemed slightly nearer the violet. 6 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES Griffiths (loc. cit.) gives the elementary composition of chlorocruorin as and the empirical formula as RESPIRATORY METAL-FREE COLORLESS PROTEINS. The absence of colored respiratory substances from certain of the invertebrates has been noted by a number of observers. Colored respira- tor}' pigments in such animals, except in certain of the very lowest forms, are doubtless represented by those without color. Griffiths (loc. cit.) has described several colorless metal-free proteins which through their behavior towards 0 and CO2 are to be regarded as being respiratory, and which he believes are widely distributed among the invertebrates. From the blood of Patella vulgata he states he obtained a colorless globulin which he dis- tinguishes as a-achroglobulin. He gives to it the formula C523H761N196S014o» and states that 100 grams at 0° and 760 mm. combine with 132 c.c. of O and 315 c.c. of C02. Its rotatory power in dilute magnesium sulphate solu- tion he found to be [a]D= -48°. From Chiton he obtained another form of respiratory globulin which he designates /3-achroglobulin, to which he ascribes the formula Its combining capacity for 100 grams he determined to be at 0° and 760 mm., 120 c.c. of 0 and 281 c.c. of C02. In dilute magnesium sulphate solution, its rotatory power was [a]D = - 55°. TABLE 1. — The achroglobulins of Griffiths, and their empirical formulas, oxygen capacities, and rotatory powers. Source. Variety of achroglobulia. Formula. Capacity per 100 grama. Rotatory power. Patella vulgata. . . Chiton Tunicates «-achroglobulin /3-achroglobulin CsasHreiNigeSO^o C62iH8i4N175S0169 C.C. 132 120 149 o -48 -55 — 63 Doris ^-achroglobulin p721S916ia194SX183 125 — 54 A third form, distinguished as y-achroglobulin, he prepared from the blood of tunicates (Ascidia, Mogula, Cyanthia). To this he gives the formula C72iH915N194S0183. Its rotatory power he found to be [a]D = - 63°. Its 0-capacity was 149 c.c. per 100 grams at 0° and 760 mm. It also com- bined with methane, CO, and acetylene. A fourth form, i5-achroglobulin, he obtained from the mollusc Doris. The formula he gives as C659H792N165SO153 and the combining capacity for O as 125 c.c. and the rotatory power as [a]D = ."> 1 . This globulin combines with methane, acetylene, and ethylene to form yellowish, greenish, and brownish compounds, respectively, which are dissociable in vacuum. Similarities and dissimilarities of composition, 0-capacity, and rotatory power are shown in table 1. It is of particular interest to note that the O-capacities compare most favorably with the 0-ca acitv of hemoglobin (134 c.c. — Hiifner). EN THE ANIMAL KINGDOM. 7 THE DISTRIBUTION OF HEMOCYANIN. Hemocyanin is closely allied to hemoglobin, physiologically and chem- ically, but it is farther removed chemically than the histohematins, echino- chrome, chlorocruorins, and the colorless respiratory proteins referred to. It is distributed solely among the invertebrates and, like hemoglobin and the allied bodies mentioned, in an erratic and as yet inexplicable way; but unlike hemoglobin it is found solely in the blood, and as far as known there are no closely related bodies in the form of compounds or deriva- tives, except oxyhemocyanin, that represent or supplement it in various body tissues and fluids. It has the respiratory function of hemoglobin, but chemically it is not nearly so closely related as chlorophyl, as will be seen by the context. Hemocyanin is colorless, and it is the analogue of reduced hemoglobin; while oxyhemocyanin is blue, and is the analogue of oxyhemoglobin, the oxygen being loosely bound as in oxyhemoglobin, but not so readily displaced. The blue color of the blood of certain invertebrates was first observed by Ermann, in 1816, in the pulmogasteropod Helix (Abhandl. d. k. Akad. d. Wissensch. z. Berlin, 1819, 199) ; he described it as an opalescence. A few years later Carus (Von d. aussern Lebensbedingungen d. weiss- u. kaltblut. Thiere, Leipzig, 1824, 85) noted the blue color of the bloods of Helix and the crayfish Astacus. Harless and von Bibra (Archiv f. Anat. u. Physiologic, 1847, 148) examined the blue bloods of Eledone, Sepia, Cancer pagurus, and Helix pomatia. They studied the influences of exposure to the atmosphere, O, N, and C02, and they also made elementary analyses. They found that when the colorless blood of Helix was exposed to the air it became blue, and that it became colorless when exposed to an atmosphere of C02; but they state that the bloods of the cephalopods Eledone and Loligo are affected in the opposite ways by these gases, becoming blue upon exposure to C02 and colorless when exposed to O, which, however, has since been shown to be incorrect. Ammonia, they found, removed the blue color, which reappeared upon neutralization with hydrochloric acid. In their analysis of the coloring matter of the blood of Helix they found £45-79^5. 05^13-23035-93 and also copper, but no iron. Genth (Annalen d. Chemie u. Pharmacie, 1852, LXXXI, 68) found that the blood of Limulus cyclops became blue upon exposure to the air, and that it contained both copper and iron. In his analyses of the ash he found in one case 0.081 per cent of oxide of iron and 0.085 per cent of oxide of copper; and in another only a trace of iron and 0.297 per cent of oxide of copper. Haeckel (Archiv f. Anat. u. Physiologic, 1857, 511) observed that the colorless blood of Homola cuvieri upon withdrawal from the animal became gradually gray, and finally an intense blue; and that the bright bluish blood of Homarus became after many hours a dark violet. Witting (Jour, f. pract. Chemie, 1858, LXXIII, 121) refers to the bluish tinge of the blood of Unio pictorum. He also examined the blood of Astacus, but failed to 8 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES note a blue coloration which was subsequently found by Krukenberg (loc. cit.) in specimens of the same species. Rouget (Jour, de la Physiologic, 1859, 660) in his studies of the colored corpuscles of the bloods of tunicates and 'Adinozoa found scarlet, orange, yellow, blue, and violet corpuscles. The bluish blood of Sepia was noted by Bert (Compt. rend. soc. biol- ogie, 1867, LXV, 300). He showed that the color belongs to the plasma, and 'that it was intensified by exposure to the air, and not destroyed by boiling. Color changes of the same kind were recorded by Rabuteau and Papillon (Compt. rend. soc. biologic, 1873, LXXVII, 135) in the bloods of Octopus and crabs./ They observed not only the effects of the air, but also (of C02, and they discovered the fact that the blue blood does not give spectral absorption bands.,. The statement by Genth that the blue blood of Limulus contains copper received confirmation in the analyses of the blue blood of Helix by Gorup-Besanez (Lehrbuch d. physiologischen Chemie, 1878, 379), and by those of Miiller and Schlossberger (quoted by Gorup- Besanez), who found copper in the bloods of Sepia and Octopus. Jolyet and Regnard (Archives de Physiologic, 1877, XLIV, 584) found two coloring matters in the blood of crabs, one blue and the other reddish, the former (hemocyanin) being precipitated by alcohol, while the latter (tetronerythrin) remained in solution. They found that agitation of the blood with air developed an ultramarine blue as seen by reflected light, and a brownish coloration as seen by transmitted light, and that upon the removal of O by the gas-pump the blood became rosy and finally yellowish, and that upon shaking the blood with 0 the blue color was restored. In opposition to the statement of Harless and von Bibra (loc. cit.) that CO2 caused the blood to become blue, they found it to be without influence. They studied the percentages of O, N, and C02 (free and combined) in the bloods of crabs, and noted the low capacity for O. In the blood of Astacus they found 3.5 per cent; in the common crab, 3 to 3.2 per cent; and in Pagurus, 2.4 to 4.4 per cent of 0. They were the first to suggest that the blue color- ing matter is in protein form. The bloods of Scorpio and Limulus were found by Ray Lankester (Quar. Jour. Microscop. Science, 1878, xxi, 453; 1881, xxiv, 151) to become blue upon exposure to the air. Tetronerythrin seems to be widely distributed: Merejkowski (Compt. rend. soc. biologic, 1881, xcni, 1029) found it in 104 species, and this list has been largely increased by the investigations of others. The term hemocyanin we owe to Fredericq (Bull, de 1'Acad. roy. de Belgique, 1878, xvi, 4; Compt. rend. soc. biolog., 1879, LXXXVII, 996), who definitely showed in the blood of Octopus that the copper, to which the blue coloration is due, is in combination with protein. He recognized the analogy between hemoglobin and its oxide and hemocyanin and its oxide, and that the copper in the hemocyanin molecule plays a similar role to that of the iron of the hemoglobin molecule. He noted that the venous blood was colorless and the arterial blood blue, and that the latter becomes decolor- ized as the weakly combined O is withdrawn in vacua or driven off by C02 or H2S, and that the color is restored by O. The statement of several previous observers that hemocyanin does not give absorption bands was IN THE ANIMAL KINGDOM. 9 confirmed. In a subsequent research (Compt. rend. soc. biologic, 1897, xvn, 47) he proved the identity of the blue coloring matter of the bloods of Octopus and Homarus, and in the latter he found two coloring matters corresponding to those described by Jolyet and Regnard (loc. cit.) in the blood of crabs. He confirmed their observations of the behavior of hemo- cyanin towards reflected and transmitted light, and he showed that the reddish coloring matter (tetrorierythrin) takes no part in the change of color caused by oxygenation and deoxygenation. Hemocyanin was found by Fredericq in the bloods of the gasteropods Arion and Helix. The list of animals whose blood contains hemocyanin was materially added to by the investigations of Krukenberg (Vergleich. physiol. Studien, 1 Rh., 3 Abth., 1880, 66; 1881, 49; 1882, 87, 182; Centralb. f. med. Wis- sensch., 1880, vni, 417), and he added information regarding the behavior of hemocyanin towards C02, CO, and H2S, and especially in the direction of indicating the existence of several forms or modifications of hemocyanin which has since received support by the investigations of Howell (p. 10), Cue"not (p. 12), and Couvreur (p. 13). He showed that the blue blood of two cephalopods (Eledone moschata and Sepia officinalis) and of a number of species of crabs (Homarus vulgaris, Carcinus moenas, Eriphia spinifrons, Portunus depurator, Graspus marmoratus, Maia verrucosa, Pilumnus villo- sus, and Squilla mantis) became more or less intensely blue upon agitation with air or oxygen, and more or less decolorized by shaking with CO2. He found that the blood of Limna>us stagnalis was scarcely affected by shaking with CO2, and he believes that in this species, and also in Helix pomatia and aspera, the coloring matter exists as a body very closely related to hemocyanin. Marked differences were noted in the degree of coloration of the blood and fixity of the O. The blood of Portunus depurator was a very light blue, while the bloods of Homarus, Eriphia spinifrons, and Squilla mantis were a deep indigo blue. In the gasteropod molluscs, crabs, and cephalopods, he noted such differences in the behavior of the hemo- cyanin towards O as to lead him to the belief that this gas is in firmer com- bination in crabs and cephalopods than in molluscs. He also compared hemocyanin and hemoglobin in their behavior towards certain gases. He found, for instance, that after decolorization of the blood with C02 the original color was restored by shaking with air, and that when subjected to S02 or sulphide of ammonium the blood of crabs and Eledone became yellowish, and that the color could not be restored by agitation with 0, both of which are the opposite to the behavior of oxy hemoglobin. He failed to find any evidence of the presence of hemocyanin in the blood of a number of molluscs. Little of importance was added to our knowledge of this important substance during the following decade. Gotch and Laws (British Asso- ciation Reports, 1884; quoted by Lankester, loc. cit.) found hemocyanin or a body closely identical with it in Limulus polyphemus; Halliburton (Journ. Physiology, 1885, vi, 300) reported hemocyanin in the crustacean Nephrops, and he gives the following list of animals in which hemocyanin, hemoglobin, chlorocruorin, hemerythrin, chlorophyl, and tetronerythrin 10 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES have been found in the blood, parentheses or brackets. A. HEMOCYANIN. Crustacea: Homarus (Fredericq). Astacus (Krukenberg). Cancer (Krukenberg). Carcinua (Jolyet and Regnard). Nephrops [Halliburton], Eripliia (Krukenberg). Squilla (Krukenberg). Maia (Krukenberg). Arachnida: Scorpio (Lankester). Limulus (Laakester). Gasteropoda: Cassidaria (Krukenberg). Fissurella (Krukenberg). Haliotis (Krukenberg). Helix (Fr&lericq). Murex (Krukenberg). Turbo (Krukenberg). Cephalopoda : Octopus (Frddericq). Sepia (Krukenberg). Eledone (Krukenberg). Loligo (Krukenberg). B. HEMOGLOBIN. Vertebrata: In special corpuscles in all except Amphi- oxus (Lankester). Leptocephalus (Lankester). Crustacea: Daphnia (Lankeater). Chirocephalus (Lankester). Apus (Regnard and Blanchard). Lernanthropus (Van Beneden). Clavella (Van Beneden). Cypris (Regnard and Blanchard). Marine parasitic crustacean, undescribed (Van Beneden). Insecta: Chironomus (Lankester). Musca domestica (MacMunn). Mollusca: Planorbis (Lankester). Area (Lankester). Solen (Lankester). Chsetopoda: Lumbricus (Lankester). Eunice (Lankester). Cirrhatulus (Lankester). Nereis (Lankester). Terebella (Lankester). Tubifex (Lankester). Arenicola (Lankester). The name of the authority is given in B. HEMOGLOBIN. — Continued. Chsetopoda — Continued. Limnodrilus (Lankester). Lurabriculua (Lankester). Nais (Lankester). Chatogaster (Lankester). Glycera (Lankester). Capitella (Lankester). Euchytrachus (Lankester). Aphrodite (MacMunn). Gephyrea: Phoronis (Lankester). Thallasena (Lankester). Hamingia (Lankester). Nemertina: Folia (Lankester). Other nemertinea (Hubrecht, 1875). Hirudinea: Nephilis (Lankester). Hirudo (Lankester). Echinodennata: An ophiurid (Foettinger, 1880). In all invertebrates hemoglobin occurs in solution in the blood plasma, ex- cept in Solen, Glycera, Capitella, Phoronis, where it is contained in special corpuscles. C. CHLOROCRUORIN. Chsetopoda: Siphonostomum (Lankester). Sabella (Lankester). Chloronema (Quatrefages). D. HEMERTTHRIN. Gephyrea: Phascolosoma (Schwalbe). Sipunculus (Krukenberg). Phoronis (Krukenberg). E. CHLOROPHYL. Insecta: Various butterflies and moths (Poulton) F. TETRONERYTHRIN. Crustacea: Homarus [Halliburton]. Carcinus [Halliburton]. Astacus [Halliburton]. Nephrops [Halliburton]. G. Various colored granules are described in the corpuscles of Holothurians and Sea-urchins (Geddes). The blood of Patella is described as being of an orange color (Krukenberg). This list has been increased by subsequent communications, as will be seen by the context. Ilowell (Studies from the Biological Laboratory, Johns Hopkins University, 1884, in, 284) studied the hemocyanin of the blood of Limulus polyphemus, Callinectes hastatus, and Cucumccria sp.?, and in comparing the condition of the respiratory oxygen he found the O to be in more stable combination in the first, and also that the hemocyanin of this organism coagulates at higher temperature. MacMunn (Quart. Jour. Microscop. Science, 1885, xxv, 469) in his studies of the chromatology of the bloods of certain invertebrates, chiefly of the spectroscopic characters, states that IN THE ANIMAL KINGDOM. 11 the blood of Helix aspersa is bluish-white by daylight and of a purplish tinge by gaslight; he found that the blood of Limnceus stagnalis on exposure to the air assumed a bluish-white color and that the blood of Paludina vivipara is of a blue color. None gave absorption bands. Griffiths (Compt. rend. soc. biol., 1892, cxiv, 496, 840, 1277; cxv, 669) made elementary analyses of the hemocyanin of the bloods of Homarus, Sepia, and Cancer, which he obtained by precipitating with magnesium sulphate, dissolving the precipitate in water, again precipitating with alcohol, and finally drying in vacuum at 60°. Fre'dericq, Krukenberg, and Henze state that magnesium sulphate causes either little or no pre- cipitation of hemocyanin. This statement is opposed by Griffiths, Couv- reur (Compt. rend. soc. biologic, 1902, LIV, 125), and Halliburton (Journal of Physiology, 1885, vi, 300). The latter states that "just as precipitation by heat is slow, so is also precipitation with salts ; to effect complete satura- tion with either of the above-mentioned salts (magnesium sulphate or sodium chloride) the serum must be shaken in an engine for 12, 24, and in some experiments 36 hours, with the finely powdered salt." Probably the difference in the results of Griffiths, Couvreur, and Halliburton from those of Fre'dericq, Krukenberg, and Henze may be explained by differ- ences in species and freshness of the blood and other incidental conditions. Griffiths' analyses showed the mean being C54-155H7. 095Ni6-268So-647 The empirical formula he calculated to be Interesting in this connection is his analysis of the brown coloring matter of the blood of the lamellibranch Pinna squamosa. This substance he describes as a body very closely related to hemocyanin, but in which there is manganese instead of copper. Krukenberg (loc. cit.) had already found that the blood of this animal was rich in manganese. Griffiths named this pigment pinnaglobulin. His elementary analysis showed C55-07H6-24Ni6-24So-8l021- and the empirical formula he calculated to be In the blood-ash of Pinna he found manganese but not iron, while in that of Sabella and Sipunculus he found iron but not manganese. The centesimal analyses and empirical formulas of hemocyanin, pinnaglobulin, echinochrome, chlorocruorin, hemerythrin, and hemoglobin are given by Griffiths as follows: TABLE 2. — Empirical formulas of certain respiratory substances, according to Griffiths. Hemocyanin ........................... Cgs? H^gs N223 Cu 84 025& Pinnaglobulin ......................... C729 H985 Ni8s Mn S4 O2io Echinochrome ......................... €102 HQQ Ni2 Fe 82 Oi2 Hemerythrin .......................... €427 H7ei NJSS Fe 82 ©153 Chlorocruorin .......................... Cgeo Hs45 ^H3 Fe 83 Oie? Hemoglobin ........................... Ceoo H9ao N1M Fe 83 Oi79 12 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES Heira (Compt. rend. soc. biolog., 1892, cxiv, 772) found in decapods that hemocyanin is not the only albuminous substance of the blood, and that dialysis does not yield a pure hemocyanin. He states that the blood of Crustacea does not contain a higher percentage of 0 than pure water, except that of Palinurus vulgaris, which contains about one-third more. He does not look upon the copper as existing in the form of an albuminate. Cuenot (Compt. rend. soc. biolog., 1891, ex, 724) found in Aplysia punctata a colorless hemocyanin (?) that is not colored blue upon exposure to the air, which he regards as being without respiratory function. He likewise found (Compt. rend. soc. biolog., 1892, cxv, 127), as had Jolyet and Regnard (loc. cit.) and others, a low oxygen capacity of hemocyanin. Fredericq (Centralb. f. Physiologie, 1899, xm, 147) in experiments with the blood of crabs showed that hemocyanin is the only protein of the blood that contains copper, that it is coagulated between 65° and 70°, that it is present in the proportion of 4.4 to 3.78 per cent and that the percentage is decreased during fasting. In a previous article (loc. cit.) he gives the coagulation point as 68° to 69°. That the blue coloring matter is a copper compound was reaffirmed by Couvreur (Compt. rend. soc. biolog., 1900, LII, 395), who found that after precipitation of the hemocyanin by mag- nesium sulphate, alcohol, or heat, the filtrate does not contain any copper. The spontaneous decolorization of hemocyanin upon keeping seems, accord- ing to Phisalix (Compt. rend. soc. biolog., 1900, LII, 729), to be due to bacterial action, for he found that this color change may be hindered by chloroform, ether, 10 per cent formaldehyde, or fluoride of sodium, and that the blood of Helix pomatia, if kept antiseptically, retained its blue color for a year. TABLE 3. — Quantities of copper in the blood of certain invertebrates, according to Dheri. Name. Copper in milligrams. Name. Copper in milligrams. 100 c.o. fresh blood. 100 grams dried substance. 100 c.c. fresh blood. 100 grams dried substance. Cancer pagurus Do... 3.5 7.5 13.5 7.5 10.5 11.0 9.5 10.5 4.0 8.0 "0 75 Helix pomatia 7.0 7.5 8.0 11.5 12.5 18.0 18.0 20.0 23.5 175 \* 205 ( 'i53 Do Do Do Palinurus vulgaris. . . Do Do Do Do Octopus vulgaris .... Homarus vulgaris. . . . Do Do Astacus fluviatilis. . Do Do * Hibernating. In studies of the distribution of copper in invertebrates and fish, Dubois (Compt. rend. soc. biolog., 1900, LII, 392; 1903, LV, 1161) found that the proportions are very variable, not only in different species and individuals, but also in different organs. Ascidians are very poor in copper, and fish contain less than invertebrates. In the blood of Palinurus vulgaris he found 22.97 mg. of copper per 100 grams, and in muscle 4.47 mg. The egg was copper-free. In the blood of Helix pomatia there were 24.39 mg. IN THE ANIMAL KINGDOM. 13 per 100 grams. Coincident with the appearance of Dubois's first article, Dhere (Compt. rend. soc. biolog., 1900, LII, 458) reported the results of his investigations of the quantity of copper in the blood of certain invertebrates and the respiratory capacity of hemocyanin. His analyses were made with 10 c.c. of fresh blood in each case, and he gives the figures shown in table 3. It will be noted that the copper content varied much, not only in different species, but also in members of the same species. He also found that the intensity of blue coloration is in relation to the quantity of copper. The respiratory capacities in relation to the quantity of copper and hemo- cyanin are shown in table 4. TABLE 4. — Respiratory capacities in relation to the quantity of copper and oxygen in the blood, according to Dhere. Name. 100 grams blood. Remarks. Helix poniatia 17° 1.45 c.c. O 19° 2.2 17° 3.0 18.5° 3.1 22° 2.4 18° /4'2 \3.9 18° { ' is° { i;6 18° | ' 6.5 mg. Cu. 11.5 9.5 10.5 8.0 28.5 23. 8.5 10.0 5.5 7.0 3.0 4.5 Blood filtered. Do. Blood defibrinated and filtered. Do. Blood with fluoride filtered. Do .. Homarus vulgaris Do Astacus fluviatilis . Octopus vulgaris . . . Carcinus moenas . Cancer pagurus . ... Maia. scjuinado .... Couvreur (Compt. rend. soc. biolog., 1900, LII, 395), in his studies of coagulative and other phenomena of the blood of the snail, states, in opposi- tion to Heim (loc. cit.) and in support of Jolyet and Regnard (loc. cit.), that hemocyanin should be looked upon as being a combination of copper with protein. In a later article (ibid., 1902, LIV, 125) he made compara- tive studies of the bloods of certain marine gasteropods (Murex brandaris, Murex trunculus, and Tritonium nodiferum) with the blood of the snail. The hemocyanin of these bloods, like that of the snail, was precipitated by saturation with magnesium sulphate. He also notes that the hemocyanin of marine gasteropods seems to be more stable than that of the snail. Couv- reur and Rougier (ibid., 1902, LIV, 1476), in comparing hemocyanin and hemoglobin, note that hemoglobin is not broken down by putrefactive processes while hemocyanin is, and that the normally blue blood becomes dark and forms an insoluble product of a dark tint which is a derivative containing copper. Dhere (ibid., 1903, LV, 1161) kept the bloods of Octopus vulgaris, Cancer pagiirus, Carcinus mcenas, and Maia squinado in sealed tubes for three years. The fluids were discolored, and upon being shaken with air only the blood of Octopus became blue, while that of the crustaceans became a slate-gray. He also made determinations of the percentages of copper. In other articles Dhe"r6 (ibid., 1903, 1012, 1338) and Couvreur (ibid., 1247) discuss the effects of dialysis, heat, alcohol, etc., on hemocyanin. Henze (Zeit. f. physiolog. Chemie, 1901, xxxni, 370) found in the hemo- cyanin of Octopus vulgaris 0.38 per cent of copper. The blood contained 14 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES 9 per cent of hemocyanin, which would give 34.2 mg. of copper per 100 c.c. of blood. According to Henze, 1 gram of hemocyanin combines with 3 to 3.7 per cent of 0, or about 0.4 c.c., which is about one-fourth the capacity of hemoglobin. Henze was the first to obtain crystals of hemocyanin. These he prepared according to the Hofmeister (Zeit. f. physiolog. Chemie, 1892, xiv, 165) and the Hopkins-Pinkus methods (Journal of Physiology, 1898- 99, xxm, 130) for obtaining crystals of egg albumin. In following out the first method the blood of Octopus vulgaris was centrifugalized and filtered, and the filtrate mixed with a sufficient amount of ammonium sulphate to cause a slight precipitation. The solution was placed in shallow vessels, when upon evaporation clusters of little needles were deposited. Much better results were obtained by the Hopkins-Pinkus method: Ammonium sulphate was added to the centrifugalized blood until the appearance of cloudiness; this slight precipitate was dissolved by the addition of distilled water; acetic acid was then added until the appear- ance of cloudiness. After standing for half a day the precipitate increased very much and settled to the bottom. A crystalline mass formed at the bottom of the vessel and consisted of excellently formed, doubly refrac- tive, microscopic prisms. There appeared in two preparations egg-shaped leaflets, some of which were 3 to 4 mm. in diameter, but which were difficult to isolate from the crystalline mass owing to their fragility. Although Henze found it possible to recrystallize hemocyanin, the second recrystal- lization was rendered impure because of amorphous admixtures. He also noted that crystallization took place perfectly only when fresh blood from healthy animals was used. The mean elementary analysis he gives is Csa-eeHr-ssNie.ogSo.seCuo.sgC^i-e? (Compare with Griffiths's analysis of the amorphous hemocyanin of Sepia, Homarus, and Cancer, p. 11.) In this article he reports studies of the manner in which the copper is in combination in the protein molecule, of the reactions, and of the O-capacity of hemocyanin, etc. He states that hemocyanin behaves in certain ways differently from hemoglobin, that the copper is in loose combination, that the substance behaves like a copper albuminate, and that the O-capacity is only about one-fourth that of hemoglobin. In a later contribution (Zeit. f. physiolog. Chemie, 1904-05, XLIII, 290) Henze reports that hemocyanin can not be separated into a protein and a component free of protein, as in the case of hemoglobin, and that it behaves like a copper albuminate from which the masked copper can easily be separated. By hydrolysis he found tyrosin, leucin, histidin, lysin, prob- ably glutaminic acid, and possibly a minimal amount of arginin. He failed to find evidence of a carbohydrate group, although the fresh blood of Octopus gave a positive reaction with Molisch's test. In determining the distribution of its N he found the following: as mono-amino-N, 10.20 (63.39 per cent) ; diamino-N, 4.45 (27.65 per cent) ; humin-N, 0.43 (2.67 per cent); and ammonia-N, 0.93 (5.78 per cent). IN THE ANIMAL KINGDOM. 15 Robert (Archiv f. ges. Physiologic, 1903, xcvm, 411), in experiments with the blood of Eledone moschata, confirmed Henze's statement of the loose combination of the copper in the form of a copper albuminate, and also the differences in the chemical behavior of hemocyanin in comparison with hemoglobin. He found that CO, for instance, does not form a com- pound similar to CO-hemoglobin, but seemingly a cyanhemocyanin, and that while hemocyanin decomposes H2O2 it does not cause a bluing of guaiac solution. He found that from hemocyanin neither hematin nor hematoporphyrin could be obtained, and he states that on this account there can not be a close chemical relationship between hemocyanin and hemoglobin. He writes that crystals from Eledone blood were examined by Prof. O. Leudecke, who states "that they are optically uniaxial and positive, apparently hexagonal." Robert reports that hemocyanin is absent from the blood of Aplysia limacina, but present in Maia verrucosa. THE DISTRIBUTION OF HEMOGLOBIN IN THE INVERTEBRATES. Hiinefeld (Der Chemismus in der thierischen Organization (prize essay), Leipzig, 1840, 160) discovered hemoglobin crystals in the blood of the common earthworm that had been placed between plates of glass. Rollett (Sitz. d. k. Akad. d. Wissensch., Wien, 1861, XLIV, 615) identified the blood crystals of the earthworm and those of the insect Chironomus with those of vertebrates. Nawrocki (Centralblatt f. Wissensch., Feb. 8, 1867, xv, 195) and Ray Lankester (Jour. Anat. and Physiology, 1867-68, n, 114) also identified the substance of the blood crystals of invertebrates with the hemoglobin of vertebrates. The latter, in a series of articles (Jour. Anat. and Physiology, 1867-68, n, 114; 1869-70, iv, 119; Archiv f. ges. Physiol- ogic, 1871, iv, 315; Proceedings Royal Soc. London, 1872-73, xxi, 70), studied the area of distribution of hemoglobin in the animal kingdom, especially in the invertebrates. In his earlier articles he notes that he detected hemoglobin in the non-corpuscular saccular fluid of annelids Lumbricus, Eunice sanguinea, and Hirudo; in the plasma of the blood of the larva of the insect Chironomus plumosus; in the plasma of the blood of the mollusc Planorbis corneus and of the crustacean Chirocephalus diaphanus; and also in larvae allied to Chironomus. In the last article he takes exception to the statement of Preyer that hemoglobin is found in all vertebrates, and he shows the absence of hemoglobin from the Leptocephalus, which pos- sesses corpuscles corresponding to erythrocytes, which animal is perfectly colorless and glass-like except the black-pigmented eye. He also failed, after repeated attempts with the spectroscope, to find evidence of hemo- globin in the Amphioxus, although, as he states, Wilhelm Miiller, of Jena, found that this vertebrate has corpuscles of a pale red color. The facts ascertained as to the distribution of hemoglobin (and myohematin) Lan- kester summarizes as follows : (1) In special corpuscles: (a) In the blood of all vertebrates, except Leptocephalus and Amphioxus (?). (6) In the perivisceral fluid of some species of the vermian genera Glycera, Capitella, and Phoronis. (c) In the blood of the lamellibranchiate mollusc Solen legumen. 16 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES (2) Diffused in a vascular or ambient liquid: (a) In a peculiar vascular system of the chstopodous annelids very generally, but with apparently arbitrary exceptions. (6) In the vascular system (which represents a reduced perivisceral cavity) of certain leeches, but not of all (Nephelis, Hirudo). (c) In the vascular system of certain turbellarians, as an exception Polia. (d) In a special vascular tissue (distinct from the general blood system) of a marine parasitic crustacean (undescribed) observed by Prof. Edouard van Beneden. (e) In the general blood system of the larva of the dipterous insect Chironomus. (f) In the general blood system of the pulmonate mollusc PlanorUs. (a) In the general blood system of the crustaceans Daphnia and Chirocephalus. (3) Diffused in the substance of the muscular tissue: (a) In the voluntary muscles generally of Mammalia, and probably of birds, and in some muscles of reptiles. (b) In the muscles of the dorsal fin of the fish Hippocampus, being generally absent from the voluntary muscular tissue of the fish, (r) In the muscular tissue of the heart of Vertebrata generally. (d) In the unstriped muscular tissue of the rectum of man, being absent from the unstriped muscular tissue of the alimentary canal generally. (e) In the muscles of the pharynx and odontophore of gasteropodous molluscs (observed in Limnccus, Paludina, Littorina, Patella, Chiton, Aplysia) and of the pharyngeal gizzard of Aplysia, being entirely absent from the rest of the muscular and other tissues and the blood of these molluscs. See as to Planorbis above (2, /) . (/) In the muscular tissue of the great pharyngeal tube of Aphrodite aculeata, being absent from the muscular tissue and from the blood in this animal, and absent from the muscular tissue generally in all other annelids as far as yet examined. (4) Diffused in the substance of the nervous tissue: (a) In the chain of nerve-ganglia of Aphrodite aculeata. Since Lankester's researches, the list of invertebrates in which hemo- globin exists has been largely increased. Hubrecht (Niederland. Archiv f. Zoologie, 1876, 11, Heft 3; Maly's Jahresbr. ii. d. Fort. d. Thierchemie, 1876, vi, 92) found by spectroscopic examination hemoglobin in the oval blood corpuscles of Drepanophorus and in the red brain ganglia of nemertean worms which are without colored corpuscles. Krukenberg (loc. cit.) found hemoglobin in Planai'bis and Apus. Van Beneden (Zoologischer Anzeiger, 1880, in, 55) reports hemo- globin in Planorbis corncus, in sea-water gasteropods, in tunicates, in para- sitical copepods (Lernanthropus and Clavelld), and in an undescribed parasitic crustacean. Foettinger (Archiv d. Biologie, 1880, i, 405) discovered hemoglobin in an ophiuridean echinoderm Ophiactus virens. Regnard and Blanchard (Compt. rend. soc. biolog., 1883, xcvn, 197) found in the blood of certain phyllopods (Apus productus and Cancriformis, and probably Branchipus), of Cladocera (Daphnia} and Ostracoda (Cypris), that the hemoglobin is dissolved in the plasma. Howell (Studies from the Biological Laboratory, Johns Hopkins University, 1884, in, 284) found hemoglobin in the blood of a holothurean echinoderm (Thyonella gemmata). IN THE ANIMAL KINGDOM. 17 MacMunn (Proc. Birmingham Philosoph. Soc., 1883, in, 385) observed hemoglobin in lamellibranchs, leeches, a turbellarian, and insects. In a later article (Quar. Jour. Microscop. Science, 1885, xxv, 469) he reports hemoglobin in Lumbricus, Arenicola, and Eunice. Eisig (Die Capitelliden; Maly's Jahresber. ii. d. Fort. d. Thierchemie, 1887, xvn, 336) in studying the bloods of a group of annelids (Capitella) obtained hemoglobin crystals, mostly in the form of four-sided prisms or rhombic plates, some of which were very large. He used methods that are employed to obtain blood crystals from the higher animals. Sometimes crystallization took place spontaneously, intraglobular and extraglobular, and more abundantly in Dasybranchus caduceus than in the other species. He also found evidence of hemoglobin derivatives. While the hemoglobin of invertebrates and vertebrates had been identi- fied microscopically and spectroscopically, Griffiths (Proc. Roy. Soc. Edinburgh, 1891 ; Physiology of the Invertebrates, 1892, 147) was the first to show by elementary analyses that the hemoglobins of invertebrates and vertebrates are comparable chemically. The blood of 500 earthworms (Lumbricus terrestris) was treated with benzene, which lakes the blood. The mixture was allowed to stand for 24 hours at 0° C., when it separated into two layers. The one containing the coloring matter was then sepa- rated from the other, and about one-sixth of its volume of pure alcohol was added. After filtration the alcoholic extract was exposed to — 12° C., when red crystals were obtained. These crystals yielded, on analysis, the figures given in table 5, which he compares with those of dog's hemoglobin recorded by Hoppe-Seyler. TABLE 5. — Analyses of crystals from blood of earthworm, compared ivith those of dog hemoglobin. i. II. III. Dog. Carbon 5391 53.85 53.85 7.02 7.10 7.32 Nitrogen 16.17 Sulphur . 041 0.37 0.39 Iron 0.39 0.43 21.84 Velichi (Inaug. Dissert., Berlin, 1900; Centralblatt f. Physiologie, 1900, xiv, 679; Deutsch. med. Wochenschr., 1900, xxvi, Juni 21, 148), with the microspectrophotometer, made determinations of extinction coefficients and the percentages of hemoglobin and oxyhemoglobin in the bloods of several annelids (Arenicola piscatorum, Terebella nebrelosa, Lum- bricus terrestris) and certain other invertebrates. In annelids he found the percentage to be similar to that of the frog, namely, 3.465 per cent. When, however, he made his determination by carbonic-oxide hemoglobin he always obtained lower values, only 3.02 per cent. From the blood of Arenicola he prepared hemin crystals. He also states that the hemoglobins of different classes of animals are not identical, because their extinction coefficients differ. He found hemoglobin in the pharyngeal muscles of gasteropods and also in the blood of certain Crustacea. 2 18 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES Dhe-nS (Compt. rend. soc. biolog., 1903, LV, 1162) records by the colori- metric method, in comparing the bloods of the dog and Planorbis corneus, that the oxygen capacity of the latter is the higher— m the dog 1.34 c.c. of 0 per gram (Hiifner) and in Planorbis 1.92 and 2.24 c.c. CAUSES OF THE PECULIARITIES IN THE DISTRIBUTION OF RESPIRATORY PIGMENTS. The extraordinarily erratic manner in which hemoglobin, hemocyanin, and other respiratory pigments are distributed among invertebrates has not unnaturally given rise to inquiries of the reasons. Ray Lankester (loc. cit.) from such an investigation writes as follows: From a consideration of the facts with regard to the mode of occurrence and dis- tribution of hemoglobin in animal organisms, the following general statements may be made, which are in accordance with the now thorough establishment, by chemical inves- tigation, of its peculiar oxygen-carrying property. Hemoglobin is irregularly distrib- uted throughout the animal kingdom, being absent entirely only in the lowest groups.* It may be present in all the representatives of a large group, with but one or two excep- tions, or it may be present in only one out of the numerous members of such a group; or, again, it may be present in one and absent in another species of the same genus. It may occur in corpuscles in the blood, or diffused in the liquor sanguinis, or in the mus- cular tissue, or in the nerve tissue. The same apparent capriciousness characterizes its occurrence in tissues as in specific forms. It may be present in one small group of muscles and absent from all the rest of the tissues of the body, or it may occur in one part only of a tissue, histologically identical throughout its distribution in the organism. The apparently arbitrary character of this distribution is to be explained (though only partially) by a reference to the chemical activity of hemoglobin. Wherever increased facilities for oxidation are requisite, hemoglobin may make its appearance in response; where such facilities can be dispensed with or are otherwise supplied, hemoglobin may cease to be developed. The Vertebrata and the annelids possess a blood containing hemoglobin in correlation with their greater activity as contrasted with the Mollusca, which do not. possess such blood. The actively burrowing Solen legumen alone amongst lamellibranchiate molluscs, and amongst gasteropods only Planorbis, respiring the air of stagnant marshes, possess blood containing hemoglobin. In the former the activity, in the latter the deficiency of respirable gases are correlated with the exceptional devel- opment of hemoglobin. But we can not as yet offer an explanation of the absence of hemoglobin from the closely allied species of Solen, and from Limnaei which accom- pany Planorbis. The crustaceans Chirocephahts and Daphnia, and the larva of Chi- ronomus, possessing, as exceptions in their classes, hemoglobin in their blood, inhabit stations where the amount of accessible oxygen must be small (that is to say, stagnant ponds), the last living in putrescent mud; whilst the possession of abundant hemoglobin in its vascular fluid may be supposed to be one of the chief properties which enables the oligochacte annelid Tubifex to hold its ground in the foul, and therefore much deox- ygenated, water of the Thames at London. The known chemical properties of hemoglobin furnish a more complete explana- tion of its peculiar distribution in tissues. That it should occur in a circulating fluid which is the medium of respiration is obviously related to those properties. Its occur- rence in the voluntary muscles of the most active of Vertebrata, and in the most active muscles of some others (as in the case of the dorsal-fin muscles of Hippocampus), is equally so; so also its occurrence in the most powerfully acting part of the intestinal *It 13 perhaps of some significance that hemoglobin has only been found in that great group of the animal kingdom which in the course of its development gives rise to a middle layer of blastodermic cells or mosoderm, and in examples from nearly every great branch of this stem. IN THE ANIMAL KINGDOM. 19 muscles, those of the rectum, and in the only rapidly and constantly acting muscles of the gasteropods, namely those used in biting and rasping. To connect its occurrence in the nervous chain of Aphrodite aculeata with its prop- erties is more difficult, since we have no knowledge that this annelid is remarkable for nervous energy. The large bulk of the animal in proportion to the size of the nervous system, and the deficient respiration, indicated by the very slightly developed vascular system and the total absence of hemoglobin from the fluids of the worm, may be a reason for the endowment of the nervous center which has to control such a large and compli- cated organism with a special facility for appropriating what little oxygen may come in its way. The complete absence of hemoglobin from Leptocephalus is an example of the submission of an auxiliary, but not an essential, structural attribute to an all-powerful necessity — that of transparency. The absence of hemoglobin from the transparent annelid Alciope may be similarly correlated. MacMunn (loc. cit.) in his studies of the distribution of hemoglobin, myohematins, and histohematins in insects attributes a relationship between the degree of activity of the musculature of the wings and the quantity of coloring matter. Likewise, Velichi (loc. cit.) found hemoglobin in the most used muscles of gasteropods, which are the pharyngeal. In a recent article (Archives de Zoologie experimentale, 1903, 31) on the respiratory pigments in relation to the alkalescence of the blood, Gautrelet states that hemocyanin replaces hemoglobin under the following conditions : (a) if the diet contains Cu instead of Fe; (6) where the exchange of O and CO2 is low (hemoglobin having about 4 times the O-capacity of hemo- cyanin) ; (c) if the salt capacity of the body-fluids is such that erythrocytes can not exist; (d) if a large liver retains the iron. One or more of these conditions may be present, but the appearance of one does not necessarily imply that one or the other of these coloring matters is present. Alkales- cence and the amount of coloring matter he found to be parallel. The distribution of hemoglobin throughout the vertebrates is univer- sal, with the exception of Leptocephalus and possibly Amphioxus, and it is invariably confined under normal conditions to the erythrocytes and the structures in which these corpuscles are formed or destroyed. In the body tissues it appears chiefly in the form of myohematins. A number of com- pounds and derivatives, normal or abnormal, may be present. A large number of coloring matters related and unrelated to hemo- globin have been found in both invertebrates and vertebrates, but a further consideration is not possible within the necessarily limited compass of this memoir. THE SOURCE OF HEMOGLOBIN PROBABLY IN CHLOROPHYL. The close chemical relationship of chlorophyl and hemoglobin suggests either that both have sprung from a common source or that hemoglobin has had its source in chlorophyl, there probably occurring in the latter case a gradual synthesis during the progress of evolution. We find in certain of the lowest organisms a modified form of chlorophyl or chlorophylloid pig- ment; in others chlorophyl; in other forms of life occur coloring matters which are identical or nearly identical with certain hemoglobin derivatives; later, bodies in the form of histohematins and myohematins; and ulti- mately, hemerythrin, chlorocruorin, hemoglobin, etc. 20 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES That hemoglobin may have its source in chlorophyl has been shown by MacMunn, who found in Helix pomatia substances intermediate between chlorophyl and hemoglobin. That substances like hemoglobin derivatives found as constant constituents of certain of the lower organisms do not represent degradation products seems likely, inasmuch as it is hardly conceivable that a molecule so exceedingly complex as hemoglobin should have appeared suddenly. Unfortunately, our data are so insufficient that we can not trace this probable synthesis or possible degradation step by step, and for the same reason we can not show the causes of the extraor- dinary gaps in the distribution of either hemoglobin or its derivative-like bodies in the invertebrates. CHEMICAL NATURE OF TYPICAL RESPIRATORY SUBSTANCES, ETC. We will now consider the functional properties of protein and other components, with especial reference to the probable misconception of the specificity of the role of the iron in the respiratory phenomenon. Specific respiratory substances are doubtless essential constituents of all living organisms, except probably only certain of the very lowest forms of plant and animal life. They may be divided primarily into two groups, metal-bearing and metal-free. The former may contain manganese, copper, or iron, and they are normally, as far as known, colored; the latter are colorless, as the achroglobulins of Griffiths. That undiscovered colorless metal-bearing respiratory substances may exist in some of the lower organ- isms, and even in the higher forms, seems more than probable. That such bodies do exist as abnormal substances (for instance, in the form of de- colorized hemoglobins or some close modification) has been ascertained by a number of investigators. It has been recorded, for instance, that hemoglobin may be crystallized and the crystals completely decolorized without change of form or elementary composition, and that the decolorized substance can even be recrystallized without alteration. Hemocyanin (reduced) is colorless. The chlorocruorins bear, as has been shown, a striking likeness to hemoglobin, spectroscopically and chemically, yet these sub- stances may appear green, yellow, carmine, red, brown, etc. From the foregoing it is obvious that it is not the mere presence of the metal, nor the kind of metal, per se, that gives to the molecule its coloration, but the peculiar arrangement of the atoms or groups of the molecule. That color- less non-metal-bearing respiratory substances do exist has been shown by Griffiths. Entirely apart from this, it must be admitted that the existence of a large number of absolutely or practically colorless organisms, inverte- brate and even vertebrate, in which the interchange of O and C02 in the blood goes on quite actively, clearly indicates that colorless (metal-bearing or non-metal-bearing) respiratory substances must have a wide distribution in animal life. Moreover, it is suspected by the physiological botanist that there may exist colorless plastids which are actively photosynthetic, like the chlorophyllous plastid. The discovery by Griffiths of colorless metal-free respiratory sub- stances in Patella, Chiton, Tunicata, and Doris (which have respiratory IN THE ANIMAL KINGDOM. 21 capacities varying from 1.2 to 1.49 c.c. per gram, or a mean of 1.31, which practically is identical with that of hemoglobin, 1.34) certainly throws grave doubts upon the universally accepted fundamental importance that is attached to the metal (iron, copper, manganese) of echinochrome, hemo- globin, chlorocruorin, hemocyanin, pinnaglobulin, etc. While the work of Griffiths has not, as far as we have been able to learn, been confirmed or disproved, it certainly has substantial support in a number of facts, espe- cially the existence of absolutely or practically colorless invertebrates and vertebrates, in which we must from analogy admit the existence of special- ized respiratory circulatory fluids, and also in the known differences in the behavior of the stromata, globin, and proteins generally, on the one hand, and of hematin, on the other, towards 0 and C02. Since it is admitted that all living protoplasmic structures are respira- tory, it seems but a short step in evolution to the differentiation of special- ized colorless respiratory substances. While it is not improbable that in some of the lowest forms of life simple forms of respiratory substances exist, it seems that (since chloroplastids, histohematins, hemoglobin, hemo- cyanin, or similar compound bodies are found in very low organisms and throughout all gradations of higher animal and plant life, and from their chemistry) we should look upon a typical respiratory substance as being a compound body which consists essentially of a protein base to which is coupled an acid radical or its analogue, the former being the active respi- ratory component and the latter serving as a go-between and probably in the nature of an energizer. Three types of O and CO2 exchange have been observed : (1) The analytic exchange that is characterized by the absorption of 0 and its utilization in the living processes in the breaking down of complex substances and the consequent formation and giving off of C02 as an effete product, a form of respiration which involves intrinsic changes: This in all likelihood is common to all forms of living matter (for even anaerobes absorb the last traces of oxygen, and even in plants this type of exchange is directly but little influenced by light). (2) The synthetic exchange, photosynthetic and chemosynthetic, the first of which is manifested actively solely through the agency of chloro- phyl and light; which is characterized by the absorption of C02 and the giving off of 0; which involves intrinsic changes; and whose intensity is in direct relation to the intensity of light up to the optimal light: This exchange of C02 and 0 is believed to take place solely in the chloroplastid, or in some primitive non-cellular form of protein-chlorophyl combination, as in certain phanerogams in which the chlorophyl is normally found in diffused form. In the chemosynthetic exchange, light energy is replaced by energy in chemico-potential form, and the process has no necessary association with chlorophyl. (3) The physico-mechanical exchange that goes on in the aeriferous system, intercellular air-spaces, etc., of plants, and in the erythrocytes, the blood plasma, and lymph, etc., of animals: This is dependent essentially upon differences in partial pressures and tensions of these gases and upon DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES mass actions, does not involve intrinsic changes, and is in effect a passive phvsico-mechanical function or a means of transport and storage. The first type must be considered as representing a property that is possessed in common by all parts of the protoplasmic mass, at least until our knowledge is different from that of the present. As to the second type, the photosynthetic exchange is almost wholly confined to chlorophyllous substances, while the chemosynthetic exchange occurs chiefly in the entire absence of chlorophyl. The view held by many that chlorophyl is per se a primary respiratory photosynthetic substance was long ago shown to be untenable. Isolated chlorophyl has been proved to be absolutely inert in the exchange of C02 and O, and the cytoplasm of the chloroplastid has been found to be functionless in photosynthesis in the absence of chlorophyl, as in etiolated plants, in which what feeble photosynthesis may be present is attributed to the etiolin. In fact, the chloroplastid is a vital mechanism which consists, broadly speaking, of cytoplasm in some form of union with chlorophyl and etiolin, which is capable of exercising its normal photosyn- thetic functions only when all of the essential structural and physiological units are intact, and whose peculiar photosynthetic properties, therefore, are dependent upon some cooperative and inseparable relationship between the cytoplasm and the pigments. The exact structural relations of the cytoplasm and chlorophyl are unknown, but the chlorophyl appears to be held in a cytoplasmic stroma in vacuolar form. Moreover, normal functional activity can be maintained only so long as normal relations exist between the chloroplastid and its habitat. The isolated chloroplastid soon becomes functionless; and notwithstanding the very close resemblance of the chloroplastids of the higher forms of plant life, it is questionable if, like the erythrocyte, these structures of one genus could continue to exist as living units in an individual of another genus. Whether or not chlorophyl is to be looked upon as being in the nature of an energizer or sensitizing agent in relation to the cytoplasm is yet a matter of speculation among physiological botanists. It is of particular interest in this connection to note that chlorophyl itself may be energized, for it has been shown in Florida that phycoerythrin, which is not assimi- lative and which apparently does not enter into either physical or chemical union with chlorophyl, markedly modifies the assimilatory curve of chloro- phyl in relation to the spectral colors, and so greatly increases the energy of chlorophyl that a very small quantity of chlorophyl will give rise to energetic photosynthesis. While iron seems to be essential in the forma- tion of chlorophyl, the latter is nevertheless iron-free, and the suggestion that the reducing action of the chloroplastid may be due to iron is regarded by the botanist as being absolutely untenable. In fact, apart from the necessity of iron in the formation of chlorophyl, this metal does not seem to be of more importance as a constituent of the chloroplastid in photo- synthesis, or in respiration, than potassium, or magnesium, or certain other inorganic constituents. The foregoing facts are, as a whole, strikingly paralleled in hemoglobin and the erythrocyte, the chief difference being found in the ways in which IN THE ANIMAL KINGDOM. 23 the corpuscle and pigment have been morphologically, chemically, and functionally specialized. The erythrocyte, in common with all living structures, must be con- ceded to be a respiratory structure of the first type by virtue of its proto- plasm, but in addition to this there exists the third type of exchange which is manifested in the continual alternating give-and-take in external and internal respiration, respectively, which is rendered possible through the feeble com- binations of O and C02 in the erythrocyte in association with the differences in partial pressures and tensions of these gases in the proximal and distal portions of the vascular system, and with mass actions, in which operations the corpuscles act as a carrier and store-house for both gases. This respira- tory phenomenon is to be attributed essentially to the agency of hemoglobin, and it will be noticed that it differs materially from the first and second types of exchange, which involve intrinsic changes, and which are manifestations of the activity of energy-transforming mechanisms. While, therefore, as has been shown in previous pages, hemoglobin and chlorophyl are intimately related chemically, and are the most important bodies in plant and animal life, respectively, in the exchange of O and CO2, it is obvious that they have become so specialized in the character of their work that the mechanisms concerned in the exchange are totally different in character and object: we observe a phenomenon which in the first instance is manifested essentially through a passive vehicle; in the second, through the operations of an energy transformer. The third type of respiratory activity, which is the preeminent prop- erty of the erythrocyte, is a property that belongs to the cell as a whole as an individual vital mechanism. Hemoglobin in solution in the plasma of the vertebrate blood has been shown to be in the nature of a foreign body; as a component of the erythrocyte it is an energetic respiratory substance, under which condition its dissociable O is more readily removed than when it is in solution ; in the erythrocyte it behaves as though it were in colloidal form. Isolated hematin is absolutely inert in relation to both O and C02, and isolated stromata and isolated globin have not been found to have respiratory energy in 0 and C02 absorption and elimination greater than protein substances generally under comparable conditions. It seems there- fore obvious that it is not the hemin, globin, or stroma, or the hemoglobin per se, that is the normal functionating substance, but a hemoglobin-stroma combination; and that from analogy, when the hemoglobin is normally in non-corpuscular form, as in certain of the invertebrates, it is probably in a primitive hemoglobin-protein combination similar to the assumed primitive non-corpuscular chlorophyl-protein combination noted in certain phanerogams. What chemical and functional relationships the hemoglobin bears to the stroma, and hematin to the globin, are not known. But from the facts that the exchange of O and C02 goes on quite rapidly in all forms of active protoplasm, that isolated hematin, like chlorophyl, is absolutely inert in relation to these gases, and that chlorophyl behaves in the nature of an energizer in relation to the cytoplasm, it seems likely that hematin is of a 24 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES like nature in relation to the globin and stroma. It has, moreover, been suspected, and even asserted, that the respiratory property of the eryth- rocyte, in so far as the give-and-take of 0 and CO2 is concerned, is inten- sified by or even dependent upon the cooperation of an oxidase in the blood corpuscles or blood plasma. Here we have a paraUel to the influence of phycoerythrin upon the energy of chlorophyl. The absorptive and carrying property of hemoglobin in relation to O has been and continues to be attributed to the atom of iron in the molecule, and this assumption has been applied to the metal components of other respiratory substances, but upon very limited and inconclusive although seemingly plausible data, which in a word are virtually the 0 affinity and capacity of iron. The absorption coefficient of hemoglobin of the bullock for oxygen has been determined experimentally by Htifner to be 1.34 c.c. at 0° C. and 760 mm. pressure, which figure is practically identical with the capacity as estimated by the percentage of iron (0.336) present; hence, the natural conclusion and the universally accepted view that the property and capacity of hemoglobin in relation to O is specific to the atom of iron. The fact, however, should not be lost sight of in this connection that the absorptive capacities, as determined by different investigators, are by no means in accord. Thus, reducing all figures to 0° and 760 mm., the values are, according to Dybkowsky, 1.57; Hoppe-Seyler, for moist crystals 1.09, for crystals dried at room temperature 0.77, and for crystals dried at 0° and powdered 0.54; Strassburg, 0.45 to 3.88; Preyer, 1.72 and 1.8; Worm-Muller, 1.38; and Hiifner, 1.59 and 1.34 (latest). Even Hiifner's latest figures vary as much as 10 per cent, and his corrections are not beyond reasonable question. It is, moreover, doubtful if the property thus attributed to the metal, or even to the hematin, is justified, for while the quantitative relation of the oxygen capacity to the quantity of iron seems convincing as an isolated fact, the deduction is not borne out by our knowledge of this and other respiratory bodies and by other facts. After all, the absorption capacity is merely the maximal capacity for oxygen that can be observed under given conditions of pressure and temperature, other things being equal; moreover, this assumed specificity of the metal of the molecule is scarcely reconcilable with the fact of the existence of metal-free respiratory substances (achroglobulins) which have practically identically the same absorp- tion capacities as hemoglobin. This quantitative coincidence of the 0- capacity of hemoglobin and achroglobulin is certainly remarkable, and it shows clearly either that the specificity attributed to the iron is wrong, or that we have a substitute in the achroglobulin which has the same quanti- tative value as an absorptive factor, but this is hardly credible. If the accepted specificity of the iron be justified, we still have to find an explana- tion for the practically absolutely identical 0-capacity of the metal-free achroglobulins. Then again, assuming that the metal is the specific 0-absorbing agent, hemocyanin should have an absorptive capacity, based upon the percent- age of copper present, of 0.66 c.c. per gram at 0° and 760 mm., while in IN THE ANIMAL KINGDOM. 25 fact, as shown by the direct experiment of Henze with the blood of Octopus vulgaris, it combines with only 0.4 c.c., which is less than two-thirds of what it should be theoretically. The low O-capacity had been previously determined by Dhe're in experiments with the bloods of Helix, Homarus, Astacus, Octopus, Carcinus, Cancer, and Maia. Low figures have also been recorded by others. From the foregoing it seems obvious that iron, manganese, or copper is an incidental rather than the essential constituent of specific respiratory substances, and that if there is a special constituent in relation to the prop- erty of respiration it is as yet not definitely known. As a specific compo- nent of these substances in relation to 0, the sulphur would seem to be of far more importance than any of the metals named : its property as an ener- getic oxidizing agent is universally recognized ; it is a universal constituent of all proteins and hence of all protoplasmic structures, which structures exhibit more or less absorptive activity towards O; the stromata of the erythrocytes, globin, leucocytes, yeast-cells, etc., energetically decompose peroxide of hydrogen. Especially energetic are the stromata of the eryth- rocytes. The facts that about 96 per cent of the hemoglobin molecule is protein, that stroma constitutes about 65 per cent of the erythrocyte, and that the globin and stroma together represent about 95 per cent of the erythrocyte of mammalian blood, strongly indicate a greater importance of the protein than of the hematin, and especially so because the proteins show affinities for 0 and CO2, while the hematin is absolutely inert. The astute Bunge long ago taught (Text-book of Physiological and Pathological Chem- istry, trans, by Starling, 1902, 22; trans, by Wooldridge, 1890, 24) that the respiratory function of hemoglobin can not be due to the iron alone and that it may be that the sulphur of hemoglobin, as of all other protein bodies, still retains its function as an oxidizing agent. In a very recent article, Carracido (Rev. d. 1. R. Acad. d. Cienc., 1906, 33; Biochem. Centralbl., 1906-07, v, 572) concludes that either the globin participates in the oxygen absorption, or that the prosthetic group of chromoprotein is not the conjectured one, since the sulphur-capacity of the hemoglobin increases with the amount of oxygen absorbed. The assumption that the respiratory property of hemoglobin pertaining to oxygen is essentially or solely a function of the atom of iron naturally led as a corollary to a belief of an inertness or practical inertness of the globin, and Bunge even suggested that "The enormous size of the hemo- globin molecule finds a teleological explanation if we consider that iron is eight times as heavy as water. A compound of iron which would float easily along with the blood current through the vessels could only be secured by the iron being taken up by so large an organic molecule." Bunge, later in his lectures, still leans to the belief of the importance of sulphur, for he states (loc. cit. 239) : If oxygen is chemically combined with hemoglobin, we would expect them to be combined in molecular proportions. It would be interesting to ascertain how many atoms of oxygen go to one atom of iron. The analyses made up to the present time are not exact enough for this purpose; they show, however, that about 2 or 3 atoms of oxygen 26 DISTRIBUTION OF HEMOGLOBIN AND ALLIED SUBSTANCES cor ^respond to 1 atom of iron. The figures so far only demonstrate that there is at least four times as much oxygen taken up in the transition of hemoglobin into oxyhemoglobin as there is in the transition from suboxid to oxid of iron, or from ferrocyanid to ferri- cyanid of potassium. Possibly the sulphur of the hemoglobin also plays a part in the loose oxygen compound, and a similar part may be assigned to the sulphur atoms in all proteins. It is noteworthy that, according to previous analyses, the animals that require more oxygen have likewise more sulphur in their hemoglobin. That the protein is really an active factor in relation to the displace- able or respiratory oxygen has, for instance, been clearly indicated by the differences in the behavior of hemoglobin when in the erythrocyte and after removal from it, and by the recent researches of Ham and Balean (Journal of Physiology, 1905, xxxn, 312), who write that- It would appear that one of the oxygen atoms in oxyhemoglobin is differently combined to the other, i.e., it is more intimately attached to the iron, and further that hematin still contains oxygen linked to the iron, only half being displaced in its forma- tion from oxyhemoglobin by the action of dilute acids. This we should expect, from the fact that it requires a strong acid to form hematoporphyrin not only from oxyhem- oglobin but also from hematin, whereas a weak acid readily effects the change in the case of reduced hemoglobin (Laidlaw). The reason for this is supposed to be that iron linked to oxygen is more stable than iron not so linked; in other words, the presence of oxygen attached to iron much increases the difficulty that acids have in removing the iron and forming hematoporphyrin. Again, since one oxygen atom, and not both, is displaced by the action of dilute acids on oxyhemoglobin in the formation of acid hematin, we are justified in supposing that the particular oxygen atom which is disso- ciated is not linked to iron in the same way as the one which is not displaced. Now the effect of dilute acids on oxyhemoglobin is not only to set free one oxygen atom, but also to split off the globin radicle. And we have found from experiment that as more and more oxygen is liberated so more and more acid hematin is formed, and there- fore more and more globin is split off. But further than this, when we have displaced half of the replaceable oxygen we find nothing but acid hematin, and therefore all the globin must have been dissociated from the oxyhemoglobin molecule. It therefore ap- pears that this displaced oxygen atom must bear some definite relation to the globin radicle, the other oxygen atom having both its oxygen affinities satisfied by the iron. The further action of the acid is then to split the bond between the oxygen atom and the globin radicle. The absorptive power of erythrocytes as regards O is accredited to the hemoglobin, and as regards C02 to the alkali of the phosphates and globulin and to hemoglobin. The combination of CO2 with hemoglobin does not give a compound that is to be classed with CO-hemoglobin and 0-hemo- globin, because the CO2 and 0 are not interchangeable in the quantitative relationship that is observed with CO and O. Bohr and his co-workers (Bohr, Festschrift f. C. Ludwig, 1887, 164; Compt. rend. soc. biolog., 1891, cxi, 243; Zentralbl. f. Physiologic, 1904, xvn, 688, 713; Jolin, Archiv f. Aunt. u. Physiologie, 1891, m, 69; Torup, Biochem. Centralbl., 1906, v, 667; Maly's Jahr. ii. d. Fort. d. Thierchemie, 1906, xxxvi, 166) have recorded that the CO2-capacity of hemoglobin is markedly higher than for 0 (which property has been found by Griffiths to be shown by the achroglobulins) ; that the C02-capacity is relatively high at low pressures and low at high pressures; that C02 is more readily dissociated than 0; that the 0-capacity of the blood may be decidedly affected by changes in the tension of C02, IN THE ANIMAL KINGDOM. 27 but that the C02-capacity is not affected by differences in the tensions of 0; and that the absorptive capacities of hemoglobin for O and C02 are higher in the hemoglobin of the dog and guinea-pig than in that of the goose. The large C02-capacity of hemoglobin is suggestive of phys- iological importance, yet it seems to be almost entirely ignored by the physiologist. Bohr, Hasselbalch, and Krogh made the important biological obser- vation that a positive relationship exists between the percentage of 0 in the blood in the presence of different tensions of C02. They made a large number of experiments in which the tension of O varied from 5 to 150 mm. of Hg, and in which the tension of C02 was 5, 10, 20, 40, and 80 mm. of Hg, the absorption being determined in fresh dog's blood at 38°. They found that when the 0 tension is high (corresponding to the pressure in the pulmonary alveoli) differences in the tension of C02 are without important influence on the quantity of 0 absorbed ; but when the tension of 0 is low (as in the blood of the capillaries of the tissues generally) an increase in the tension of C02 has a very depressing effect on the absorption of O. Assuming the tension of 0 in venous blood to be 25 mm. of Hg and that CO2 is absent or without influence, only about 24 per cent of the O of the hemoglobin would be given off, but if the tension of C02 were 40 and 80 mm. of Hg, as much as 60 and 78 per cent, respectively, would be given off without the tension of the O falling below 25 mm. of Hg. It seems from this that the high tension of the CO2 in the tissues must have an important influence on the rapid dis- sociation of O from the oxyhemoglobin. THE NON-IDENTITY OF HEMOCYANINS. Krukenberg (page 9) has found that hemocyanins from different sources show differences in their behavior towards 0 and CO2; Howell (page 10) noted difference in hemocyanins as regards the condition of the respiratory 0 and the temperature of coagulability; Cuenot (page 12) refers to a hemocyanin which is colorless, which does not become blue upon exposure to the air, and which he regards as being not respiratory; and Couvreur (page 13) noted differences in the degree of stability. THE IDENTITY OR NON-IDENTITY OF CORRESPONDING RESPIRATORY SUBSTANCES. As regards the identity or non-identity of corresponding respiratory substances, it seems probable, from the literature on the subject: (1) that chlorophyl (pigment), like hematin, is an identical substance from whatever source; (2) that there are several forms of achroglobulin ; (3) that hemocyanin is not a uniform substance, and that the same conclusion applies to hemo- globin, echinochrome, and chlorocruorin. CHAPTER II. SPECIFICITY OF THE BLOOD OF VERTEBRATES IN RELATION TO ZOOLOGICAL DISTINCTION. A large number of facts bearing upon generic and allied differences of the blood are scattered throughout the voluminous literature of the bio- logical sciences, but these with few exceptions as isolated facts seem to be of so little importance as not to attract more than passing notice. When, however, they are considered collectively and in connection with the pecu- liarities pointed out or suggested in the preceding chapter, and with our discoveries of the specific peculiarities of the hemoglobin crystals shown in subsequent chapters, they will be found to be so positive in their mean- ing as to leave no doubt that we are on the threshold of a specialization so sensitive as to justify the prediction that the blood of each family, genus, species, and individual will be found to be absolutely specific. While it has not been possible for us to make an exhaustive collection of such data, we have brought together sufficient in the following paragraphs to show clearly that we may not only generalize but also specialize, and that with a number of determinant facts and with the present progress of research we are fast approaching the time when not only genera and species but also races, and even individuals of a race or species, can with as much or with greater certainty be distinguished by the peculiarities of their bloods as by the conventional methods of the zoologist. Moreover, we believe, from even the limited studies we have made, that the zoological distinctions indicated by peculiarities of the blood will be found to be paralleled by similar peculiarities of other of the more important body fluids and solids. THE QUANTITY OF BLOOD IN RELATION TO BODY-WEIGHT IN REFERENCE TO GENERA. The investigations of Welcker and others show that, while the pro- portions of blood to body-weight in both warm-blooded and cold-blooded animals, excepting certain of the amphibia and fishes, do not as a rule vary greatly, the differences in the various orders, classes, etc., are of zoological significance. The methods of estimation are not exact, so that the figures recorded are to be regarded as being approximate. The discrepancies in the records of different observers in the proportions in members of a given species are to be accounted for in a measure in this way, and in part in variations due to age, sex, general condition, and the changes that arise from various incidental normal and abnormal states. Notwithstanding the crudity of the methods generally, the records are sufficiently in agreement to indicate specific but not important zoological distinctions. 29 30 SPECIFICITY OF THE BLOOD OF VERTEBRATES TABLE 6.— Proportion of blood to body-weight in different animals, according to figures of Welcker. Kind. No. of experi- ments. Proportion to body-weight. Minimum. Maximum. Mean. Mammalia: 3 5 1 2 1 5 3 5 3 3 1 5 10 4 5 5 4 2 1 1 13.4 1 15.1 1 12.4 1 12.6 1 13.1 1 13.5 1 15.1 1 12.0 1 16.1 1 18.1 1 16.5 1 13.1 1 12.4 1 10.9 1 20.4 1 16.8 1 16.8 1 15.4 1 18.3 1 17.4 1 15.9 1 63 1 19.4 1 49 1 20.5 1 17.2 1 12.1 1 14.7 1 19.4 Dno- gat 1 13.8 1 10.4 Goat (young) 1 20.8 1 17.9 1 15.7 1 13.1 1 13.1 1 16.4 1 15.8 1 11.8 1 11.8 1 9.0 f°r Aves: Reptilia, Amphibia, and Pisces: T)n Do 1 18.4 1 14.3 Coluber natrix and Anguis f ragilis 1 26.7 1 20.4 1 17.3 1 74 1 12.3 1 15.3 1 14.1 1 53 Comparison of different classes. Fish 3 4 4 4 8 1 1 74 1 33 1 20.4 1 13.1 1 18.1 1 19.4 1 15.9 1 15.4 1 10.9 1 12.0 "Scaly" amphibians Birds Mammals While it will be seen (tables 6, 7, and 8) that the proportions recorded by different observers in studies of a given species differ (differences insepa- rable from the methods of determination and the variations that occur in the quantity of blood in any given individual even under perfectly normal conditions), the mean figures may be regarded as being sufficiently approx- imate for purposes of comparison. The range of mean values for the differ- ent classes is between one-forty-ninth (2 per cent) for fish and one-twelfth (8.3 per cent) for birds ; in other words, in proportion to body-weight fish have less than one-fourth the quantity of blood found in birds. Comparing monkey and man, the proportion in the monkey is distinctly lower. Only two monkeys were studied, one young and the other veiy young, the values being 1 : 11.1 (9.02 per cent) and 1 : 13.6 (7.35 per cent), respectively. Among the members of the different orders, it will be found that the figures are higher in birds than in carnivora, herbivora, and rodents ; higher in her- bivora, especially in the horse, than in carnivora and rodents ; and higher in carnivora than in rodents. While Welcker's table, showing a comparison of different classes, leads to the assumption that, with the exception of birds, the proportion of blood is higher as the animal is higher in the scale of life, it will be seen by comparing the values in tables 7 and 8 that such relationship is open to so many exceptions as to be untenable. When, however, comparisons are restricted to an order or class, without reference to the scale of life, the values give the impression of zoological distinctions. IN RELATION TO ZOOLOGICAL DISTINCTION. 31 The differences between carnivora, herbivora, etc., have already been noticed, and if now we compare certain of the members of different classes it will be seen, for instance, that there is a marked difference between the dog 1 : 13.5 (7.4 per cent) and the cat 1 : 15.4 (6.5 per cent) ; between the bullock 1 : 13 (7.7 per cent), the sheep 1 : 12.5 (8 per cent), the goat 1 : 16.1 (6.2 per cent), and the horse 1 : 10.3 (9.7 per cent); between the rabbit 1 : 16 (6.2 per cent), the guinea-pig 1 : 16.7 (6 per cent), and the mouse 1 : 12.3 (8.1 per cent), etc. These differences are certainly of sufficient defi- niteness to be suggestive of importance. TABLE 7. — Proportion of blood to body-weight in different animals, according to various observers. Kind. Proportion. Authority. Primates: Man 1 13.1 Welcker. 1 13.5 Bischoff. Man 1 20.5 Haldane and Smith. 1 11.5 to 1 : 12.6 Kottmann. Woman 1 13 Do. 1 13 6 to 1 : 11.1 Sherrington and Copeman Chiroptera: Bat 1 13.5 Welcker. Carnivora: Cat 1 15.1 Welcker. Cat 1 21 Ranke Cat 1 12 to 1 10.4 Steinberg. Cat 1 17 to 1 15 Jolyet and LarTont. Cat .... 1 14 2 to 1 13 3 Brozeit Dog 1 13.5 Welcker. Dog . 1 18 to 1 12 Heidenhain. Dog 1 15 to 1 12 Panum Dog 1 15.1 Ranke. Dog . 1 14 to 1 11.2 Spiegelberg and Gscheidlen. Doe 1 12 5 to 1 112 Dog 1 13 to 1 12 Doe 1 14.5 to 1 14 9 Sherrington and Copeman Ungulata: Bullock . . . 1 13 Sheep . 1 12 5 Do Horse 1 10.3 Do. Rodentia: Mouse (common) Dormouse 1 13.1 1 16.5 Welcker; Brozeit. Welcker Guinea-pig 1 22 to 1 17 Guinea-pig 1 17.3 Ranke 1 12 to 1 12 Guinea-pig ... 1 18 Rabbit 1 18 1 Rabbit 1 20 to 1 15 Rabbit 1 22 to 1 17 Rabbit 1 21 Rabbit 1 13 3 to 1 12 3 Rabbit 1 18 Rabbit 1 17 2 to 1 12 4 Rabbit 1 157 to 1 13 4 Rabbit buck 1 206 Rabbit, doe .... 1 188 Do Aves: Small birds 1 109 Pigeons 1 18 to 1 • 11 97 Welcker's investigations (Prager Vierteljahressch. f. d. prakt. Heilk., 1854, iv, 63; Zeit. f. rat. Medicin, Ser. 3, 1858, iv, 147) are of especial value because of the number and variety of the species and orders represented 32 SPECIFICITY OF THE BLOOD OF VERTEBRATES (table 6). It is of interest to note that his records for the human being were made on executed criminals — one hanged and two guillotined. Those of Bischoff (Zeit. f. wissensch. Zoologie, 1855, vn, 331; 1857, ix, 65) were also made on guillotined men. Welcker's figures show that the proportion of the blood is in relation to zoological classification, it being higher in warm- blooded than in cold-blooded animals; higher in birds than in mammals; higher in "scaly" amphibia than in "naked" amphibia; higher in amphibia than in fish; and veiy low in fish, the value being only about one-third of that in the mammals. TABLE 8. — Means of the proportions of blood to body-weight in different animals, according to the figures of Welcker and others. Kind. Proportions to body- weight. Authority. Primates: 1 20.5 1 13.3 1 12.3 1 12 1 15.4 1 13.9 1 13 1 12.5 1 16.1 1 10.3 1 12.3 1 16.5 1 16.7 1 16 1 12.9 1 12.4 Per cent. 4.9 7.5 8.1 8.3 6.5 7.2 7.7 8.0 6.2 9.7 8.1 6.1 6.0 6.2 7.8 8.0 Haldane and Smith. Welcker and Bischoff. Chiroptera: Bat Carnivora: Cat . Dog Ungulata: Bullock Sheep Goat Horse. Rodentia: Mouse (common) Dormouse . Guinea-pig Rabbit Aves: Pigeon Small birds The investigations subsequent to Welcker's have added materially to our list of genera and species among warm-blooded animals (Heidenhain, Archiv f. physiolog. Heilk., N. F., Ser. 1, 1857, 507; Panuin, Archiv f. path. Anat. u. Phys., 1864, xxix, 241, 481; Brozeit, Archiv f. d. ges. Physiologie, 1870, in, 353; Ranke, Die Blutvertheilung u. d. Thatigkeitswechsel der Organe, Leipzig, 1871; Spiegelberg u. Gscheidlen, Archiv f. Gynacologie, 1872, iv, 530; Steinberg, Archiv f. ges. Physiologie, 1873, vn, 101; Gscheid- len, Physiologische Methodik, 1877, 333; Jolyet et Laffont, Gazette medicale, 1877, 349; Heissler, Arbeiten a. d. path. Institut z. Mi'mchen, 1886, 322; Haldane and Smith, Journal of Physiology, 1899-1900, xxv, 331; Doug- lass, Journal of Physiology, 1906, xxxin, 493; Sherrington and Copeman, Journal of Physiology, 1893, xiv, 74; Kottmann, Archiv f. exper. Path. u. Pharm., 1906, uv, 356). It would be a natural assumption that in those animals in which the activities of the general metabolic processes are the most intense the proportion of blood would likewise be the highest; yet in fact this is true to only a limited extent, and even then probably only incidentally so. In warm-blooded animals the value is higher than in the cold-blooded, IN BELATION TO ZOOLOGICAL DISTINCTION. 33 and among the former it is higher in birds than in mammals, which is in accord with what we should expect. On the other hand, it is higher in the horse than in the human being, notwithstanding the fact that the intensity of the metabolic processes, as expressed by the intensity of oxidation per kilo of body-weight, is lower in the horse than in man. Likewise do we note an inverse relationship when we compare the dog and the guinea-pig, and small and large birds. From this it is manifest that there must be some factor or factors coupled with the relative blood-volume which compen- sate for the discrepancies between the proportional volume and the rela- tive degree of tissue activity. Such incongruities might in some instances be accounted for in adaptations in the speed with which the total volume of blood is forced through the vascular system, but the chief explanation is doubtless to be found in differences in the composition of the blood, especially as regards hemoglobin and other proteins. TABLE 9. — Specific gravities of the blood as determined by different observers. Kind. Specific gravity. Authority. Primates : Man 1059 Jones. Monkey . . . 10549 Sherrington and Copeman Carnivora: Dog 1060 Pfluger Dog; . . 1059 Nasse. Cat 1054 Do Cat 1054.6 Sherrington and Copeman. Ungulata: Bullock 1061 Davy Calf 1058.3 Sherrington and Copeman. Sheep 1042 Goat 1062 Sherrington and Copeman Horse 1062 Nasse. Ass 1042 Do Pig 1060 Do Rodentia: Mouse 1059 Rat 1056 Do 1059 Do Rabbit 1049 Rabbit 1053 1 Sherrington and Copeman Aves: Turkey ... 1061 Davy Chicken / ben. . . 1063.6 Sherrington and Copeman. \ cock . . Blackbird / hen. • • 1064 1062 Do. Jones. \ cock . . Sparrow /hen. . . 1066 1063.5 Do. Do. \ cock . . Hedge sparrow 1074 1059 Do. Do Greenfinch ... . 1068 Do Pigeon 1067 3 Sherrington and Copeman Reptilia, Amphibia, and Pisces: Snake 1055 Do Frogs (14 winter) f 1034 min. Jones. Frogs (1041 mean 10556 Do. Sherrin(rton and Copeman. Skate 1035 to 103S If we compare the proportions of blood and the percentages of hemo- globin and plasma proteins of the bloods of man and the frog, it will be noted that the proportion of blood in the former, according to Welcker, is 7.69 34 SPECIFICITY OF THE BLOOD OF VERTEBRATES per cent, and in the latter 6 per cent, so that in the human being the pro- portion is but little more than in an animal comparatively low in the scale of life and in which the metabolic processes in comparison with those of man are at a comparatively low level. A partial explanation of this incon- sistency becomes at once obvious in the differences in the content of the bloods as regards the constituents just referred to, the blood of the former containing about 12.5 to 13.5 per cent of hemoglobin, and about 7.6 per cent of proteins in solution in the plasma, while in the latter there are about 2.5 to 3 per cent of hemoglobin and 2.54 per cent of plasma-proteins. Likewise, mammals generally have a lower proportion of blood than birds generally, but the percentages of hemoglobin and plasma-proteins are notably higher in the former. The blood of all animals having nucleated corpuscles, if not actually poorer in erythrocytes than the blood of mam- mals, is usually or invariably poorer in hemoglobin and proteins. The meanings of the differences in the proportions of blood in different genera, etc., are as yet undetermined; but it seems that the proportion of blood in relation to body-weight, the proportions of vital constituents of the blood in relation to body-weight, and the rapidity with which the total volume of blood is driven through the vessels, should collectively show a definite and close relationship to the individual's position in the scale of life and to the intensity of its metabolic processes. TABLE 10. — Mean specific gravities deduced from the records of table 9. Kind. Specific gravity. Kind. Specific gravity. Primates: Man 1059. Rodentia: Rat 1056 Monkey 1054 9 1059 Guinea-pig 1059 Carnivora: Rabbit 1053 1 Cat 10543 Dog 1059 5 Turkey 1061 Chicken 1064 Ungulata: Blackbird 1064 Bullock 1061 1068 2 Sheep 1042 1059 Goat 1062 1068 Horse 1060 1067 Ass 1042 Pig 1060 1055 Free. . . 1055.6 THE SPECIFIC GRAVITY OF THE BLOOD IN RELATION TO GENERA. So many conditions, especially age, diet, general nutritive state, parturition, etc., may affect to even a marked extent the specific gravity of the blood, that decided variations must be expected not only among individuals of the same species, but also in any given individual from day to day and hour to hour. Notwithstanding the difficulties of obtaining accurate data under such conditions, the results of the investigations of Lloyd Jones and of Sherrington and Copeman and others (Davy, Researches Anatomical and Physiological, London; Lloyd Jones, Journal of Physi- ology, 1887, vm, 874; 1891, xn, 299; Pfliiger, Archiv f. ges. Physiologic, IN RELATION TO ZOOLOGICAL DISTINCTION. 35 1886, L, 75; Nasse, Wagner's Handworterbuch, Das Blut, i, 134; Gscheidlen, Physiologische Methodik, 1877, 328; Sherrington and Copeman, Journal of Physiology, 1893, xiv, 52; Harris, Journal of Physiology, 1903, xxx, 319) show in a general way at least that differences exist in the bloods of differ- ent species which indicate zoological distinctions. Examining the records of tables 9 and 10 it will be seen that specific gravity is higher in warm-blooded than in cold-blooded animals; higher in birds than in mammals; higher generally in herbivora than in man and monkey, rodents, and carnivora; highest in birds, and probably lowest in carnivora. The high standard in the frog and the snake are particularly noteworthy. Among herbivora, the specific gravity of the ass and sheep are particularly low; that of the cat is distinctly lower than that of the dog; that of the rabbit decidedly lower than that of the mouse, rat, and guinea-pig. There is not in the differences of specific gravity the quantitative demar- cation between warm-blooded and cold-blooded animals that was found to exist in the proportions of blood to body-weight, nor does there appear any approach to the class distinctions there noted. The cause or causes of the differences in the specific gravities of the bloods of different animals are of course of much more importance than the specific gravity per se, because, while we may in any two or more instances find the same specific gravity in related or unrelated members of an order or class, etc., for instance, the horse and pig, or the bullock and goat, or the rabbit and cat, etc., thus expressing the same percentage of solids, gravity gives no indication as to how those solids are constituted — that is, as to how they may vary in kind and proportions in the different bloods. Since hemoglobin and other proteins represent nearly the whole of the solids, we infer that differences in specific gravity express somewhat closely corre- sponding differences in the percentage of one or the other, or both, of these constituents. In fact, in human blood, under normal and certain abnormal conditions, the relationship between specific gravity and the percentage of hemoglobin is so constant within narrow limits that the clinician makes use of specific gravity tables which indicate quite accurately the percentage of hemoglobin present. Thus, a blood having a specific gravity of 1.059 (water taken as 1.000, as determined by the hydrometer) will be found to contain about 14 per cent of hemoglobin; at 1.056, about 11.2 per cent; at 1.052, 9.8 per cent, etc. While there is thus an unquestionable relationship between these factors in human blood, it does not follow that if in different species we find the same specific gravity there will be the same or even nearly the same per cent of hemoglobin. Thus, while the specific gravities of the bloods of the bullock and pig are the same, the hemoglobin percentages are 10 and 14 respectively; the specific gravities of the blood of the rabbit and cat are about the same, but the percentages of hemoglobin are 12.3 and 14.3 respec- tively. The blood of the dog has a higher specific gravity than that of the cat, but a lower hemoglobin content; and birds have the highest specific gravity of all animals examined, yet a relatively low proportion of hemo- globin. In the bloods of different species the protein content of the plasma 36 SPECIFICITY OF THE BLOOD OF VERTEBRATES is far less variable quantitatively than hemoglobin, but there is no definite relationship between the increase or decrease of one and the changes in the other. THE ALKALINITY OF THE BLOOD IN RELATION TO GENERA. The reaction of the blood from the standpoint of modern physico- chemistry is neutral, inasmuch as the blood does not contain a larger quan- tity of hydroxyl ions (OH—) than water. Moreover, this neutral state is maintained with remarkable persistency, as is shown by the fact that a very much larger quantity of sodium hydroxide is required to cause a given intensity of reaction than when added to water. This peculiarity is owing, according to Friedenthal, to the acid character of the proteins. When, however, the blood is tested with litmus, lacmus or lacmoid, or by titration with a weak acid, such as tartaric or phosphoric acid, a marked degree of alkalinity will be found. In the case of the human blood, for instance, it will be noted that 100 c.c. have an alkaline equivalent of from 250 to 300 mg. of NaOH, or in other words an alkalinity corresponding to a 0.25 to 0.3 per cent aqueous solution of sodium hydroxide. The alkales- cence thus expressed is the measure of the amount of bases in combina- tion with weak acids in the form of weak basic bodies, such as certain of the proteins, disodium phosphate, and sodium carbonate. That the degree of alkalinity must of necessity be variable, within certain limits at least, seems apparent in the fact of the unceasing chemical changes that take place within the blood, and in the continual passage of substances of varying reactions between the blood and the tissues. It has been shown that the intensity of the reaction may be affected to even a marked degree by the character of the diet, by muscular exercise, and by various other conditions, normal and abnormal; it decreases rapidly in the shed blood and during the process of coagulation, and the more markedly as the alkalinity was previously high; it is higher in the plasma than in the serum and highest in the coagulum, and it is very high in laked blood; and it varies within limits so wide in different species that the equivalent of one may be as much as or more than twice as high as in another species. The alkalescence of human blood has been studied by a large number of investigators, chiefly clinicians, and the values are far from being in accord, the reason for which is not far to seek when one considers the crudi- ties of some of the methods and the fact that the reaction alters within a period so short as a couple of minutes after the blood is shed. According to Strauss (Zeit. f. klin. Mi-dicin, 1896, xxx, 327) the unavoidable errors may range as high as 30 mg. of NaOH per 100 c.c. of blood. The alkalinity of human blood, based upon a study of the records of different observers, may be taken as corresponding to 250 to 350 (mean 280) mg. of NaOH to 100 c.c. of blood. In the lower animals, Zuntz (Hermann's Handbuch der Physiologie, 1880, iv, 2 Th., 73; Beitriige z. Physiologie des Bluts, 1868, 13; Centralblatt f. med. Wissensch., 1867, v, 801) found in a pig a value of 330 mg. of Na2C03 and in 10 dogs values ranging from 133 to 274 mg. of Na2CO3. In one ex- IN RELATION TO ZOOLOGICAL DISTINCTION. 37 periment on the dog, Lassar (Archiv f. ges. Physiologic, 1874, ix, 45) found a range of 164.4 to 169.7 and a mean of 166.1 mg. of NaOH. In 6 cats this same observer records a mean of 187.3 mg. of NaOH; and in 20 rabbits, 10 German and 10 French, he records mean values of 146.3 and 164.5 mg. of NaOH respectively. Loewy (Archiv f. ges. Physiologic, 1894, LVIII, 462, 507, 511) found alkalinity higher in man than in the horse, and higher in the horse than in the dog. Zuntz has shown in his experiments with the bloods of the horse, dog, and calf not only marked differences in the alkaline equivalents of these bloods, but marked differences in the alkalinity of the serum and clot. His determinations were made by means of dilute phosphoric acid, each cubic centimeter of which neutralized 5 mg. of Na2CO3. The accompanying statement (table 11) shows the amount of dilute acid required to neutralize 100 c.c. of serum of blood and clot of blood of certain animals. TABLE 11. Serum of blood. Clot of blood. Kind. Amount required to neutralize 100 c.c. Kind. Amount required to neutralize 100 c.c. Dog 17.75 C.C. 27.7 c.c. 38 c.c. Dog 43.75 C.C. 46.4 c.c. 64 c.c. Horse Horse Calf Calf From these figures it will be seen that when the alkalescence is ex- pressed in milligrams of Na2CO3 the values for the sera of these animals are 88.75, 138.5, and 190, respectively; and for the clots, 218.75, 232, and 320, respectively; and that the mean values for the sera and clots are for the dog 153.7, for the horse 185.25, and for the calf 225. It will also be noted, as has been shown in human and other bloods, that the alkalinity of the serum is always less than that of the whole blood and of the clots; and also that the ratios between the sera and clots are not the same in the different species, these ratios being for the dog 1 : 2.47 and for the horse and the calf 1 : 1.68. Comparisons of the values (table 12) obtained for different species show the existence of marked zoological distinctions. It is shown that the alkaline equivalent is decidedly the highest in omnivora (man and pig), and then in the following order: herbivora, carnivora, and rodents, the value in the last being only about half that of the omnivora. Comparing the figures for the calf and the sheep with that of the horse, it seems as though the value for ruminants would be found to be higher than for other ungulates, except those belonging to the pig class. Further zoological differences are suggested by the different values of the cat and dog, of the calf and sheep, and of the German and French rabbits. Whether or not the differences in the ratios of alkalinity of sera and clots are of significance is problematical, yet the correspondence between the horse (1 : 1.68) and calf (1 : 1.68) on the one hand, as contrasted with that of the dog (1 : 2.47), is suggestive that this may be worthy of inquiry. In human blood the 38 SPECIFICITY OF THE BLOOD OF VERTEBRATES records of Brandenburg (Zeit. f. klinische Medicin, 1898-99, xxxvi, 280) indicate that the serum has half the alkalinity of the whole blood (blood 330 to 370; serum 160 to 190). This gives human blood a lower ratio between serum and clot than in the dog, and higher than in the horse and calf. TABLE 12. — Alkaline equivalents of the bloods of different animals. Kind. Alkaline equivalent. Authority. Primate: Man 250 to 300 (280) mg. NaOH Jakseh. Carnivora: Cat 170 3 to 207.2 (187.3) mg. NaOH Lassar. 133 to 274 mg. of Na2COs Zuntz. L>Og 164 4 to 167.9 (166.1) mg. NaOH Lassar. Ungulata: f'olf 255 mg of NaoCOs Zuntz. 190 5 mg. of NaOH Lassar. 185 25 mg of Na ••••• 6,115,375 Otto. 5,799,520 Do. 9,638,000 Worm-Muller. Rodentia:. 8,410,000 Welcker. 4,430,000 Do. 6,293,000 Claisse and Josud. Rabbit 4,407,000 Vierordt. Rabbit 4,866,000 Stdlzing. 4,720,076 Otto. 3,605,140 Do. Rabbit 6,426,750 Worm-Muller. Rabbit 4,410,000 Malassez. Rabbit 6,502,433 Sherrington and Copeman. Aves: Chicken . 3,100,000 Malassez. Chicken 3,860,000 Stblzing. Turkey 2,700,000 Malassez. 1,600,000 Do. 3,400,000 Do. Duck 2,800,000 Do. Swan 2,300,000 Do. Bullfinch 2,660,000 Welcker. 2,010,000 Do. Amphibia: Frog (temporaria) 404,000 Do. Proteus 36,000 Do. Triton cristatus (newt) 103,000 Do. Pieces: Osseous fishes: Sole 2 000 000 Malassez. Eel 1 100000 Do. Cartilaginous fishes: Skate 230 000 Do. Skate 350 000 Torpedo 140 000 Malassez. Lamprey .... 133 000 Welcker Reptilia: Lizard (Lacerta agilis) 1 420 000 Do Lizard (Lacerta muralis) .... 960 000 Do Salamander (maculata) SO 000 Do IN RELATION TO ZOOLOGICAL DISTINCTION. 53 Worm-Miiller, Transf. u. Pleth, Christiania, 1875; Otto, Archiv f. ges. Physiologic, 1884, xxxiv, 233; Vierordt, Daten u. Tabellen, 1906, 205, 206; Ellenberger, Physiologie d. Haussaugethiere, 1890, 181; Claisse et Josue", Compt. rend. soc. biologic, 1896, XLVIII, 1020; and Harris, Journal of Physiology, 1903, xxx, 319). See table 20. The erythrocytes are, except in the elephant, more numerous per cubic millimeter in mammals than in birds, amphibia, reptiles, and fish, and gen- erally they are much more numerous. In birds they are more numerous than in cold-blooded animals, and they are least numerous in the salamander and certain of the amphibia, in which the proportion may fall to a mere fraction of the average in warm-blooded animals. Among mammals the number is highest in the camel tribe, next highest in the sheep and goat, and lowest in the elephant. There is not any numerical distinction of the ruminants from other classes of ungulates, nor does there appear to be anything definite in the way of numerical differences between the ungu- lates, carnivora, and rodents. Considerable addition to our data must be made before any figure can be accepted as the mean for any species, except possibly in the case of the human being. Among the cold-blooded animals the differences are in some instances so marked as to be positive in showing generic peculiarities, as, for instance, the differences in the members of the group of Amphibia, the differences between the osseous and cartilaginous fishes, and the differences between the lizard and salamander. The num- ber of corpuscles is, however, probably of less importance than the per- centage of hemoglobin within them. THE SIZE OF THE ERYTHROCYTES IN RELATION TO GENERA. While the erythrocytes of specimens of blood from different individuals of a given species may vary as much as 40 per cent or more in either direc- tion from the mean diameter, a very large proportion in most if not all bloods of mammals falls within narrow limits of the mean measurements, and in different individuals of the same species the mean measurements are of such uniformity as to justify their acceptance as reliable standards of comparison and differentiation. The cells of the new-born have a some- what larger diameter than those of the adult, and they have a larger range of measurement; the range in the female is greater than in the male. The mean diameters of the red corpuscles of different species vary within wide limits (table 21), the smallest corpuscles thus far examined being those of the musk-deer (2.1 (i, Gulliver), and the largest those of the amphiuma (69.8 to 41.4 p, Gulliver), which may be seen by the unaided eye. In many instances, however, the differences may be so slight, even in species and genera far removed from one another, as to be valueless of themselves in zoological differentiation. Nevertheless, it is probable that in no two species are the mean diameters exactly the same, and even when they are so close as to be practically identical there may be certain peculi- arities, such as the extent of the range in size, the constant occurrence of erythrocytes of unusual dimensions, obscure appearances in the cells which have been expressed by the term "individuality," etc., which may be determining. 54 SPECIFICITY OF THE BLOOD OF VERTEBRATES TABLE 21. — The sizes of the erythrocytes in different genera according to the measurements of Gulliver, Wormley, Treadwell, Formad, Welcker, and Malassez.* Kind. Gulliver. Wormley. Treadwell. Formad. Welcker. Malassez. Primates: f 7.9 f 7.9 f 7.9 P 7.9 f f 7.5 7.1 7.5 6.4 Chiroptera: 5.9 6.4 Bat (noctula) 5.8 Fruit bat 6.6 Carnivora: Cat 5.8 5.8 5.5 6.5 5.9 5.8 5.9 6.1 Ocelot 6.0 6.6 6.8 7.0 Wolf 7.1 7.5 7.4 Dog 7.2 7.1 6.9 7.1 73 7 1 pox 6.5 Bear 6.9 7.0 6.4 6.2 Ungulata: Bullock 5.9 6.0 5.4 6.0 6 0 Sheep 4.8 5.2 4.7 5.1 8 0 Goat 4.0 4.1 3.2 4.2 4 1 3 5 Ibex 3.9 Reindeer 4 5 Elk 6.5 5.8 5.6 Musk-deer 2.1 25 Llama f long diameter. . 7.6 7.9 8.0 9.0 ( short diameter. Camel, double-hump / long diameter. . \ short diameter. Camel, single-hump . / long diameter. . \short diameter. Vicugna . . i long diameter. . 4.1 8.1 4.3 7.8 3.7 7.1 4.0 4.8 4.8 4.0 4.5 10.0 4.5 \shortdiameter. Alpaca.. f long diameter. . 3.9 7.6 \short diameter. Peccary 4.0 5.7 Tapir 6.4 6.1 Hyrax 7.7 Rhinoceros 6.8 7.0 Hippopotamus 7.1 7.4 Pie 59 59 6.1 6 0 5 9 Horse 55 6 0 5.5 5.9 59 Mule 6.8 5.7 Ass 6 4 7 0 63 7 0 Klephant (Indian) 9.2 9 3 9 4 Cetacea: Whale (boops) 82 Whale (caaing) 7 9 . . . ... Porpoise 92 ... Rodentia: 64 6 1 6 6 Squirrel (gray) 7 6 Squirrel (ground) 7 6 6 0 6 8 Beaver 7.6 67 6 8 6 0 6 2 *A few additional measurements by Schmidt (1848), Mallinin (1875), the French Medico-Legal Society (1873), Masson (1885), Schmid (1878), and Woodward (1875) are quoted by Formad (loc cit.). IN RELATION TO ZOOLOGICAL DISTINCTION. TABLE 21 — Continued. 55 Kind. Gulliver. Wormley. Treadwell. Formad. Welcker. Malassez. Rodentia — Continued : Rat f1 6.8 ^ 7.0 u 6.5 ^ f f 7.2 7.7 7.3 7.5 7.2 7.9 7.5 7.5 7.9 Capvbara 8.0 8.0 Rabbit 7.0 7.0 6.4 6.9 6.9 7.0 7.3 7.3 Edentata: Sloth 8.9 7.7 Ant-eater 9.2 Marsupialia: 7.4 7.4 7.1 8.1 Wombat 7.3 6.4 Aves: Chicken . 1 lonS diameter. . . . 12.1 12.2 12.1 13.0 \ short diameter. . . Turkey . . 1 '?DS diameter. . . . 7.3 12.4 7.3 13.4 7.2 13.9 6.5 13.0 \ short diameter. . . Duck f long diameter. . . . 7.1 13.1 7.4 13.0 7.0 12.9 6.5 13.0 \ short diameter. . . Pigeon / IonS diameter. . . . 7.5 12.9 7.3 13.4 8.0 14.7 7.0 \ short diameter. . . Goose . 1 IonS diameter. . . . 7.0 13.8 6.7 6.5 1 short diameter. . . Quail ( long diameter. . . . 6.0 10.8 \ short diameter. . . Dove /long diameter.... 7.3 12.7 L short diameter. . . Sparrow . . t long diameter. . . . 7.5 11.9 11.9 \ short diameter. . . Q 1 1 long diameter. . . . 7.3 14.4 6.8 \ short diameter. . . Ostrich / l°D& diameter. . . . 6.2 18.6 \ short diameter. . . Swan / long diameter. . . . 9.0 14.0 \ short diameter. . . Spoonbill 1 lonS diameter. . . . 7.0 15.0 \ short diameter. . . Bullfinch. . . . { long diameter. . . . 12.4 7.0 \ short diameter. . . Reptilia: Tortoise . . . . / long diameter. . . . 20.3 20.3 7.5 \ short diameter. . . Turtle . f long diameter. ... 11.5 20.6 11.5 \ short diameter. . . Boa . . J lonS diameter. . . . 13.5 17.6 20.4 \ short diameter. . . Viper . . f long diameter. . . . 10.4 19.9 10.0 I short diameter. . . Lizard (agilis)....!^^^... Lizard (muralis) . . / long diameter. . . . \ short diameter. . . Amphibia: Fro™ / long diameter 14.1 16.3 9.2 22.9 23.3 ... ... 15.8 9.8 15.4 10.3 22.3 ... \ short diameter. . . Toad f long diameter — 13.9 24.4 14.1 15.7 30.2 \ short diameter. . . Proteus / Ion8 diameter. . . . 12.7 63.5 18.2 58.2 I short diameter. . . 35.1 33.7 56 SPECIFICITY OF THE BLOOD OF VERTEBRATES TABLE 21 — Concluded. Kind. Gulliver. Wonnley. Treadwell. Fonnad. Welcker. Malaseez Amphibia — Continued : Triton ............ f long diameter.. \short diameter. Amphiuma.. - Salamander ....... / long diameter. . \short diameter. c;rpn I long diameter. . •\ short diameter. Pisces: Trout ............. Mong diameter. . \ehort diameter. p h f long diameter. . ' \ short diameter . f 30.0 19.6 69.8 41.4 709 409 f ' \ long diameter. . short diameter . j long diameter. . short diameter. Lamprey* Sole ( long diameter. . ' \short diameter. Skate (long diameter., (.short diameter. Torpedo i long diameter. . I short diameter. Sea-horse* 16.7 10.3 12.1 9.0 12.7 7.1 14.6 8.9 9.0 29.3 19.5 51.2 31.7 37.8 23.8 41.0 29.8 15.0 120 90 25 14 27 200 150 * Circular. The figures recorded by different observers (Gram, Fortschr. d. Medicin, 1884, n, 33; Georgopulus, Zeit. f. klin. Medicin, 1906, LXIII, 322; White and Treadwell, Reference Handbook of the Medical Sciences, 1901, n, 84; Gulli- ver, Proc. Zoolog. Society, London, 1875, 474; Wormley, Microchemistry of Poisons, 2d ed., Phila., 1888; Welcker, Zeit. f. rat. Medicin, Ser. 3, 1863, xx, 257; Malassez, Compt. rend. Acad. d. Sciences, 1872, LXXV, 1528; Formad, Comparative Studies of Mammalian Blood, Phila., 1888, and Journal of Comparative Medicine and Surgery, July, 1888, etc.) in their studies of given species differ in many instances. As a rule, Wormley's figures are somewhat higher than Gulliver's, while Formad 's and Tread- well's are lower. These differences are not of importance, since on the whole they are remarkably close and entirely in accord in their indications of generic peculiarities. Human corpuscles have been more thoroughly studied than those of any other species. The extreme limits of measurements probably lie within 4 to 10 jt/, but the ordinary range may be placed at about 6 to 9.5 fi. The mean is from 7.9 to 8 (i. The remarkably large proportion that measure close to the average is shown by the figures of Gram, Georgopulus, White, and others : Gram found that 82 per cent were of about the average meas- urements, 13 per cent larger, and 5 per cent smaller; Georgopulus records 73 per cent between 7 and 7.5 ,«, 10 per cent between 8 and 8.5 n, and 17 per cent between 6 and 6.5 /u; and White, 79.5 per cent between 7.5 and 8.5 f/, 12 per cent between 8.5 and 9.25 /.i, and 8.5 per cent between 6.25 and 7.5 u. IN RELATION TO ZOOLOGICAL DISTINCTION. 57 The diameters of the erythrocytes of different species have been made the subject of study especially by Gulliver, Wormley, Schmidt, Welcker, Malassez, Formad, and Treadwell. Gulliver's investigations extended over a period of 35 years and included studies of about 650 species. He frankly states that his tables can not pretend to absolute exactness, and are only offered for what they may be worth, and that in the estimation of their value allowance should be made for errors, whether instrumental or personal, more or less inevitable, notwithstanding the greatest care, in observations so extensive, and that the relative value of the measurements, though probably not unexceptionable, may be entitled to more confidence as fair approximation to the truth. He further states that in spite of little mis- takes or of variations in the dimensions of the corpuscles of this or that species, the comparative results will appear sufficiently uniform. Gulliver's measurements are so closely in accord with those of later observers that they are to be accepted as being sufficiently accurate to serve for purposes of comparison. His investigations were made from the point of view of the biologist, and he claims that the differences in the measurements of the erythrocytes of different species constitute an important means of zoo- logical distinction. Thus, he states: If we compare the red corpuscles of species of one order or family, e. g., Tragulus and other ruminants, the corpuscles of the former animals will constantly prove the smallest; so, too, in Paradoxurus and Cam's, in Hippopotamus and Elephas, in Mus and HydrochoeTus, in Dasypus villosus and Orycteropus capensis, in Rhea americana and Casuarius, in Zootica vivipara and Anguis fragilis, in Bujo viridis and Bufo vulgaris, in Osmerus eperlanus and Salmo salar. And in like manner the facts are equally clear in comparison of the different orders, so that the corpuscles are smaller in the Rumi- nantia than in the Rodentia, in the Marsupialia than in the Edentata, in the Graminivora than in Rapaces, in Anura than in Urodela, in Sturiones than in Plagiostomi. Notwithstanding the foregoing positive statement, there seems to be a general, if not universal, belief that the size of the corpuscles is without much zoological importance, which is indicated by the very infrequent, casual, and scanty references to this subject. There is no doubt that the figures show unequivocally that the mean diameters of the erythrocytes of different genera, related or unrelated, may be practically the same, as, for instance, those of the monkey, lipped bear, hyena, and rhinoceros; and again, those of man, opossum, dingo, dog, wolf, whale, armadillo, beaver, capybara, guinea-pig, muskrat, etc. Nevertheless, it is clear that even among the members of a given order, or tribe, or genus, etc., the differences may be sufficient to be positively distinctive, and at times to have some other and more special zoological significance. Thus, in the primates there is seemingly an increase in the size of the corpuscles as the individual is higher in the scale of life, as, for instance, man, 7.9^; chim- panzee, 7.4 [i ; monkey, 7.1^; lemur, 6.4 p. This relationship may be in rela- tion to differences in the sizes of the species (page 58) . Another interesting relationship is noted in the horse, mule, and ass, the mule being a hybrid and the corpuscles having an intermediate measurement. Then again, comparing representatives of classes of different orders, such as ruminants, felines, canines, etc., not only may each class be readily distinguished 58 SPECIFICITY OF THE BLOOD OF VERTEBRATES from the others by the mean sizes of the corpuscles, but even individuals belonging to each class. (Plate A.) The nearness of the diameters of the erythrocytes of certain of the domesticated animals to those of man is a matter of considerable impor- tance, chiefly because of its medico-legal bearing, and it is yet an open question if the corpuscles of the dog, and especially of the guinea-pig, can with positiveness be distinguished from those of man. The mean measurements found by White (expressed in (i) are: man 8.01, guinea-pig 7.47, dog 6.87, pig 6.07, ox 5.44, sheep 4.75, and goat 3.69. Not only are these measurements sufficiently different to be significant, but the variations in the ranges in the sizes in the different species are peculiar. Particularly striking is the wide range in the pig and the narrow range in the goat, the limits of the former being 3.75 to 8.50 and of the latter only 3 to 4.5. 80.5 per cent of the corpuscles of man ranged between 7.5 and 8.5, 90 per cent of the dog between 5 and 7.5, 80 per cent of the pig between 5 and 7.25, 95 per cent of the ox between 4.75 and 6.25, 89 per cent of the sheep between 4 and 5.25, and 96 per cent of the goat between 3.45 and 4.25. There are also certain relationships between the mean size of the cor- puscles and the size of the species. Gulliver states that, if "we confine the observations to small natural groups of the class, such a relation will plainly appear in a rule that the largest corpuscles occur in the largest species and the smallest corpuscles in the small species of a single order or family. This relation is well shown in ruminants, rodents, and edentates, and even in fera3, which offer some exceptions; the largest corpuscles are found in the big seals and the smallest in the little viverras and paradox- ures. In fine, though this rule is applicable only to single orders or lower sections of apyrensemata, it extends to the whole class of birds, but neither to the reptiles, batrachians, nor fishes, except in partial instances, which seem to be rather indeterminate or accidental than regular." Attempts to trace a relationship between the number and size of the corpuscles and the speed of the animal's movements have proven negative. The very small and numerous corpuscles of the chevrotain have been associated with the fleetness of the animal ; while, on the other hand, the enormously large and comparatively few corpuscles in the amphiuma have been associated with sluggishness. Such assumptions have been founded upon insufficient or erroneous data. There is, as a rule, an inverse relationship between the number of corpuscles per cubic millimeter and the mean diameter, but even in closely related genera this relationship may not exist. CERTAIN PROPERTIES OF THE ERYTHROCYTE IN RELATION TO GENERA. There are certain peculiarities shown by the erythrocytes of different species which are of zoological significance. The well-known property of the erythrocytes of mammalian bloods to form rouleaux after the blood is shed has not been observed in the case of bloods having nucleated cells, except in the lamprey. This difference may be purely mechanical, and due to the nuclei preventing the approximation of the sides of the erythrocytes. There are certainly differences in the specific gravities and coloration of the erythrocytes of different species. PLATE A I. MAN. II. QUADRUMANA. III. CHEIROPTERA. OOO OOO OOO IV. FER/E. OOOOQOOOOO pqts t uwiiyz V. CETACEA. VI. PACHYDERMATA. OOO Oooooo VII. RUMINANTIA. be d e f h i k I m n ' VIII. RODENTIA. IX. EDENTATA. X. MARSUP. XI. MONOTR. OOOO OOO OO O XII. AVES. 345 6 78 9 10 11 12 13 14 XIII. REPTILIA ET BATRACHIA. Gymnopodus. Crocodil. Lacert. Anguis. Coluber. Python. Bufo. XIV. Lissotriton . Perca. Tinea. Esox. Salrno. Gymnotus. Squalus. Arocooccetes. „, •j^gth of an Inch I l I I _ i I i I i i j * 900 G.SuLliVerad nat. del. 10000 Gulliver's micrometry of red blood corpuscles, all drawn to a uniform scale. an^la?™3? •"' ?I1Vl XT' ^VI' XVI1' and XVI11 «P«sent red blood corpuscles of Reptilia n .. ,e, of ,i ' Whl'f UDd^r flgUre, X1X those of the fishes are given. In all these figures the pfa£ ? It ^^J^^^^ed upon the plate, and do not require any comment at this be perceived by the naked eye corPusclt* of the Amphiuma are so large that they can 7.9 7.4 7.1 G.4 A. — VERTEBRATA APVREN.EMATA. I. Homo (man): 1. Corpuscles lying flat 2. The same on edge 3. Membranous base of same after removal by water of coloring matter; it shows diminu- tion in diameter on account of acquired spherical shape II. Quadrumana (monkeys}: 4. Simla troglodytes (chimpanzee) 5. Ateles ater (black-faced spider monkey) 6. Lemur anguanensis III. Cheiroptera (bats}: 7. Cynonycteris collaris (fruit bat) G.6 8. Vespertilio noctula (large bat) 5.8 9. Vespertilio pipistrellus (.common bat) 6.9 IV. Fera (beasts of prey): (/)) 10. Sorex tetragonurus (shrew) 5.6 ("j:N^ 67 68 GENERAL CHEMICAL AND PHYSICAL CHARACTERS in which G represents the globin radical (page 26). According to Zinoffsky (Zeit. f. physiolog. Chemie, 1886, x, 16), the molecule may be regarded as consisting of two molecules of globin and one molecule of hematin. Whether or not globin and hematin are thus combined, or the hematin is linked with one or several molecules of globin; whether the globin is a simple or com- pound body; whether the hematin may be combined with polymeric or isomeric forms of globin; whether the hematin is with certainty a uniform substance, etc., are still open questions. If, as Miescher states, the albumin molecule with its 40 atoms of carbon may have as many as a billion stereo- isomers, what may be the possibilities of hemoglobin or globin molecules with their hundreds of carbon atoms? Whatever may be the chemical relations between globin and hematin, they are so peculiarly associated that undecomposed hemoglobin gives neither albuminous nor iron color reactions. It is of incidental interest to note that, except the iron in hemoglobin, nearly all of the iron of the tissue cells is contained in the nucleoproteins, and that while these substances, unlike hemoglobin, yield the protein color reactions, they, like hemoglobin, do not yield iron reactions, show- ing that in both the iron is in a non-ionic or "masked" state. We are also in doubt as to the state or states in which hemoglobin exists in the erythrocytes, especially as to whether it is in a liquid, semi- liquid, or solid form, and as to the nature of the compound or compounds it probably forms with other constituents of the erythrocytes. The red corpuscles consist of a stroma and hemoglobin with other substances. The former is elastic, non-contractile, seemingly homogeneous, colorless, transparent, and albuminous. According to some, the stroma is in the form of minute sacs which contain hemoglobin and other substances in solution. According to others, it is in the form of a protoplasmic mass, throughout which the hemoglobin and other substances are distributed. That the hemoglobin is not in either crystalline or amorphous form has been shown by microscopic examination with high powers; and that it is not in solu- tion in a free state seems obvious from the fact that in the case at least of the very insoluble forms of hemoglobin, as in the guinea-pig, squirrel, rat, necturus, etc., not only are the water and the inorganic salts of the corpuscles wholly inadequate to dissolve or keep in solution the hemo- globin, but even the entire blood plasma is altogether insufficient to hold the hemoglobin in solution when freed from the corpuscles. The assumption of Preyer that the hemoglobin is held in solution in the corpuscles by virtue of potassium salts because of the presence of a relatively high percentage of those salts in comparison with the percentage in the plasma, and because of the higher solubility of the hemoglobin in water when these salts are present, is not worthy of consideration, inas- much as in certain bloods, for instance in those of the dog and cat, the per- centage of potassium in the corpuscles is practically the same as in the plasma, and yet in the dog crystallization takes place rapidly in the plasma upon the laking of the blood. Rywosch (Centralbl. f . Physiologic, 1905, xix, 388) believes that the hemoglobin is present in the corpuscles in a free state. He found, after destruction of the erythrocytes by grinding in sand, OF HEMOGLOBIN, AND ITS SPECIFICITIES. 69 that by mixing the pulp with an isotonic salt solution the hemoglobin was dissolved. This he holds would not occur " if the hemoglobin was in com- bination with the stroma. " However, his method may have been the means of breaking up a hemoglobin-stroma union. Moreover, Stewart (see below) has found, in his experiments on the influences of various agents on the osmotic properties of the erythrocytes, that the hemoglobin can not exist in the corpuscles in ordinary aqueous solution. Hoppe-Seyler (Physiologische Chemie, 1877, 381; Zeit. f. physiolog. Chemie, 1889, xm, 477) attempted to show, by various facts and arguments, that such differences exist between the behavior of the coloring matter of the blood as it exists in corpuscles and hemoglobin in solution that they can not be identical. Most of his deductions have, however, been found to be untenable. He distinguishes between the coloring matter of the blood, oxyhemoglobin, and reduced hemoglobin, regarding both oxyhemoglobin and reduced hemoglobin as cleavage products. He looks upon the " coloring mat- ter" of the blood as consisting of combinations of oxyhemoglobin and hemo- globin with lecithin, forming firm chemical unions. The coloring matter of arterial blood he distinguishes as arterin and that of venous blood as plebin, the only difference between these two substances being a feebler combina- tion of oxygen in the former. While Hoppe-Seyler's hypothesis seems to have received a tacit acceptance, it has been opposed by Gamgee (Scha- fer's Text-book of Physiology, 1898, 1, 190) and questioned by others as being untenable ; but it has been defended by Robert (Das Wirbeltierblut, etc., Stuttgart, 1901, 5). Bohr (Zentralbl. f. Physiologic, 1904, xvn, 682, 688) believes that the coloring matter of the blood, which he terms hemochrome, is not identical with hemoglobin (which he prepared without the addition of alcohol), because the latter has a lower oxygen capacity. Recent evidence that hemoglobin exists in the corpuscles in some peculiar form of combination has been recorded by a number of investi- gators. Thus, Stewart (Journal of Physiology, 1899, xxiv, 211; Amer. Journal of Physiology, 1902, vm, 103) found, in a very interesting study of the effects of taking agents, " that the relations of the hemoglobin and the electrolytes of the corpuscle to some of the other constituents of the cor- puscle or to the envelop are such that under certain conditions hemoglobin may be liberated while the electrolytes are retained; while under other conditions electrolytes may pass through an envelop which refuses passage to the hemoglobin, although in general it is easier for the hemoglobin, in spite of the great size of the molecule, to escape from the corpuscles than it is for the electrolytes. " He also found that, while hemoglobin may pass from the corpuscle, hemoglobin dissolved in the serum would not pass into the corpuscle. In explanation of these phenomena Stewart proposes four hypotheses as to the condition of the hemoglobin and the electrolytes in the corpuscles: (1) A portion of the electrolytes and of the hemoglobin is in solution as such; and the rest is in solution as compounds with other substances, such compounds being unable to pass through the envelop. 70 GENERAL CHEMICAL AND PHYSICAL CHARACTERS (2) A portion of the electrolytes and of the hemoglobin is in solution as such, and the rest exists in a solid or semisolid form united to some constituent of the stroma. (3) A portion of the electrolytes, but none of the hemoglobin, is in solu- tion as such; the whole of the hemoglobin and the rest of the electrolytes being in solution in the form of such compounds as are mentioned in (1). (4) A portion of the electrolytes, but none of the hemoglobin, is in solution as such; the rest of the electrolytes and all of the hemoglobin are united in the stroma. The last hypothesis, he thinks, best takes account of the facts of laking. Oxygen, it seems, serves as a connecting link not only between globin and hematin, but also between the stroma and hemoglobin. The removal of oxygen from the blood causes hemolysis. This phenomenon might, at first thought, be regarded as a mechanical effect due to the rapid dis- charge of 0 from the erythrocytes when the blood is subjected to the vac- uum pump, but this is negatived by the fact that hemolysis occurs just the same when a continuous stream of CO2 is passed through the blood and the O thus driven off gradually. Even the linkage between globin and hematin may be broken by C02. That the hypothetical union between hemoglobin and the stroma must be a feeble one is evident in the readiness with which it is broken, by the removal of O from the blood, by minute quantities of foreign serum, snake venom, and certain bacterial products, by repeated freezing and thawing, etc. While it thus seems probable that the hemoglobin of the corpuscles is essentially or solely in some form or forms of union with the stroma, it is also probable, from the investigations of Hiifner (Archiv f. Anat. u. Physiologie, 1894, 135, 176), that the combination does not, in opposition to Hoppe-Seyler's statements, effect a marked alteration in the chemical nature of hemoglobin in so far as pertains to its relations to oxygen and to light, for he found that its behavior to oxygen and its spectropho- tometric properties are the same as when the hemoglobin is free, provided the solution be of the same degree of concentration. On the other hand, it is positive that at least the degree of solubility in relation to the plasma and the crystallizability are lessened to a marked degree, so much so that the crystallization may occur in the plasma of partially laked blood and not in the corpuscles, even though in the latter the concentration of the hemoglobin may be greatly higher. The corpuscles of the dog contain about 33 per cent of hemoglobin, while the highest percentage that could exist in the laked blood is about half of this; but while crystallization does not occur in the corpuscles, it does occur rapidly in the laked blood. (See Chapters V and XV.) THE ELEMENTARY COMPOSITION OF HEMOGLOBIN. The determinations of the centesimal composition of hemoglobin of different species of animals differ sufficiently to indicate that all hemoglobins are not alike ; but these differences are not on the whole greater than those noted in the analyses of specimens of blood from individuals of the same OF HEMOGLOBIN, AND ITS SPECIFICITIES. 71 species, and are therefore of little significance in indicating positive non- identity (table 25). In fact, the analyses, as a whole, are so discrepant that it must be admitted that hemoglobin is not a uniform substance, TABLE 25. — The centesimal composition of hemoglobin, according to various observers. Kind. Centesimal composition. Authority. C. H. N. S. Fe. O. Carnivora: Cat 54.60 53.91 54.57 53.85 54.00 54.15 53.64 54.66 54.87 54.76 54.40 51.15 54.56 54.75 54.40 54.17 54.71 54.09 54.12 52.47 54.26 54.77 53.91 53.86 7.25 6.62 7.22 7.32 7.25 7.18 7.11 7.25 6.97 7.03 7.20 6.76 7.15 6.98 7.25 7.38 7.38 7.39 7.36 7.19 7.10 6.99 7.02 7.10 16.52 15.98 16.38 16.17 16.25 16.33 16.19 17.70 17.31 17.28 17.61 17.94 17.33 17.35 17.51 16.23 17.43 16.09 16.78 16.45 16.21 17.07 0.62 0.542 0.568 0.39 0.63 0.67 0.66 0.4 0.65 0.67 0.65 0.39 0.43 0.42 0.45 0.66 0.479 0.59 0.58 0.859 0.54 0.38 0.41 0.37 0.35 0.333 0.336 0.43 0.42 0.43 0.43 0.447 0.47 0.45 0.47 0.335 20.66 22.62 20.93 21.84 21.45 21.24 20.03 19.543 19.73 19.81 19.67 23.42 Abderhalden, Physiologischen Chemie, 1906, 596. Jacquet, Zeit. f. physiol. Che- mie, 1888, xii, 285. Jacquet, Zeit. f. physiol. Che- mie, 1890, xiv, 289. Hoppe-Seyler, Med. chem. Un- tersuch., 186S, Heft 3, 366. Hufner, Jour. f. prakt. Chemie, 1S80, xxii, 362. Schmidt, Preyer, Die Blut- krystalle, 1872, 65. Schmidt & Bottcher, Ueber Blutkrystalle; Inaug. Dis- sert., Dorpat, 1862. Hufner, Beitrage z. Physiol., C. Ludwig, Leipzig, 1S87, 74. Kossel, Zeit. f. phvsiol. Che- mie, 1878-9, n, 149. Otto, Archiv. f. ges. Physiolo- gie, 1883, xxxi, 240. Hufner &Bilcheler, Zeit. f. phys- iol. Chemie, 1884, vm, 358. Zinoffskv, Zeit. f. physiol. Che- mie, 1880, x, 16. Schulz, Zeit. f. physiol. Che- mie, 1898, xxiv, 449. Abderhalden, Zeit. f. physiol. Chemie, 1903, xxxvn, 494. Jutt, Inaug. Dissert., Dorpat, 1894; Maly's Jahresbr. u. d. Fort.d.Thierchemie,1895,128. Otto, Zeit. f. phvsiol. Chemie, 1882, vin, 57. " Hufner, Beitrage z. Physiol., C. Ludwig, Leipzig, 1887, 74. Hoppe-Seyler, Med. chem. Un- tersuch., 1S68, Heft 3, 366. Hoppe-Seyler, Med. chem. Un- tersuch., 1868, Heft 3, 366. Jacquet, Zeit. f. physiol. Che- mie, 1888, xiv, 289. Hoppe-Seyler, Med. chem. Un- tersuch., 1868, Heft 3, 366. Bardachzi, Zeit. f. physiol. Chemie, 1906, xux, 465. Griffiths, Physiology of the In- vertebrata, 1892, 147. Dog Doe Dos Doe Doe.. Doe ... Ungulata: Bullock Horse . . Horse Horse. . Horse... Horse Horse . 0.38 0.393 0.426 0.399 0.40 0.48 0.335 0.43 0.41 20.12 19.85 21.634 19.602 21.44 20.68 22.50 20.69 Horse Pig Pig Rodent ia : Squirrel Guinea-pig. . . . Aves: Chicken* . Goosef Reptilia: Sea-tortoise. Invertebrata: Earthworm | II [III. 0.39 = 0.1973 = 0.770 even in individuals of a given species, or that there are important sources of fallacy in the methods of analysis, or that the methods of preparation are so faulty as to yield either an impure or a partially decomposed sub- stance. It seems so very improbable that the hemoglobin from normal 72 GENERAL CHEMICAL AND PHYSICAL CHARACTERS individuals of a given species is not of uniform composition, that this pos- sible source of difference need scarcely be considered. That important errors in analysis have occurred seems evident, as, for instance, in the very low C, Fe, and S percentages found by Zinoffsky in his analyses of the hemo- globin of the horse, and in the differences in the percentages and ratios of Fe and S shown by the record of different analyses of the hemoglobin from individuals of the same species (table 26). Another source of error is to be found in the different methods for determining the N content, but doubt- less the most important source is in abnormalities of the substance itself which have been due to the methods of preparation. The attempts to ob- tain pure hemoglobin by repeated crystallization have, instead of yielding a pure product, given rise to artifacts, each recrystallization adding another step in the denaturalization and disintegration of the molecule. TABLE 26. — The ratios of Fe to S according to the analyses of various observers. Kind. Ratios of Fe toS. Authority. Kind. Ratios of Fe toS. Authority. Carnivora: Cat 1 1 771 Abderhalden. Ungulata — cont'd: Horse 1 1.145 Jutt. Do" 1 1 628 Pig 1 1.549 Otto. Doe 1 1 660 Do Pig 1 1.150 Hufner. Doe 1 0907 Hoppe-Seyler. Rodentia: Dog 1 1.500 Hufner. Squirrel 1 1.475 Hoppe-Seyler. Doe 1 1 558 Guinea-pie 1 1.208 Ungulate: Bullock 1 0853 Hufner. Aves: Goose 1 1.488 Do. Horse 1 1 383 Chicken 1 2 564 Jaccjuet. Horse 1 1.489 Otto Reptilia1 Horse 1 1 383 1 0927 Bardachzi. Horse 1 1 161 Horse 1 1 105 Earthworm 1 1 Griffith. The great instability of the hemoglobin molecule has been shown in various ways, and the tenacity with which this and other proteins mechan- ically or chemically cling to or combine with certain substances has like- wise been proved. Hoppe-Seyler (Archiv f. path. Anat. u. PhysioL, 1864, xxix, 223) in his earliest researches on hemoglobin found that neither concentrated solutions nor crystals of hemoglobin remain unchanged for even 24 hours at ordinary temperature; that hemoglobin undergoes partial decomposition when dried by the aid of an air-pump and sulphuric acid; and that with each recrystallization there is formed an insoluble residue in the form of a derivative; Halliburton (Chemical Physiology and Pathol- ogy, 1891, 287) states that, even when hemoglobin is dried in a Torricellian vacuum at 40°, not only is hematin and an insoluble protein formed but some of the water of crystallization is driven off. He also found that repeated crystallization of the hemoglobin of the squirrel ultimately changes the form of the crystals from hexagonal plates to rhombic prisms, or a mixture of these with rhombic tetrahedra. Moreover, the crystals of hemoglobin that have been analyzed have been prepared by the "alcohol method," and presumably purified by repeated recrystallization, a method which of itself makes it practically absolutely impossible to obtain a normal hemoglobin. Alcohol denaturalizes hemoglobin, as it does other proteins. OF HEMOGLOBIN, AND ITS SPECIFICITIES. 73 Dr. S. Weir Mitchell (Proc. Acad. Nat. Sciences, Philadelphia, 1858, x, Biolog. Dept. 2) found that the color of hemoglobin crystals could be washed out with alcohol and water without injury to their form, and that the crystals may even be redissolved in water and again obtained devoid of color but without change in crystalline type. Preyer (Archiv f. ges. Physiologic, 1868, i, 395) records that hemoglobin crystals are rendered less soluble after standing in dilute alcohol, and that they are converted into pseudomorphs when dried or when in alcohol; Nencki (Archiv f. exp. Path. u. Phar., 1885, xx, 332) states that hemoglobin crystals through the influence of alcohol are converted into an insoluble " parahemoglobin, " which has the same elementary composition as hemoglobin ; Struve (Berichte d. d. chem. Gesel., 1881, xiv, 930) completely deprived hemo- globin crystals of their color by treating and rendering them insoluble with alcohol and water, and without changing their form, and he also found (Jour. f. prakt. Chemie, N. F., 1884, xxix, 304) that fresh blood crystals in strong alcohol became completely insoluble in dilute alcohol. Loewy (Zentralb. f. Physiologie, 1899, xm, 449) and Hiifner (Archiv f. Anat. u. Phys., 1901, Supplement, 187) both have determined that alcohol so alters the hemoglobin molecule as to render the readily dis- sociable 0 less readily removed, and therefore render it like methemo- globin. In fact, Hiifner has in recent years insisted upon the importance of avoiding the use of alcohol in the preparation of hemoglobin crystals. Kupffer (Inaug. Dissert., Dorpat, 1884), in experiments with the hemo- globin from the dog, and Kriiger (Zeit. f. Biologie, 1887, xxiv, 47), with crystals from the blood of the horse, have found that with each crystal- lization the absorption coefficient in relation to the spectrum is altered, the absorptive ratio becoming higher and higher, which is the opposite to that which should be expected if recrystallization means merely purification. The foregoing facts, together with others which will be found in subse- quent pages, show clearly not only that alcohol is injurious but also that each step in recrystallization means probably the stripping off of extremely un- stable or feebly combined radicals which are normal constituents of the mole- cule and which contribute in giving the molecule its distinctive properties. The tenacity with which protein molecules hold impurities has been convincingly shown by Schulz and Zigmondy (Beitrage z. chem. Phys. u. Path., 1902, in, 137), who experienced much difficulty in obtaining egg- albumin free from colloidal substances, and that recrystallization from 5 to 7 times was often necessary to obtain a pure substance. While such recrystallization does not affect this protein, according to these observers, it without doubt, as stated, markedly affects the hemoglobin. Abderhalden (Zeit. f. phys. Chemie, 1903, xxvii, 484) states that the hemoglobin of the horse once crystallized may yield as much as 0.62 per cent of glycocoll, the presence of which he attributes to contamination with serum globulin. Since serum globulin yields a little over 3 per cent of glycocoll, there would therefore be about 15 per cent of serum globulin present. He did not find glycocoll after the second crystallization. Even after purification, protein crystals may mechanically take up foreign substances from solution, as has 74 GENERAL CHEMICAL AND PHYSICAL CHARACTERS been shown by Wichmann (Zeit. f. physiolog. Chemie, 1899, xxvn, 575), who compares protein crystals to a sponge. Moreover, in the preparation of crystals of albumin, globulin, phycoerythrin, phycocyanin, hemocyanin, and hemoglobin by the "salting-out" process, in the case of all excepting possibly hemoglobin the crystals are not free substances, but some form of combination. Phosphorus according to some observers is a contamination, but ac- cording to others it may be or is a normal constituent. While it can be removed from the hemoglobin of mammalian bloods by repeated crystal- lization, this was not found possible by Hoppe-Seyler and Jacquet in the case of the hemoglobins of bloods that contain nucleated erythrocytes, yet Bardachzi (Zeit. f . physiolog. Chemie, 1906, XLIX, 465) has obtained from the sea-tortoise crystals of hemoglobin that were free from phosphorus. The fact, however, that phosphorus has been removed from mammalian hemo- globins is not proof of its being a contamination, because it may have been stripped from the molecule. Inoko (Zeit. f. phys. Chemie, 1894, xvm, 57) regards the phosphorus as a normal constituent existing in the form of nucleic acid which is in combination with hemoglobin. Jacquet (Zeit. f. phys. Chemie, 1888, xn, 285) also regards it a normal constituent, but Gscheidlen (Archiv f. ges. Physiologic, 1878, xvn, 421) and Gamgee (Scha- fer's Text-book of Physiology, 1898, 1, 206) look upon it, doubtless correctly, as a contamination. [Since the foregoing was put in type Abderhalden and Medigreceanu (Zeit. f. physiolog. Chemie, 1909, LIX, 165) have shown, by their analyses of the hemoglobin of the goose, that phosphorus is an impurity.] THE MOLECULAR FORMULA AND WEIGHT OF HEMOGLOBIN. The molecular formulas and molecular weights of proteins, especially of the coagulable proteins, are admittedly high, and are particularly high in the chromoproteins. Owing, however, to the difficulty of obtaining these substances of uniform purity, the estimates must be regarded as being purely tentative. Vaubel (Jour. f. prakt. Chemie, 1899, LX, 55) gives the following figures which he compiled from the records of different investi- gators, which records were obtained by various methods of determination: Egg albumin 4618 to 6542; serum albumin 4572 to 5135; myoalbumin 4572 to 5135; casein 6500 to 6542; plant albumin 5050 to 6690; plant globulins 5257 to 8848; globin 15000 to 16086; and hemoglobin 15000 to 16730. Inasmuch as we have not a rational formula for hemoglobin, the empirical formulas and weights must be regarded sub judice. The calcu- lation of the molecular formula of the hemoglobin of the horse by Schulz was based upon the sulphur content (table 27). Preyer (Die Blutkrystalle, 1871, 65), Hiifner (Jour. f. prakt. Chemie, 1880, xxn, 362; Zeit. f. physiol. Chemie, 1884, vm, 361), Zinoffsky (Zeit. f. physiol. Chemie, 1885, x, 16), and Jacquet (Zeit. f. physiol. Chemie, 1889, xiv, 289) made determinations based upon the percentages of Fe; and Hiifner verified Jacquet's figures for the hemoglobin of the dog by determinations of the combining proper- ties of hemoglobin with O and CO. Hufner and Ganser (Archiv f. Anat. u. OF HEMOGLOBIN, AND ITS SPECIFICITIES. 75 Physiologie, Phys. Abth., 1907, 209) have determined the molecular weights by means of osmotic pressures. Jutt's (Inaug. Dissert., Dorpat, 1894; Maly's Jahr. li. d. Fort. d. Thierchemie, 1895, 128) estimates were founded upon the combinations of hemoglobin with heavy metals. Kulz (Zeit. f. physiolog. Chemie, 1883, vn, 384) based his calculation upon the com- bining power of hemoglobin with CO. Hiifner and Ganser made use of the hemoglobin of the horse and bullock, freshly produced, free from alcohol, and crystallized three times without alcohol. The mean value of the hemo- globin of the bullock they found to be 16321, and of that of the horse 15115. They state it is doubtful whether the molecular weights of hemoglobins of the bullock and horse are the same or different ; that the results of these latest experiments agree with the mean values formerly obtained; and that the molecule of oxyhemoglobin is composed of 0 and of reduced hemoglobin. TABLE 27. — The molecular formulas and iveights of hemoglobin, according to various observers. Kind. Molecular formula. Molecular weight. Authority. Doe 13 332 Preyer Dog 16669 Jacquet Dog 14,129 Hiifner. Bullock 16 640 Bullock 16321 Hiifner and Ganser. Horse 12 042 Hiifner and Biicheler. Horse 16,730 Zinoffsky. Horse 15 260 Jutt. Horse 15 115 Hiifner and Ganser. Pig 13,513 Kulz. While the formulas given vary materially, the most striking differ- ence will be noted to be in the ratios of the percentages of Fe to S (table 26). Otto (Zeit. f. physiolog. Chemie, 1882, vn, 65) found that the hemo- globins of the dog and pig are practically identical as regards elementary composition and their coefficient of extinction, and that a close if not complete identity exists in the combining power with 0. These hemo- globins, he calculates, each contain 1 atom of Fe to 3 of S. Kulz estimated the same ratio for the hemoglobin of the pig, and Preyer the same ratio in the hemoglobin of the dog. But in the case of the horse and bullock the ratio is 1 of Fe to 2 of S. THE SOLUBILITY OF HEMOGLOBIN. The most marked differences noted in the hemoglobins of different species have been in the degree of solubility and in the quantity of water of crystallization. While the determinations of solubilities are extremely limited and far from satisfactory, because of obvious impurities of the sub- stances experimented with and the failure at times to record temperatures, they nevertheless show clearly very wide differences. Crystals of bullock's and pig's blood, for instance, are soluble in their water of crystallization at ordinary room temperature, while the crystals of raven's blood are practically insoluble in cold water, and between these extremes there are all gradations. 76 GENERAL CHEMICAL AND PHYSICAL CHARACTERS Hoppe-Seyler (Archiv f. pathol. Anat. u. Physiolog., 1864, xxix, 233) states that the dry hemoglobin crystals of the dog are soluble in the pro- portion of 2 per cent at 5°. Schmidt and Bottcher (Preyer, Blutkrystalle, loc. cit.) with an impure preparation found that water-free hemoglobin of the dog was soluble in 12.2 parts per 100 at 18°, and the dry crystals in the proportion of 15.59 parts per 100 at 18°. Lehmann (quoted by Preyer) records that the impure dry hemoglobin of the dog is soluble in the pro- portion of 0.4 to 3.1 per cent, and the solubility of guinea-pig crystals 1 part in 597, or 0.167 per cent. With the crystals of the horse Otto (Archiv f. ges. Ph)rsiologie, 1883, xxxi, 240) was unable to obtain concordant results, but Hiifner and Biicheler (Zeit. f. phys. Chemie, 1884, vm, 358) found a solubility of 2.614 per cent at 1° and 14.375 per cent at 20°. Standing in dilute alcohol renders the crystals less soluble (Preyer and others). THE QUANTITY OF WATER OF CRYSTALLIZATION. The percentages of water of crystallization given by different investi- gators, and even those noted by a given investigator with hemoglobin of a given species, show marked discrepancies. These differences are owing chiefly to the use of impure substances, to differences in methods of drying, and to certain difficulties incidental to accurate determinations. Schmidt and Bottcher (loc. cit.) found 13.49 per cent of water of crystallization in the crystals of dog's blood that had been dried by standing many days over sulphuric acid. Lehmann (loc. cit.) found in air-dried crystals of guinea- pig in two instances 19.9 per cent, and in others 15 per cent and 16 per cent. Preyer (loc. cit.) notes that after the crystals of the dog's hemoglobin were dried in the usual way, and then powdered and subjected to a temperature of 100° C., they lost 4.17 per cent. This powder upon standing in a glass case (not air-tight) for 3 days increased in weight 10.93 per cent, and when dried again at 100° C. decreased in weight 10.71 per cent. Lehmann re- corded figures for dry guinea-pig crystals which agree with these. At 15° they absorbed on an average 11.19 per cent of water in five experiments, according to which the air-dried guinea-pig hemoglobin would still contain 10.06 per cent of hygroscopic water, while the air-dry dog hemoglobin contains, according to Preyer's experiments, 9.67 per cent. Later Lehmann found that the air-dry dog crystals (which, however, were not pure) lost in weight in vacuo 9.79 per cent, and that the crystals dried in vacuum at 15° absorbed in 14 clays 9.54 per cent of water, so that the air-dried sub- stance would contain 8.71 per cent of water. They lost 9.09 per cent in weight at 120° C. Proyer states that his and Lehmann's figures agree very well when one considers that Lehmann worked with impure material and that he (Preyer) used a pure recrystallized substance. Hoppe-Seyler (Med. chem. Untersuch., 1868, Heft 3, 366; Chemischen Analyse, 1883, 292) noted the following percentages of water in crystals dried at 100° C. with the aid of an air-pump: Dog 3 to 4, goose 7, guinea- pig 6, and squirrel 9.4 per cent, In another publication (Physiologische Chemie, 1877, 377) his figures for guinea-pig and goose hemoglobins are 7 and 9.4 per cent, respectively. Otto (Zeit. f. physiolog. Chemie, 1882, OF HEMOGLOBIN, AND ITS SPECIFICITIES. 77 vii, 57) gives the percentage for pig's hemoglobin crystals dried over sulphuric acid, then at 115°, as 5.9, and for the dog 4. He also reports (Archiv f. ges. Physiologic, 1883, xxxi, 240) that he did not obtain con- cordant results with the crystals of horse's blood. Hiifner and Biicheler (Zeit. f. physiolog. Chemie, 1884, vin, 358) dried horse crystals at 0° over sulphuric acid and anhydrous phosphoric acid, and then found them to contain 3.94 per cent of water. Jacquet (Zeit. f. phys. Chemie, 1889, xiv, 289) records that air-dried crystals of the dog lost 11.39 per cent of water at 115°, and those of the chicken 9.333 per cent. Hiifner (Archiv f. ges. Physiologie, 1894, 130) gives the water of crystallization of bullock's blood as being 9.98 per cent. Bohr (Exper. Untersuch. u. d. Sauerstoffaufnahme d. Blutfarbstoffes, Copenhagen, 1885) found that the percentage in bullock's blood varies from 1.2 to 6.3 per cent, which variations may be due, in part at least, to impurities of his preparations. THE EXTINCTION COEFFICIENTS AND QUOTIENTS. Vierordt, Hiifner, and others have found that reliable extinction co- efficients can not be obtained by measurements of a single spectral field unless the solution contains but a single coloring matter; because, while solutions of a single coloring matter affect the light intensities of the differ- ent regions of the spectrum in constant relationship to each other, irrespec- tive of the strength of the solution, the presence of a second coloring matter alters or destroys this relationship. Therefore, two fields must be measured, and the fields to be selected should be those which are most readily influenced by the differences in the strength of the solution. Moreover, the two coefficients thus obtained serve as mutual checks. The quotient obtained from these coefficients, as shown by Hiifner (Archiv f. Anat. u. Physiologie, Physiolog. Abth., 1894, 130, and 1900, 39) in his studies of oxyhemoglobin, reduced hemoglobin, methemoglobin, and CO-hemoglobin, is absolutely con- stant and distinctive for each substance, and, therefore, departures in extinc- tion coefficients show, according to him, not only the presence of impurities but also the quantity of each coloring matter present. Hiifner measured the extinction coefficients of these substances for the mid-region between A and B absorption bands (the interval between the wave lengths 554 and 565 fifi), and for the darkest portion of band B (the interval between 531.5 and 542.5,wu). In such determinations with fresh bullock's blood and solutions of the crystals of bullock's oxyhemo- globin he found constant results. The quotient for oxyhemoglobin was 1.578, for reduced hemoglobin 0.7617, for CO-hemoglobin 1.095, and for fresh rabbit's blood 1.579. Zeynek (Archiv f. Anat. u. Physiologie, Phys. Abth., 1899, 460) by the same means determined for methemoglobins of the horse 1.187, and of the pig 1.183. Hiifner in the later article states that the oxyhemoglobin quotient (1.578) and the reduced hemoglobin quotient (0.762) have each the same value independent of the degree of concentration of the solution and the species of blood. Von Noorden (Zeit. f. phys. Chemie, 1880, iv, 9) found the mean quo- tient for pure oxyhemoglobin of the dog to be 1.324, for the guinea-pig 78 GENERAL CHEMICAL AND PHYSICAL CHARACTERS 1.357, and for the rat 1.337. He also found the like value in the case of man, the cat, and the owl. Otto (Zeit. f. physiolog. Chemie, 1882, vn, 57) noted with solutions of the oxyhemoglobin of the dog and pig a quotient of 1.33; and in a later research, with an improved form of spectrophotometer, obtained a quotient of 1.352 for horse oxyhemoglobin and 1.34 for that of the dog. Sczelkow (Archiv f. ges. Physiologic, 1887, XLI, 373) calculated, by means of Hiifner's spectrophotometer, a quotient of 1.336 for horse hemoglobin. For dog's hemoglobin he recorded 1.305, which he looks upon as not being correct, and he remarks that coefficients obtained by him differed from each other and from the mean value much more than did Otto's, which differences he explains upon the assumption that the concentration of the solutions used by him differed more than those employed by Otto. Hiifner, in the more recent investigation referred to, found these quo- tients so constant and specific that he formulated tables by aid of which the quantity of oxyhemoglobin, reduced hemoglobin, methemoglobin, or CO- hemoglobin, or the quantities in mixtures of oxyhemoglobin and reduced hemoglobin, or of methemoglobin and CO-hemoglobin may be determined. Moreover, he states that these quotients, as well as the O, CO, and the iron capacities of the coloring matter of the blood, are not only the same in related but also in unrelated species, and that when this coloring matter is freed from water it has in all of the higher animals the same molecular weight and the same capacity for O and CO. In opposition to Hiifner's assertion of the constancy of the extinction coefficients and quotients of the same and of different bloods, we find evidence in the results of a number of investigations. The quotients given by von Noorden, Otto, and Sczelkow already noted are far off. Korniloff (Zeit. f. Biologic, 1876, xu, 513) determined the extinction coefficients in relation to the second absorption band (B) of the coloring matter of the blood of 110 vertebrates, comprising 44 species. He made determinations by other regions of the spectrum, but these values deviated considerably from those obtained from the second band. Inasmuch as he did not make his determinations by the effects on two bands, which is necessary to obtain accurate results, and as the bloods doubtless contained variable proportions of oxyhemoglobin and reduced hemoglobin, and in some instances probably methemoglobin if not also other coloring matters, his figures must be looked upon as representing only approximate values. Accepting them as approx- imations, they differ so much as to indicate that the coefficients in at least different orders of animals are very far from being identical, as will be seen by the figures in table 28. Kruger (Zeit. f. phys. Chemie, 1898, xxv, 256) found the quotient of cat's oxyhemoglobin to be 0.128, and for that of the dog 0.137. Velichi (Inaug. Dissert. Berlin, 1900; Centralbl. f. Physiologie, 1900, xiv, 679; Dcutsch. med. Wochenschr., 1900, xxvi, 148) found such differences in the extinction coefficients that he states that the hemoglobins of all classes of animals are not identical. Dreser (loc. cit.) gives 1.557 as the quotient for human oxyhemoglobin, and Saint Martin (Compt. rend. soc. biologic, 1901, LIII, 302) obtained the following quotients: Human 1.60, bullock 1.62, dog OF HEMOGLOBIN, AND ITS SPECIFICITIES. 79 1.61 and 1.63. Miiller (Archiv f. ges. Physiologie, 1904, cm, 541) in studies of freshly drawn blood, after three years' experience with the spectrophotom- eter, has cast doubt upon certain of Hiifner's teachings regarding extinction coefficients, etc. He found that the relations of the extinction coefficients of the blood taken directly from the animal are not as constant as Hiifner states, and in opposition to Hiifner he holds that values which differ from 1.56 are not necessarily wrong. He goes on to state that Hiifner looks upon all values which differ materially from 1.56 as wrong, which Hiifner explains by the formation of methemoglobin. This explanation seems to Miiller to be untenable, since he found in examinations of freshly drawn blood from the ears of a dog a value of 1.47 to 1.49, which figure Hiifner would designate as incorrect, and yet it was fresh blood taken from the healthy animal, so that we must either accept the presence of methemoglobin in the apparently normal animal, or else material individual differences in the optical con- stants of oxy hemoglobin. The second probability seems to Miiller more likely, and he goes on to state that it should not be left unmentioned that Torup observed by means of the Glan photometer, after the addition of a little sodium bicarbonate to the diluted hemoglobin solutions, a shifting of the point of strongest absorption; and also, as Bohr states, an insignificant change of the hemoglobin, which has no influence whatever either upon the molecular weight or the amount of absorbed oxygen, may give an entirely different value in light absorption. Changing alkalinity of the blood seems therefore to have a disturbing influence, as has been found by others. TABLE 28. — Extinction coefficients in different orders of animals. Orders. No. of animals. Extinction coefficients. Fishes . . . 16 03564 Amphibia 13 0.3889 Reptiles 13 0.4328 Birds 17 07814 Mammals 22 0.9366 In a more recent inquiry with fresh blood, Aron, Hans, and Miiller (Archiv f. Anat. u. Physiologie, 1906, Suppl. Bd., 109) throw even more serious doubts upon Hiifner's assertions as to the constancy of the extinc- tion coefficients. The average quotient they found to be about the same in different species (dog, horse, cat, ox, and rabbit), but 55 out of 142 cal- culations differ much more from the average value than can be explained by the greatest possible errors that can be accounted for in errors of method or by variations in the strength of solution. Moreover, the average value was found to be 1.47, whereas Hiifner's is 1.578. Light absorption they found to be in direct relation to the quantity of iron, and approximately the same for the blood of the rabbit, ox, and dog, but varying somewhat for the blood of the horse. They suggest that methemoglobin normally exists in the blood, which may account for differences in Hiifner's and Bohr's results in their studies of oxyhemoglobins. They also point out that by the regular method of defibrinating the blood there occurs a loss of hemoglobin which does not occur if the defibrinization be effected by agi- tation in a closed vessel. 80 GENERAL CHEMICAL AND PHYSICAL CHARACTERS Bardachzi (Zeit. f. phys. Chemie, 1906, XLIX, 465) determined the mean quotient for the fresh blood of the sea-tortoise (Thalassochelys corticata) to be 1.561, and for the oxyhemoglobin crystals in solution 1.569. The mean quotient for methemoglobin he records as 1.184. The injury to the hemoglobin molecule caused by the methods of prep- aration has been referred to a number of times in preceding pages. Interest- ing in this connection are the results of the investigations of Kupffer (Inaug. Dissert., Dorpat, 1884) and Kriiger (Zeit. f. Biologie, 1887, xxiv, 47), both of whom have found that recrystallization notably and injuriously affects the extinction coefficients. Kupffer determined that the oxyhemoglobin of the horse and the dog showed a higher extinction coefficient when crystal- lized three times than when crystallized twice; while Kriiger, using the Hiifner spectrophotometer, found that even a single crystallization gives rise to a higher absorption ratio. Inasmuch as this is the opposite effect to that which should be expected if recrystallization means merely puri- fication, it is clear that the molecule has been altered. He also calls attention to the fact that the addition of ammonia increases the solubility of the crystals about twice. The accompanying table (table 29) from Kruger is of interest : TABLE 29. — The effects of recrystallization and alkali upon the extinction coefficient of hemoglobin, according to Kruger. OxyhemoglobiD crystals — Number of times crystallized. 1 2 3 4 From defibrinated horse blood with NH, 0.1297 .1321 .1259 .1417 .1429 0.1372 .1372 .1317 .1435 .1453 Do 0.1266 From uncoagulated horse blood with NH, From dog blood without NH, .1337 .1401 0.1498 .1452 From dog blood with NH, Hoppe-Seyler in his earlier investigations (Med. chem. Untersuchungen, 1868, Heft 3, 366) noted that the intensity with which hemoglobin absorbs the light of definite portions of the spectrum is not alike for different species. Thus, to have solutions of like absorptive intensity, the following quantities were necessary in a liter of solution: 1.641 grams of goose hemoglobin, 1.682 grams of dog hemoglobin, 1.703 grams of guinea-pig hemoglobin. This has received confirmation in the researches of Abderhalden (Zeit. f. physiolog. Chemie, 1898, xxiv, 545), who found that in order to obtain the same colorimetric value 10 c.c. of a standard solution of dog hemoglobin must bo diluted with 8 c.c. of water to have the same intensity as the same standard solution of cat hemoglobin. THE DIFFERENCES IN THE DECOMPOSABILITY OF THE HEMOGLOBIN OF DIFFERENT SPECIES. While hemoglobin is, in comparison with proteins generally, extraor- dinarily resistant to the putrefactive organisms, excepting practically in so far as concerns the conversion of oxyhemoglobin into methemoglobin and reduced hemoglobin, it readily undergoes decomposition, especially so OF HEMOGLOBIN, AND ITS SPECIFICITIES. 81 in the presence of any reagent which causes a separation of the globin and hematin, or which converts the hemoglobin into methemoglobin or reduced hemoglobin. The degree of decomposability varies in relation to different species, and in individuals of the same species under abnormal conditions. Korber (Ueber Differenzen des Blutstoffes, Inaug. Dissert., Dorpat, 1886; Centralblatt f. med. Wissensch., 1867, v, 117) found by the aid of the spec- troscope in a study of the bloods of 11 species of warm-blooded animals (man, horse, bullock, sheep, pig, dog, cat, hare, goose, chicken, and crow) and 3 species of cold-blooded animals (frog, pike, and lote) interesting differences in the behavior towards certain reagents. Normal human hemoglobin was found to be more readily decomposed by acetic acid than by soda, but the opposite was noted under pathological conditions. In febrile states in the human being and in inanition in the dog decomposability was increased. The hemoglobin of typhus blood decomposed 18 times sooner than normal blood. Under given conditions, the hemoglobin of the chicken was decom- posed 150 times more quickly than that of man; pig's was decomposed 780 times more slowly than that of the bullock and 350 times more slowly than that of the dog, etc. Rabbit's hemoglobin was decomposed 150 times more quickly through acetic acid than by soda, but the hemoglobin of the pike was affected much more readily by soda than by acetic acid. With a proportion of 0.5 gram of sodium hydrate to 20 c.c. of a 1 per cent solution of blood, he found that the decomposition of hemoglobin began at time intervals shown in table 30. TABLE 30. — Time intervals of beginning of decomposition of hemoglobin. Kind. Time interval. Kind. Time interval. Typhus Sheep 36 mins. Doe 51 miiis. Lote 60 mins. Pike 45 sees Chicken 65 mins. Cat Pig 3 hours. Bullock Over 3 but less Goose 4 & minfi than 16 hours Kruger (Zeit. f. physiolog. Chemie, 1888, xxiv, 318), in similar spectral examinations with the bloods of the dog and horse, found support to Kor- ber's results. He states that the resistances of the hemoglobins of the dog and horse against acetic acid and sodium hydrate differ, that they vary considerably, and that the variability is due to the chemical condition of the hemoglobin itself; that the difference in the decomposability increases with the quantity of decomposing agent ; and that sodium hydrate is more effective than the acid. The results of the foregoing investigations have received support in the comparatively recent investigations of Magnanimi (Bull. d. soc. Lancisiana d. osped. di Roma, 1898; Jahr. li. d. Fort. d. Thierchemie, 1898, xxvin, 144) and Ziemke (Vierteljahresschr. Med., 1901, xxii, 77). Magnanimi by the aid of a Kriiss spectrophotometer examined the bloods of 4 men and 1 woman, and also the bloods of the dog, horse, calf, pig, wether, and lamb. He found upon the addition of sodium hydrate that the bands of human blood vanish in 38 minutes, of the dog after 110 6 82 GENERAL CHEMICAL AND PHYSICAL CHARACTERS. minutes, and in other animals only after 3 hours. In the case of blood stains 6 to 60 days old, the older the stains the less resistance of the hemo- globin, but the relationship between the different bloods remains — that is, the bands of human blood disappear sooner than those of dog's blood, and those of dog's blood sooner than those of the others. Ziemke states that in order to show differences in resistance to alkali the use of the spec- trophotometer is not necessary, but that by colorimetric measurements dried blood and blood stains of human blood can be distinguished from those of domesticated animals, even though methemoglobin has been formed. IS THE OXYHEMOGLOBIN OF THE BLOOD OF ANY INDIVIDUAL A SINGLE SUBSTANCE? That there is always in the normal blood a mixture of oxyhemoglobin and reduced hemoglobin in varying proportions has long been established, but to what extent and constancy, if any, such substances as methemo- globin, C02-hemoglobin, and CO-hemoglobin may be present has not been determined; nor has it been shown that either oxyhemoglobin or reduced hemoglobin is a single homogeneous substance in any species of blood. Hoppe-Seyler (Zeit. f. physiol. Chemie, 1878, n, 139) found that the oxy- hemoglobin of the horse appears in the form of minute needles and prisms which differ in solubility, which difference he believes may be due to dif- ferent amounts of water of crystallization. Otto (Archiv f . ges. Physiologic, 1883, xxxi, 240), in repeating Hoppe-Seyler's experiments, also noted these two forms, but he found that one form could not be separated from the other by washing with dilute alcohol, as stated by Hoppe-Seyler. Bohr (Cornpt. rend. soc. biologie, 1891, cxi, 243), in his studies of the homogeneity of hemoglobin, found that the portion of hemoglobin in the mother-liquor after crystallization showed a lower combining power with o\ym-ii than that which crystallized out; and in a previous article (Ceutral- blatt f. Physiologie, 1890, iv, 242) he reported from the results of his experi- ments that the oxyhomoglobin of the blood is not a simple substance, but a mixture of oxyhemoglobins, and that there may exist in the blood four forms, all giving the same oxyhemoglobin spectrum, but differing in their absorption coefficients, in their percentages of iron (0.35 to 0.46), and in their combining with () (0.4, 0.8, 1, and 2.7 c.c. O per gram). He believes that there are also several forms of CO2-hemoglobin. Iliifner lAivhiv f. Anat. u. Physiologie, 1894, 130) seems, however, to have demonstrated that Bohr's several forms of oxyhemoglobin were mix- tures of oxyhemoglobin with varying amounts of methemoglobin and other decomposition products which resulted from his methods of preparation. Hiif net's suggestions are borne out by the investigations of Marchand (Archiv f. path. Anat. u. Physiol., 1879, LXXVII, 488), and even by the early work of Hoppe-Soyler and by the researches of others. In the blood of cattle, Hlifner states, there is but one oxyhemoglobin, and likewise in all the higher vertebrates. By the crystallographic method, however, we have found several kinds of oxyhemoglobin in certain bloods. CHAPTER IV. THE PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS PREVIOUS TO THE INVESTIGATIONS OF PREYER. Crystals of hemoglobin were first discovered by Hunefeld (Die Chem- ismus in der thierischen Organization, Leipzig, 1840, 160), who found, upon exposing the blood of the earthworm between plates of glass, that there were deposited bright red table-form crystals having sharp borders. He also refers to crystals from the blood of man and of the pig. While Htine- feld's article is the first on record, the real foundation of our knowledge of hemoglobin was laid by the discovery of K. E. Reichert (Miiller's Archiv f. Anat. u. Physiologie u. wissensch. Medicin, 1849, 198), during the summer of 1847, of tetrahedral crystals of hemoglobin in the fetal membranes and in the mucous membrane of the uterus of a guinea-pig which had suddenly died, and which was examined 6 hours after death. The uterus contained 4 fetuses, and in all four placentas the crystals were found. The crystals were regular tetrahedra of various sizes. The inclination of the planes towards each other amounted to 70° 31' 43", and that of the planes towards the edge, 54° 44' 8.5". He noted truncation in rare cases, which he thought might be due to outside mechanical force. Reichert undoubtedly recog- nized from the studies of the chemical reactions that these crystals were albuminous. This contribution appears to have at once aroused interest in the study of the blood crystals, as is indicated by the appearance of a number of contributions during the next few years. Leydig (Zeit. f. wissensch. Zoologie, 1849, i, 116) found crystals of the blood of Nephelis in the stomach of Clepsine. The corpuscles, he states, became decolorized and disappeared, and in the plasma were found red tabular leaflets and rods and columns, small and large, single and aggregate. He also noted that if water entered the stomach the crystals dissolved. Kolliker (Zeit. f. wissensch. Zoologie, 1849, i, 266), in the records of his histological studies of the blood corpuscles, describes red crystals in the blood of the dog, river perch, and python, and states that the crystals were within the corpuscles and also in the plasma of the blood of the spleen and liver. Crystals of human blood were observed by Budge (Sitz. d. Niederrh. Gesellsch. f. Natur- u. Heilkunde, 12 Dec., 1850, and Koln. Zeitung, No. 300, 1850; quoted by Preyer, loc. cit.) in the stomach of leech. Shortly after this appeared the first article by Funke (Zeit. f. rat. Medicin, 1851, N. F., 1, 185), which was almost immediately followed by his second and third contributions (ibid., 1852, N. F., 2, 198, 288). To Funke is due the credit of being the first to devise methods for preparing blood crystals, which crystals had heretofore been obtained solely by 83 84 PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS accident. His chief method, still in use and known as "Funke's method," is most simple and very satisfactory for bloods that are readily crystalliz- able. Funke prepared crystals from the blood of man, the horse, bullock, dog, fish, cat, pig, and pigeon. He states that if some water is added to a drop of blood (which in consequence of free evaporation has already begun to dry) spread out on an object-glass, and the edges of the prepara- tions are observed, it can be seen that the corpuscles suddenly change. While some of the blood corpuscles vanish the others acquire dark, thick outlines, become angular, and develop into small, sharply defined rods. In this way are formed an enormous number of crystals which are too small to have their form accurately ascertained. These crystals quickly increase in length, while their diameter remains unchanged or increases only slightly, and finally the whole field is a thick network of needle-shaped crystals crossing in all directions. This process goes on so extraordinarily quickly that it is difficult to follow with the eye the first formation, as well as the steps of gradual development, on which account, he states, he could not convince himself that the crystals really arise from the corpuscles themselves. The whole phenomenon, he writes, can be most beautifully observed if the cover-glass is shifted after water has been added to the concentrated drop of blood, and then those places observed where before, on the edges of the cover-glass, thicker layers of blood were in the process of drying. Occasionally crystals form in clots of splenic venous blood upon evapo- ration, but in this way there arise very incomplete crystalline formations, ordinarily a small row of pale-red leaflets or rods arranged palisade-like, without any recognizable ciystal form. When a drop of blood is mixed with ether it changes almost at once into an entangled heap of scale-shaped and leaf-shaped crystals, which are suspended in a homogeneous fluid. By the addition of alcohol Funke succeeded in producing crystals of so enormous a size that they could be recognized by the naked eye, although for the most part the crystals were badly formed. During the first minute, he states, the alcohol coagulated the blood to thick red clots. After evaporation, however, there appeared in isolated spots long, broad, sword-shaped leaves of intense red color, with irregular, often saw-shaped and indented, splintered ends. Only a few of the crystals were 4-sided prisms. Funke made measurements of the angles of the crystals by the aid of a goniometer; but upon insufficient data he accredits the crystals to certain systems. He noted that the forms and solubilities of the crystals of dif- ferent species are not alike, and therefore that species may thus be differ- entiated. (See Chapter VII.) He also noted that, in the bloods of the three species of fish examined, all of the corpuscles changed into crystalline form, and that upon the addition of water a great part of them were changed I nick to corpuscles under the eye of the observer. The foregoing investigations, especially those of Funke, because of his being the first to prepare blood crystals, may justly be regarded as consti- tuting the foundation for the rational study of hemoglobin. At that time (1851-2) the precise nature of the substance of the crystals was unknown. PREVIOUS TO THE INVESTIGATIONS OF PREYER. 85 Reichert looked upon the crystals as being albuminous; Kolliker refers to them as "globulin" crystals; and Funke states that the crystalline sub- stance is the chief constituent of the blood corpuscles, which substance he regards as being a combination of globulin and hematin. Since then various terms have been suggested, such as hematoglobulin, hematocrystallin, cruorin, hemochrome, hemoglobin, etc., but the last, suggested by Hoppe- Seyler, is the universally accepted term at the present day. Immediately following Funke, Kunde (Zeit. f. rat. Medicin, 1852, N. F., 2, 271) obtained, by a slight modification of Funke's process, crystals from the blood of the bullock, horse, dog, guinea-pig, squirrel, rat, mouse, bat, rabbit, pigeon, and tortoise. He also found crystals of human blood in the stomach of leech. He observed that the crystals from different species are not identical, from which he concludes that the form of the crystals is pecu- liar to each species. Remak (Archiv f. Anat. u. Physiologie, 1852, 115) found crystals in blood of the tench, perch, and roach 24 to 48 hours after death. Parkes (Medical Times and Gazette, 1852, xxvi, 103) accidentally found crys- tals in human blood that had putrefied. Lehmann (Berichte konigl. sachs. Gesellsch. d. Wissensch. in Leipzig, math.-phys. Klasse, 1852, 23, 78; 1853, 101 ; Chem. pharmac. Centralblatt, 1853, 98) was the first to make an ele- mentary analysis of the blood crystals. He diluted the blood with 1 to 1.5 volumes of water and prepared crystals from the bloods of the guinea-pig, squirrel, and hedgehog. He gives the following figures from his analyses : C53-4-54-lH7-7.3Ni5.5-j6.2Sx. 2 Teichmann (Zeit. f. rat. Medicin, 1853, N. F., 3, 375) obtained crystals from the blood of man and from that of dog, bullock, pig, rabbit, pigeon, and fish. He observed also decolorized crystals. He opposes the conclu- sion of Funke and Kunde that differences in the forms of the crystals are peculiar to species, for he found that even from the same blood various crystalline forms may be obtained, from which he concludes that the form of the crystal is accidental and due to exterior conditions. Berlin (Neder- landsch. Lancet, 1853, in, 16, and 1855-56, v, 734; Archiv f. d. Holland. Bei- trage z. Natur- u. Heilkunde, 1858, 1, 75) describes crystals of the lion and python, and he also found crystals of human blood in the leech. Robin and Verdeil (Traite de chim., anatom. et physiol., Paris, 1853, n, 335) doubted the albuminous nature of the blood crystals, which they thought were phosphates rendered impure by contaminating albuminous substances. Kolliker (Microscop. Anat., 1854, n, 2 Aufl., 280) again reported instances of intraglobular crystallization, and also of his having prepared crystals from the bloods of several species already reported. Bissegger and Bruch (Verhandlungen d. Baseler Naturforschenden Gesellschaft, 1857, i, 174) isolated crystals of the rat, and they also found crystals within some of the corpuscles. Meckel (Archiv f. d. Holland. Beitrage, 1858, i, 90) obtained crystals from the blood of man and the pig. Dr. S. Weir Mitchell (Proc. Acad. Natural Sciences, Philadelphia, 1858-59; Proc. Biolog. Dept., 2), who was the first American to report studies of hemoglobin crystals, examined the crystals of the sturgeon, 86 PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS guinea-pig, and man. He obtained crystals by Funke's method, and also by allowing the blood to stand in an open vessel exposed to light at a tem- perature of 60° to 70° F. to putrefy. He found that at any time after 48 hours a drop of this blood would yield by slight evaporation, without added water, the most beautiful crystals. He states that those of sturgeon's blood are hexagonal columns and tablets; that their color may be washed out with alcohol and water without injury to their form, and that the decolorized crystals may be dissolved in water and again obtained devoid of color, but unchanged in crystalline shape. Crystals in the form of hex- agonal plates were frequently seen "within the envelope of the corpuscles." When a glass slide containing a group of crystals was kept for some months, the crystals were altered in their color so as to exhibit beautiful tints, such as yellow, orange, purple, and various shades of green, recalling "very strikingly the alterations of tint undergone by the leaf in the autumn." Dr. Mitchell found that the crystals are of the same form from whatever part of the body they are obtained; but he also makes a note of the fact that Dr. Johnson obtained tetrahedral forms from the splenic blood of the opossum but rhombic crystals from the blood of other vessels. He also observed that human blood of the male, the female, the fetus, and the placenta, and the blood in many diseased conditions, such as dysenteiy, measles, cholera, typhoid fever, yellow fever, pneumonia, etc., give in each case the same form of blood crystal. Bottcher (Preyer, Die Blutkrystalle, Dorpat, 1871, 14; Archiv f. path. Anat. u. Physiologic, 1863, xxvn, 465) prepared crystals from the blood of dogs, cats, and other animals by anesthetizing the animals, then inject- ing cold water into the veins, and finally killing with chloroform. The blood was diluted with an equal volume of water and subjected to a tem- perature kept down to freezing-point for two days. Schmidt (Archiv f. path. Anat. und Physiologie, 1863, xxvii, 465) analyzed crystals of dog's blood that had been dried at 110° C. He gives the following figures: C53-64H7.11N16.19So-66020-03Fe0.43 also alkali and alkaline earth (0.04 per cent) and phosphoric acid (0.91 per cent), which latter shows that he had a very impure preparation. Rollett (Verstiche u. Beobachtungen am Blut, etc., Wien, 1862; Sitzungsberichte d. math.-natur. Classe d. Kaiser. Akad. d. Wissensch., Wien, 1862, XLVI, Abth. 2, 85) studied, with the help of von Lang, the crys- tallographic and optical characters of the blood crystals. Rollett prepared crystals from the bloods of the guinea-pig, squirrel, cat, man, rabbit, pig, and frog. Crystals from man, the rabbit, dog, cat, and guinea-pig they describe as belonging to the rhombic system, while those of the squirrel they assign to the hexagonal system. Rollett made use of several means of Inking the blood to facilitate crystallization, which will be found referred to in other pages. Von Wittich (Konigsberger medicinische Jahrbiicher, 1862, in, 332) obtained crystals from the blood of the rat, guinea-pig, and dog by breaking down the corpuscles with ether, but he did not succeed with the blood of man, rabbit, chicken, or frog. PREVIOUS TO THE INVESTIGATIONS OF PREYER. 87 Bursy (Inaug. Dissert., Dorpat, 1863; Ber. ii. d. Fort. d. Anatomie u. Physiologie, 1862, 293) studied the influences of various salts upon the crystallizability of hemoglobin. He found that sodium sulphate, sodium phosphate, sodium acetate, magnesium sulphate, and potassium sulphate favor crystallization; that potassium carbonate, potassium sulphate, sodium borate, barium nitrate, and sal ammoniac have little favorable effect; that sodium chloride, ammonium nitrate, calcium chloride, and alum were without influence; and that sodium nitrate appeared to hinder crystallization. Ankersmit (Inaug. Dissert., Groningen, 1863; Ber. ii. d. Fort. d. Anat. u. Physiologie, 1863, 268) prepared crystals from human venous blood, which crystals he found became decolorized under certain conditions, and he therefore believed, as did Lehmann and others before him, that the crystals are only mechanically colored or stained. Klebs (Centralblatt f. med. Wissensch., 1863, i, 268) reports having found crystals in the cor- puscles of the guinea-pig, rabbit, pig, sheep, and bullock, and also in man. Kiihne (Centralblatt f. med. Wissensch., 1863, i, 851) recorded a process for preparing hemoglobin crystals by the addition to the blood of bile salts. He prepared in this way crystals from the blood of the horse and dog. In a later article (Archiv f. path. Anat. u. Physiologie, 1865, XLIII, 423) he states that he prepared crystals of reduced hemoglobin from the blood of the dog. He also noted the fact that the alkaline serum hinders the crystal- lization of hemoglobin. Valentine (Untersuch. z. Naturlehre, etc., 1863, ix, 129) obtained crystals from the blood of a marmot that had been hiber- nating for a long time. At this time (1863) there still existed much difference of opinion as to the exact nature of the blood crystals, and Bojanowski (Zeit. f. wissensch. Zoologie, 1863, xn, 312), in going over certain unsettled points, concluded that only the contents of the corpuscles participate in the formation of the crystals, that the crystals are merely stained, and, therefore, that the name given by Kolliker, "globulin crystals," is entirely justifiable. His reasons for reaching this last statement were based partly on the reports of Leh- mann, Teichmann, and others of having obtained decolorized crystals, and partly from his own experience. He states that if the blood crystals are allowed to stand for a time in the air they always retain their form, but become clearer and clearer, and finally completely colorless and transparent. The same is observed if to the crystals is added a strong sugar or gum solution. Bojanowski prepared crystals from the blood of man, and from the rabbit, mouse, dog, cat, hedgehog, river bream, pike, horn-fish, herring, lark, raven, and pigeon. Crystals of human blood he obtained from the stomach of the leech, and also from venous blood. The latter was 36 hours old, and crystallization was completed within 3 to 4 hours without anything being added. The addition of water causes crystallization, but more spar- ingly and irregularly; but on the addition of alcohol and ether he failed in 15 experiments to obtain crystals. Crystals of the river bream he ob- tained without any treatment of the blood, and he states that this blood 88 PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS crystallizes with extraordinary rapidity. To the blood of a mouse taken from the animal 20 hours after death he added a mixture of equal volumes of alcohol and ether, and obtained, within a few minutes, numerous irreg- ular six-sided plates and also rod-shaped crystals, occasionally in star- shaped groups. From the blood of the dog he always obtained crystals within 15 to 20 minutes after the addition of the mixture of alcohol and ether. From the cat he obtained three-sided prisms, which in most cases appeared a bright red, occasionally a bright yellow, and at times com- pletely colorless. He also obtained crystals after the addition of water, and also after putrefaction had set in. The blood of a hedgehog that had been chloroformed 24 hours previously yielded crystals without treatment, but the crystals were much better when a mixture of alcohol and ether (1 : 4) had been added to the blood. Crystals of the lark he secured by the addition of a mixture of alcohol and water (1:1). Crystals from the horn- fish formed in the blood with or without the addition of water, and in the same way he obtained similar crystals from the blood of the pike. The blood of the herring he states crystallizes extraordinarily quickly, and the crystals almost always appeared colorless and possessed a shimmer similar to mother-of-pearl. From the blood of the raven he obtained crystals only after the blood had stood for 8 days in a cool place, and by the addition of chloroform and ether (1:3); but he failed to obtain crystals by the addi- tion of distilled water, alcohol, gum solution, or sugar solution. The crystals he describes as partly colored bright yellow, partly completely colorless. Similar crystals were obtained from the pigeon by the addition of distilled water. Bojanowski notes from his investigations that the crystals of dif- ferent species have something specific and characteristic about them, so that occasionally he could designate the species from which the crystals were derived. The characteristic absorption spectrum of hemoglobin was discovered by Hoppe (Archiv f. path. Anat. u. Physiologie, 1862, xxm, 446; Hoppe- Seyler was known as Hoppe previous to 1864), who states his belief that it is the same for the bloods of all vertebrates. He showed that hematin, which until then had been almost universally regarded as the coloring matter of the blood, is an abnormal constituent, and a product of decom- position of hemoglobin. He identified hemoglobin with the blood crystals described by Himefeld, Reichert, Funke, and others, and he showed that while certain reagents were without effect on hemoglobin, others gave rise to a decomposition into an albuminous substance and hematin. No differ- ence was noted in the spectra of arterial and venous blood, which was doubtless owing to the rapid oxidation of his preparations of venous blood, as he did not know, until some time later, of the difference in the coloring matter of arterial and venous blood and of the rapid oxidation of reduced hemoglobin when exposed to the air. In Hoppe-Seyler's second contribution (Archiv f. path. Anat. u. Phy- siologie, 1864, xxix, 233, 567; Centralblatt f. d. med. Wissensch., 1864, u, April 16, 261, 817, 834) he proposed the terms "hemoglobin" and "hemato- globin" to distinguish the coloring matter of the blood. This substance, he PREVIOUS TO THE INVESTIGATIONS OF PREYER. 89 states, constitutes, excepting a few traces of other matters, the only con- stituent of the red corpuscles in man and the dog, while in birds and several mammals considerable quantities of albuminous substances are present in the corpuscles. Hemoglobin crystals of bloods of man, the dog, ox, sheep, guinea-pig, rat, mole, hedgehog, mouse, goose, pigeon, hen, frog, adder, and turtle, and probably of the bloods of all vertebrates, he states, contain no other substance than hemoglobin, and particularly is no hematin present in them. While Hoppe-Seyler failed to note in his previous research any differ- ence in the spectra of arterial and venous blood, he records that if the solution of hemoglobin is freed from 0 by a current of C02, or by decom- position, it shows a spectrum that is somewhat different from that of a solution that has been shaken with air. He called particular attention to the readiness with which hemoglobin in crystalline form, or in solution, undergoes decomposition. A feeble alkaline reaction of the solution, the presence of albuminous bodies, and a temperature at or below 0° preserve the hemoglobin; but the higher the temperature the quicker the decom- position. No concentrated solution remains undecomposed for 24 hours at ordinary temperature, and, as the spectrum shows, the crystals also become decomposed with like quickness. Dilute solutions are somewhat less readily decomposed than strong solutions. Even dry crystals can not be kept undecomposed, and in the presence of albumin decomposition goes on quickly. In every instance the decomposition of the hemoglobin still present in concentrated solution takes place at first very quickly, and grad- ually less and less rapidly. Therefore, in concentrated solutions, even after many weeks and months, some undecomposed hemoglobin remains. In his third contribution (Archiv f. path. Anat. u. Physiologic, 1864, xxix, 597; Centralblatt f. med. Wissensch., 1865, in, 38) Hoppe-Seyler calls attention to the loose combination of 0 with hemoglobin, to the combination of CO with hemoglobin, and to his centesimal analyses of hemo- globin and hematin. His mean figures for the dry hemoglobin of the dog and goose are : C54.2H7.2N16Fe0.42 He did not record phosphoric acid and other inorganic constituents, but in later analyses he found phosphoric acid in goose hemoglobin, but not in the hemoglobin of mammals. In later publications (Medicinisch-chemische Untersuchungen, 1866, Heft 1, 151, 1867, and Heft 2, 293; 1868, Heft 3, 366, 386) he gives a process for preparing hemoglobin, together with much matter pertaining to the chemistry and to other properties of hemoglobin. His process is as follows : In order to obtain crystals of pure hemoglobin, the blood is defibrin- ated and mixed with 10 volumes of salt solution, which consists of 1 part of saturated solution of chloride of sodium and 9 parts of water, and set aside at 0° until the corpuscles have sunk to the bottom. The supernatant fluid is then drawn off and the corpuscles are washed as often as four times in this way. To the washed corpuscles are added merely enough water to dissolve the hemoglobin and afterwards an equal volume of 90 PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS ether, the whole is shaken, and then the excess of ether is poured off. The solution is quickly filtered. The solution of dog, guinea-pig, squirrel, and rat blood corpuscles crystallizes without further treatment. With bloods that do not crystallize readily, the nitrate is cooled to 0° and mixed with one-fourth of its volume of 80 per cent alcohol which has also been cooled to 0°, and the mixture subjected to a temperature of —5° to —10°. After crystallization, the crystals are collected on a filter paper at a tem- perature near 0°, and washed in the cold (at 0°) with a cold mixture (at 0°) consisting of 1 volume of alcohol to 4 volumes of water. The crystals are dried between filter paper by slight pressure. To recrystallize, the crystals are dissolved in 3 volumes of distilled water by heating to 30° to 40° C., the solution is filtered and cooled to 0°, one-fourth volume of absolute alcohol cooled to 0° is added, and the mixture subjected to —5° to —10°, as before. In this way crystals from the blood of man, of the pig, bullock, sheep, rabbit, duck, pigeon, and goose were obtained. Appearing shortly after Hoppe-Seyler's second contribution, an article of epochal importance in physiology was published by Stokes (Proceedings of the Royal Society, London, 1864, xin, 355, June and November), to whom is due the credit of the discovery of the "respiratory function" of the coloring matter of the blood, and also the specific differences in the spectra of oxyhemoglobin and reduced hemoglobin. Stokes writes that it was to him " a point of special interest to inquire whether we could imitate the change of color of arterial into that of venous blood, on the supposition that it arises in reduction. He found upon adding to a solution of blood a reducing agent ["Stokes's reagent"] the color almost immediately changed to a much more purple red as seen in small thicknesses, and a much darker red than before as seen in greater thickness. The change of color, which recalls the differences between arterial and venous blood, is striking enough, but the change in absorption spectrum is far more decisive." When the purple solution was exposed to air in a shallow vessel it changed imme- diately into its original condition. He states that the addition of a reducing agent caused reduction as before and exposure to air a return to the original condition, and that these phenomena could be repeated a number of times. From such facts he inferred that the coloring matter of the blood, like indigo, is capable of existing in two states of oxidation, distinguishable by a difference in color and a fundamental difference in the action on the spec- trum. Hematin having been shown by Hoppe-Seyler in his first communi- cation to be a decomposition product, and Stokes being obviously unaware of this communication (published in Archiv f. path. Anat. u. Physiologic and elsewhere, loc. cit., several months previously), and therefore not knowing that Hoppe-Seyler had proposed terms for the coloring matter of the blood, proposed, at the suggestion of Dr. Sharpey, the term cruorin; and in order to differentiate the two states of oxidation he suggested the terms scarlet cruorin and purple cruorin. Stokes observed that the change in color from arterial to venous blood is in the direction of a change from scarlet to purple cruorin, and that the blood is reoxidized in passing through the lungs and deoxidized while passing through the tissues generally. He PREVIOUS TO THE INVESTIGATIONS OF PREYER. 91 also noted independently of Hoppe-Seyler the feeble state of the combina- tion of the oxygen, for he notes that shaking the blood with CO2 removes the O, and he states that if, as we have reason to believe, this oxygen is for the most part chemically combined, it follows that carbonic acid acts as a reducing agent, and that we are led to regard the change of color not as a direct effect of the presence of carbonic acid, but a consequence of the oxygen. He records certain differences between effects of carbon dioxide and the "real" reducing agents, and he notes that while the former no longer acts on a dilute and comparatively pure solution of scarlet cruorin, the latter acts just as before. He infers that scarlet cruorin is not merely a greedy absorber and carrier of oxygen, but also an oxidizing agent, and he states that "as the purple cruorin in the solution was oxidized almost instantly on being presented with free oxygen by shaking with air, while the tin solution remained in an unoxidized state, so the purple cruorin of the veins is oxidized during the time, brief though it be, during which it is exposed in the lungs, while the substance derived from the blood may have little disposition to combine with free oxygen. As the scarlet cruorin is gradually reduced, oxidizing thereby a portion of the tin salt, so part of the scarlet cruorin is gradually reduced in the course of the circulation, oxidizing a portion of the substances derived from the food or of the tis- sues; the purplish color now assumed by the solution represents the tinge of venous blood, and a fresh shake represents a fresh passage through the lungs." Immediately following Stokes's article a contribution by Hoppe-Seyler appeared (Centralblatt f. med. Wissensch., 1864, n, 817, 834) in which he refers to the work of Stokes as follows: "The observation of Stokes coin- cided fully with my observation earlier, but in addition there are phenom- ena described by him with which I was already familiar, but only showed in my lectures." The author then makes reference to various experiments he had carried out in this particular direction of inquiry, and also in con- nection with hematin. Kuhne (Archiv f. path. Anat. u. Physiologic, 1865, xxxm, 79) iden- tified the spectrum of the coloring matter of muscles with that of the blood described by Hoppe-Seyler. In a later article (ibid., xxxiv, 423) he reports having crystallized reduced hemoglobin. He made a concentrated solution of crystals of dog's blood in very weak ammonia, and then subjected the solution in a gas chamber to pure dry hydrogen. Crystallization occurred as evaporation proceeded. Oxyhemoglobin of the dog, he notes, is very insoluble, while the reduced hemoglobin is very soluble, and he points out that the difficulty experienced in preparing reduced hemoglobin crystals is owing to their great solubility. He also noticed intraglobular crystal- lization. Rollett (Sitzungsb. d. k. Akad. d. Wissensch., Wien, 1866, LII, 2 Abth., 246) shortly afterward prepared reduced hemoglobin crystals by agitation with reduced iron. Schultz had (Archiv f. mikros. Anat., 1865, xxxi, 1) the year before reported crystals of monkey's blood which he obtained by the addition of water and alcohol to the blood, and which were doubtless reduced hemoglobin. 92 PREPARATION AND STUDY OF HEMOGLOBIN CRYSTALS. Schmidt (Preyer, De hsemoglobino observationes et experimenta, Bonn, 1866) gives the following analysis and molecular formula for the hemoglobin of the dog: During this same year Moleschott (Pathologic u. Physiologie, 1866, 42) made note of the occurrence of six-sided plates in guinea-pig blood. Nawrocki (Centralblatt f. med. Wissensch., 1867, xv, 195) and shortly after Ray Lankester (Jour. Anat. and Phys., 1868, u, 114) established the identity of the coloring matters of the bloods of the worm and vertebrates. Hoppe-Seyler (Medicinisch-chemische Untersuchungen, 1867, Heft 2, 1, 215, 293; Heft 3, 366, 394) at about that time prepared crystals from a number of bloods of different species, and made elementary analyses and observations in various directions. His elementary analyses of the hemo- globins of the dog, goose, guinea-pig, and squirrel will be found in the table on page 71. Kuhne (Lehrb. d. physiol. Chemie, 1868, 198) recorded, among other kinds of hemoglobin crystals already obtained, that of the polecat. At this time Ray Lankester began the publication of a series of articles on the distribution of hemoglobin in the animal kingdom, which articles have been referred to in Chapter I. In a research on the cellular structure of the red blood corpuscles, Richardson (Trans. American Med. Association, 1870, xxi, 261) studied intracorpuscular crystallization in the menobranchus. The corpuscles being very large and the hemoglobin readily crystallizable lend condi- tions extraordinarily favorable to such observations. He deposited a drop of blood upon a slide, allowing it to remain uncovered about 10 minutes, or until a mere line of desiccation appeared at the margin, and then cover- ing it with a thin glass. On examination with a power of 200 diameters, he found that numerous corpuscles along the edge of the drop, where the liquor sanguinis had become concentrated, contained one, two, or more crys- tals; and under favorable conditions this process of crystallization went on until the contents of every corpuscle assumed a crystalline form, either wholly or in part. The crystals were frequently irregular, but their typical form appeared to be that of a quadrangular prism with dihedral summits, the angles being sometimes truncated. CHAPTER V. THE INVESTIGATIONS OF PREYER ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. The monograph of Preyer (Die Blutkrystalle, Jena, 1871, 263 pages, 3 plates) , which continues to the present day to be the leading authority on the crystallography of hemoglobin, had for its foundation several contribu- tions that were published a few years previously, and which were chiefly with reference to the optical and chemical properties of hemoglobin. His first serious contribution to the crystallography of hemoglobin appeared in 1868 (Archiv f. ges. Physiologic, 1868, i, 395), in which he expresses his surprise that so little is known of hemoglobin. He writes that while blood crystals have been obtained from 47 vertebrates (23 mammals, 7 birds, 4 reptiles, 1 amphibian, and 12 fishes), in only 10 of these was the crystal system ascertained. The crystals of the human being, the dog, the guinea- pig, and the rabbit were found by von Lang to be rhombic; those of the cat according to Rollett, those of the horse according to Funke, and those of the lion, jaguar, and marbled cat according to Preyer, are also rhombic; while those of the squirrel, according to von Lang, are hexagonal. Preyer goes on to state that in addition to those mentioned the following are prob- ably rhombic: those of the monkey, bat, hedgehog, sheep, pig, harfang (owl), and frog. The blood of the monkey yielded rhombic plates which crystallized with difficulty and which were readily soluble, even in the cold. The blood of the bat crystallized in thin plates with very pointed angles. Hedgehog blood produced right-angled elongated prisms, which even in the cold are readily soluble. Sheep's blood crystals were obtained only once in gas-free blood, and they were prisms. Pig's blood crystallized with extraordinary difficulty, mostly intraglobular (in every corpuscle a prism). The blood of the harfang (Strix noctua) crystallized readily, but that of the frog with difficulty. The former yielded 4-sided plates; the latter, thin prisms, which appeared to be 4-sided. Of the other blood crystals pre- viously seen, those of the mouse and the hamster, he states, might be hex- agonal. Preyer obtained from the blood from the heart of a mouse only fine needles. The hemoglobin crystals of the fox, polecat, mole, marmot, ox, raven, sparrow, pigeon, goose, duck, lark, rat, and fish were produced, but their crystallographic investigation gave little satisfaction; most of them appeared to be rhombic. Preyer, in his review, writes that Lehmann's statement that occasion- ally regular octahedrons are found in guinea-pig blood is incorrect; and also that Hoppe-Seyler's assertion that guinea-pig blood crystals are tetragonal is wrong. He also states that the opinion of Funke that human and cat 93 94 INVESTIGATIONS OF PREYER hemoglobins crystallize in the monoclinic system has long been refuted. Accordingly, as Preyer writes, all the hemoglobins accurately investigated crystallographically up to the present time are rhombic, with the single exception of that of the squirrel. The remaining portion of Preyer's article, which is very largely a record of his own researches on the physical, chemical, and optical properties of hemoglobin and methemoglobin, is included practically in full in his mono- graph, which was published three years later. Owing to the authoritative character of this publication, its indispensable value to the physiological chemist, the practical impossibility of obtaining copies, and the rareness of the work in the libraries of this country, we have deemed it advisable to embody in this memoir a rather full and free translation of his chapters on the methods of preparation and on the descriptions of the crystals. This extract constitutes the remainder of this chapter. PROCESSES USED BY PREYER FOR OBTAINING CRYSTALS IN LARGE QUANTITIES. (I) Lehmann's process has the advantage over the others which fol- low in that a very low temperature is not necessary. The solvent of the coloring matter of the corpuscles is water. The fresh blood is allowed to coagulate and the coagulum to shrink, the serum is drawn off, and the coagulum is ground up. The fluid is separated from the clot by straining through linen, and to this fluid are added from 1 to 1.5 volumes of water. O is then passed through this diluted extract of the clot for about half an hour, and then CO2 for 10 or 15 minutes. The formation of crystals begins within a few minutes and a rich mass of crystals has separated after 2 hours. By this method crystals were obtained only from the blood of the guinea- pig, rat, and mouse. In order to prepare crystals from dog's blood, and from other blood that is not readily crystallizable, small quantities of alcohol are added to the blood before and during the passage of the gases. The solution very quickly becomes cloudy with crystals and congeals to a crystal pulp. Instead of alcohol, ether can be used in part, but it does not suffice alone. The crystals obtained in this way are, however, not pure. Since recrystallization is necessary in order to obtain pure crystals, and as this can not be done except at low temperatures, there is no advantage in this method if it is necessary to secure a pure product. Preyer found, moreover, that it is only necessary to pass for many hours diy or moist atmospheric air free from carbonic acid through the defibrinated blood of the dog in order to cause crystallization, which occurred abundantly at room temperature, or at about 35° to 38° ('. (II) As a solvent for the coloring matter of the corpuscles Rollett made use of freezing. He placed a platinum vessel containing defibrinated blood in a freezing mixture, and after about half an hour the blood was allowed to thaw slowly. It was then poured into shallow vessels to a depth of about 15 mm. These vessels were placed in the cold at even temperature to crystallize. Within an hour or so a deposit of crystals had formed. In this way guinea-pig and squirrel blood quickly yielded well-formed ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. 95 crystals, but cat's blood only after a longer time. With dog's blood crystal- lization proceeds from the surface. The crystalline mass may be lifted off, then a new one forms, and so on. Very much more time is required for human and rabbit's blood. Pig's and frog's blood gave no crystals, yet the hemoglobins of these bloods are capable of being crystallized. By repeated freezing and thawing of the blood all the blood corpuscles could be com- pletely decolorized, but this requires larger quantities of blood, repeated freezing and thawing, and much time. Moreover, it is necessary to con- centrate this laked blood by evaporating at a low temperature. It is im- material in this process whether or not the blood be exposed to the air during the process. This method, which is particularly convenient in winter, is worthy of recommendation only where absolute purity is not necessary, as in making comparative crystallographic and optical investigations of the hemoglobins of different animals. (III) Into the animal from whose blood the crystals are to be pro- duced, Bottcher injected intravenously a quantity of cold water during chloroform narcosis, and then the chloroform was administered until death occurred. The blood is obtained immediately after death from the heart and vessels, and it is readily crystallizable. If mixed with an equal volume of water and alcohol, and the mixture placed in the cold, a magma of crys- tals will form. This method is not recommended, because of the difficulty of obtaining sufficient blood from the dead animal. (IV) The solvent for the hemoglobin which Kiihne recommends is the taurocholate and glycocholate of sodium. Thiry also employed bile salts to obtain hemoglobin crystals from bloods that crystallize with difficulty. 600 c.c. of horse blood are collected in a cylindrical vessel and cooled. As soon as the plasma has separated from the corpuscles, it is removed, together with the layer of white corpuscles, and the remaining mass of red corpuscles is mixed with 0.5 per cent solution of crystallized ox bile. The corpuscular mass is then allowed to coagulate. The fibrin which has formed has entangled the decolorized corpuscles which have not dissolved, so that the deep-red lake-colored solution, which may be drained off, contains none of the corpuscles. To this solution is added, during continual stirring, and as long as the precipitate that forms is dissolved, 90 per cent alcohol which contains a very little acetic acid. After several hours the preparation is converted into a crystal pulp, which may be collected on a filter, then washed with dilute alcohol, and subsequently with iced water. Or 100 c.c. of dog's blood are allowed to coagulate in a shallow vessel; the coagulum is then detached from the walls of the vessel and allowed to stand in a cool place for 24 hours, until the serum has separated as much as possible. The serum is removed; the coagulum is washed with water and broken up in 50 c.c. of water by means of a syringe. After 24 hours it is strained through linen and the clot is washed out with 10 c.c. of water. To the mixture of the washing from the clot and the fluid obtained by straining are added 2 c.c. of a solution consisting of 1 part of crystallized bile and 3 parts of water. After 24 hours the solution is filtered through 96 INVESTIGATIONS OF PREYER many thicknesses of filter-paper. On the addition of 20 c.c. of 90 per cent alcohol to 100 c.c. of the filtrate a firm crystalline pulp is soon formed, which is collected on a filter and washed, at first with a mixture of 4 parts of water and 1 part of alcohol and then with iced water. According to this method fully 5 grams of pure recrystallized dry hemoglobin are obtained. Recrystallization yields, according to Kiihne, a pure preparation only if the first crystallization contained no corpuscles. This method is somewhat troublesome. The addition of crystallized bile, as well as of acetic acid, Preyer believes, may give rise to decompositions of the hemoglobin. (V) Defibrinated dog's blood is mixed with about its own volume of distilled water, and to every 4 volumes of the blood solution there is added 1 volume of alcohol. The mixture is left for 24 hours at a temperature of 0°, or lower. The crystals that have separated are collected on a filter, pressed, dissolved in the smallest amount of water at 25° to 30°, cooled to 0°, the solution mixed with one-fourth its volume of alcohol and left stand- ing for 24 hours at 0°, or better at — 10° to — 20°. The entire fluid becomes converted into a crystalline mass without freezing. This recrystallization can often be repeated. From the bloods of several rodents, for example the guinea-pig and the rat, blood crystals were obtained on the addition of pure water after defibrinization, because the crystals are not soluble in cold water, yet they can also be recrystallized by dissolving in water at 30° and cooling, or evaporating over sulphuric acid in a rarefied atmosphere, and they can be dried at 0° without decomposition. (Method of Hoppe-Seyler.) Preyer occasionally observed that fresh defibrinated blood of the dog, after dilution with pure water, yields the most beautiful crystals upon evaporation. Once he mixed 5 c.c. of blood with 4 c.c. of distilled water, poured the solution into a shallow porcelain vessel, and let it stand over night at a temperature between 19° and 20°. On the dried marginal por- tions of the solution, after about 15 hours, exceptionally beautiful crystals, 5 to 6 mm. in length and intensely red, were formed. Yet he did not always succeed in producing blood crystals from dog's blood in this way. Of the five methods described, the last, according to Preyer, is decidedly the best. Yet, he states, it also needs improving. He proceeded, therefore, in the production of pure hemoglobin crystals on a large scale from any blood selected, in the following way: (VI) The blood is collected in a vessel and allowed to coagulate and to stand for several hours (or, better, for a day) in a cool place. Then the serum with the white corpuscles and the fat which has collected on top are removed and the coagulum washed with distilled water and then cut into very small pieces, and these pieces in turn are repeatedly washed with cold distilled water. Then the clot is comminuted, best by freezing and reduc- ing the frozen mass to powder. This powder is placed on a filter-paper and washed with cold distilled water until the filtrate no longer gives a very strong precipitate with bichloride of mercury. The coagula are extracted ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. 97 by water heated to 30° to 40°, and filtered, and the filtrate is collected in a large cylindrical vessel standing in ice. A small measured portion of the red solution thus obtained is gradually mixed during constant agitation with small quantities of alcohol until a slight precipitate forms. This determines how much alcohol may be added to the whole solution without a precipitate appearing. A slightly smaller proportion of alcohol is now added to the remaining filtrate and the mixture is placed in a cooling medium. Even after a few hours the crystals separate in great abundance. The crystals are, owing to the volume of water used, very easily filtered off in the cold. They are then washed with cold water containing a little alcohol until the filtrate yields only an insignificant cloudiness upon the addition of acetate of lead or corrosive sublimate. The product yielded is a very large one. The crystals may be purified by repeated washing by decanta- tion until the wash-water does not become cloudy with bichloride of mer- cury, acetate of lead, or silver nitrate. They are then nearly pure, and the ash is free of phosphoric acid and consists of pure iron oxide. If this is not the case, then they must be dissolved in warm water and recrystallized as directed. At a temperature of less than 0° the crystals can be dried in the air without becoming decomposed. This method differs essentially from (V) only in that instead of defibri- nated blood a diluted extract of the clot is used. But in this there is a great advantage, because the crystals can be obtained pure very much more easily and quickly owing to the very small quantities of serum albumin that can adhere to them. Furthermore, the fluid, because of the lack of serum albumin, filters more quickly. It will therefore be noticed that by the coagulation of the blood, by the treatment of the clot with water, by the freezing of the same, and by the longer interval from the time of bleeding to the mixing with alcohol, the degree of crystallizability increases. Preyer obtained larger crystals by this method than by any of the others. Of all kinds of blood, that of the horse, he states, is best adapted for the production of very large quantities of pure hemoglobin. It is defibri- nated and the corpuscles are allowed to settle in a high cylindrical vessel, the serum is drawn off, and the corpuscles are frozen, etc. If crystallized hemoglobin is to be produced quickly from the defibri- nated blood of the dog, it is best to mix the blood with its own volume of distilled water, add 1.5 volumes of absolute alcohol to 4 volumes of the mixture, and then place the solution in a cylinder in a cooling mixture. After a few hours the fluid has changed to a crystal pulp. By frequent washing, by decantation, or by centrifugalization with diluted alcohol (4 volumes of water to 1 volume of absolute alcohol), crystals are obtained pure, but of course with great loss. This very convenient method has the disadvantage that the corpuscles by their remaining longer in dilute alcohol become difficult of solution in water. If the preservation of the normal solubility is disregarded an abundant crystallization can be obtained at 8° to 10° from dog's blood by mixing 1 volume of fluid with an equal volume of water and a little more than one-fourth of the whole volume of absolute alcohol. The mixture in one case had formed a thick crystal pulp in 9 7 98 INVESTIGATIONS OF PREYER hours, so that a large amount of nearly pure crystals can be obtained by decantation with a mixture of 4 volumes of water and 1 volume of absolute alcohol. Nevertheless, this method does not always give such favorable results. PROCESSES GIVEN BY PREYER FOR OBTAINING CRYSTALS IN SMALL QUANTITIES. One of the simplest processes for obtaining crystals from the blood of several animals is by heating. Max Schultze (Archiv f. mikrosk. Anat., 1865, 1, 31) found that the corpuscles were dissolved at a temperature of about 60°, forming a lake-colored blood solution. Every drop then evapo- rated yields crystals, Preyer observed this also in guinea-pig's blood when he gradually heated a drop on a slide to about 60°, and then allowed it to cool and to evaporate slowly ; and also when a large quantity of blood was warmed in a water-bath to at least 60°. With squirrel's, calf's, and human blood crystallization did not succeed. On the other hand, Preyer writes, by no other method could there be obtained from horse's blood such well- formed and large crystals. The temperature must be at least 60°, but it should not go beyond 64°. Preyer proceeded in the following way : Horse's blood was collected in a vessel, defibrinated by agitation, and decanted. The defibrinated blood was separated after several minutes into two por- tions— an upper layer of serum and a lower dark-red layer of corpuscles. The serum was pipetted off and the corpuscles heated in a water-bath at 60°. This produced a lake-colored solution, of which every drop, upon being cooled and evaporated, yielded extraordinarily beautiful crystals. This crystallization, which is brought about by warmth causing a separation of the coloring matter from the corpuscles, is not to be con- founded with one earlier reported by Bojanowski (Zeit. f. wissensch. Zool- ogie, 1863, xn, 323), who evaporated the diluted extract of the coagulum of rabbit's blood at 50°, and who noticed in so doing that the upper surface of the blood was covered with a delicate crust composed of prismatic crys- tals. In this process the coloring matter had been extracted by water, so that the effect of warmth can only be looked upon as an aid to rapid evapo- ration. The favorable influence of slight warmth as a means of hastening evaporation has been repeatedly misunderstood and denied, and generally it has been regarded as a hindrance to crystallization. If an extract of the coagulum of dog's blood prepared with cold dis- tilled water is shaken with sufficient ether so that it smells of it, and then a little alcohol added, and then very gradually heated in a very shallow vessel until the margin of the fluid or the drop on the object-glass begins to dry, evaporation proceeds quickly and regularly, and crystals form as the blood cools. In this way a most beautiful preparation can be obtained in a short time. However, without artificial heating as many hours are necessary to obtain crystals as minutes are required with it. Electric shocks have a similar effect in causing a solution of the hemo- globin from the corpuscles, as was found by Rollett. A. Schmidt had already noted that a like effect is caused by the galvanic current. By both ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. 99 the corpuscles are decolorized and the hemoglobin (of human, cat, dog, and guinea-pig blood) crystallized in the lake-colored solution, it being immaterial whether the oxygen from the air has been admitted or not. Yet this method is not well adapted for the production of crystals on a large scale. An observation made by Pasteur (Compt. rend. soc. biolog., 1863, LVI, 739) is very noteworthy. He allowed dog's blood to stand in a balloon of heated air at a constant temperature of 30° C. After 4 to 6 weeks the air contained 2 to 3 per cent less of oxygen and just as much more of car- bonic acid. A large mass of hemoglobin crystals was formed. After several weeks not a single corpuscle was present. The clot was colorless and very elastic, and associated with an incalculable number of crystals. Julius Bernstein (1866) conducted atmospheric air through a small amount of chloroform into defibrinated blood. He noticed that the blood soon became lake-colored, and that he could no longer find any corpuscles in it. Every drop produced crystals when evaporated. Preyer supple- mented this procedure by treating the diluted extract of the coagulum in the same way. This yielded crystals, but not in great masses. Kunde had already observed (1852) the favoring influences of chloroform, ether, and alcohol on crystallization. Alexander Schmidt found that crystallization followed upon the addi- tion of pure alcohol to dog's blood. He mixed fresh blood with one-half to two-thirds its volume of alcohol until albumin began to separate and then left the mixture undisturbed. After a time it became laked and crystalline. Ether causes the very same thing. Defibrinated dog's blood is shaken with ether until the blood is laked and smells of ether. If it is allowed to stand for 24 hours in the cold crystals can be seen microscopically in every drop. Later A. Schmidt saw that dog's blood and horse's blood became lake-colored when the fresh blood was shaken with a definite amount of turpentine containing ozone, each time ascertaining by testing the amount necessary. He could then cause crystallization with alcohol (only in the clog), or ether, or sodium sulphate, or by water extraction in a vacuum. Several times it has been observed, continues Preyer, that an addition of certain neutral salts to bloods which can be crystallized hastens crys- tallization. According to Bursy the salts favoring crystallization are in the order of their value as follows: Sodium sulphate, sodium phosphate, sodium acetate, potassium acetate, magnesium sulphate, and potassium nitrate. Less energetic are potassium carbonate, potassium sulphate, sodium borate, barium nitrate, and sal ammoniac. Sodium nitrate ap- peared to hinder crystallization when the blood was alternately frozen and thawed. Sodium chloride, ammonium nitrate, calcium chloride, and alum were without effect. For the production of crystals on a large scale the addition of salts is not to be recommended, because of the introduction of such foreign substances. Only chloride of sodium seems to be of value in the isolation of the corpuscles according to Hoppe-Seyler's method. 100 INVESTIGATIONS OF PREYER The crystals thus obtained can be recrystallized 5 or 6 times, or as long as the hemoglobin remains undecomposed. This plan, nevertheless, has reference only to the kinds of blood which crystallize easily. These crystals, even after several recrystallizations, show the bright-red color of arterial blood. Another means by which the blood can be crystallized is by extracting the gases. In dog's blood from which the gases had been extracted, Rollett found that the blood was lake-colored, very dark, and that it produced hemoglobin crystals immediately. Preyer states that he found that dog's blood and sheep's blood freed from gases crystallize by evaporating a drop on an object-glass, the colorless stromata of the corpuscles still being visible. The addition of a little dilute solution of oxalic acid aided crys- tallization. Preyer then tried to decide experimentally if all of the three blood- gases (carbonic acid, oxygen, and nitrogen) were necessary for the preser- vation of the normal condition of the blood corpuscles in the circulating blood. He investigated the blood of asphyxiated animals and found micro- scopically that there was some disintegration of the corpuscles. Previous to this experiment he had found that blood made rich in carbonic acid and freed of oxygen by a continuous stream of carbonic acid crystallized very slightly, and that therefore the absence of oxygen alone (without taking into account the nitrogen) is sufficient to cause a partial decomposition of the blood corpuscles into colorless stromata and hemoglobin. He then made the following experiment: The A. carotis dextra, the V. jugularis externa sinistra, and the trachea of a little dog were laid bare, and into the vessels glass cannulse were tied. The trachea was then clamped so as to prevent the entrance of air to the lungs. The moment the conjunctiva became insensible to the touch the ligatures on the vessels were loosened and the blood drawn into separate receptacles. The blood was dark red, and that of the artery could not be distinguished from that of the vein. A drop of each kind of blood showed under the microscope a rich crystal formation within the first minute after it was caught. Under the eye of the observer the crystals increased in thickness, length, and number as long as the evaporation of the drop on the object-glass lasted, but more slowly if the drop had been covered with a cover-glass. By gently shaking in the air the blood became bright red again. All the manipulations mentioned here by which crystals can be obtained are somewhat troublesome, and are not used for purposes of microscopic preparations. C. Bojanowski placed a drop of blood on an object-glass, exposed it to the air several minutes, breathed upon the preparation several times, then covered it with a cover-glass, and allowed it to evaporate slowly. He found that a small addition of alcohol or ether is occasionally necessary. Bojanowski obtained microscopic crystals without adding anything to the blood by merely allowing it (as it comes from the veins, or, better yet, as it is found in them after death) to stand in a vessel 2 to 4 days in a cool place. The blood coagulum partly dissolves and the blood becomes thick and dark red. A drop of the same is allowed to stand several hours between ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. 101 the cover-glass and the object-glass without being heated. If the blood is too thick some distilled water is added to it. In order to produce hemoglobin crystals in a short time from fresh blood chosen for microscopic purposes, the following method is best : Several cubic centimeters of defibrinated blood are mixed with just enough water to yield a clear solution. A drop of the mixture covered with a cover-glass crystallizes on being subjected to cold. If this is not the case, about one- fourth the volume of alcohol is added to the solution and the mixture is placed in a platinum or silver vessel in a cooling mixture. Crystals are always obtained. Almost all kinds of blood yield crystals by merely allow- ing the blood to freeze, even ox blood. THE FORMS AND SYSTEMS OF CRYSTALLIZATION OF HEMOGLOBINS. Upon this subject Preyer writes: Of the six crystal systems only five are to be taken into consideration in the classification of crystals of hemo- globins, namely, the regular (tesseral), the tetragonal, the rhombic, the monoclinic (clinorhombic, monoclinohedric), and the hexagonal. Crystals belonging to the triclinic system have not been claimed to have been found by anyone. Of the remaining five systems the regular and the tetragonal are to be eliminated — the regular because all hemoglobin crystals are doubly refractive, and the tetragonal because the only crystals assigned to this system were those of the guinea-pig by Hoppe-Seyler. Hoppe-Seyler's statement is without facts to justify it, and it has been disproved by the accurate investigations of guinea-pig crystals by Victor von Lang and others. There remain then three systems — rhombic, hexagonal, and mono- clinic. The last named is also to be eliminated because Funke is the only scientist who asserts that hemoglobin crystallizes in this system, and he does not support his statement. He only asserts that human and cat hemo- globins crystallize in the monoclinic system, yet in another place he him- self calls human blood crystals rhombic, and as a matter of fact so are those of the cat. Monoclinic crystals have not, then, up to this time been shown to exist. On the other hand, crystals belonging to the rhombic and hexagonal systems have been shown to exist beyond any question. The fact that crystals of different species are assigned to two systems is a matter that is not to be ignored. It is firmly settled that squirrel's blood yields crystals that belong to the hexagonal system, while dog's blood yields crystals that with as little doubt belong to the rhombic system. The observations of V. von Lang are: Squirrel's, 6-sided plates formed from a 6-sided "prism, showing the basal surface. These crystals belong undoubtedly to the hexagonal system, because when observed through the basal surface between crossed nicols they remained dark in all azimuths. In agreement with this, the crystals when observed through the prism surface showed double refraction. All the rays, then, are not of like intensity. The vibrations parallel to the optical and crystallographic axes are less absorbed than those which are perpendicular. Preyer states that he confirms von Lang's observations: Between nicol prisms squirrel's blood crystals only show color when their optic 102 INVESTIGATIONS OF PREYER axis does not lie parallel to the direction of the polarized light ray. Preyer goes on to state that if future investigations should show that all blood crystals are either rhombic or hexagonal, which is probably true, then it could not be maintained that the difference in the forms of the crystals is a case of polymorphism or dimorphism, because chemical identity is lacking. The hexagonal system, as A. Schrauf (Jahrbuch f. Mineralogie, 1865, 46) has_shown, is conceived of as being a peculiar combination of the rhom- bic (P.P oo , with the single condition that oo P: oo P= 60°), therefore the mis- take could be made of looking upon the crystallographic distinction as being a material one. The statement of Schrauf does not, however, bear the test, because even if the hexagonal forms are still the simple combination of the rhombic, the fundamental optical distinction of both systems can not be denied. Optically the rhombic crystals are biaxial and the hexagonal are uniaxial. There exist other distinctions outside of the crystallographic systems. The peculiar crystalline form in relation to each kind of animal (of the guinea-pig, the sphenoidal; of the dog, 4-sided prisms; of man, these and rhombic plates, and so forth) is so constant and definite that only these forms could be obtained from the blood referred to. After recrystallization repeated ever so often the same form always appears, which is peculiar to each kind of animal, and which can not be changed to another (see Chapter VII). The same applies to solutions of hemoglobin. Yet little importance is to be attached to statements on the crystallographic differences of the hemoglobin of different animals, because neither is the same method of crystallization always used, nor is the blood always capable of being com- pared, nor has a measure of the crystallizability of any optional substance been found. It is the same with decomposability as with the crystallizability of hemoglobin. Both vary according to the species of animal, but the investi- gations undertaken in this direction suffer from so many and such large errors that they prove nothing beyond what has long been known — the different quantitative determinations of the blood-coloring matter in various animals and individuals. Such matters lead to the desire to follow up through a series of animals the several properties of hemoglobin — for example, the coagulation-point, the crystalline form, and the capacity of water of crystallization — in order that the question concerning their great differences might be more closely approached : whether the various hemo- globins are present as so many different substances which only agree in cer- tain characteristics, or whether they are entirely identical in derivation and their differences come about solely because of combining with other sub- stances, or are properties of the crystals. Perhaps an explanation could be obtained by transfusion, as, for example, of squirrel's blood to guinea-pig, or vice -versa. The following table (table 31) is from Preyer, and is an excellent sum- mary of our knowledge of the crystallography of hemoglobin up to the tune of the publication of his memoir. ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. 103 TABLE 31.—Preyer's table showing the source of hemoglobin crystals, crystalline form, crystalline system, etc. Name of kind. Crystal form. Crystal system. Appearance. Solubility in water. Crystallizability. Remarks. \jfln Elongated rect- angles, rbombi and 4-sided prisms. Acute angles of the rhombi 54° 6' (von Lang) Small rhombic plates (Preyer) Thin plates with very acute an- gles Right-angled elon- gated prisms Rhombic (Funke von Lang) In blood suckec by the leech 6 to 8 weeks previously (Budge, Bo- janowski); ex- traglobuiar in venous blood (Funke) ; intra- globular (H. Meckel) Extraglobular Do 1 Fresh from ven- ous blood extra- ordinarily easily dissolved ; that from the leech, in the cold quite difficultly, in warmth very readily soluble The fresh crystals readily soluble in cold (Preyer) Crystallizes with difficulty Crystallizes with difficulty (Prey- er) Illustrations in Funke's Atlas, x, 1 and 2. Compare (1) Funke, Journ. f. prakt. Chemie, 1852, p. 384, and Zeitschr. f. rat. Med., 1852, 205. (2) V. Lang, Sitz- ungsber. d. Wiener Akad., XLVI, 1 Abth., 1862, with illustrations of the crystals of Rollett's own treatise. (3) Bojanowski, Zeit- schr. f. wiss. Zool., xn, 332, taf. 30, 1 and 3. (4) Kunde, Zeit- schr. f. rat. Med., 1852, taf. ix, 1. Fuuke found the angles 73°0' to 73° 35' and on the almost SR° *W Monkey (Cynocepha- iae babiiin) Bat Budge, Verhandlgn. d. naturhist. Vereins der Rheinl. u. Westph., 1850. (Koln. Zeitung, No. 300, Ankersmits Diss., p. 5 ) Also, Berlin (Nederlandsch. Lancet, 1853 and 1854, 3 ser.. 16-34) investigated the formation of the crystals in leeches. Meckel, Archiv f. d. Holland. Beitr. zur Natur- u. Heil-Kunde, 1, 90, 1858. I produced the crystals by the ad- dition of water and alcohol to the blood of a monkey poisoned with santonin. (Max Schultze in his Archiv, 1866, p. 195. Illust., plate in.) I have only once seen a poor pre- paration. Kunde first produced the crystals (needles). Zeitschr. f. rat. Med., 1852, p. 285. Illustration, Zeitschr. f. wiss. Zool., xii, plate xxx, fig. 8; Lehmann saw the hedgehog crystals 1853. I obtained prismatic crystals from the blood of a chloroformed hedgehog. F. Hoppe-Seyler, Handb. d. phys- iol. u. pathol.-chem. Analyse, 2 Aufl., Berlin, 1865, p. 201. Illustration, Zeitschr. f. wiss. Zool., XII, plate xxx, fig. 7, and Funke's Atlas, x, 3. Compare Funke. Journ. f. prakt. Ch., 1852, LVI, 195, and Rollett, Sitzungs- ber. d. Wien. Akad., 1862. Funke's statement that the crys- tals are clinorhombic is incorrect (Zeitschr.f. rat. Med., 1852.291). The crystals were produced in 1806 by Theodor Deecke in Liibeck. Berlin already saw lion crystals in 1856 (Nederlandsch. Lancet, v, 734) 1 investigated the very beautiful Deecke preparations, which, however, after 4 months became completely useless. In- stead of the crystals only fine grains appeared and the spec- trum was that of oxygen-freed hemoglobin. The crystals were kept at room temperature in- stead of in the cold. Produced 1866 by Deecke. The same. Hoppe-Seyler, Med. chem. Unters., u, p. 182, 1867. Kiihne, Lehrb. d. physiol. Chemie, p. 198, 1868. Illustration of the crystals appar- ently colorless, but in reality not appearing red because of their thinness, in Funke's Atlas, ix, 5. Funke saw also rhombic plates (in venous splenic blood) fiO° with j^ (Zeitschr. f. rat. Med., 1851, p. 190). Compare Kunde in Zeitschr. f. rat. Med., 1852, p. 271. Kolliker shows intraglob- ular crystals (Mikroskop. Anat., 1854, II, 2 Halite, fig. 271, p. 280). Hedgehog (Erinaceus europaus) Mole (Talpa ewropcra) Cat (Fdia domeatico.) Lion (Fdia leo) Probably rhom- bic (Preyer) Do Extraordinarily readily .soluble in cold water (Bojanowski) Crystallizes read- ily from the blood of the chloroformed animal 4-sided prisms truncated by 1 or 2 obliquely placed planes 1-sided prisms which term i- nate in 2 ob- liquely placed truncating planes (Preyer) Prisms as in the lion (Preyer) Do Rhombic (Rol- lett) Rhombic (Preyer) Do Extraglobular . . . Do.. In cold water not readily soluble; in warm very readily (Boja- nowski) Crystallizes read- ily Cat (Felit marmorota) Cougar (Feli» puma) Fox.... Do Do Do Polecat.... D°e (Cants famili- -sided prisms bounded by a perpendicularly or an obliquely placed plane (Preyer) Rhombic ntraglobular and extraglobular In cold water not readily soluble, in warm very readily soluble Crystallizes easily 104 INVESTIGATIONS OF PREYER TABLE 31. — Preyer's table showing the source of hemoglobin crystals, etc. — Continued. Name of kind. Crystal form. Crystal system. Appearance. Solubility in water. Crystallizability. Remarks. Guinea-pig (Cavia Tetrahedral (sphe- Rhombic (v. Lang) Intraglobular and Very difficultly Crystallizes very Lehmann 's statement (Chem.- cobaya) noids) only ap- extraglobular. soluble readily pharm. Centralbl.. 1853, p. 98) parently regular Illustrations that occasionally regular octa- because the an- made by Beal hedrons are also found rests on gles deviate (Quart. Journ. a gross error; so also is Hoppe's slightly from 60° of Microscopic statement that the crystals are (von Lang) Be., 1864, 32-43. tetragonal incorrect. Slole- The crystals schott's information (Pathologic have a tendency u. Physiologic, Giessen, 1866, p. to lie beside each 42) that he also obtained 6- other in saw- sided plates from guinea-pig tooth form blood can be explained in this way, that the tetrahedrons are so abundantly packed together that they apparently result in planes bounded by 6 sides. Il- lustration in Funke's Atlas, x, 4. Rpichert, Miiller's Archiv, 18-19, taf. ii, fig. 6. Kunde, Zeitschr.f. rat. Med.. taf. rx, fig 2 (1852). Squirrel (Sciurus vul- 6-sided plates and Hexagonal (von Extraglobular . . . Very difficultly Crystallizes easily Illustration in Funke's Atlas, x, 5. garis) 6-sided prisms. Lang, Rollett, soluble Lehmann's statement, that the often grouped Kunde, Preyer) crystals do not belong to the rosette-like hexagonal system, is incorrect. Kunde gives an illustration in Zeitschr. f. rat. Med., taf. ix, fig. 3 (1852); also Kiihne, Lehrb., p. 200. Mouse (Mut m uscu- G-sided plates Hexagonal . Do . . Very readily sol- Crystallizes easily Illustration, Zeitschr. f. wi?s. Zool., IU8) (Bojanowski). uble (Bojanow- (Bojanowski xn, taf. xxx, fig. 5. From the Tetrahedrons(?) ski). and Lehmann) heart blood of the mouse I ob- (Lehmann). Fine needles Very difficultly soluble (Leh- tained only small prismatic crys- tals. Kunde (Zeitschr. f. rat. (Kunde) mann) Med., N. F., n, 1852, 285) ob- tained with water, without any additional mixture, needles and "prismatic plates." Rat (Musrattus, Mua Tetrahed rons lutraglobular .... Very difficultly Crystallizes very Hoppe-Seyler (Handbuch, 1865, p. decumanus) (Kunde, 1852). soluble (Leh- easily (Leh- 202) obtained crystals by simply Tetrahedrons mann) mann) diluting the blood with water. (L e hm a n n , Compare Kunde in Zeiuchr. f. 1853). rat, Med., 1852, N. F., u, 276, Prisms (Biseg- Bisegger and Brach found the ger 1852) crystals "prismatic." (Ver- handl. d. naturforsch. Ges. IU Basel, 1, 1857, 174.) Rabbit (Lepus cuni- culus) Right-angled, elongated rhom- Rhombic (v. Lang) Extraglobular. . . . Extraordinarily readily soluble Crystallizes rath- er difficultly Illustration in Zeitschr. f. wiss. Zool., xn, taf. 30, 2, and in Rol- bi, prisms (Bojanowski) lett, Vera. u. Beob. am Blute, Wien, 1862. Compare the same, p. 25. Kunde (Zeitschr. f. rat. Med., 1882, p. 284) obtained the crystals simply by addition of water, as also did Tcichmann (same place, 1853, 370). Budge, Spec. Physiol. 8 Aufl., p. 250. Hamster (Cricetua R hombohedrons Hexagonal (?) Do Illustration in Funke's Atlas, ix, 6. vulgaria) and 6-sided Kunde also saw the crystals. plates (Leh- mann). Angle: 60° /T , Y20S CLebmann, 1853) Marmot (Arctomys Column-shaped Crystallizes not Valentin, in Moleschott's L'nters. marmotta) crystals (\ al- easily z. Naturl., 1863, ix, 131. entin) Horse 4 -sided prisms Rhombic (Funke) Ext raglobular. Readily soluble. Crystallizes easily W. Kiihne Med. Centralbl., 1863, and rhombic 1851 No. 53, p. 833. Illustration in plates the Zeitschr.f. rat. Med., N. F., 1 Bd., 1851, taf. 1, figs. 4, 5, 6. Funke obtained the crystals from diluted venous splenic blood, Kunde (same, 2 Bd., 1852. p. 285) from jugular ven- ous blood. Funke found the angle 60° 9* and 119° 32'. Sheep Prisms Do Crystallizes with T \ ' rv-tftls in difficulty the wether's blood after removal of the gases. But it is very dim- cult to bring the blood to crys- tallization in any other way. Ox Lit tie colu mns Most probably Do Very readily sol- Crystallizes with See A Schmidt in Virchow's placed beside rhombic (Prey- uble in cold extraordinary Arch'., xxrx, p. 'l, 1864. Funke each other in palisade fash- ion(A. Schmidt). Needles with er) water difficulty also saw the crystals. Kunde obtained them by means of ether (Zeitschr. f. rat. Mod., 1852, p. 284), Teichmann (ibid.. 18SJ, double end 376) by allowing the blood to planes (Kunde, evaporate after dilution with 4 1852). Prisms to 5 times its volume of water. (Preyer) ON THE CRYSTALLOGRAPHY OF HEMOGLOBIN. 105 TABLE 31. — Preyer's table showing the source of hemoglobin crystals, etc. — Continued. Name of kind. Crystal form. Crystal system. Appearance. Solubility in water. Crystallizability. Remarks. Pig (Susscrofadomes- tica) Owl (Strix noctua) . . . Crystallizes with extraordinary difficulty Crystallizes easily (Preyer) Crystallizes with difficulty Crystallizeseasily Compare Funke, Journ. f. prakt. Ch., LVJ, 195, and Zeitschr. f. rat. Med., 1852, 201, and Klebs, Med. Centralbl., 1863, No. 54, p. 852. I also saw the crystals in every blood corpuscle. Funke refers to nets of crystal rods in the compounds. Meckel (Archiv f. d. Holl. Beitr. z. Nat.- u. Heilkunde) saw the intraglobu- lar crystals also. Teichmann obtained them by the evapora- tion of diluted blood (Zeitschr. f. rat. Med., 1853 376). I obtained owl-blood crystals by letting a drop of blood, 2 days old, stand between the object- glass and cover-glass at room temperature. Illustration in Boianowski, Zeit- schr. f. wiss. Zoo!., xii. taf. 30, fig. 12. I obtained very large crystals from frozen heart blood. The crystal form is not clearly recognizable from the illustra- tion in Bojanowski, Zeitschr. f. wiss. Zool., xii, taf. 30, fig. 9. The crystals were produced by Bo- janowski, Zeitschr. f. wiss. Zool., XII, 334. Bojanowski found the pigeon- blood crystals similar to the raven-blood crystals, foe. «(., p. 335. Hoppe-Seyler finds that dove-blood crystals are more easily produced pure than dog- blood crystals. Kunde, Zeit- schr. f. rat. Med., N. F., n, 285 4-sided plates (Preyer) Most probably rhombic (Preyer) Very probably rhombic Same as above. . . Do Very difficultly soluble in cold, not readily sol- uble in warm water (Bojan- owski) Crow (Corvus corone) Lark (Alauda criatala) Sparrow Rhombic plates and comb-shap- ed and f a n- shaped grouped prisms (Preyer) Needle-shaped crystals ending very pointedly Like the lark crys- tals Do Do Very difficultly soluble in cold, very readily soluble in warm water (Bojan- owski) Pigeon Domestic goose Large 4 or 6-sided rhombic plates Prisms Rhombic (?) and Teichmann (in the same place, 1853, 376). Hoppe-Seyler finds that goose- blood crystals, according to his method, can be more easily pro- duced pure than dog-blood crys- tals. According to Kolliker. Kunde, Zeitschr. f . rat. Med., N. F., n, p. 285. Berlin found, 1856, the blood of python crystallizable. He also saw crystals in the stomach of the Amblyomma exornatum, a blood-sucking parasite which the snake had brought with it from Senegal to Europe (Neder- landsch. Lancet, 3 serie, 5 Jaar- gang, 1855-56, p. 739). Zeitschr. f. wiss. Zool., 1849, 1, 266 (Kolliker). Illustration in Virchow's Archiv, xxx, taf. 15, fig. 4, and Bullet, de 1'Acad. de St. Pe'tersboure, vill, 561-572. Teichmann (Zeit- schr. f. rat. Med., 1855, p. 379) obtained the crystals by mixing the defibrinated blood with very much water and allowing it to evaporate at. a low temperature, but since he obtained them color- less, it is doubtful if they con- sisted of hemoglobin. I saw the crystals in extravasated blood in the lymph-sac. Illustration in Funke's Atlas, taf. x, fig. 6. Funke saw the direct change of the blood corpuscles to crystals, and on the addition of water blood corpuscles were again formed. Lacerta Turtle (Testudograca) Python (Python schneideri) Python (Python bivii- latus) Frog (Rana esculenta) White-fish (Leuciacus dobula) Need les and plates Prisms and plates Intraglobularand extraglobular Prisms Crystallizes with much difficulty Crystallizes very easily Do Intraglobularand extraglobular 106 INVESTIGATIONS OF PREYER. TABLE 31. — Preyer's table showing the source of hemoglobin crystals, etc. — Concluded. Name of kind. Crystal form. Crystal system. Appearance. Solubility in water. Crystallizability. Remarks. Seal y-s h a p e d Crystallizes very Funke, in Zeitschr. f. rat. Med pro) crystals (Funke) Prisms . easily on the aa- dition of water 1851, 191. Kunde, ibid. 1852 p. 286. Remak (Muller's Arch 1852 121) prinus erythroph- thalmus) Barbel (Barbus flu- viatilis) White bream (.46ra- Spindle- and nee- dle-shaped crys- tals (Remak) and intraglobular (Funke) Extraglobular . . . ily soluble than the tench-blood crystals (Re- mak) easily found the crystals 2 hours after death in the blood-veeaeJB. Funke (Zeitschr. f. rat. Med., 1852, 200) saw the change of the crystal containing blood cor- puscles into the ordinary ones on the addition of water. Kolliker, Mikroskop. Anat., 1854, ii, 2 Halfte, p. 281. mis blicea) and extraglob- ular (Funke) readily Do corpuscles into crystals and the rechanging of the same on the addition of water, and could recrystallize them 3 to 4 times on the object-glass. Remak (Muller's Archiv 1852 sitis) tapering at both ends Do very easily in water (Remak) 121) saw the crystals always 24 hours after the death of the ani- mal in thick bundles in the vessels and in the heart. His statement that they are readily soluble in ether and alcohol rests on delusion (see Zeitschr. f. rat. Med., 1852, 213). The crystals can be recrystallized on the object-glass. nits fcrama) Needles schr. f. wiss. Zool., xn, t. 50, 4. Kolliker saw the crystals, 1849. fluvial His) Herring (C lup ea haTfnyus) Plates and rods (Bojanowski) Very probably rhombic and intraglob- ular Extraglobular roach (Remak) Very readily solu- ble easily Crystallizes with extraordinary difficulty (Todd's Cyclop, of Anatomv and Physiology, 1849, pt. 36. LoncL p. 792, "Spleen"). Remakfound them 2 hrs. after death in the blood-vessels (Muller's Archiv, 1852, 121). Illust. in Kolliker's Handbuch der Gewebelehre, 1863, 4 Aufl., p. 627). Illustration in Zeitschr. f. wiss. Zool., xn. taf. 30, 11 (Bojanow- ski). garis} Pike (Esox Indus') . . . 4-sided prisms . . . Do Probably rhom- bic Do Extragtobular. . . . Do Illustration in Zeitschr. f. i\is.-. Zool., xn, 334, fig. 10 (Bojan- owski). rostrata) Earthworm (Lumbri- Very delicate nee- Do ble ily Zool., xii, taf. 30, 10 (Bojan- owski). cus terrestris) Horse leecli (?) (Ne- dle-shaped crys- tals (Prever) Small tabular of earthworm blood is allowed to evaporate slowlv. phelis) plates, little rods and col- umns (Leydig) Clepsine. 116. Illustration in the same. Levdig, Lehrb. d. Histologie, 1857, 446, taf. 8, fig. 34 B. CHAPTER VI. THE PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS SINCE PREYER'S INVESTIGATIONS. Since the appearance of Preyer's monograph very little progress has been made either in the methods of preparing the blood crystals or in the study of their crystalline characters, although much has been added to our knowledge of hemoglobin in certain other directions. Such simple methods as have been described for preparing crystals in small quantities, together with Hoppe-Seyler's method for preparing them in large quantities, have proved so satisfactory for general laboratory purposes that there has been little encouragement to seek new processes; while Preyer's long list of crystals from different species, and the assignment of all of them to the rhombic system, except those of the squirrel, seem to have discouraged research along the lines of crystallography. In fact, with rare exceptions when crystals from a new species have been isolated, the observer has been content without further inquiry to record them as being rhombic. We are therefore now, so far as the crystallography of hemoglobin is concerned, virtually where we were when Preyer's monograph was published (1871). The method of furthering crystallization by the putrefactive process, already pursued by a number of observers, was adopted by Gscheidlen (Archiv f. ges. Physiologie, 1878, xvi, 421), who placed defibrinated blood in a glass vessel with little air, and kept it in an incubator until the absorp- tion spectrum showed the absence of oxyhemoglobin. When a drop of this blood was placed on an object-glass, allowed to evaporate slightly, and then covered with a cover-glass, crystals appeared under the eye of the operator. From dog's blood which had stood for several days in the incubator he obtained crystals from 3 to 4 mm. long. The rapid crystallization of the blood thus prepared he found to be due to putridity, since blood kept in sterilized vessels under the same conditions showed far less power of crys- tallization. In guinea-pig's blood kept in the incubator with the admission of air he found not only large tetrahedra, but also rhombic plates and prisms. By this method Gscheidlen prepared crystals from the blood of the dog, guinea-pig, sheep, bullock, rabbit, and goose. He also noted that blood kept in hermetically sealed tubes for several years crystallized in a short time upon exposure to the air. The readiness with which putrid blood crystallizes had already been noted by Schmidt, Bottcher, Blebs, and others. A method by Kiihne and Gamgee (Gamgee's Physiological Chemistry, 1880, 87) is as follows: 500 c.c. of defibrinated dog's blood are treated with 31 c.c. of ether and the mixture shaken for some minutes. It is then set aside in a cool place. After a period varying from 24 hours to 3 days the liquid becomes converted into a thick magma of crystals. The crystals 107 108 PEEPAEATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS may be separated by placing the mixture in tubes and using the centrif- ugal apparatus. The cakes of crystals thus obtained are mixed with water holding one-fourth its volume of alcohol, and again centrifugalized. By repeating this process, the crystals are said to be obtained free from serum- albumin. If requisite, the crystals are recrystallized by dissolving them in as small a quantity of water as possible at 25° to 30°, cooling the solution to 0°, and adding a fourth of its volume of alcohol. It is better to place the fluid in a freezing mixture at a temperature of --10° to —20° for 24 hours. Crystals of reduced hemoglobin were prepared by Hiifner (Zeit. f. physiol. Chemie, 1880, iv) from human blood, diluted or not, by placing the blood in tubes from which air is excluded. After standing for a month or two at summer temperature the blood became of a beautiful purple color, and in many spots on the inner wall of the tubes there could be seen whole layers of purple-red crystals, which upon spectroscopic examination were found to give the characteristic bands of reduced hemoglobin. Wedl (Archiv f. path. Anat. u. Physiologie, 1880, LXXX, 172) obtained reduced hemoglobin crystals expeditiously by subjecting a solution of fresh or dried blood in a confined atmosphere in the presence of a solution of pyrogallic acid. The acid absorbs the oxygen and thus reduces the hemoglobin. In this way crystals of reduced hemoglobin were prepared within 24 hours from the blood of man, the rabbit, hare, deer, pig, and sheep. Crystals of reduced hemoglobin were prepared in large quantities by Nencki and Sieber (Berichte d. d. chem. Ges., 1886, xix, 128, 410), who, however, make the erroneous statement that no one had up to that time prepared crystals of reduced hemoglobin. Kiihne (Archiv f. path. Anat. u. Physiol., 1865, xxxiv, 423), and shortly after Eollett (Sitzungsb. d. k. Akad. d. Wissensch., Wien, 1866, LII, 246), obtained crystals of reduced hemoglobin by reduction of concentrated solutions of oxy hemoglobin. Kiihne used a very concentrated solution of oxyhemoglobin in very dilute ammonia, which he subjected to a stream of pure dry hydrogen in a glass chamber. As evaporation proceeded crystals formed. Rollett (loc. cit.} prepared reduced hemoglobin by the aid of iron filings. Gscheidlen in 1878 (loc. cit.) and Hiifner (toe. cit.) and Wedl (loc. cit.) in 1880 also prepared crystals of reduced hemoglobin. Nencki and Sieber proceed in this way: Pure oxyhemoglobin crys- tals from the blood of the horse are dissolved in lukewarm water; the solution is then mixed with several cubic centimeters of decaying blood in a flask that is provided with an india-rubber stopper having two perfora- tions for tubes leading to and from the flask. The mixture is then freed from air by the passage of a stream of hydrogen, after which the two tubes are sealed by heat, and then the flask is set aside at a temperature of 20° to 25° for 8 to 14 days. After a time every trace of oxygen has dis- appeared, the fluid is of a beautiful violet-red color, and contains only re- duced hemoglobin. The solution is now cooled to 0°, an india-rubber tube is for some distance slipped over the outlet tube of the flask, and the other end of the tube is dipped in cold absolute alcohol. The flask is gently heated by immersing in lukewarm water, the end of the glass tube within SINCE PREYER'S INVESTIGATIONS. 109 the rubber tube is broken off, and by alternate cooling and heating of the flask sufficient alcohol is introduced so that the solution contains about 25 per cent of alcohol. The free end of the rubber tube is now closed by a screw clip and glass stopper and the solution is subjected to a temperature of 5° to 10°. After 12 to 24 hours the reduced hemoglobin has crystallized into glittering plates and prisms. When examined under the microscope at 0° in the mother-liquor, the crystals for the most part appear as 6-sided plates, of which some were from 2 to 3 mm. in diameter. In the micro- spectroscope every crystal showed only the one band of reduced hemo- globin. The prismatic crystals are doubly refracting. The color of the larger plates is a beautiful violet red; the smaller thin plates appeared greenish in transmitted light. The crystals were very sensitive to oxygen and warmth. At room temperature they quickly melt, and as quickly they lose their violet color and show by the microspectroscope the bands of oxy hemoglobin. In absolute alcohol they remain unchanged, at least in so far as their form is concerned. If the hemoglobin solution is mixed too soon with alcohol, before the bacteria have taken up the last traces of oxygen, both reduced-hemoglobin and oxyhemoglobin crystals are formed. Besides the differences they describe in the color and spectroscopic behavior Nencki and Sieber also make note of differences in the forms of oxyhemoglobin and reduced hemoglobin. From horse's blood they obtained oxyhemoglobin in long 4-sided columns, and the reduced hemoglobin in thin 6-sided plates which are more soluble in water than the oxyhemoglobin. In horse's blood which has decomposed in well-closed or sealed vessels, the reduced hemoglobin separates as a thick crystal pulp on the addition of alcohol after standing several hours at a temperature under 0°. Gamgee (Schafer's Text-book of Physiology, 1898, 1, 232) gives a method for preparing reduced hemoglobin which he states he employed 20 years previously, and which seems to him to possess some advantages: A magma of pure oxyhemoglobin crystals and a small quantity of the mother-liquor are placed in a glass tube so as nearly to fill it, and the tube sealed and heated for some days in an incubator at about 35° and then set aside in a cool place. After some weeks of exposure at winter temperature crystals of reduced hemoglobin will be found. Crystals of reduced hemo- globin have also been prepared by Ewald, Frey, Uhlik, Copemann, Dono- gany, and others, as will be shown by subsequent references. The changes in solubility of crystals of hemoglobin that are caused by alcohol were studied by Struve (Ber. d. d. chem. Ges., 1881, xiv, 930; Jour. f. prakt. Chemie, 1884, xxix, 304), who found that fresh crystals placed in strong alcohol immediately became darker, without change of form, and insoluble in water. Upon treating crystals with dilute alcohol, they became faintly yellowish or completely decolorized. These and other phenomena led Struve to resurrect the long since abandoned view that the blood crystals are composed of a colorless albuminous substance which is stained or colored mechanically. After leeches have sucked blood and crystallization has begun, speci- mens of hemoglobin crystals may be obtained from time to time, as shown 110 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS by Stirling and Brito (Jour. Anat. and Physiology, 1882, xvi, 446), by causing the leeches to disgorge. To do this they applied pressure, an 8 per cent salt solution, weak to strong acetic acid, 2 to 1000 solution of sulphuric acid, or galvanic or faradic shocks. Within 20 days, hemoglobin crystals appeared, which was much earlier, they state, than was noted by Budge and Bojanowski; but no hemin crystals were found, as were thought by Bojanowski to be present at tunes. Even after a year and a half they found dusky-red purplish crystals of reduced hemoglobin of human blood in the form of 4-sided prisms, some of them nearly equal-sided, while others were oblong. From the stomach of the leech they obtained crystals from the blood of the common gold-fish, and also obtained crystals from sealed microscopic preparations of the diluted fish blood. From the blood of the frog the}' secured both colored and colorless crystals of exactly the same form. The former they describe as being very variable in size, highly refractive, acicular, and pointed at one extremity like the point of a pen. Stirling and Brito note that colorless crystals of frog's blood had also been discovered by Teichmann (loc. cit.), who mixed the defibrinated blood with water and evaporated at low temperature. Besides obtaining these crystals from the blood of the stomach of the leech they also prepared them by mixing 5 or 6 drops of the freshly drawn blood from the heart with one or two drops of distilled water, and then sealing up the preparations with gold size. They state, however, that exposure to the air favors the formation of the crystals, which first form around and in the neighborhood of coagula. In the case of one of the leeches, on exposing some of the blood on the fourth day, they obtained blood which, when sealed up and allowed to stand, developed beautiful colored crystals of exactly the same shape as those which are colorless. The sole difference, they state, was in the color, and they therefore were inclined to regard the latter as being closely related to hemoglobin, if not identical with it. They did not find any crystals from the blood of the newt that had been ejected by the leech. Studies were also made of the influences of certain reagents on the crys- tallization of rat's blood. Stirling and Brito found that common salt and urine prevented the diffusion of hemoglobin from the corpuscles, and there- fore prevented crystallization; but a weak solution of pure urea behaved exactly like water, liberating the hemoglobin and thus permitting of crys- tallization. From this they conclude that the presence of common salt in the urine is sufficient to neutralize the effect of the urea. The crystals found in the solution were exactly the same as those formed after the addition of water. Crystals appeared in a few minutes when chloroform was freely mixed with a drop of rat's blood on a slide and covered and examined in the usual way, but the ordinary flattened prisms with beveled ends were shortened so as to be hexagonal. They also made the interesting observation that the passage of a galvanic current causes a deposition of crystals equally well at both negative and positive poles, but that the induced current was without effect. The use of chinolin to increase crystallizability was reported by Otto (Zeit. f. physiolog. Chemie, 1882, vn, 57). He employed an alcoholic solution SINCE PREYER'S INVESTIGATIONS. Ill or an aqueous solution of the hydrochlorate of chinolin, and by its aid pre- pared crystals of pig's blood. He notes that Hiifner previously found that the blood of the pig mixed immediately with one-third of its volume of a 1 per cent alcoholic solution of chinolin crystallizes beautifully when sub- jected to cold, the mixture after several days containing a mass of needles and plates which liquefied within an extremely short time when exposed at room temperature under the microscope. Otto used chinolin solutions and blood in varying proportions, as follows: (a) 100 c.c. of blood, 40 c.c. of 1 per cent chinolin hydrochlorate solution, and 30 c.c. of alcohol; (b) 100 c.c. of blood, 30 c.c. of chinolin solution, and 30 c.c. of alcohol; (c) 100 c.c. of blood, 25 c.c. of chinolin solution, and 25 c.c. of alcohol. The mass of crys- tals which had collected during 8 days was washed on a filter-paper with alcohol (diluted 4 times) and then dissolved in a small quantity of water. Adding to this solution one-eighth its volume of alcohol, the mixture was again placed in the cold, whereupon crystals sometimes separated within a few days. As a rule, the second crystallization failed to occur, and instead a mass separated out in from 8 to 14 days, which was found to be met- hemoglobin. The unsatisfactory results of this method led Otto to adopt what is practically the Hoppe-Seyler method : The blood was diluted with salt solution and stood in a cylindrical vessel for two days. The corpuscles were collected and dissolved in the smallest possible quantity of water at 50°, 300 c.c. of water being sufficient for the solution of the corpuscles from 1 liter of blood. Owing to the unusual solubility of the crystals of pig's blood, which liquefy at room temperature, it is very important, as Otto states, to avoid an excess of water in dissolving the corpuscles. The solution is filtered, cooled, mixed with cold absolute alcohol in the usual proportion of 4:1, and then subjected to cold. As a rule, after only one day a thick mass of fine, bright-red needles was found at the bottom of the cylinder. For the purpose of recrystallization, the crystals were collected upon a folded filter- paper, washed, and crystallized 3 times with dilute alcohol in the ice-chest. He prepared dog's crystals in the same way. The crystals were finally spread upon plates and dried under a bell-jar over sulphuric acid in the cold. The crystals of pig's blood thus prepared were then powdered, heated to 115°, and subjected to a stream of hydrogen, when they gave off 5.9 per cent of water. Those of the dog similarly treated lost only 4 per cent of water. Both kinds of crystals were subjected to elementary analyses (page 71). Otto also analyzed the methemoglobin of the pig. Studies were also made of the extinction coefficients (page 77) and of the oxygen capacities. The crystals were determined by the spectroscope to be oxyhemoglobin. In a later research (Archiv f. ges. Physiologic, 1883, xxxi, 240) Otto pre- pared crystals of horse's blood, which he also subjected to elementary analysis and spectroscopic examination. In his former investigation he determined that the extinction coefficients of the oxyhemoglobin of the pig and dog are the same (1.33), and in this inquiry* the extinction coefficient of horse oxyhemoglobin was found to be 1.352. His elementary analyses are given on page 71. He also noted the observation of Hoppe-Seyler 112 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS (Zeit. f. phys. Chemie, 1878, 11, 149) that the crystals of horse hemoglobin appear to be of two kinds (needles and prisms) which differ in solubility — a difference which Hoppe-Seyler thought likely to be due to differences in the amount of water of crystallization. Otto states that there continually appeared, besides little needles which are in relatively greater abundance in recrystallization, long, thick prisms which prevail in the first crystallization. He endeavored, but failed, to separate the needles from the prisms by wash- ing with dilute alcohol, as Hoppe-Seyler states could be done. He also tried to determine differences in the water of crystallization, but he failed to obtain concordant results. Crystals from horse's blood were prepared by Hiifner and Biicheler (Zeit. f. phys. Chemie, 1884, viu, 355) by the ordinary alcoholic method and recrystallized three times in a refrigerator. Generally needles were obtained from 2 to 3 mm. long and 0.5 mm. wide. Once they found hex- agonal tablets of reduced hemoglobin, which changed quickly upon coming in contact with the air. Dried at 0° over sulphuric acid and anhydrous phosphoric acid, the crystals retained 3.94 per cent of water, which came off when the crystals were subjected to a stream of hydrogen at 115°. They made elementary analyses (page 71), calculated the molecular weight and formula (page 75), and determined the oxygen capacity. A new method for preparing hemoglobin crystals was reported by von Stein (Centralblatt f. med. Wissensch., 1884, xxn, 404; Archiv f. path. Anat. u. Physiologic, 1884, xcn, 483), which is applicable to small quan- tities of blood that are readily crystallizable. A drop of defibrinated blood or blood squeezed from a clot was placed on an object-glass and exposed to the air until it began to dry up at the margins. Canada balsam was then applied, first around the drop of blood, in order to prevent any possible escape, and then the remaining space above it was filled. It is to be observed, von Stein states, that the center of the drop of blood is pushed off to the periphery. In this way a clear space is made for crystallization, otherwise the crystals are so small that their outlines can not be made out. Too thick a layer of blood is to be avoided, because the balsam does not penetrate to the deeply lying portions. Von Stein proceeded in another way, by not allowing the blood to evaporate, and by treating it immediately with the reagent and covering the mixture with a cover-glass. Canada balsam is best when it appears yellow and not entirely clear. In liquid balsam the crystals form more quickly, and sometimes have larger dimensions, but they soon become brown (in one or two days), then dull and black, and in a short time are fissured to small pieces. Preparations can be made which retain their form and color for years if the balsam has been exposed to the air for a long time, or is evaporated to such a consistence that it can be drawn out into transparent but not milky threads when lifted with a glass rod. Whichever method is used, it is important that the preparation be left uncovered in the air until the crystallization has been completed, and until the odor of the balsam has completely disappeared, which lasts ordinarily a few days. Then with a knife immersed in ether, turpentine, or oil of cloves (little should be used of either), the upper portion of the balsam is removed, SINCE PREYER'S INVESTIGATIONS. 113 and the whole covered with a cover-glass and sealed with asphalt or balsam. Crystals from human, horse, guinea-pig, and rat blood were obtained by the above methods. Von Stein's methods were extended by Smreker and Zoth (Sitzungsber. d. Wiener Acad., 1886, xcni, Abth. in; Maly's Jahr. ii. d. Fort. d. Thier- chemie, 1886, xvi, 102), who used Canada balsam, turpentine, Peru and other balsams; solutions of colophony, damar, and mastic dissolved in xylol; fixed oils; xylol solutions of rosin; fatty acids, etc. The doubt as to whether or not hemoglobin is a chemical individual, together with the fact of the discrepancies in the centesimal analyses of hemoglobin, led Zinoffsky (Zeit. f. physiol. Chemie, 1886, x, 16) to prepare crystals of hemoglobin in several ways and to make careful determinations of the iron and sulphur contents. In preliminary experiments he found that the washing of the corpuscles by common salt solution, according to the directions of Hoppe-Seyler, is not only superfluous, but also undesir- able, because the washing introduces the danger of decomposition, owing to the fact that from 3 to 5 days are required in the process, and because it is not of importance in removing the small quantity of protein in solution. In experiments in relation to the separation of the hemoglobin from the stromata he found that, when the corpuscle pulp is heated to 35° with 3 volumes of distilled water, the hemoglobin dissolves and crystallizes and that the stromata remain undissolved and cling so tenaciously to the hemoglobin crystals that they can not be removed by nitration. They must, therefore, be dissolved before the crystallization of the hemoglobin, either (1) by the addition of very little ammonia to the fluid heated to 35°, which must then be carefully neutralized with dilute hydrochloric acid (according to the direction of Schmidt), or (2) by the addition of ether (30 c.c. of ether being sufficient for 9 liters of blood). To crystallize the hemoglobin the solution was cooled to 0°, mixed by titration with one-fourth its volume of absolute alcohol, and left standing for 72 hours. The crystals were washed by decantation with a mixture of 1 part of alcohol to 4 parts of water cooled to 0°. To obtain pure crystals, the crystals were dissolved in 3 volumes of distilled water at 35°, the solution was filtered, and the filtrate was mixed with dilute alcohol as before. Tests showed that two recrystallizations of the first product sufficed to obtain pure crystals. Zinoffsky also makes note of the fact that the drying of the crystals in vacua at 0°, according to the directions of Hoppe-Seyler, is an exceptionally lengthy process, and that the crystals can be dried in about 8 hours at 18° to 20° without being placed in a vacuum. After these preliminary investi- gations he prepared crystals from horse blood by three methods, viz : First method: 20 liters of horse's blood were defibrinated; the blood- corpuscle pulp, which after 3 hours' standing in the cold had been deposited, was separated from the serum and mixed with 8 volumes of a 2 per cent solution of common salt. After 3 days the corpuscles were collected and placed in 3 volumes of distilled water at 35°, to which were then added 16 c.c. of one-tenth normal ammonia solution. After 5 minutes the ammonia was neutralized by titration with a very dilute hydrochloric acid. The 114 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS mixture was quickly cooled to 0°, to which was then added 1 volume of absolute alcohol at 0° to every 4 volumes of the solution. After 3 days' standing in an ice-and-salt mixture, the crystals were collected and washed twice with alcohol and water (1 :4) at 0°, and then, in the way stated, recrys- tallized twice and finally dried by the aid of the air-pump. This preparation yielded 200 grams. Second method: The blood-corpuscle pulp obtained by decantation from 10 liters of blood, and without washing with salt solution, was dis- solved in 3 volumes of water at 35°, and then treated as in the first method. The yield was 520 grams. The very much larger quantity thus obtained led Zinoffsky to believe that washing with salt solution reduces the yield. Third method: The blood-corpuscle pulp from 9 liters of blood was dissolved immediately in 3 volumes of distilled water at 35°, then cooled; 30 c.c. of ether were added instead of ammonia, as in the first method, and then the solution was treated as in the first method. The product by the third process was the purest, the ash containing but a trace of chlorine, no alkalies, and only imponderable quantities of phosphorus, lime, and magnesia. The product by the first process con- tained 0.0235 per cent of phosphoric acid. The second preparation was the least pure. It contained 0.0401 per cent of phosphoric acid, 0.0097 per cent of CaO, and 0.0131 per cent of MgO. TABLE 32. — Table from Zinoffsky, showing the percentages of sulphur and iron, the number of atoms of sulphur to each, atom of iron, and the amount of sulphur and iron in the ash of hemoglobin. Material and author. s. Fe. Atoms of S to each atom of Fe. Amounts of S and Fe in the ash of hemoglobin. S. Fe. Dog's blood: Schmidt Per cent. 0.66 .375 .448 .359 .6532 .6443 Per cent. 0.43 .45 .42 .42 .46370 .47238 .46720 .47 .45 2.686 1.60 2.427 1.6637 1.9972 1.4033 1.8915 1.8481 \ 1.8132 1 1.6637 1.7062 2.5122 4.7855 0.9439 Hoppe-Seyler Do Do Horse's blood: Bucheler Do Do Kossel .65 .67 2.42 2.60 2.2S76 Otto 4.0S66 Zinoffsky in the earlier part of his article shows (table 32) the marked discrepancies in the results of the analyses by different observers of speci- mens of bloods from different individuals of the same species. They are also of particular interest in connection with the figures obtained by Zinoff- sky in this research. The two sulphur determinations of the first preparation were 0.3902 and 0.3916 per cent; of the second preparation, 0.3583 and 0.3658 per cent; and of the third preparation 0.3899 and 0.3881 per cent. In the determi- nations of iron he found in the first preparation 0.325 to 0.327 per cent and in the third preparation 0.334 to 0.338 per cent. These results show, he SINCE PREYER'S INVESTIGATIONS. 115 states, that there are 2 atoms of sulphur to 1 atom of iron. The mean of his elementary analyses is C5i.i5H6.76 and the molecular formula C7i2 Comparing Zinoffsky's percentages with those of other analysts (see page 71), it seems as though there must be errors in his carbon and hydro- gen estimations. Moreover, his iron and sulphur determinations differ materially from those of others, yet his analyses were conducted in such a way as to warrant confidence in these figures. The low C content is cer- tainly suggestive of imperfect combustion, or, according to Hiifner, of contamination with stromata. Zinoffsky's work has been reviewed and supplemented by Hufner (see later). The optical properties of oxyhemoglobin, reduced hemoglobin, met- hemoglobin, hemin, and CO-hemoglobin were studied by Ewald (Zeit. f. Biologie, 1886, xxn, 459). He laked the blood by repeated freezing and thawing, and then spread layers of varying thickness upon microscopic slides. The margins of the preparations soon dry, and then a cover-glass is placed directly on the blood or supported by a wedge of glass. If the preparations are examined immediately, only oxyhemoglobin crystals will be found; but after several days violet-purple spots appear which consist of reduced hemoglobin, but which soon pass into solution. He also obtained crystals of reduced hemoglobin by letting the blood stand in tubes for several days; in the deeper layers the oxygen disappears and crystals of reduced hemoglobin form. He found the crystals of oxyhemoglobin and reduced hemoglobin to be doubly refracting and pleochroic, and that the pleochroism is much more marked in reduced hemoglobin. In a research to determine whether or not the 6-sided crystals of certain rodents really belong to the hexagonal system, and to find an explanation of the difference in crystalline form that hemoglobin presents in different animals, Halliburton (Jour, of Physiology, 1886, vn, Proc. Physiol. Soc. No. 1 ; Jour. Microscop. Science, 1887-88, xxvni, 181) carried out a series of observations chiefly with the bloods of the rat, guinea-pig, and squirrel. The rat was taken as a type of animals whose crystals are rhombic; the guinea-pig, of those whose crystals are tetragonal; and the squirrel, of those whose crystals are hexagonal. Halliburton notes that Lehmann states, without giving any reason, that although the crystals of the squirrel are hexagonal in form they do not belong to the hexagonal system, and that von Lang, Kunde, and Preyer state that they do. In examinations of squirrel's crystals by polarized light he found evidence, as he believes, of their being true hexagons instead of their being, according to Lehmann, rhombic plates with an "hexagonal habit." It had already been found by von Lang that the tetrahedra of the guinea-pig belong to the rhombic system. In experiments instituted to show whether differences in crystalline form are due to some agency extrinsic to the hemoglobin or to some property 116 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS inherent in the hemoglobin, he in some instances mixed serum, or the serum and the stromata, or the blood of one species of animal with the blood of another, or solutions of hemoglobins of different species with one another. The presence of the foreign serum, or serum and stromata, was without influence on crystalline form, and, while mixed bloods, or mixed hemoglobin solutions, did not affect crystalline form, they sometimes caused modifica- tions in crystalline habit. Thus, in case of the bloods or solutions of hemo- globins of the rat and guinea-pig the crystals of the rat were rhombic with hexagonal habit, no needles or tetrahedra being present. (See table 33.) TABLE 33. — Forms of hemoglobin crystals in case of mixed bloods (from Halliburton). Blood of— Mixed with that of — Rat. Rat. Squirrel. Dog.. Dog. Squirrel. . Guinea-pig. Guinea-pig. Form of hemoglobin crystals from the mixture. Squirrel. . . . Guinea-pig. Both rhombic prisms and hexagons present. No rhombic prisms of the shape usu- ally seen in rat's blood present. No tetrahedra. Crystals are all rhom- bic prisms with hexagonal habit. Hexagonal plates and tetrahedra both present. Many tetrahedra imper- fect. The tetrahedra were all re- duced to about half the size of those prepared from the unmixed blood of the same guinea-pigs. Fine rhombic needles and hexagonal plates both present in abundance. The greater number of the crystals formed are very small tetrahedra, about a quarter the size of those prepared from the blood of the same guinea-pigs. The optical properties are, however, the same. Rhombic prisms very slender, like those of dog's blood, also seen. In another set of experiments Halliburton tried to break down the hexagonal constitution of the hemoglobin of squirrel's blood, first, by draw- ing off the water of crystallization and then adding water; second, by converting the hemoglobin into methemoglobin, and then by reducing agents to form once more hemoglobin, and to obtain crystals from this. Both attempts were unsuccessful. In opposition to the statement of Preyer that recrystallization does not alter the form of the crystals, Halliburton found that by recrystalliza- tion of squirrel's hemoglobin, after 3 or 4 recrystallizations no 6-sided crystals were obtained, but a mixture of rhombic needles and tetrahedra, and that in some cases the latter were absent. In conclusion, the author states that the difference between the various forms of hemoglobin can not be a very deep or essential one, and that it seems to narrow itself down to this, either we have a case of polymorphism or the cny-stalline forms are due to the combination with varying proportions of water of crystallization. SINCE PREYER'S INVESTIGATIONS. 117 In the second contribution referred to, Halliburton adds the following to our crystallographic data: Opossum (Didelphis cancrivora). — Very large dark crystals can readily be obtained. They belong to the rhombic system. Kangaroo (Macropus giganteus). — Crystals are more soluble, and so less readily obtained. They are rhombic prisms, slenderer than in the opossum. Sugar squirrel (Belideus breviceps — a marsupial). — Crystals similar to those of opossum. Seal (Phoca vitulina) .— Rhombic prisms, many of them very short and simulating hexa- gons. Easily obtained. Bear (Ursus syriacus). — Bunches of rhombic needles, easily obtained. They are slen- derer than those obtained from dog's blood, as a rule, some being almost silken in appearance. White-bellied beaver rat (Hydromys leucogaster) . — Rhombic prisms. White-whiskered swine (Sus leucomytax). — Rhombic prisms. Water vole (Arvicola aquatica). — Crystals are obtained easily by adding water to the blood. They are of the usual rhombic shape. The analyses of hemoglobin of horse's blood by Zinoffsky (loc. cit.) differed so much from those of previous observers that Hiifner (Beitrage z. Physiologie, Fest. f. Carl Ludwig, 1887, 74) was led to review and supple- ment Zinoffsky's work. Hiifner prepared hemoglobin crystals by a process that is a modification of Zinoffsky's to the extent essentially of separating the stromata of the corpuscles by mechanical instead of chemical means, that is by centrifugalization, so that the crystals could be freed from the stromata dissolved or undissolved and more expeditiously prepared. Crys- tals were obtained from the bloods of the pig and ox by centrifugalizing the corpuscles, extracting the hemoglobin from them by distilled water at 30° to 40°, cooling to 0°, centrifugalizing and treating by the usual method. After the crystals have formed they are centrifugalized in the cold to pre- vent their solution, and the hemoglobin is then three times crystallized by the usual method, and finally dried in an atmosphere at 0°. The ash of 10 grams of this product contained only an imponderable amount of phosphoric acid. The mean figures of his elementary analyses are as follows: Pig's oxyhemoglobin, C54.71H7.38N17.43So.479Feo-3390i9-602 Ox's hemoglobin, C54.66H7.25N17.76So.447Fe0.4oOi9-543 In comparing these figures with those of Otto (loc. cit.}, Hiifner states that the complete removal of the stromata in his preparations causes a higher percentage of C and N, Otto having found C54.17 and N16.23. Zinoffsky's C content (51.15) was very much lower than Hiifner's. Hiifner's analyses show the same ratio of S and Fe in both pig and dog hemoglobins, i.e., 2 of sulphur to 1 of iron, the same as Zinoffsky found with horse hemoglobin. The elementary analysis of the hemoglobin of the dog which was reported the following year by Jacquet (Zeit. f. physiol. Chemie, 1888, xn, 285) was of crystals prepared as follows: The corpuscles were centri- fugalized, then mixed with 2 volumes of water warmed to 35°, then cooled and shaken with ether and treated according to the Hoppe-Seyler process. The crystals were twice recrystallized, and then analyzed according to the methods pursued by Zinoffsky, but he endeavored to eliminate certain possible fallacies in the iron determinations. His analyses gave a mean 118 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS These figures do not agree with those of Zinoffsky for the hemoglobin of the horse, or with those of Hiifner for the hemoglobins of the pig and ox. The relation of Fe to S in clog's hemoglobin, Jacquet found to be 1 : 2.85; Zinoffsky found 1:2 for the horse ; and Hiifner found 1:2 for the pig and ox. Jacquet believes his sulphur value to be too small, and that there is 1 atom of Fe to 3 of S. Later (Zeit. f. physiolog. Chemie, 1889, xiv, 289) he analyzed crystals of the hemoglobin of the dog, which he prepared by a modification of his previous process. The earlier method was used, except that the solution of the corpuscles after the addition of the ether was centrifugalized in a machine running at from 1,600 to 2,000 revolutions, whereby the stromata could be partly done away with. The hemoglobin 3 times crj^stallized contained only a trace of phosphoric acid, the quantity not being estimated. The mean of his analyses was £54-57-^7. 22Ni6-38So-56sFe0-336020-93 The amount of water of crystallization was 11.39 per cent. The ratio of Fe to S was 1 : 2.96, which was higher than in his preceding investigation. From the values obtained he gives the formula C758H1203N195Fe021s, and the molecular weight as 16669. He also analyzed the oxyhemoglobin of the chicken. In the prepara- tion of the crystals he made a special effort to get rid of the large amount of phosphoric acid (0.77 per cent) shown in the preparations of goose crystals by Hoppe-Seyler. He found that he could not treat the blood in precisely the same way as dog's blood, because when the corpuscles are agitated with ether a gelatinous mass is formed which could not be filtered. The corpuscles were therefore treated with an equal volume of water and one-third volume of ether. The mixture when heated to 35° formed into dark-red, gelatinous lumps which were separated from the fluid by centrifugalization. The clear fluid thus obtained was readily filtered, and by the customary treatment very soluble needles of hemoglobin were formed. By reciystallization both rhombic plates and prisms were obtained. The three times crystallized hemoglobin was found upon analysis to have the following composition : The water of crystallization was 9.333 per cent, and the ratio of Fe to S 1 : 4.485. If the molecule be doubled the ratio is 2 : 9. In comparing the analyses of the oxyhemoglobin of the dog, chicken, and horse, he states that although these hemoglobins are different they have a similar iron capacity, which warrants the conclusion that the iron-containing group in the various hemoglobins is the same. Jacquet made ineffectual attempts to prepare crystals from fresh salmon blood, but succeeded when the blood was left to rot, there appearing clusters of crystals and beautiful single rhombic prisms. Jolin (Archiv f. Anat. u. Physiologic, 1889, 265) records that the hemo- globins of the dog and guinea-pig differ from that of the goose in their absorptive rapidity in relation to O, as well as in the volume of O absorbed. The increased crystallizability of putrid blood has been noted by a number of observers and referred to in previous pages, and Bond (London SINCE PREYER'S INVESTIGATIONS. 119 Lancet, 1887, n, 509, 557) has added to our knowledge in this particular by showing a relationship between crystallizability and septic conditions in the body. He found that if a drop of blood were taken from the cleansed finger of a patient who is suffering severely from absorption of the prod- ucts of putrefaction, and that if such drop be placed between a slide and a cover-glass and allowed to remain at room temperature (60° F.), in the course of 20 to 30 hours crystals of reduced hemoglobin of prismatic and needle form will be found, while within some corpuscles little bars and needles may plainly be seen, apparently distinct from the enveloping stroma. He also found that adding putrid blood facilitates crystallization, and that in cases of pernicious anemia crystallizability seemed to be increased. The increased crystallizability of human hemoglobin in pernicious anemia that was pointed out by Bond (loc. cit.} was later noted by Cope- mann (Journal of Physiology, 1890, xi, 401), who found that when a drop of blood from the finger of a patient thus affected was allowed to fall on a glass slide, the edge of the drop allowed to dry, and a cover-glass placed on the blood, crystals of hemoglobin gradually formed in from 10 to 48 hours. The only exception to this was in the case of patients who had been treated with arsenic for some days, although crystals were obtained upon the discontinuance of the arsenic. To imitate the influence of septicemia, as was also shown by Bond, Copemann treated the blood with decomposing serum. This method he found to be successful in the case of the bloods of the bullock, sheep, pig, dog, and cat, but unsuccessful for the blood of man, the monkey, rabbit, and squirrel. Except in the case of man and monkey the crystals were of oxyhemoglobin, and this notwithstanding that the decomposing serum invariably brought reduction of the oxyhemoglobin as it diffused from the corpuscles into the plasma. He states that this occurred to the greatest extent just inside of the edge of the cover-glass, but not extending to the edges where the layer is kept oxidized; and that it is in this intermediate zone of fully reduced hemoglobin that crystals are to be found in the great- est quantity, both in case of human and monkey blood and of that of the rabbit and squirrel; but in the latter the crystals are of oxyhemoglobin, while in the former they are of reduced hemoglobin. He also made the interesting observation that in specimens of squirrel's blood (species not stated) the crystals were in every instance in the form of fine needles and rhombic prisms, the needles sometimes being collected into bundles, while the usual hexagons were absolutely absent. Copemann also prepared crystals from the blood of the horse, bullock, sheep, pig, dog, cat, squirrel, rabbit, guinea-pig, rat, mouse, and chicken by the following simple process: The blood is shaken with ether (16 : 1) and then kept under an atmosphere of ether for some time, which may be accomplished by performing the agitation of the blood with ether in a stoppered bottle and gradually allowing the air to escape as the ether is volatilized. By this means the contained air is gradually replaced by ether vapor, while at the same time the small portion of blood which is forced out around the stopper of the bottle on drying fixes it in its place and so prevents 120 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS the ingress of air again. It seems also that it is better to leave the bottle in a room at ordinary temperature than to put it in a cool place, as advised by Gamgee. After a variable time, in case of most animals at least two days, a drop of blood is placed upon a slide, and when the margin of the drop is slightly dry a cover-glass is gently lowered on the surface of the drop. The formation of crystals will often be seen within an hour or so. Human blood subjected to the same process does not usually yield crystals, but when crystals do appear they invariably present the appearance of reduced hemoglobin. Copemann also obtained crystals from human blood by the use of bile — preferably, as he states, cat's bile. Two methods for preparing hemoglobin crystals given by Mayet (Compt. rend. soc. biol., 1890, cix, 156), and stated by him to be improve- ments on the method of Hoppe-Seyler, are as follows: First method: The corpuscles are washed with sodium sulphate solu- tion (1.5 per cent solution of the anhydrous salt) instead of sodium chloride solution. To wash the corpuscles, a glass vessel having the capacity of 5 liters is used, the upper part of the vessel being of cylindrical form and tapering conically, the lower part being in the form of a narrow cylinder which holds about 80 c.c. The latter part has at the bottom an opening which can be closed by a glass stopcock; a second opening, capable of being closed, is located where the upper conical and the lower cylindrical parts join. The treatment of the corpuscles with ether (one-fifth volume) is also performed in a special vessel consisting of a cylinder 3.5 mm. in diameter and 35 cm. long and extended by a conical part in the form of a narrow tube provided with a glass stopcock. To the blood solution is added one-fifth volume of absolute alcohol. This mixture is cooled at least 3 times for 12 hours at --14°. The crystals are separated and dissolved in water at 35°, the solution mixed with alcohol as before, and the hemoglobin at least 3 times crystallized by cooling to —14°. In this way crystals 1.5 mm. long were obtained from the bloods of the dog, horse, and ass. Second method: The corpuscles are washed as above, the corpuscle pulp is shaken with water (1 volume) and pure benzine (one-fifth volume), and kept 24 hours at 5° to 8°. Then the solution is gradually mixed with one-fifth volume of absolute alcohol and treated in the usual way. The yield by the second process is the greater. A study of the influences of various reagents upon the crystallization of oxyhemoglobin and reduced hemoglobin was made by Donogany (Math- ematikai es termeszettudomanyi ertesito, 1893, 11, 262; Maly's Jahr. ii. d. Fort. d. Thierchemie, 1893, xxin, 126), who prepared crystals from the bloods of the dog, cat, pig, mouse, ox, rabbit, duck, guinea-pig, horse, and man. Donogany tested the usefulness of a number of the methods used for preparing oxyhemoglobin and reduced hemoglobin crystals, and he also made some examinations of the crystalline forms. Several of the methods were modified. To obtain oxyhemoglobin crystals from dog's blood, the "Canada balsam method" (loc. cit.) is recommended. A method of his own, which he believes equally as good, is as follows: A drop of blood is treated with a little ethyl bromide, methylene chloride, or ethylidene chloride. SINCE PREYER'S INVESTIGATIONS. 121 From cat's blood, Donogany states, oxyhemoglobin crystals can be obtained by any of the usual methods except the methods of Gscheidlen, Rollett, and Wedl, by which only reduced hemoglobin can be produced. From horse's blood good results were recorded with Canada balsam, damar varnish, chloroform, amyl alcohol, pental, xylol, colophonium dissolved in amyl alcohol, pyrogallic acid, or by freezing. The crystals are doubly refracting, and they consist for the most part of oxyhemoglobin. With the methods of Gscheidlen and Wedl, crystals of reduced hemoglobin were obtained. If the Rollett method is used, combined with distilled water, a mass of reduced-hemoglobin and oxyhemoglobin crystals is formed. The blood of pigs, which is looked upon as crystallizing with difficulty, he found crystallized readily by the use of ethereal oils. The formation of crystals went on slowly, and the crystals were large and well developed. The crystals were doubly refracting and consisted of oxyhemoglobin. From the blood of white mice crystals could not be produced by the aid of Canada balsam, distilled water, chloroform, ether, alcohol, or xylol. Ox blood did not crystallize by treatment with Canada balsam, damar varnish, ether, amyl alcohol, xylol, chloroform, pental, ethereal oils, or pyrogallic acid. By freezing and by Gscheidlen's method, combined with Canada balsam or damar varnish, only small needles could be obtained. From their light color, Donogany believes that they were probably oxyhemo- globin. They were doubly refracting. The coloring matter of the blood of rabbits also crystallized with difficulty. The addition of ether, Canada balsam, chloroform, pental, ethereal oils, and acetone gave negative results. With the method of Gscheidlen and with damar varnish, only small needles could be obtained. With Rollett's method rather large needles were formed. The best result was obtained with pyrogallic acid. The crystals formed, he states, consisted of reduced hemoglobin. The blood of the duck treated with damar varnish, xylol, ether, amyl alcohol, Canada balsam, chloroform, colophonium solution, distilled water, and by quick cooling, scarcely yielded crystals, and even in the most favorable instance only stunted ones. Gscheidlen's method, he writes, can be used with much better results, although here, too, crystallization goes on slowly. The crystals were purple- red, almost blue, needles or prisms, and consisted of reduced hemoglobin. Later these crystals, under the influence of atmospheric air, changed to flesh-colored rhombic, even 6-cornered tablets, which were doubly refract- ing, and consisted, perhaps, of oxyhemoglobin. From guinea-pig blood Donogany produced crystals by means of Canada balsam. They formed quickly, and also became quite large if the Canada balsam used was not very thin and the preparation stood in a cool place. If form and size are not important good results can be obtained, he states, by means of ethylidene chloride. With damar varnish crystallization goes on somewhat slowly and at the sacrifice of sharp edges. Pyrogallic acid and valerian oil did not cause crystallization. Ether, chloroform, xylol, amyl alcohol, acetone, Canada balsam dissolved in xylol, freezing, a mixture of water and alcohol, and repeated treatment with Canada bal- sam gave only poor results. With ethyl bromide, after the course of an 122 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS hour, the whole mass became crystalline, yet the crystals, because of their smallness, were unsuited to investigation. The most beautiful crystals could be obtained with ethylidene chloride according to the following method: A drop of blood is thoroughly mixed with an equal amount of ethylidene chloride, the cover-glass is placed on it, and the preparation set aside in a cool place. After the course of 10 to 12 hours it is entirely filled with crystals. With amyl nitrite or pyridine forms similar to these could not be obtained. All the crystals were oxy hemoglobin. Regarding the form of the oxyhemoglobin crystals of the guinea-pig, Donogany adheres, on the basis of the geometric and optical characteristics, to the view of von Lang that they are sphenoids belonging to the rhombic system. In a later article (Zeit. f. Krystallographie, 1894, xxm, 499) Donogany publishes the results of his crystallographic studies of hemoglobin crystals, which will be referred to in later chapters of this memoir. Crystals were easily obtained from human blood by pyrogallic acid and by the aid of putrefaction. Donogany first reduced the hemoglobin with a 10 per cent solution of sulphide of ammonium, which, however, is not necessary when using old decaying blood. After an interval of 5 to 6 hours crystals separate in the form of rather thick, flesh-colored or purple- red needles. After 12 to 24 hours the individual crystals are pretty well formed. Contrary to Wedl's assertion, Donogany observed that the crystals can not be kept, since they burst in the course of 2 to 3 months in spite of being properly sealed. He states that the crystals produced in decaying blood are of reduced hemoglobin and that they may be changed into oxy- hemoglobin without change of form. He succeeded in producing only reduced hemoglobin directly from the human blood, and he believes it doubtful whether by the influence of atmospheric air these crystals can be changed to oxyhemoglobin. Oxyhemoglobin was prepared by means of Canada balsam, xylol, damar varnish, chloroform, alcohol, amyl and methyl alcohols, acetone, valerian oil, methylene chloride, and ethvlene chloride. Pyrogallic acid and freezing gave only reduced hemoglobin. The crystals, he states, belong to the rhombic system. Wedl had produced reduced hemoglobin crystals by means of pyrogallic acid from dried blood 3 days old, and Donogany modified this method for the production of hemoglobin crystals from dry blood powder (1 year old). The powder was dissolved in a 5 to 10 per cent solution of sulphide of ammonium, pyrogallic acid was added, and crystals appeared after 10 to 12 hours. After the course of 24 to 48 hours crystallization had ceased. The crystals obtained from horse, cat, and rabbit blood in this way were very beautiful, and large crystals (1 cm. long) were not rare. The crystals were chiefly thin needles, broad prisms, and rhombic plates. In human blood, besides these forms, there appeared right-angled truncated prisms and forms similar to hexahedrons. Experiments with bloods of other animals gave less favorable results. The crystals were doubly refracting and consisted of reduced hemoglobin. The sulphate of ammonium process devised by Hofmeister (Zeit. f. physiol. Chemie, 1890, xxiv, 165) for preparing crystals of egg albumin, and subsequently used by Giirber (Wiirzburger physiol. medizin. Ges., SINCE PREYER'S INVESTIGATIONS. 123 1894, 113) and others for crystallizing serum albumin, has been used by Dittrich (Archiv f. exper. Path. u. Pharm., 1892, xxix, 250) and others for preparing crystals of hemoglobin. Owing to the rapid conversion of hemo- globin into methemoglobin by this process, Dittrich used it also to prepare the latter. The blood of the horse was subjected to the Hoppe-Seyler process for preparing the blood-corpuscle pulp. The corpuscles were then dissolved in ether, the solution filtered, and then mixed with two volumes of a cold saturated solution of ammonium sulphate, filtered again, and then placed in flat vessels in the cold. Generally within 24 hours crystallization begins, but occasionally only after 2 to 3 days. The crystals could be recog- nized microscopically in transmitted light as glittering elongated prisms or broad plates. The crystals of the first crystallization were not pure; moreover, the mother-liquor contained, besides crystals, an amorphous precipitate which often could be separated only by repeated recrystalliza- tion. Generally a separation of "globulites" and spherocrystals preceded the formation of crystals. The most of the crystal mass of oxy hemoglobin changed on standing in the air, and through the processes of recrystalliza- tion, gradually and completely into methemoglobin. No further change, for example the formation of hematin, took place. The crystal pulp, recrystallized several times from ammonium sulphate solution, was finally pressed between absorbent paper, and when dry was saved in this condi- tion. This method of production of methemoglobin renders superfluous the use of ferricyanide of potassium or any other agent, the ammonium sulphate in large quantities being sufficient to change the hemoglobin to methemoglobin. Finally, the crystal pulp with the contained ammonium sulphate is permanent, and its solubility is not lost. If, however, the preparation is completely dried over sulphuric acid in vacuo, the largest part of the methemoglobin is changed to an insoluble modification. Schulz (Zeit. f. physiol. Chemie, 1899, xxiv, 454) used essentially the same process for preparing oxyhemoglobin for his studies of globin. Horse's blood was rendered incoagulable by ammonium oxalate, the corpuscles were collected by decantation and then diluted with 2 volumes of water. If the solution obtained in this way is mixed with a like volume of cold saturated ammonium sulphate solution, there is formed an abundant precipitate which consists essentially of fibrinogen and serum globulin. The precipitate after a time becomes so compact that it can be separated by filtration, but, since the hemoglobin begins to crystallize immediately, filtration is rendered difficult because the pores of the filter become quickly clogged. In the completely clear filtrate crystallization soon begins, but the quantity thus obtained is small because of the separation on the filter. If the hemoglobin solution and the ammonium sulphate solution are warmed to 40° before mixing, the separation of crystals takes place less quickly, so that the filtrate obtained is almost completely free from blood-coloring matter. If, on the other hand, both solutions are cooled in an ice-chest before the mixing, and the solution after the mixing is allowed to stand until the albuminous precipitate has completely settled, the crystallization of the hemoglobin is almost completely prevented before filtration. If the 124 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS solution is filtered in the ice-chest, a clear, dark-red filtrate is obtained, which contains most of the coloring matter, and when brought to room temperature soon yields a rich crystal formation, which increases if a little concentrated ammonium sulphate solution is added. After several days the separation is complete — so complete that the filtrate appears almost colorless. It is purely crystalline, without amorphous admixtures. The crystals are without exception little rhombic plates, some of very consider- able size, while ordinarily crystals of horse hemoglobin, produced according to Hoppe-Seyler, separate in the form of long 4-sided prisms. The precipi- tate is filtered on a Biichner filter by the aid of a Sprengel pump and finally freed from the mother-liquor by pressing between filter-paper, then dis- solved in water, and again separated by the addition of an equal volume of a saturated solution of ammonium sulphate. In this way the hemoglobin can be recrystallized with ease several times. Ammonium sulphate efflo- resces on the surface of the firm cake that had been obtained by pressing, and can easily be removed. The cake when dried in the air can be crushed to a fine powder, which readily dissolves in water. The solution shows a pure oxyhemoglobin spectrum. Schulz states that in this way the hemoglobin may be separated from other proteins. Fibrinogen and serum globulin separate completely in a half- saturated ammonium sulphate solution, while the albumin separates only by a higher concentration of the ammonium sulphate than was used here. While as mentioned the oxyhemoglobin, according to the method used by Dittrich (loc. cit.}, changes to methemoglobin, even during the recrystalliza- tion, a pure oxyhemoglobin can also be obtained by this method. The preparation thus obtained is, however, limited in stability; in one case it contained after about one year considerable methemoglobin. The limit of the quantity of ammonium sulphate required for the precipitation of the hemoglobin in the amorphous condition, incidentally noticed, is distinctly higher than that for crystallization. An amorphous precipitate occurred only when in 10 c.c. of the solution there were 6.5 c.c. of concentrated am- monium sulphate solution. In the tests which contained 5, 5.5, and 6 c.c., respectively, of the saturated ammonium sulphate solution in 10 c.c., no amorphous separation occurred, but after longer standing crystallization gradually took place. This method of preparation, according to Schulz, is good because of its convenience for experiments not depending on preparations free from salt. The ammonium sulphate method was also used by Spiro (Zeit. f. physiol. Chemie, 1899, xxvni, 182). The corpuscle pulp was obtained from oxalated horse's blood by decantation, diluted with 2 volumes of water, cooled in an ice-chest, after which the solution was agitated with ether in the proportion of 1,000 c.c. of blood-corpuscle pulp to 50 to 70 c.c. of ether. During continual stirring a saturated solution of ammonium sulphate in the proportion of 700 c.c. to 1 liter of blood corpuscles was gradually added, the ammonium sulphate solution having the same temperature as that of the blood corpuscles. After 5 to 10 minutes the voluminous precipitate which has formed begins to rise ; but if this does not occur more ether must be added, care being exercised to avoid a great excess, since hemoglobin SINCE PREYER'S INVESTIGATIONS. 125 may be precipitated by it. Within several hours a light-red deposit has formed on the surface, while the fluid below appears clear and a dark granite-red. The mixture is filtered, and the filtrate is kept in an ice-chest. After 2 days only an insignificant quantity of crystals has formed. These crystals are suspended on the top of the mixture and are filtered off. The filtrate, which contains almost all of the hemoglobin, is poured into large porcelain vessels and set aside at room temperature. The hemoglobin sepa- rates at first as red and later as brownish crystals. After 3 days almost all of the hemoglobin has crystallized, so that the filtrate appears to be colored only slightly brownish. Microscopically investigated, the crystals contain only slight impurities which can eventually be eliminated by recrystalliza- tion. The hemoglobin is best drained on Biichner filters until the forma- tion of firm cakes. To recrystallize, the crystals are dissolved in the least possible amount of water and mixed with ammonium sulphate (100 c.c. of the hemoglobin solution to 80 c.c. of saturated solution of ammonium sul- phate). The yield from 5 liters of horse's blood was 1,500 grams. Fluoride of sodium was added by Arthus and Huber (Compt. rend. soc. biolog., 1893, XLV, 970) to the list of inorganic salts that favor the crystal- lization of hemoglobin. They found that when to normal or defibrinated blood there was added an equal volume of a 2 per cent solution of fluoride of sodium, and the solution allowed to stand at room temperature, crystals of oxyhemoglobin could be obtained within a few days. They also state that crystallization is accelerated by the addition of 0.1 to 0.5 per cent of hydrochloric acid and by increasing the temperature to 40°. Crystals were prepared from the bloods of the dog, horse, cat, and guinea-pig. Guelfi (Rif . med., 1897, No. 10; Maly's Jahr. ii. d. Fort. d. Thierchemie, 1897, xxvn, 149) also reports success with fluoride of sodium. He obtained crystals from the bloods of the dog and guinea-pig by the addition of an equal volume of a 2 per cent solution of this salt and maintaining the mixture at a tem- perature of 40°. This method, he states, failed in the case of both arterial and venous human blood. The statement by Bohr (toe. cit.) of his belief that oxyhemoglobin is not a homogeneous substance, and that it consists of a mixture of oxy- hemoglobins which differ in elementary composition, molecular weight, and combining capacity with O, has been shown by Hiifner (Archiv f. Anat. u. Physiol., 1894, 130) to be untenable. Hiifner's researches proved that Bohr's methods for producing the several forms of oxyhemoglobin gave rise to mixtures of oxyhemoglobin with variable amounts of decom- position products. Hiifner made new studies of the photometric constants of oxyhemoglobin, reduced hemoglobin, and carbon-monoxide hemoglobin, and determined the absorption coefficients for O and CO. He concluded, from the constancy of the extinction coefficients, the O and CO capacities, and the percentage of iron, that in healthy fresh bullock's blood there is only one hemoglobin present, and that the blood-coloring matters of the higher animals have all, when freed from water, the same molecular weight and with it the same capacity for carbonic oxide and oxygen. Hiifner also noted that when horse's blood is crystallized in closed cylinders there appear in great abundance dark-red 6-sided plates, together with the well-known 126 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS prisms. He states that if one observes under the microscope a drop of the fluid in which the crystals are suspended, before it is covered with the cover- glass, it will be seen that these hexagonal plates quickly melt, and that where they dissolve bundles of fine, bright-red, prismatic crystals suddenly shoot out. The dark-red hexagonal plates are crystals of reduced hemo- globin, as Nencki showed several years ago, while the bright-red prisms are of oxyhemoglobin. He found that horse's blood is particularly inclined to give crystals of reduced hemoglobin, and that in preparing crystals of horse's hemoglobin by the regular method, without particular exclusion of air, both forms appear at the same time. Hemoglobin crystals from the bloods of the horse, ox, pig, and dog were prepared by Frey (Inaug. Dissert., Wurzburg, 1894; Jahr. ii. d. Fort. d. Thierchemie, 1895, xxv, 108) by means of the dialyzing method of Giirber. The corpuscles were separated from the defibrinated blood by centrifu- galization, mixed with 2 volumes of water, and placed in a dialyzer which was suspended in 30 to 70 per cent alcohol. Beautiful crystals were obtained after 3 to 24 hours. If a drop of blood be placed on a slide under a cover- glass, crystals form (primary crop) which dissolve as the blood becomes fully laked, when occasionally a second crop forms. By reduction the blood became yellowish, and after 3 or 4 hours it was violet-red and venous, and at the same time granules appear which finally separate towards the margin as distinct crystals. These crystals, Frey states, are of reduced hemoglobin and in addition to these are clusters of colorless crystals. Horse's blood crystallized most readily, and then that of the dog, and finally that of the pig with difficulty. Kobert (Das Wirbelthierblut in mikrokristallographischer Hinsicht, 1901, 25) used the Giirber method to prepare crystals from the bloods of the dog and cat. Arthus (Compt. rend. soc. biolog., 1895, XLVII, 686) employed a similar method to obtain crystals of the horse and dog. The blood was prevented from coagulating by the addition of oxalate, and after the corpuscles had been separated by decantation they were mixed with 2 volumes of water and placed in a Kiihne membrane dialyzer suspended in 90 per cent alcohol. Large masses of crystals were formed. Arthus in a later research (Zeit. f. Biolog., 1897, xxxiv, 444) modified his previous method: The corpuscles from oxalated blood were dissolved in 2 volumes of water and filtered, and the filtrate was placed in a parchment-paper tube which was suspended in 17 to 33 per cent alcohol. At room temperature oxyhemoglobin crystals 7 to 8 mm. long with sharp edges were formed. When stronger alcohol was used the crystals were impure owing to an amorphous precipitate. Studies of the crystallographic characters of crystals from blood of the silkworm were made by Panebianco (Zeit. f. Krystallographie, 1897, xxvin, 198) . These crystals were colorless and it is doubtful if they were hemoglobin. Crystals from horse, dog, and cat were prepared by Abderhalden (Zeit. f. physiol. Chemie, 1898, xxiv, 545). Success in the production of crystals, he states, depends upon using the least possible amount of water necessary to dissolve the blood corpuscles which have been freed as much as possible from serum. To horse's corpuscles he added 3 volumes of water; to dog's SINCE PREYER'S INVESTIGATIONS. 127 corpuscles, 2 volumes; and to cat's corpuscles, 1 volume. On the addition of alcohol in the cold, etc., according to the Hoppe-Seyler method, and twice recrystallization, he obtained, he states, absolutely pure crystals. His elementary analysis of cat oxyhemoglobin will be found on page 71. The very simple and satisfactory "Canada balsam" method of von Stein (loc. cit.) for preparing small quantities of crystals from readily crystallizable bloods was again used by Mm in a later research (Archiv f. path. Anat. u. Phys., 1900, CLXII, 477) in a determination of the effects of certain reagents on crystallization of guinea-pig's blood. The addition of sodium chloride up to 2 per cent aided crystallization, while quantities beyond this hindered; calcium chloride, sulphureted hydrogen, and nitrous oxide hindered crystallization. Von Stein also noted variations in crystalline form from the typical tetrahedra to forms ranging from truncated tetra- hedra to 6-sided plates. A painstaking study of the crystallography of the crystals of pigeon's blood was made by Schwantke (Zeit. f. physiolog. Chemie, 1900, xxix, 486). His results will be referred to fully in subsequent chapters. A new method of getting rid of the stromata, which whether in sus- pension or in solution hinder the crystallization of hemoglobin, was devised by Schuurmanns-Stekhoven (Zeit. f. physiolog. Chemie, 1901, xxxin, 296). The blood corpuscles are washed with 1 per cent salt solution by centri- fugalization, and then shaken violently for 2 hours with asbestos wool. The blood-coloring matter passes into solution, while the stromata for the most part cling to the asbestos and are removed by nitration. By this method the hemoglobin is not brought in contact with ether. The hemoglobin solution is placed in a parchment-paper dialyzer, which is suspended in 45 per cent alcohol, and put in an ice-chest. As soon as crystals begin to form on the wall of the dialyzer (after 24 to 48 hours) the contents of the dialyzer are placed in a cylindrical vessel and then set in an ice-chest until crystallization has been completed. The hemoglobin is not brought in contact with any more alcohol than is necessary for the crystallization. The crystal pulp is as far as possible freed by pressure from the mother-liquor, after which the crystals are dissolved in the smallest possible amount of water at 37°. This solution is again dis- solved and placed in the dialyzer in 45 per cent alcohol. Crystallization begins much more quickly than the first time. After crystallization has been completed the crystals are separated from the mother-liquor and dried, first on porous plates and then in a porcelain bowl over chloride of calcium at room temperature. In the monographs by Schulz (Krystallization von Eiweisstoffen und ihre Bedeutung fur die Eiweisschemie, Jena, 1901) and Robert (loc. cit.) much of the literature on the processes for preparing crystals of hemoglobin is referred to. The latter gives an account of blood crystals which he pre- pared from various species, and he attempts the support of the hypothesis of Hoppe-Seyler regarding the existence of the blood-coloring matter in the form of "arterin" and "phlebin." Stewart (American Journal of Physiology, 1903, vm, 102), in his studies of the actions of laking agents on the blood, found that intraglobular 128 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS crystallization of necturus blood is very readily obtained by the action of various hemolytic agents. A quick method for preparing crystals of oxyhemoglobin was reported by one of us (Reichert, American Journal of Physiology, 1903, ix, 97), who also made studies of the effects on crystallization by mixing the bloods of different species, etc. It was found that if to the blood of the dog there be added, before or after laking, from 1 to 5 per cent of ammonium oxalate, crystallization invariably begins immediately, and that any quantity of crystals can be obtained within a few hours at ordinary room temperature. If a drop of this blood be placed under the microscope, crystals will be seen to form at once near the margin of the drop, and to be deposited so rapidly that a solid mass is formed in a few minutes. The blood of the horse does not yield quite so readily to this treatment. If a drop of blood so prepared be examined under the microscope, it will be found that crystallization will not begin usually at room temperature until after from 15 to 20 minutes or more, and that it will proceed slowly. Better results can be obtained if the blood be oxalated and centrifugalized, or set aside for the corpuscles to subside. The supernatant liquid is then poured off, and the remaining corpuscles are laked with ether. Defibrinated blood of the rat, laked with water on a slide, and covered with a cover-glass after the margin of the drop has become dried, usually crystallizes very readily, as is well known. Quicker results can be obtained if the blood be oxalated before or after laking, and even more rapid crystal- lization occurs if the blood be laked with ether instead of water. Crystals form so rapidly in the oxalate-ether blood that a magma is formed in the test-tube within a few minutes. The oxalate-ether process applied to the blood of the guinea-pig gives most satisfactory results. Crystallization does not proceed quite so rapidly as in rat's blood, yet within a minute or two innumerable tetrahedra appear, and practically complete crj'stallization can be obtained within a couple of hours. The blood of the necturus crystallizes readily when so treated. The crystals resemble in form those of the triple phosphates. The rapidity with which crystallization begins and proceeds was found to be influenced decidedly both by the method of laking and the percentage of oxalate. Ethyl ether is a much better laking agent than water, and acetic ether is stronger than ethyl ether. The presence of any quantity of oxa- late up to saturation increases crystallizability, but he found from 1 to 5 per cent to be the best; the larger the quantity the more is crystallization hastened. When more than 5 per cent is used, the oxalate also tends to crystallize upon the slide. If the blood be prevented from drying, as in the test-tube, the oxalate remains in solution. Asphyxial blood yields crystals more readily than normal blood. If to the blood of one species, the blood, plasma, or serum of another species be added, the laking of the blood may be retarded, accelerated, or unaffected, according to the character of the mixture. The period required for laking may be prolonged 5 minutes or more. The crystallization of the oxyhemoglobin may be hindered or prevented in such mixtures. Thus, by varying the proportions of a mixture of the bloods of the dog and guinea- SINCE PREYER'S INVESTIGATIONS. 129 pig crystals from one or both may appear, but the process is invariably retarded, sometimes to a marked degree. If crystals of both kinds of oxy- hemoglobin are deposited, those of one usually begin forming some time before those of the other, and the crystallization of one may be seemingly complete before crystals of the other are seen. The interesting observation was also made that the typical forms of the crystals of certain kinds of oxyhemoglobin may be modified or com- pletely changed when the bloods of two species are mixed. Thus, if to the blood of the rat there be added a definite percentage of the blood of the guinea-pig, crystals of the rat's oxyhemoglobin may appear in unaltered form, but most, if not all, of those from the guinea-pig's blood are changed; in fact, if any perfect tetrahedra are found, they will have been formed at the very end of the crystallization. If the proportions of the mixture be properly modified, not a single crystal of what can be identified as rat's oxyhemoglobin will appear, and all the crystals will be modified tetrahedra, spindles, and transitional forms between these. The spindles resemble Char- cot's crystals in form, but not in color; they vary in size, some being very large, and some may have small spindles attached to them; they can be obtained having sharp edges if crystallization has not been too rapid. This complete change in the form of the crystals of oxyhemoglobin when the bloods of two species are mixed, and the spindle-shaped form of the crystals, are, he believes, unique facts in the crystallography of this most important substance. (See Halliburton, page 115.) Moser (Vierteljahresschr. f. gerichtl. Medizin, 1901, xxn, 44) asserts that differences in crystalline form afford a positive means of recognition of the origin of the blood, and that positive distinction can be made between human and animal blood. He examined blood stains of the fresh blood of man and 10 species of mammals and fish, and gives drawings of their appearance under the microscope. From the differences in shapes he infers differences in crystallization, which he reasons indicate differences in chem- ical constitution. No descriptions of the crystallographic or optical char- acters are given, and, as differences in the shapes of the crystals do not necessarily imply differences in crystal system, his conclusions are based upon insufficient data. Bonnel (These de Paris, 1903; Jahr. ii. d. Fort. d. Thierchemie, 1903, xxm, 182) showed, however, that the method of Moser is not worthy of recommendation because by this method he obtained from human blood crystals of different shapes. Friboes (Archiv f. ges. Physiol- ogic, 1903, xcvni, 434) also found that human blood treated in the manner described by Moser crystallizes in various forms. He notes that the bloods of certain animals show crystalline shapes which, with the exception of the bat and the goat, are distinguishable from human blood. He describes the different forms of the crystals he observed, but gives no definite crystallo- graphic data by which they may be recognized. (See Chapter VII.) In experiments with the blood of the horse, Uhlik (Archiv f. ges. Phys- iologie, 1904, civ, 64) found that as putrefaction progresses the usual rhombic crystals of oxyhemoglobin are replaced by hexagonal, holohedral crystals of reduced hemoglobin. Table 34 indicates the influences of the condition of the blood and temperature upon crystallization. 130 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS. TABLE 34. — Effects of the condition of the blood and temperature upon crystallization according to Uhlik. Condition of the blood. Temperature and crystal systems. 0° 5° 10° 15° 20" Fresh . . . . ... Rhombic, abundant Rhombic, scarce or none Hexagonal, scarce or none Hexagonal, abundant Do Rhombic.. . Rhombic, scarce Rhombic. Hexagonal and rhom- bic. Partly reduced; not any decomposi- tion Reduced; beginning to decompose. . Hexagonal, abundant Do Hexagonal and rhom- bic Hexagonal, abundant Hexagonal Uhlik also notes that Pregl found that a thrice-crystallized hemoglobin appeared as hexagonal crystals. Crystals of reduced hemoglobin have been prepared and described by a number of investigators, as stated in previous pages. The last hemoglobin to be obtained in crystalline form, excluding our own preparations, was prepared by Bardachzi (Zeit. f. physiolog. Chemie, 1906, XLIX, 465) from the blood of the sea-tortoise (Thalassochclys corti- cata). The blood was centrifugalized, the corpuscles mixed with water, and then set aside for several hours at 50°. The solution was then filtered, one-fifth volume of alcohol added to the filtrate, and the mixture placed in an ice-chest. Crystallization occurred quickly and abundantly in the form of plates. The crystals were soluble with difficulty in cold water. For the purpose of analysis the crystals were dissolved in water at 40°, and after cooling one-seventh volume of alcohol was added, and crystalliza- tion obtained as before. The crystals were then centrifugalized off and dried in vacuum. The mean values of the elementary analyses were C54-77He-99Ni7. 0780-38^60-41 The absence of phosphorus is striking, since previous observers failed to obtain hemoglobin free from phosphorus from bloods that contain nucleated erythrocytes. The optical investigation by means of the Hiifner spectro- photometer showed decided agreement with the blood-coloring matter of such other animals as have been closely investigated up to this time. The average quotient was e' : e = 1.561, while Hiifner found the quotient to be 1.578. The calculation of the extinction coefficients and quotients of hemo- globin and methemoglobin agreed, he states, with those of other oxyhemo- globins and methemoglobins, so (hat the coloring matter of the blood of the tortoise, Bardachzi holds, is identical with that of mammals. Abderhalden and Medigreceanu (Zeit. f. physiol. Chemie, 1909, LXIX, 165) report their preparation of crystals of goose hemoglobin free from phosphorus. CHAPTER VII. CRYSTALLOGRAPHY OF HEMOGLOBIN IN RELATION TO SPECIES, ACCORDING TO PREVIOUS INVESTIGATORS, WITH EXPLANATIONS OF VARIOUS CONTRADIC- TORY STATEMENTS, ETC. As early as 1852 Kunde (Zeit. f. rat. Medicin, 1852, N. F., n, 271) and Funke (ibid., 288) in coincident articles stated that the hemoglobin crystals of different species are different. Kunde prepared crystals from the bloods of a number of species, including the bat, dog, ox, horse, guinea- pig, squirrel, rat, mouse, rabbit, pigeon, and tortoise, and published some figures illustrating the shapes of the crystals. From these differences in the shape and from the differences in solubility he concluded that the blood crystals obtained from different species are not identical, but distinct and characteristic of the species. Funke was led to the same conclusion from the examination of the crystals from the blood of the horse, ox, pig, dog, cat, and several species of fish. While making no attempt to give an exact crystallographic description, Funke records a number of angles ob- served in two of the species examined. These contributions were almost immediately followed by an article by Teichmann (ibid., 1853, in, 375), who states that from the same blood, and even in the same preparation, crystals of various forms may be obtained, from which and for other reasons he concludes that the differences are not in relationship to species, but accidental and due to exterior conditions. Teichmann's statement seems to have arrested further interest in this subject until 10 years later, when it was taken up by Rollett (Sitzungsb. Math.-nat. Klasse d. k. k. Akad., Wien, 1862, XLVI, Abth. n, 85), and shortly after by Bojanowski (Zeit. f. wiss. Zoologie, 1863, xn, 312). Rollett pre- pared crystals from the bloods of man, the guinea-pig, dog, rabbit, squirrel, and cat, all of which preparations, with the exception of the last, he sub- mitted to von Lang, a crystallographer, for crystallographic investigation. Von Lang's examinations were made with the microscope, and in some cases the optical characters were examined and a few angles recorded. Von Lang determined the crystal system in each case, and from his data Rollett concluded that while the crystals from different species are different they may all be included in two crystal systems, the orthorhombic and the hexagonal. The descriptions of von Lang are very brief, and no attempt at giving all of the crystallographic constants is made, but these are the first definite determinations on record of the systems of crystallization of hemoglobin. Bojanowski reviewed the literature of hemoglobin crystals and pre- pared crystals from the blood of rabbit, mouse, dog, cat, hedgehog, river 131 132 CRYSTALLOGRAPHY OP HEMOGLOBIN IN RELATION bream, pike, horn-fish, herring, lark, raven, and pigeon, and of man. He records that hemoglobin of various animals crystallizes in various forms and systems, and that he always obtained rhombic plates from the blood of man and many species of lower animals, regular 6-sided plates from the blood of the mouse and squirrel, tetrahedra from the blood of the guinea-pig, and prismatic crystals from the blood of the rabbit. Crystals from various kinds of blood which appear to possess a similar form still showed unmistakable differences in the sizes of the angles. From his investigations he reached the conclusion that the bloods of individual species have something specific and characteristic about them, so that it is occasionally even possible to determine the species of animal from whose blood the crystals were derived. Where, as in the case of human blood, as described by Funke, there appear to be two or more kinds of crystals in the same blood, Bojanowski considers that one of them is the characteristic form and the others undeveloped crystals. Thus, in human blood what he describes as the "right-angled plate" is, he believes, the characteristic form, while the "prisms and rhombic plates" are regarded as undeveloped forms of the right-angled plate. The descriptions given are very brief and incomplete: thus, the crystals from the dog are described as "rod-like crystals forming closely woven nets," and from the cat as "very regular three-sided rods," etc. The description of the crystals of dog's blood would apply equally well to any species whose hemoglobin crystals are rather insoluble, if the hemoglobin crystallized in prisms, for such hemoglobins form felted masses of capillary or long pris- matic crystals. The prisms of reduced hemoglobin of the cat are not 3- sicled, but nearly rectangular in section. After a latent period in the study of the crystallography of hemoglobins for the 5 succeeding years the first contribution by Preyer appeared (Archiv f. ges. Physiologic, 1868, i, 395), which was shortly followed by his now classic and authoritative memoir (Die Blutkrystalle, Jena, 1871). When the former contribution was published blood crystals from 47 species of vertebrates had been recorded, and of these in only 10 cases had the crys- tal system been recorded. In his memoir these 47 species are enumerated and the data concerning them are given. Preyer evidently regarded the crystals obtained from different species as differing from one another, but he concluded with Rollett that they may all be included in the two crystal systems, the orthorhombic and the hexagonal. He states that "besides the crystal system there are other distinctions, as, for instance, the sphe- noidal crystal of the guinea-pig, the 4-sided prisms of the dog, the 4-sided prisms and rhombic plates of man. These peculiar morphological shapes are obtained only from each animal, even after repeated recrystallizations; a definite form is peculiar to each animal and can not be changed to another form. The same holds good with solutions of hemoglobin. Yet little im- portance is to be attached to statements on the crystallographic differences of the hemoglobin of different animals, because neither is the same method of crystallization always used, nor is the blood always capable of being compared, nor has the measure of the crystallizability of any optional sub- stance been found. ^It is the same with decomposability as with crystalliz- TO SPECIES, ACCORDING TO PREVIOUS INVESTIGATORS. 133 ability — both vary according to the species of animal; but the investigations undertaken in this direction suffer from so many and such large errors that they prove nothing beyond what has long been known, that is, the different species and individuals. " Preyer's statement that the form of the crystals can not be altered by repeated recrystallization, and that there is a constant and peculiar form in relation to each kind of animal, has been shown to be wrong by the records of Halliburton (page 115), Copemann (page 119), von Stein (page 127), Bonnel (page 129), Friboes (page 129), Moser (page 129), and Pregl (page 130). The work of Preyer was so painstaking and exhaustive that his con- clusions seem to have been accepted without question, and his dictum that all hemoglobins crystallize in the orthorhombic system with an exception which crystallizes in the hexagonal system seems to have absolutely dis- couraged investigation in the crystallography of hemoglobin, and such studies as have since been made have been chiefly with the view of dis- tinguishing human blood from that of domesticated animals, for medico-legal purposes. Of the papers treating of the crystallography of hemoglobin in rela- tion to species from this standpoint, those of Guelfi (Giornal di Med. Legale, 1898; Maly's Jahr. ii. d. Fort. d. Thierchemie, 1898, 145) and Moser (Viertel- jahr. ger. Med., 1901, xxn, 44) may here be noticed. Guelfi obtained " tetra- hedral crystals" from guinea-pig's blood and "prismatic crystals" from dog's blood, using both fresh and dried blood in each case. Comparing these with crystals obtained from partly dried human blood, which crystals he describes as "needle-shaped," he states that they can be distinguished from each other so that "it can be definitely stated that neither the tetra- hedra from the guinea-pig blood nor the prisms from the dog blood were from human blood." Moser describes crystals obtained from the blood of about a dozen species of vertebrates including mammals and fish. His article is illustrated with drawings made from the appearances of the crystals under the micro- scope, but these are not accompanied by any exact crystallographic descrip- tions. The differences in the shapes of the crystals led him to the conclusion that differences in the forms of the crystals afford a positive means of recog- nition of the origin of the blood, and that in this way positive distinction can be made between human blood and the blood of other animals. The descriptions of the crystals are very brief and relate to their general mor- phology; this is true also of the drawings. No correlation of the different shapes of crystals found in the same species is attempted, and what are evidently different views of the same crystal are shown as different forms. It is obvious that he is distinguishing the different crystals merely and hazardously by their morphology. Moser's article has been the subject of adverse criticism, as will be pointed out. Various observers have studied the shapes of the ciystals obtained from the bloods of different species, and in a few instances the crystal system has been determined by crystallographic study, and from these data they have arrived at the conclusion that the bloods of different species 134 CKYSTALLOGRAPHY OF HEMOGLOBIN IN RELATION may be distinguished by an examination of the hemoglobin crystals. On the other hand, this conclusion has been contradicted by many observers. Teichmann, for instance, as already stated, asserts that from the same blood, and even from the same preparation, he has obtained various crystal forms, and that still other forms may be produced by varying the method of prepa- ration, from which he naturally concludes that the form of the crystal is something entirely accidental and dependent upon exterior conditions and not an essential character of hemoglobin. Others have made the observa- tion that in the same blood several forms of crystals may be found. It has also been pointed out that crystals from the blood of a given species, as recorded by different investigators, are of different forms. Thus, Leh- mann described the crystals from the guinea-pig as isometric tetrahedra, he also describes them as isometric octahedra; Moleschott states that they are 6-sided plates. Von Lang writes that they are only seemingly isometric, and that, while the angle of the triangular face is so near 60° that they can not be distinguished from isometric tetrahedra, the optical characters make them orthorhombic. Donogany measured the three angles of the triangular face of these crystals and records them as 64°11', 60°50', and 55°45', which three angles it will be noted do not add up to 180°. Of course the explana- tion of the record of tetrahedra in the one instance, of octahedra in another, and of 6-sided plates in a third is veiy simple. All of these observers were examining crystals of the same substance, and all were, as von Lang and Donogdny state, orthorhombic sphenoidal: in the case of the simple "tetra- hedra" the right or left sphenoid only was observed; in the case of the "octahedra" the crystal was the combination of the right and left-handed sphenoids in approximate equilibrium; and in the last instance, of the 6- sided plates, the form seen was this combination observed normal to a sphenoid face upon which the crystal is flattened, causing the outline to be hexagonal. The outline of an octahedron looked at as it lies on one of its faces is hexagonal, but if it become flattened parallel to the face upon which it lies it appears at a casual glance to be a hexagonal plate. Many such cases as that of the guinea-pig crystals have been noted, where the blood of the same species by varying the treatment, or even according to different observers, furnished crystals of diverse form; and many observers have been led to the conclusion that was reached by Teich- mann, that the forms of hemoglobin crystals are variable in the same species, are perhaps even identical in different species, and that the differ- ences are not to be relied upon for distinguishing the source of the blood in any given case. When the article by Moser appeared it apparently revived interest in the subject of the differentiation of the crystals of different species, but his results were soon attacked by Bonnel (These de Paris, 1903; Maly's Jahr. ii. d. Fort. d. Thierchemie, 1903, xxni, 182) and by Friboes (Archiv f. ges. Physiologie, 1903, xcvin, 434). Bonnel argued that because human blood treated in the way described by Moser crystallizes in different shapes the method is of no value. He points out that the method is not to be recom- mended for the purpose of distinguishing human and animal blood (although TO SPECIES, ACCORDING TO PREVIOUS INVESTIGATORS. 135 differences between these are to be detected), because it is only applicable to fresh blood and can not be applied to blood stains, at least if they are more than two weeks old. Friboes also attacks Moser's conclusion that the crystals serve to distinguish between human and animal blood. He states that normal human blood treated in the way described by Moser crystallizes in various forms, and that the crystals from dried human blood are different from any of these. Human blood obtained from the splenic vein and the umbilical vessels is again different from these, so that a uniform crystal shape for human blood does not exist. Thus, from fresh human blood are obtained 4-sided doubly refracting prisms, also sharp-angled rods split into brush-like forms at the end, and very characteristic rectangular plates arranged in step-like aggregates. From the blood of a young child he obtained long rectangular plates which he regards as still different. From the splenic vein he found crystals showing composite aggregates of the step- like arrangement of the rectangular plates. From the blood of the umbilical vessels he prepared rosette aggregates of ray-like crystals, and in this same blood he also noticed sheaf-like bundles of crystals and also isolated irregular crystals. The blood of other animals showed still other forms, which, however, are usually distinguishable from the crystals obtained from normal human blood, with the exception of those from the blood of the bat and goat. The distinction from human blood depends, he states, upon having a sufficient supply of blood and in obtaining it before it becomes dry. The article by Friboes is illustrated by excellent photomicrographic reproductions of some of the blood crystals examined, but his descriptions of the crystals are very brief and in many cases incorrect. Thus, in the description of the crystals from the cat he enumerates three kinds of crys- tals and illustrates them by two photomicrographs. These three types are (1) long, 3-sided prismatic rods, single or in bundles; (2) 4-sided prisms, rhombic ; (3) fine needles. He points out in the photomicrographs what he designates the "3-sided rods," which are evidently only an edge view of what he reports as " 4-sided prisms. " The fine needles are simply the same crystals in capillary form. All of these belong to the long prismatic type of crystal of cat reduced hemoglobin, and he appears not to have observed the short prismatic type nor the parallel growth aggregates that are usual in the preparation from cat's blood. His "fine needles" are generally the first crystals to appear, and his other two types, which he regards as distinct (one trigonal, the other rhombic), are but two views of the same crystal. The foregoing is simply an example of how an expert microscopist who is not a crystallographer may be misled by different appearances that he is unable to reconcile. The objections recorded in opposition to the conclusion of Kunde and others that the blood crystals from different species are not identical and that they are characteristic of the species may be summarized briefly as fol- lows : The form of the crystal of any species may be entirely accidental and dependent upon exterior conditions, and hence can not be characteristic of the species. In the same species different forms of crystals may be seen 136 CRYSTALLOGRAPHY OF HEMOGLOBIN IN RELATION even in the same preparation, and by varying the method of preparation many forms of crystals may be obtained from a given species. Different forms of crystals have been obtained from the blood of different vessels of the same species or the same individual. Different observers have pro- duced quite different crystals from the blood of a given species, some of these closely resembling or seemingly identical with those obtained from the blood of other species. There can not, therefore, be any one form of crystal that is characteristic of a given species. Preyer himself, while recognizing that crystallographic differences exist between the hemoglobins of different species, states that little importance is to be attached to statements on the crystallographic dissimilarities of the hemoglobin of different species, because neither is the same method of crystallization used nor is the blood always capable of being compared. He might have added, that in very few cases have the crystallographic descriptions been at all adequate or even accurate, but this he probably failed to recognize. His statement that all hemoglobins crystallized in the orthorhombic s}rstem excepting that of the squirrel was doubtless taken by many as an argument in favor of the assump- tion of the identity of the blood crystals obtained from different species. We thus see that equally expert observers, working with the same data, have arrived at very diverse conclusions. Before attempting to reconcile these conflicting conclusions it will be of advantage to examine certain other observations that have been made on hemoglobin crystals. A number of the earlier investigators, including Lehmann, Teichmann, Weir Mitchell, and Bojanowski, and several of the later ones, such as Struve, and Stirling and Brito, have noted that the crystals obtained from the blood may be nearly or quite colorless, or may become so on standing; or, according to several of them, the deep-red crystals may be decolorized by washing them with alcohol, or with alcohol and water, or with other reagents. Thus, Bojanowski states that the blood crystals exposed to the air retain their form, but become paler and paler and finally completely colorless. The addition of sugar or gum produces the same result. Teich- mann had made similar observations on the loss of color of the deep-red crystals. Bojanowski's statement is a fairly accurate description of the paramorphous change of crystals of oxyhemoglobin to metoxyhemoglobin, many examples of which will be found in the records of this research. The color of the crystals of metoxyhemoglobin is very pale as compared with that of oxyhemoglobin, and when the crystals are thin they appear almost colorless. The very strong pleochroism of metoxyhemoglobin makes the crystals appear quite colorless in some positions. Weir Mitchell made similar observations on oxyhemoglobin crystals exposed to the air. The "colorless" crystals retain the form of the original oxyhemoglobin crystals, but after the change they are a different substance, and are in fact pseiido- morphs of the original oxyhemoglobin crystals, and if dissolved and recrys- tallized the form would probably be altered only slight!}^ not sufficiently to be noticed by casual observation. From the blood of the raven that had stood exposed to the air for 8 days, Bojanowski obtained " crystals which were partly bright yellow and TO SPECIES, ACCORDING TO PREVIOUS INVESTIGATORS. 137 partly colorless." This is a description of the method of producing crystals of metoxyhemoglobin, and the colors described are such as would be found in metoxyhemoglobin crystals that were rather insoluble, as these are described as being. He made a similar observation upon the crystals from the cat. Weir Mitchell describes the production of crystals of oxyhemoglobin from the blood of the sturgeon, and states that their color may be com- pletely removed by alcohol and water without injury to the form, and that these decolorized crystals may be dissolved in water and recrystallized in the original form. Struve (Ber. d. d. chem. Ges., 1881, xiv, 930) decolorized blood crystals by treating them with dilute alcohol, but without causing any change of form. In a later communication (Jour. f. prakt. Chem., N. F., 1884, xxix, 304) he gives a more detailed description of his observations: Fresh blood crystals placed in an excess of alcohol change their color to a darker tint, without change of form, and become insoluble in water and alcohol. This, he states, is due to a loss of water of crystallization and going over into an amorphous condition. These altered crystals by treatment with ammoniacal alcohol, by glacial acetic acid, or by concentrated sulphuric acid are decol- orized without change of form. Struve did not dissolve and recrystallize them. The color extracted he regards as a hematin derivative, which he names hematin acid. His conclusion is that hemoglobin crystals are a col- orless albuminous substance, mechanically mixed with a coloring matter. On reading the descriptions of Struve it seems evident that the treat- ment with alcohol changes the crystals of hemoglobin by hardening them, an effect of alcohol upon albuminous substances generally ; and if he started with oxyhemoglobin the darkened crystal treated with alcohol was already a different substance, a pseudomorph in fact. Such a pseudornorph might retain its form even though the substance of which it was composed should be the original material decomposed. In inorganic substances we find for instance crystals of pyrite, FeS2, changed by pseudomorphism into limonite, Fe403(OH)6 without the slightest change in outward form; fluorite, CaF2, in this way is changed to quartz, Si02. The colorless crystals obtained by treatment of the alcoholized crystals with the agents mentioned above are but skeletons of the original oxyhemoglobin crystals, and may have quite a different composition. As Struve states, they are amorphous and not really crystals at all. But Weir Mitchell's recrystallized colorless crystals are not of this kind, and are not to be explained in the light of our present knowledge. Colorless blood crystals are (with the exception of the recrystallized colorless forms described by Weir Mitchell) to be accounted for by a change of oxyhemoglobin to metoxyhemoglobin, by pleochroism, or by pseudo- morphism in case of chemically treated crystals. Besides colorless and slightly colored crystals, other variations from the typically colored oxyhemoglobin crystals have been observed. Thus, we find records of "bluish," "purple," and "pink" crystals that are evidently reduced hemoglobin; and "yellowish" and "brownish" crystals that may be methemoglobin. The failure to distinguish between methemoglobin and 138 CRYSTALLOGRAPHY OF HEMOGLOBIN IN RELATION metoxyhemoglobin has given rise to much confusion. It is clear that dif- ferent observers of blood crystals have examined crystals of oxyhemoglo- bin, reduced hemoglobin, metoxyhemoglobin, and methemoglobin in many instances without making any distinction between them. Since these sub- stances in a given blood may form quite different crystals, a source of the variations in the recorded crystals of a given species is obvious. As has been shown, equally expert observers working with bloods of the same species have arrived at very different conclusions as to the specifi- city or non-specificity of hemoglobin crystals in relation to species, some claiming that the crystals are occasionally specific, others that they are always specific, and others that they are not specific because the same blood may yield crystals of very different forms and that the differences are probably accidental. Crystals of various colors and varying forms have been obtained from the same blood. It has been held in favor of specificity that recrystallization, even when frequently repeated, does not effect any change in form; but this has been contradicted by observers who point to final evidence to the contrary. How are these diverse conclusions to be reconciled? In the first place, it is evident that the substance under investigation was not always the same: sometimes it was oxyhemoglobin, or reduced hemoglobin, or metoxyhemoglobin, or methemoglobin, etc. Any one of these substances may appear in several forms of crystallization in the same blood, often as many as three of them in the blood of a given species; and it is even probable that there are other forms of hemoglobins present which have not yet been isolated. But much more important even than these sources of variation in the crystals was the failure of the observer to in- terpret correctly his observations. The same crystal viewed in different aspects presents different appearances, and the same crystal combination may exist in different shapes due to the variation in crystal habit. The expert microscopist might learn to interpret the different aspects presented by a single crystal, but no one who is not a crystallographer would be likely to suspect that a long rod-like crystal and a thin tabular crystal might be the same combination of crystal forms. It was such failure to interpret the forms observed that has caused the confusion between the apparent octahedrons and the apparent 6-sided plates of the guinea-pig oxyhemo- globin crystals that have been mentioned. An octahedron lying on one of its faces and observed normal to this face has a hexagonal outline, and if it grows lying on this face it will develop into a 6-sided (or a 3-sided) plate, because it grows twice as fast parallel to the plane on which it lies as it does normal to that plane, since it can not grow at all on the bottom plane. A tabular crystal seen on edge looks like a rod or prism, and has been so described by many observers. Actual errors in observation are very common. For instance, Bojan- owski, owing to the nearly square prisms of the cat hemoglobin when seen on edge, looks upon them as being 3-sidod prisms; and Friboes falls into the same error, and even shows photomicrographs of the nearly square orthorhombic prisms of the same substance, and refers to them as "3-sided." TO SPECIES, ACCORDING TO PREVIOUS INVESTIGATORS. 139 Kunde and Lehmann observed "tetrahedra" in the "hemoglobin" of the black rat. These were doubtless the ^-oxyhemoglobin crystals, which are iso- metric, and appear as the three-sided plates that develop from the flattening of the octahedron. Such a crystal seen on edge would be described as a prism. When the different habits that the same crystal combination may assume are considered, the difficulty of interpreting the observations increases enormously. Thus, crystallization may begin with the formation of needle-like or capillary crystals, and these may later become short prisms. Friboes describes these two forms of the same crystal as two kinds of crys- tals in the case of cat hemoglobin; and, as has been stated, by looking at the same crystal in two aspects at 45° to each other, he sees two kinds of prisms, thus making three kinds of crystals of the same identical crystal combination. In certain species of the cats the hemoglobin occurs in all of these variations of the prismatic type of crystal and also in the tabular form, yet the crystal forms shown may be the same in prism and plate. Under less pressure the crystals form as prisms; under greater pressure they form as plates. Crystals from the blood of the black rat have been described as tetra- hedra, prisms, elongated plates, and hexagonal plates. The tetrahedra have already been referred to, and they are evidently, as stated, /3-oxyhemoglobin. The prisms, elongated plates, and hexagonal plates are all the same combi- nation of crystal forms, the prism and macrodome, and are our a-oxyhemo- globin. When symmetrically developed the crystal is the squarish prism terminated by the dome. Flattening of the crystal on two opposite prism faces produces the "elongated plates" of Hoppe-Seyler, and shortening of this flattened prism produces the apparently hexagonal plate. Careful focusing would show at once that this plate is not bounded by vertical sides and that the angles are not hexagonal angles. All of these forms we have observed in the crystals from the blood of the common rat. The crystals are frequently interfered with by the slide and cover pro- ducing false planes, so that a tabular crystal on edge, thus confined, becomes a "prism." Many examples of crystals with such false planes have been figured, even as late as the work of Moser (1901). When it comes to the determination of the crystal system, we find that most of the observers make no attempt at it. Preyer states that in his table (page 103) five of the six crystal systems are recorded, the triclinic being the only one not included. The isometric, he writes, may be ruled out because all hemoglobin crystals are doubly refracting and because isometric crystals can not be doubly refracting. Crystallographers now recognize that the tetartohedral class of the isometric system is doubly refracting, and, as will be shown later, we have found singly refracting isometric crys- tals of hemoglobins. The tetragonal system he eliminates because the statements of Hoppe-Seyler in regard to the tetragonal character of the guinea-pig crystals were disproved by von Lang. Similarly he excludes the monoclinic because he states that Funke, who claims to have observed monoclinic crystals in the case of the cat and man, "supports his state- ment by nothing. " This leaves only the orthorhombic and the hexagonal. 140 CRYSTALLOGRAPHY OF HEMOGLOBIN IN RELATION TO SPECIES. Preyer also states that of the 47 species examined and recorded the system of crystallization is known in 10 instances, in only one of which are the crystals accredited to the hexagonal system. In fact, von Lang seems to have been the only professional crystallographer who examined blood crystals up to the time of Preyer, and his descriptions, as has been stated, are very brief. Since von Lang found only two crystal systems, so Preyer concludes there can be but two crystal systems to which the hemoglobin crystals belong. Nevertheless the five ciystal systems mentioned by Preyer as having been recorded by various observers, of which he rejects three, are all represented by us in the hemoglobins included in this research. When we try to find how these investigators arrived at their conclusions as to the crystal system we are met by short, very incomplete descriptions, and we are led to the conclusion reached by Preyer in the case of Funke's monoclinic crystals, that "they support their statements by nothing." The work of von Lang was evidently accurate, although his crystallographic notes are brief; Donogany confirmed von Lang's findings in the case of guinea-pig's crystals, but, as we have already pointed out, he records three angles of a triangle which sum up to 180° 46'. The only contribution that has appeared giving the crystallographic constants and an accurate descrip- tion of hemoglobin crystals is that of Schwantka (Zeit. f. physiol. Chemie, 1900, xxix, 486) on the oxy hemoglobin of the pigeon, which will be found referred to at length under that species in a later chapter. The foregoing is in effect a brief statement of the status of the crystal- lography of hemoglobins at the inception of this research and up to the present time. CHAPTER VIII. METHODS FOR PREPARING, EXAMINING, AND MEASURING CRYSTALS OF THE HEMOGLOBINS EMPLOYED IN THIS RESEARCH. METHODS FOR PREPARING CRYSTALS OF HEMOGLOBIN. The necessarily limited quantities of blood that have been furnished us led, as a consequence, to the study of only such methods as are especially applicable to very small supplies, such for instance as 1 to 5 c.c. of fluid or clotted blood, although several of our processes may be used to advan- tage in the preparation of very large quantities if a method be selected that is suited to the species and to the condition of the blood. In only a few instances were we unsuccessful in obtaining crystals, and when we failed it was owing to an inadvertent selection of a wrong method or to attendant conditions over which we had no control. Our difficulty was not so much in the way of securing crystals as it was in the preparation of specimens that were adapted to the peculiar requirements of our investiga- tion. We found, as we gained experience with the bloods of different species, that, while the blood of each species must be treated as an individ- ual, we could nevertheless depend with some confidence upon the guidance of certain generalizations in the selection of the best method to be pursued. Thus, we found that usually the hemoglobins of Rodentia and Canidce crystallize with great readiness, those of Marsupialia very readily, those of Felidce readily, those of Ungulata not readily, those of Aves with difficulty, etc. ; but there were so many unexpected exceptions that we were often misled, and, as a consequence, obtained inferior results, as a number of our photomicrographic reproductions show. Even in the case of species closely related, as, for instance, certain of the rats, we found striking exceptions: The blood of the common albino or white rat (Mus norvegicus var. albus)* and that of Mus decumanus Pall. (Mus norvegicus Erxleben — brown rat) crystallize with such readiness that we found it desirable to use a restrainer to obtain crystals of desirable size for study; on the other hand, the bloods of Mus rattus (black rat) and Mus alexandrinus (alexandrine rat) crystallize much less readily, and hence should be treated in an entirely different way. We absolutely avoided the use of alcohol, because, notwithstanding the fact that it has proven one of the most widely used and most valuable agents in the preparation of hemoglobin crystals, it so deleteriously affects the hemoglobin molecule that even when present in dilute solution it lessens * Hatai (Biological Bulletin, Wistar Institute of Anatomy and Biology, Philadelphia, 1907, xn, 266) states, upon morphological grounds, that the albino rats of Chicago and Philadelphia are a variety of Mus norvegicus. 141 142 METHODS FOR PREPARING, EXAMINING, AND MEASURING solubility, alters the extinction coefficient, gradually decolorizes the crystals, and doubtless affects the water of crystallization. Alkalies and mineral acids have likewise been avoided, because of their pernicious influences. Hemoglobin, whether in crystalline form or in solution, especially when in concentrated solution, undergoes rapid alteration; we therefore made our studies as soon as possible after we obtained satisfactory crystals, usually within a few hours. In none of our examinations have we used recrystal- lized hemoglobin. Our specimens have been too small in quantity to permit of satisfactory recrystallization, and, moreover, the disadvantages of recrystallization, especially in so far as the methods of our investigation are concerned, quite outweigh the advantages. The injurious effects of recrystallization have been fully referred to in previous pages. At the inception of our research it seemed to us that the best results, on the whole, were to be obtained by the use of fluid blood, either defibri- nated or rendered incoagulable by oxalate, fluoride, or other anticoagulant, so that in the case of bloods which do not crystallize readily the corpuscles could be collected from the serum or plasma by centrifugalization, and thus eliminate certain substances in these fluids which retard crystallization and at the same time obtain a concentrated solution of hemoglobin. Since it seemed impracticable to obtain defibrinated blood, owing to the circum- stances under which our specimens were to be collected, and since one of us (Reichert, page 128) had already found that the presence of an anti- coagulant, such as neutral oxalate, was not only not injurious but actually beneficial, we made use of oxalate of ammonium in all of our preparations except in a very few instances, when for some special reason its absence was desirable or necessary. The addition of oxalate, in the proportion of 1 to 5 per cent of the dried powder, it was found, very much favors crystal- lization ; the larger the quantity up to the point of saturation the better the effect, saturation not being a disadvantage beyond the appearance of crystals of oxalate, which, however, are readily distinguishable from those of hemoglobin. In fact, in several instances these crystals appeared to be of advantage, because hemoglobin crystals formed on them, but not in other parts of the preparations. When we had defibrinated blood or clots to work with, oxalate was added at the proper time during our procedures of preparation. Since the presence of foreign bodies may, as is well known, not only augment or hinder crystallization, but also affect crystallization in other and even more important ways, we made appropriate tests to determine especially if the presence of the oxalate in any particular quantity affected either the type of the crystals or the optical properties of hemoglobin. The optical properties were not in any way appreciably affected. The habit of crystallization, as in the case of \ta and the crystal is positive. From the characters of these methemoglobin crystals, it is very likely that the sub- stance is really a mimetic twin and only pseudohexagonal. It is not very permanent, but decomposes and produces a granular brownish precipitate, leaving the monoclinic crystals (usually now changed to reduced hemoglobin) unaltered. M FIGS. 13, 14, 15. Alosa iapidiesima Oxyhemoglobin. 156 CRYSTALLOGRAPHY OF THE HEMOGLOBINS These methemoglobin crystals were found in blood of shad that had died in the air, and in freshly drawn blood that had been exposed to the air, and seem to be due to a separation of the metoxyhemoglobin into methemoglobin and oxyhemoglobin, which latter may be afterwards changed to reduced hemoglobin before the methemoglobin disappears. The formation of the pure methemoglobin, which crystallizes in these hex- agonal plates, is probably due to the further oxidation. CARP, Cyprinus carpio. Plates 5 and 6. Blood of the carp was obtained from live fish caught at Gloucester, New Jersey. It was oxalated, ether-laked, and slides prepared within a few hours after it was collected. The blood had a brownish color, and was probably the metoxyhemoglobin mixture. After standing in a test-tube for 24 hours it was practically all converted into reduced hemoglobin. Preparations of this were also made and examined. Both the metoxyhemoglobin and the reduced hemoglobin crystallized readily, but without any separation of pure methemoglobin. They are isomorphous, having apparently almost the same axial ratio, and perhaps the axial ratios are actually identical. (1) Metoxyhemoglobin of Cyprinus carpio. Orthorhombic: Axial ratio, a : b : c= 0.949 : 1 : 1.03. Forms observed: Prism (110), base (001), and, from twins on the macrodomes, also macrodomes (301), (201), (302). Angles: 110 A 110= 93° (87° normals). 110 A 001 =90°; from twins 302-302 = 68° 30'; 201 A 201=55° 15'; 301 A 301 =37° 30'. Habit generally tabular, nearly square plates, formed by flattening on the base (001) in combination with the prism (110) (text figures 16 and 17); also long prismatic, formed by development of the prism in the same combination (text figure 1 8) . The prismatic crystals are the first to ap- pear; these are gradually absorbed as the plates develop. Twins are common in the prismatic habit, apparently on the macrodomes noted above; they are not so common in the tabular habit, but apparently the twin on (302) oc- curs. Parallel growths are common in the tabular form; perhaps also homo- geneous regular growths occur. This parallel growth produces a piling up of the plates, and composite crystals and groups result. Pleochroism is marked ; the colors 1 are shades of brownish-red. Orienta- ls. 10. 17, 18. Cyprinu, carpio Metoxyhemoglobin. tiOD °f *** elasticity axes Was made Fio. 19. Cyprinta carpio Reduced Hemoglobin. Out US follows: fl=0, D=a, C=C. The interference figure was not observed. Extinction is symmetrical on the plates when examined on the base (001) and straight when examined on edge. (2) Reduced Hemoglobin of Cyprimis carpio. Orthorhombic : Axial ratio, a : b : c =0.949 : 1 : 1.098. Forms observed: Prism (110), base (001), macrodome (401). Angles: 110 A 110=93° (87° normals); 110 A 001 =90°; 401 A 501=27°. 17 b- OF PISCES, BATRACHIA, AND REPTILIA. 157 Habit at first prismatic, long lath-shaped crystals consisting of the prism (110) and the base (001) ; these later develop the acute macrodome (401), showing then the com- bination (110) and (401) with sometimes the base also (text figure 19). They gradually give place to the tabular form, analogous to the second form of the metoxyhemoglobin. The tabular form consists of the prism (110) and the base only, beginning as very thin plates, but gradually becoming thick tables or blocks, due to the elongation of the prism. The rods grow into sheaf-like tufts, but do not appear to twin on the macrodome as in the metoxyhemoglobin form. The tabular crystals also aggregate into groups by parallel growth on the base. Pleochroism is rather strong; a rose-pink, 6 rose-red, c deep red. Orientation of the elasticity axes is as in the metoxyhemoglobin, a = b, b=a, c = deeper red than e. e is the axis of greater elasticity, and hence the optical char- acter is negative. These small crystals of ^-oxyhemoglobin appeared in the blood received from Washington several days after the slides had been prepared, this being usual with bloods developing several forms of oxyhemoglobin. Fio. 30. Python molurut /5-Oxy- hemoglobm. TABLE 35. — Characters of crystals of reduced hemoglobin, oxyhemoglobin, etc., of the Pisces, Batrachia, and Reptilia examined. Name of species. Axial ratio. Angle/3. Prism angle. Extinction angle. Optical character. System. Substance. Pisces: 0.6008 : 1 : 4024 0 90 0 / 62 0 0° Negative Orthorhombic OHb. 90 0° Do. OHb. Alosa sapidissirna 1.804 : 1 : A 68 58 0 aAa = 6° Positive Monoclinic OHb. Do 1.786 : 1 : t 70 58 30 aAa = 10° Do. Do. MOHb. Do 1.786 : 1 : A 70 58 30 a Aa=14° Do. Do. Hb. Do 90 60 0 0° Do. Hexagonal MHb. 0 949 • 1 • 1 03 90 87 0 0° Orthorhombic MOHb. Do 0.949 : 1 : 1.098 90 87 0 0° Do. Hb. Batrachia: Necturus maculatus. . . . Reptilia: Python molunis 0.6494 : 1 : 1 0.900 : 1 : k 90 65 66 0 84 0 0° aAo=21° Negative Positive Do. Monoclinic OHb. a-OHb. Do 1 : 0.537 90 90 0 0° Negative Tetragonal ,3-OHb. CHAPTER X. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. The hemoglobin crystals of 10 species of birds were examined, 2 species belonging to the subclass Ratitce or flightless birds, and the remainder to the subclass Carinatce or flying birds. The Ratitce examined were the African ostrich, a representative of the Struthionidce ; and the cassowary, a member of the Casuariidce. The carinate birds represented 4 orders, and were distributed as follows : Anseres, 3 species, the goose, trumpeter swan, and whistling swan; Gallince, 3 species, chicken, Virginia quail, and guinea- fowl; Columbce, 1 species, the carrier pigeon; and Passcres, 1 species, the crow. It will be noticed that this is not a representative list of birds, but it includes examples from the two principal subclasses, the Ratitce and the Carinatce. Of the 23 orders of living birds ordinarily recognized but 5 are represented in this list. In the case of the Anseres the 3 species are closely related and 2 belong to the same genus, thus permitting of close compari- son. In the Gallince, too, are 3 species usually regarded as closely related. Two of these, the chicken and quail, will be seen to resemble each other closely, but the third, the guinea-fowl, is quite far removed from them as shown by its hemoglobin crystals. Indeed, the crystals of the guinea-fowl show closer resemblance to those of the African ostrich, one of the Ratitce, which in its zoological relations is generally regarded as far removed from the Gallince. The chicken and quail crystals, however, show some resem- blance to those of the Anseres, and even to the Columbce. The one passerine bird studied gave crystals that were quite different from any of the others examined. The table given at the end of the chapter shows some of the characters of the crystals of the oxy hemoglobin of the birds examined, and it will be noticed that, with two exceptions, they are either orthorhombic or tetrag- onal. In the detailed descriptions which follow it will be shown that the orthorhombic crystals have a tendency to become pseudo-tetragonal (or not distinguishable from tetragonal) by mimetic twinning ; so that it seems very likely that the two species recorded as tetragonal may in reality be only pseudo-tetragonal. AVES. AFRICAN OSTRICH, Struthio camelus. Plate 8. The sample of blood was received from the National Zoological Park at Washington in an exceedingly putrid condition, and clotted. The clot was rubbed in sand, with addition of ether, and the mixture centrifugalized. But a small quantity of the clear liquid was obtained, from which slides 11 161 162 CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. were made. After 48 hours at near a freezing temperature crystals appeared in one of the slides; they appeared to be reduced hemoglobin, but the deep-colored solution prevented spectroscopic determination. Later they improved somewhat and then appeared to be methemoglobin. They were very small — too small for obtaining an interference figure. They did not appear to dissolve readily when once formed, although they formed very slowly. Methemoglobin (?) of Struthio camelus. Orthorhombic : Axial ratio a : b : 6 =0.5658 : 1 : 6. Forms observed: Prism (110), base (001). Angles: 110 A 110=59° (normals); 110 A 001 =90°. Habit in small rhombic tables, very thin (text figures 31 and 32); occurring singly and not twinned, as is common in these rhombic plates. Pleochroism rather strong; a colorless, b yellowish-red, c deep red. Extinction was straight on all edge views and symmetrical on the base. Orientation of the elasticity axes is a =6, b=a, c=cl; plane of the optic axes is the macropinacoid. No interference figure could be obseved. CASSOWARY, Casuarius galeatus. Plate 8. The specimen was received from the National Zoological Park at Washington, District of Columbia. The blood was in the form of a small clot and was somewhat decomposed. Owing to the small quantity, it was only treated with ether to lake it, but it was at once converted into a jelly. After being ex- posed to temperatures ranging above and below the freezing-point for 24 hours, some fluid was ob- tained and 5 slides were prepared. The crystals formed gradually, but >J 3-1 were very poor. They did not appear to be very soluble. The spectro- scope showed a mixture of oxyhemoglobin and methemoglobin in the serum, and the crystals were rather brownish-red, but probably were oxyhemoglobin. The serum was highly colored, and photographs were obtained with some difficulty. The crystals formed mainly along the edge of the cover-glass. Oxyhemoglobin of Casuarius galeatus. Monoclinic: Axial ratio not determined, /? = 64° 30'. Forms observed: Clinopinacoid (010), orthopinacoid (100), and clinodome (Oil). Angles: 010 A 100=90°; 100 A edge of Oil =a A ci=/? = 640 30'. The angle of the dome was not satisfactorily measured. a/, Q! "~ 0_ FIGS. 31, 32. Struthw camelus Methemoglobiii. FIGS. 33, 34. Casuarius galeatus Oxyhemoglobin. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. 163 Habit prismatic by development of the two pinacoids (100) and (010) in the zone of c; the termination is oblique and is produced by the clinodome (Oil), text figures 33 and 34, usually only one end of the crystal being seen. Pleochroism rather strong; a pale yellowish, b deep red, c deep red. Extinction on (100) is straight; on (010) it is oblique with the angle c A w and the optical character is positive. When examined on edge, the crystals polarized as a whole and did not indicate any appearance of twinning, but from the fact that similar tetragonal characters are produced by what appears to be homogeneous regular growth in the case of the whistling- swan blood — Olor columbianus — which is distinctly orthorhombic, it is quite possible that these blood crystals are only pseudo-tetragonal. TRUMPETER SWAN, Olor buccinator. Plates 9 and 10. This specimen of blood was received from the Philadelphia Zoological Gardens. The blood was oxalated, ether-laked, and centrifugalized, and preparations made as usual. The slides were kept at a temperature near the freezing-point, but no crystals developed until after 24 hours. Even then only scattered crystals appeared, excepting in two slides. The crystals 164 CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. were very soluble and had to be kept at about 0° C. during the examination and photographing. Several other trials were subsequently made with the same blood, but they failed to yield crystals. The crystals obtained were found to be oxyhemoglobin by spectroscopic examination. Oxyhemoglobin of Olor buccinator. Tetragonal or pseudo-tetragonal: No axial ratio determined, as no pyramidal planes developed. Forms observed: Prism (110) and base (001). Angles: 110 A 110=90°; 110 A 001=90°. Habit thin to thick tabular, by development of (001) and (110) (text figure 36); in single crystals and in groups, often arborescent by growing together on the base or on a pyramid; also in clusters, but without definite appearance of twinning. Some of the large crystals were evidently composite, but did not show any appearance of twinning when examined on edge in polarized light. Pleochroism faint, apparently abnormal; the absorption for the direction of greater elasticity appears to be slightly greater than for the direction of less elasticity. Colors are deep oxyhemoglobin red; somewhat paler for u>. Uniaxial, singly refracting on the base in parallel polarized light, and showing a faint dusky cross in convergent light. Seen on edge the double refraction is very weak, but is observable with the aid of the quartz wedge, etc. ; when it is seen that u> is the direction of less elasticity and hence a> > s and the optical character is negative. The fact that the double refraction is so weak would favor the suspicion that the crystals are composite and really only pseudo-tetragonal, as is the case with some crystals in the next species, Olor columbianus. FIG. 36. Olor buccinator Oxyheinoglobin. FIGS. 37, 38, 39. Olor columbianus Oxyhemoglobin. WHISTLING SWAN, Olor columbianus. Plate 10. The specimen was received from the Zoological Gardens at Washington and was clotted and in a very putrid condition. The clot was ground up with sand, etherized, and the liquid obtained oxidized by exposing it to the action of pure oxygen. It was then centrifugalized, and slides prepared as usual, the drops being allowed to become very concentrated before cover- ing. In only two slides out of some two dozen prepared did crystals appear. The crystals were rather dark, but were oxyhemoglobin. Oxyhemoglobin of Olor columbianus. Orthorhombic: Axial ratio a : b : 6 =0.9057 :!: b > a. The colors are shades of the oxyhemoglobin red, a being yellowish-red. The orientation of the elasticity axes is a=a, b=c, c=6; the optic axes being in the plane of the basal pinacoid. No interference figure appears therefore on the flat view (001) ; but on edge, when looking nearly along a, one brush of the figure is seen. In the composite crystals this also is visible, the arrangement of their elasticities being as shown in text figure 39. From these figures it is seen that the axis b keeps its position in the regular growth, and the axes a and c alternate in the successive layers. In case of very thin layers in the composite crystals, so that the layers become too thin to show by the microscopic examination, this averaging of a and c would greatly reduce the amount of the double refraction, so that it might become almost zero, which is the condition in the species of swan examined, and leads to the suspicion that in the blood of Olor buc- cinator the crystals are only pseudo-tetragonal. From the position of the brush of the interference figure it is evident that the acute bisectrix Bxa=n, and the optical character is negative. The mimetic crystals produced by the homogeneous regular growth are singly refracting when examined on (001) and are not strongly doubly refracting when examined on edge view, especially when the individual layers are thin and not of the same thick- ness throughout; they are hence in some cases truly pseudo-tetragonal. The averaging of the angles of 88° and 92° to 90° makes them strictly tetragonal in form. CHICKEN, Gallus domestica. Plate 11. Blood was obtained from the living chicken, oxalated and centrifu- galized, and only the corpuscles used. The corpus- cles were treated by the usual method and crystal- lized at a temperature near the freezing-point. All examinations had to be made at the same low tem- perature, the room being kept at about the freezing- point or below. Very few slides showed crystals, and they were usually isolated or in small groups. Blood from two different birds was examined at different times, but the habit was about the same in both cases. The crystals were oxyhemoglobin. Oxyhemoglobin of Gallus domestica. Orthorhombic : Axial ratio a : b : c =0.949 : 1 : c. Forms observed: Prism (110), base (001). Angles: 110 A 110=87° (normals); 110 A 001 =90°. Flos' 40' 4J 166 CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. Habit tabular, the square tables (text figures 40 and 41) aggregated into groups by piling up of the plates or perhaps by twinning on an axis normal to the edge (110-001); also by what appears to be twinning on a dome; the crystals usually occur in isolated clusters. Sometimes skeleton crystals are seen that look tetragonal, but are orthorhombic according to their optical characters. Pleochroism is not very marked, hardly noticeable on the flat view, but stronger on the edge view, especially when looking along 6. Orientation of the elasticity axes, a =6, b=a, (=6. The plane of the optic axes is the macropinacoid; Bxa=a, hence the crystal is optically negative. Absorption c > b > a. On looking along a in conver- gent light the interference figure is seen with the brushes rather widely separated. QUAIL, Colinus virginianus. Plate 12. The blood was obtained from the living bird, and prepared in the usual manner. Corpuscles were used for extraction of the oxyhemoglobin which was tested by the spectroscope. The crystals form sparingly and melt readily at a little above 0° C. Oxyhemoglobin of Colinus virginianus. Orthorhombic: a : b : 6 =0.9657 : 1 : t. Angles: 110 A 110=88° (normals); 110 A 001=90°. Forms observed: Prism (110), base (001). Habit thin to thick square tabular; the tables consisting of the above combination and varying in thickness from one-fourth to one-half of the width of the plate (text figures 42 and 43) ; the cry- stals grew singly and in groups, but did not grow in the radiating form of the chicken oxyhemoglobin. Perhaps they twin on the axis normal to the prism-base edge, as the plates pile up on the base and overlap somewhat irregularly. Examined on (001), the crystals show no perceptible pleochroism; on edge the pleochroism is weak, but notice- able. The angle of the prism is so near 90° and the plates so irregular, due to overlapping, etc., that it is difficult to determine the exact orientation of the optic axes; the extinction is straight on the edge view and symmetrical on the (001) view. One of the diagonals is readily made out by the quartz-wedge to be an axis of greater elastic- ity than the other, but on edge views it is seen that c= a. Pleochroism: c and 6 43 Flos. 42, 43. Colinus virginianut Oxy- hemoglobin. character is hence negative. deep red, a paler red. GUINEA-FOWL, Numida melcagris. Plate 12. The blood was obtained by bleeding the living bird and was oxalated and prepared in the usual manner. Crystals formed readily and did not appear to be very soluble, as they remained in perfect condition at room temperature. The oxyhemoglobin crystallizes readily at ordinary temper- ature in well-formed crystals, in contrast to the crystals obtained from the bloods of most of the birds examined. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. 167 Oxyhemoglobin of Numida meleagris. Orthorhombic : Axial ratio a : b : 6 =0.554 : 1 : 6. Forms observed: Prism (110), base (001); and, in twins, unit pyramid (111). Angles: 110 A lTO=58° (normals); 110 A 001 =90°. The angle of the prism sometimes appears to approximate 60°, but ran down as low as 57°, and 58° seems to be the best measurement. 44 "Tb Fios. 44, 45, 46. NumiJa meleaffrii Oxyhemoglobin. Habit thin to thick tabular; the rhombic plates varying in thickness from one- tenth to one-fourth of the long diagonal of the prism; the crystals consist simply of the short prism (110) and the base (001) (text figures 44 and 45). Twinning on the normal to the prism-base edge as twin axis (horse-type) common (text figure 46) ; also twins on the pyramid and perhaps a macrodome were observed. The crystals usually occur singly or in twins, not in more complicated groupings. Pleochroism strong; a very pale yellow-red, b moderately deep scarlet-red, c very deep red. Extinction on the base is symmetrical and on edge is straight in all positions. The plane of the optic axes is the macropinacoid; the orientation of the elasticity axes is a=6, b=a, c=(S. On the base, in convergent light, the biaxial interference figure is seen with the axes rather widely separated; hence Bxa = t and the optical character is positive. CARRIER PIGEON, Columba livia var. Plate 13. The bird was killed and bled and the blood allowed to form a clot. This clot was then ground in sand, with excess of ether, and the mixture centrifugalized ; afterwards 2 per cent of ammonium oxalate was added and the preparations made as usual. Crystals began to form in about 2 hours. These crystals were Oxyhemoglobin, and formed only in the cold. When taken into a warm room they melted rapidly. Another preparation with and without oxalate was tried, but crystals did not form except when the oxalate was present. In the slides from the first preparation containing the a-oxyhemoglobin crystals (crystal a) there was formed after some days a second crop of crystals (crystal 6) of metoxyhemoglobin and the a-crystals changed to methemoglobin by paramorphism. The solution meanwhile had changed to reduced hemoglobin as shown by the spectroscope, and finally needle- like crystals of reduced hemoglobin (crystal c) appeared in the slides. By the time that the reduced-hemoglobin crystals had formed, the 6-crystals were converted by paramorphous change into pure methemoglobin. The Oxyhemoglobin and the metoxyhemoglobin also were both converted into pure methemoglobin in the presence of an increasing amount of reduced hemoglobin. 168 CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. (a) a-Oxyhemoglobin of Columba livia var. Orthorhombic: Axial ratio a : b : t= 0.9856 : I : t; and by twinning this becomes pseudo-tetragonal. Forms observed: Prism (110) and base (001). Angles: 110 A 110 = 90° 50'; 110 A 001=90°. The angle of the prism of 90° 50' (89° 10' normals) becomes by twinning exactly 90°. Ib FIGS. 47, 48, 49, 50. Columba livia o-Oxyhemoglobin. Habit tabular, in square plates (text figures 47 and 48) , which pile on one another by twinning; also growing into somewhat arborescent groups by parallel growth in the direction of a crystal axis. Twins on an axis normal to the prism-base edge, with the base as the composition face (text figure 49) ; this being repeated produces, by mimetic twinning, a composite crystal that is practically tetragonal, being isotropic on the flat and nearly so on the edge view (text figure 50). Pleochroism is rather marked; a pale yellowish-red, b deeper yellowish-red, c deep red. In the twinned crystals, that show little double refraction on the edge view, the absorption c > b or a is still noticeable. Extinction is straight on all edge views and symmetrical on the base. The orientation of the elasticity axes is a =b, b=a, (=6. The axis of least elasticity c appears to be the acute bisectrix; Bxa=t, hence the optical character is positive. These crystals were gradually converted by paramorphous change into pure methe- moglobin, giving the absorption band 630 pp to 605 pp and extending to 680 pp in the red; this change to pure methemoglobin appeared first in these crystals, although pure methemoglobin was later seen in the form of 6-crystals also. (b) Metoxy hemoglobin of Columba livia. Orthorhombic: Axial ratio a : 6 : 6 =0.4615 : I : 6. Forms observed: Prism (110), base (001). Angles: 110 A 1TO=49° 33'; 110 A 001=90°. Habit tabular, in rather acute rhomboidal plates (text figures 51 and 52), usually occurring singly, elongating on the macrodiagonal by parallel growth or sometimes on the brachydiagonal; but not twinning in the way commonly seen in these tabular crystals, on an axis normal to the prism-base edge. After the crystals of this metoxyhemoglobin 6-type had passed into the pure methemoglobin, they formed a sort of regular growth with the tufts of needles of the reduced hemoglobin, the needles being arranged in tufts growing nearly normal to the prism-base edge of the crystals (see plate 13, fig. 77). These crystals showed a laminated structure parallel to the plane of symmetry, perhaps indicating a cleavage in that direction (see plate 13, fig. 76). The color of these crystals is reddish-brown, rather dark, and the spectrum showed absorption bands at 640 pp to 615 pp, rather faint (the methemoglobin red band); and stronger bands at 580 pp to 565 pp and 550 pp to 530 pp, the oxyhemoglobin bands. When they finally became converted to methemoglobin, the oxyhemoglobin absorption bands disappeared entirely. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. 169 Pleochroism is very strong; a colorless or nearly so, b deep brownish-red, c very deep brownish-purple. The absorption for c and b is very strong. Double refraction strong; the extinction is straight on edge views and symmetrical on the base. Orienta- tion of the elasticity axes is a = 6, 6 = a, c=6. On the base, traces of an interference figure are seen, but the brushes pass out of the field; looking along a the complete figure is seen, showing that the acute bisectrix Bxa = a, and the optical char- acter is negative. This was confirmed by observations with the quartz wedge upon the interference figure. 52 Flos. 61. 62. Colamba lima Metoxyhemoglobin. Flo. 53. Columba livia 3-Oxyhemoglobin. 5.1 Reduced Hemoglobin of Columba livia. Orthorhombic (?). In fine needle-like crystals, grow- ing in tufts, often on the 6-type of crys- tals in a sort of regular growth, also not connected with other types of crystals. The double refraction is rather strong; the extinction is straight. The length of the needles appears to be the direction of greatest elasticity ; the pleochroic color of a is rose-pink. The directions of less elasticity normal to this show deep purplish-red colors. The crystals were not well enough formed to make out much as to their characters. Schwantka (Zeit. fur physiolog. Chem., 1900, xxx, 486) examined crystals of oxyhemoglobin of pigeon's blood that were prepared by A. Kossel, which were sufficiently large to be measured on the reflecting goniometer. The examination was conducted at a room temperature of between 5° and 10° C. Schwantka's description furnishes the following data : ^-Oxy hemoglobin of Columba livia. Tetragonal sphenoidal: Axial ratio a : 6 = 1 : 1.175. Forms observed: Unit prism (110), unit sphenoid (111). Angles: Prism angle 110 A 110=90°; prism to sphenoid 110 A 111=31°; sphe- noid faces over pole 111 A III = 118° 6'; sphenoid faces 111 A lTT = 106° 39' (calculated 108° 18'). The crystals consisted of the unit prism and the sphenoid, with somewhat prismatic development, elongated on the vertical axis and sometimes flattened on two opposite prism faces, making the crystal somewhat tabular (text figure 53) . In many crystals a face produced by contact with the vessel in which the crystals were grown was seen; this was vicinal to the prism face (110), but it did not give good reflections. The develop- ment of this accidental face caused a distorted appearance. What seems to be parallel growth was also observed. The crystals were determined to be uniaxial and showed only a weak pleochroism, changing from a brighter to a duller red. They extinguished parallel to the vertical axis. The optical character is not recorded. CROW, Corvus americanus. Plates 14 and 15. The fresh blood was obtained from the living bird and was allowed to clot. This clot was ground up with sand and with a large excess of ether and centrifugalized ; afterwards the clear fluid was oxalated. The prep- arations were made as usual and the crystals began to form soon after the slides were covered. The slides stood for several days before they were 170 CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. photographed, and reduced hemoglobin developed along with the oxy- hemoglobin. Other preparations were made from the clear fluid, prepared as above; and were recorded while containing only oxyhemoglobin. Upon long standing, the crystals in the slides were converted into metoxyhemo- globin. These crystals were all very soluble, but the oxyhemoglobin much more so than the others, and when removed from the cold into a warm room the crystals of oxyhemoglobin dissolved rapidly. Owing to this ready solu- bility the examinations had to be made in a room kept a temperature near the freezing-point. Measurements were made of the three forms observed, and the substantial identity in form of the oxyhemoglobin, reduced hemo- globin, and metoxyhemoglobin was made out. Oxyhemoglobin of Corvus americanus. Monoclinic: Axial ratio a : b : t = \ : b : 1.044; /? = 50°. Forms observed: Orthopinacoid (100), clinopinacoid (010), base (001),orthodome (T01), and a prism (mm 0). Angles: 100 A 001=^ = 50° (130°); 100 A 010 = 90°; 001 A 010 = 90°; TlO A 001 = 63°. The angle of the flat prism (clinoprism) was not observed. a V> Flos. 54, 55, 56. Corrui americanua Oxyhemoglobin. Flo. 57. Corvus americama Reduced Hemoglobin. Habit thick or thin tabular by development on the plane of symmetry (010), the usual crystal showing only the three pinacoids, but elongated along the vertical axis (text figure 54) and some crystals showing a clinoprism in this zone. In a few crystals the full combination was observed, of three pinacoids, prism, and positive hemiorthodome (text figure 55), and some showed this orthodome without the prism. The direction of the vertical axis is shown by inclusions of the mother-liquor and by cracks parallel to the vertical axis. Perhaps a prismatic cleavage is indicated. "Twins" form with a twinning axis about the normal to the orthodome and with the orthopinacoid of one member in contact with the base of the other member (text figure 56). Pleochroism strong; a pale yellow, b and c deep red. In thick crystals the color of a rises to pale yellowish-red. All show the oxyhemoglobin spectrum. Extinction on the edge view, zone of (100) and (001) is straight; on the plane of symmetry (010) the extinction is oblique, nearly bisecting the acute angle of the plate, a A 6=22°, the extinc- tion angle, measured from the edge 100-010. Orientation of the elasticity axes is a A t = 22° in the acute angle, BA a =62° in the obtuse angle, c=6; the plane of the optic axes is normal to the plane of symmetry. On the clinopinacoid the two brushes of the inter- ference figure are seen, but pass out of the field; the acute bisectrix, therefore, probably lies in the plane of symmetry and Bxn =a, or the crystal is optically negative. It will be noticed that the symmetry is nearly orthorhombic, but the parting or cleavage along the axis t and the habit of the crystal, especially when the prism is developed, all indicate monoclinic symmetry. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF AVES. 171 Reduced Hemoglobin of Corvus americanus. Monoclinic: Ratio of a : 6 =1 : 1.044; 0=50° 30'. Forms observed: Orthopinacoid (100), clinopinacoid (010), base (001), positive hemiorthodome (101). No prism was definitely observed. Angles: 100 A 001=0 = 50° 30' (129° 30'); 100 A 010 = 90°; 001 A 010=90°; 100 A 001=63°. Habit more distinctly orthorhombic than the oxyhemoglobin crystals; tabular by development on (010) and nearly equidimensional in many cases, not elongated verti- cally, but in nearly regular rhombic plates (text figure 57). It forms regular growths with the oxyhemoglobin (see plate 15, fig. 88). Color the usual reduced hemoglobin purplish-red. The optical characters were not determined for this form of crystal and perhaps it may be orthorhombic. The cleavage in the direction of the vertical axis is not notice- ably stronger than parallel to a, hence it might be a prismatic cleavage in the orthorhombic system. TABLE 36. — Some of the characters of the crystals of the oxyhemoglobins of the Aves. Name of species. Axial ratio a:b:t. Angle 0. Prism angle. Extinction angle. Optical character. System. Ratitae: 0 5658 • 1 • A 0 90 0 / 59 0 o 0 Orthorhombic. Negative Monoclinic. Carinatee : Anseres : Anser anser 90 90 0 0 Positive Tetragonal. Olor buccinator 90 90 0 0 Negative Do. Olor columbianus . . 0.9657 : 1 : 4 90 88 0 0 Do. Orthorhombic Gallinse: Gallus domestica . . 0 949 : 1 : k 90 87 0 0 Do. and pseudo- tetragonal. Orthorhombic. Colinus virginianus 0 9657 : 1 : t 90 88 0 0 Do. Do. 0.554 : 1 : t 90 58 0 0 Positive Do. Columbse: Columba livia, var 0 9856 : 1 : t 90 89 10 0 Do. Orthorhombic Passeres : 1 • 1 • 1 044 50 aAi = 22° Negative and pseudo- tetragonal. Monoclinic. CHAPTER XI. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE MARSUPIALIA, EDENTATA, AND SIRENIA. Nine specimens of the Marsupialia were examined, including repre- sentatives of the following families: Didelphyidce: 1 species, the common opossum, Didelphis virginiana. Dasyuridce: 4 species, the Tasmanian devil, Sarcophilus ursinus; the spotted dasyure, Dasyurus maculatus; the Australian cat, Dasyurus viverrinus; and the Tasmanian wolf, Thylacynus cynocephalus. Phalangeridce : 1 species, the vulpine phalanger, Trichosurus vul- pecula. Macropodidce : 3 species, the rat-kangaroo, Mpyprymnus rufescens; the kangaroo, Macropus giganteusC?) ; and the rock-kangaroo, Petrogale sp. The normal crystals of every species of marsupial examined are mono- clinic, and only in 2 of the 9 species examined were crystals of other systems observed. One of these was a /3-form of oxyhemoglobin found in the opos- sum and the other a /3-form of oxyhemoglobin which developed in the blood of the Tasmanian wolf. Comparing the crystals of the Pohjprolo- dontia, to which the opossum, dasyurus, Tasmanian devil, and the Tas- manian wolf belong, a close resemblance can be traced in the form of all the species except the Tasmanian wolf, and in this species the development of the crystals was such that direct crystallographic comparison with the other species .was not possible, as no axial ratio could be determined. In the species in which this constant was determined it was found to vary for the axis a from 1.7856 in the opossum and Australian cat to 1.8047 in the Tasmanian devil for oxyhemoglobin, a variation that was considerably less than the difference between the a-oxyhemoglobin and the reduced hemoglobin in the opossum (1.7856 and 1.963). The optical character of all of these species is the same, with the exception of the Tasmanian wolf. The crystals of the Diprotodontia (including the phalangers and the kangaroos) showed the kangaroos (Macropodidce) forming one group, and the phalanger apparently closer related to the Didelphyidce and the Dasyu- ridce in ratio, while the phalanger form recalls the Tasmanian wolf crystals of which no ratio was obtained. The kangaroos examined belonged to the genera Macropus, JEpyprymnus, and Petrogale, and they naturally showed some variation, but formed a fairly close group. In this chapter there is also included the description of one species of the Edentata and one of the Sirenia which naturally do not show any very close resemblance to the Marsupialia nor to each other. 173 174 CRYSTALLOGRAPHY OF THE HEMOGLOBINS MARSUPIALIA. OPOSSUM, Didelphis virginiana. Plates 15-17. The first specimens were received from the Philadelphia Zoological Gardens, and the blood was putrid. The blood was oxalated and ether- laked, and yielded crystals very readily, the crystals forming soon after the slides were covered. These crystals were reduced hemoglobin. Later, living animals were procured, from which fresh blood was obtained. The animal was bled into oxalate in each case, and preparations were made of the whole blood, of the corpuscles alone, and of the corpuscles with various diluents. From these, oxyhemoglobin was obtained; but in several cases reduced-hemoglobin crystals appeared in the slides soon after the oxyhemo- globin began to form, and these developed along with the crystals of the first-formed oxyhemoglobin called cc-oxyhemoglobin. Later the oxyhemo- globin, and eventually even the reduced hemoglobin crystals, dissolve, and a second form of oxyhemoglobin called /3-oxyhemoglobin begins to appear, but all three, a-oxyhemoglobin, reduced hemoglobin, and /3-oxyhemoglobin, may be seen side by side in the same slide (see plates 16 and 17). These three forms of crystals are produced independently of each other, and there is no paramorphous change of the one into the other, as may readily be seen from the difference in angles of the crystals, and also by the difference in crys- tallization between the /^-oxyhemoglobin and the other two forms. These three kinds of crystals were obtained in several different preparations of the fresh blood. Carbon-monoxide hemoglobin was also made from the opos- sum blood, and it was found to crystallize in two forms analogous to those of the oxyhemoglobin a and /?. The old blood was regenerated, after it had stood for some weeks and become putrid, by shaking with oxygen, and also by addition of a diluted solution of commercial hydrogen peroxide. The treatment with oxygen was much more satisfactory, because if the hydrogen peroxide is used in too concentrated a form the result is methemoglobin ; and if it is very much diluted, the solution becomes too dilute to crystallize well. But the corpuscles, after laking, make such a thick preparation that it was usually found better to dilute somewhat with normal salt solution. The procedure of regenerating was usually carried out as follows: The brownish stale blood was oxidized by shaking in a flask with oxygen gas until the color changed to bright oxyhemoglobin red; if corpuscles alone were used, to two parts of the corpuscles laked with ether was added one part of normal salt solution and one-fourth part of ether, and the whole briskly shaken, and then centrifugalized in a small hand machine for about 2 minutes. The solution was perfectly clear after this treatment, and a thick amorphous mass rose to the top of the liquid, which carried all of the precipitate and granular matter, leaving the solution as clear as if centrifugalized for 2 hours in the ordinary process of treat- ment. The crystals of oxyhemoglobin thus obtained were very sharp and fine, and of exactly the same form as those prepared in the usual way. The preparations of CO-hemoglobin were made in a similar manner, the blood being first shaken with oxygen gas under slight pressure, and then laked with ether. After the ether-laking it was shaken with illuminating OF THE MARSUPIALIA, EDENTATA, AND SIRENIA. 175 gas and the oxygen displaced by CO as shown by the spectroscope, after which it was centrifugalized. Most of the preparations examined were made in the usual manner of laking the oxalated blood with a few drops of ether and centrifugalizing for several hours. From the fresh blood the first crystals to form were always a-oxyhemoglobin, but from stale blood reduced hemoglobin formed. The regenerated stale blood gave only a-oxy- hemoglobin at first, but later the crystals of the other hemoglobins appeared. a-Oxyhemoglobin of Didelphis virginiana. Monoclinic: Axial ratio a : b : 6 =1.7856 : 1 : 2.6685; /3=48° (about). Forms observed: Prism (110), base (001), orthopinacoid (100), pyramid (111), orthodome (T01). Angles: Prism-base edges 110-001 A lTO-001 =58° 30'; 001 A 010=90°; 001 A 100 =/3 =48°, or perhaps somewhat less; T01 A 001 =about 90°. The angle of the ortho- dome to the base was not exactly determined. The angle ft was only determined approxi- mately, and some of the crystals measured showed the angle to be apparently about 43°. FIGS. 58, 59, CO, 61, 62, 63, 64. Didelphit virginiana a-Oxy hemoglobin. FIG. 65. Didelphis virginiana 0-Oxyhemoglobin. Habit tabular parallel to the base (text figures 58 and 59), the first crystals to form being very thin and very symmetrical rhombic tables; as they increase in size they develop other planes besides the simple prism and base, which is the usual crystal. The first plane to appear, besides these two, is generally the positive hemiorthodome (T01) which cuts off the ends, or sometimes mainly one end, of the rhombic plate (see plate 16, fig. 91) . The best crystals were obtained from the laked corpuscles and these showed in some cases the negative hemipyramid (111), in addition to the orthodome, beveling the edges of the plate in the obtuse angle (text figure 60) . Composite groupings formed by parallel growth are common, especially in the direction of b. Twins are of several kinds, but are not so frequently observed as is usually the case in these rhombic plates of the monoclinic sys- tem. A common form is a twin with the twinning axis parallel to the prism-base edge 110-001, and the base as the composition face (text figure 61). These form the symmet- rical twin shown on plate 16, fig. 92. The similar twin on the base as composition face and the twinning axis normal to prism-base edge and lying in the base, and the plane of twinning normal to the base, was also observed (text figure 63). This twin with the composition face the base is the usual type of twin in such monoclinic rhombic plates (text figure 62) (horse-type; see description of this twin under Horse). A third type is 176 CRYSTALLOGRAPHY OF THE HEMOGLOBINS shown on plate 17, fig. 99, with the orthodome (101) as the plane of twinning and the com- position face (text figure 64). This form was rather common. From an examination of such twins it would seem that this orthodome makes an angle of exactly 90° with the base. From this, if /? were accurately determined, the value of 6 could be easily calcu- lated. Taking /? at 48° the value of 6 becomes 2.6685. Pleochroism is very marked, a pale yellowish-red, b deep red, c very deep red. The plane of the optic axes is the plane of symmetry; the orientation of the elasticity axes is a A a = 17° in the obtuse angle; b=&; c A 6 = 25° in the obtuse angle. On the flat the extinction is symmetrical; on edge view, looking along b, the extinction angle is 17° from the edge 001-010 or from the trace of 001. On the flat the interference figure is seen, somewhat unsymmetrically arranged, and the acute bisectrix, Bxa=c. The optical character is hence positive. y hemoglobin of Didelphis virginiana. Hexagonal or pseudo-hexagonal. No axial ratio determinable. Forms observed: Prism (1010), base (0001). Angles: Prism angle 1010 A 0110=60° (120°), prism to base 1010 A 0001=90°. Habit tabular, thick or thin plates consisting of prism and base, with great develop- ment of the base (text figure 65). The /?-oxyhemoglobin crystals appear after the a-oxy- hemoglobin crystals, and the appearance of the former is accompanied by the resolution and disappearance of the latter. They form first in the protein ring, but later may appear anywhere in the slide. Apparently the solution of the a-oxyhemoglobin is due to the action of bacteria, but frequently the /?-oxyhemoglobin crystals appear growing on the a-oxyhemoglobin crystals as regular growth and with the a-crystal unaffected. The orientation of the regular growth appears to be such that the edges of the plate of the a-crystal are approximately normal to the edges of the /3-crystal in some cases, and par- allel to them in others. Etching figures are seen, elongated normal to the edges of the /3-oxyhemoglobin plates. Some of them appear to be composite crystals (see plate 17, fig. 98), as in the mica twins that are nearly uniaxial, and yet on edge the layers of the twin are so thin, or so intergrown, that polarized light does not seem to show any trace of composite character. No regular twins of these crystals occur, but parallel growth is common. The color of these /3-oxyhemoglobin crystals is brighter red, more scarlet than the color of the a-oxyhemoglobin crystals, but this is evidently due to the fact that they show little or no pleochroism; and the spectroscope does not show any difference from the normal oxyhemoglobin spectrum. Pleochroism is not noticeable on the basal view, and there is practically no pleochroism or absorption on the side or edge view, looking normal to the vertical axis. There is no double refraction that can be detected on the basal view; on edge view the double refraction is easily seen and the extinction is straight. The vertical axis is the axis of least elasticity, e > w, and the crystal is positive. On the base, in convergent, light, the uniaxial cross is readily seen; in some cases the crystal is slightly biaxial, as in the nearly uniaxial micas, and the axis of least elasticity is normal to a prism face. The biaxial crystals are also distinctly positive. The separation of the brushes is only very slight, the angle 2E is very small. While these crystals are seen in all sizes, and do not appear to be composite, there can be little doubt that they are really mimetic hexagonal only and are twins of the a-oxyhcmoglobin on the base in one of the two forms of twinning that have been described under the a-oxyhemoglobin. If the twin laminae were thin enough, the polarization test would not show the composite character and this would be especially true if, as is usually the case in these twins, the same layer did not run as a plane entirely across the basal surface. In looking through from side to side the different orientation of the layers would hence average, and neutralize each other. Of course, this averaging would happen on the flat view to a still greater degree, and the elasticity axes a and b in different orien- tation would completely extinguish each other, making a uniaxial effect. This may be done artificially with only three plates of mica, twinned as these a-oxyhemoglobin crystals OF THE MARSUPIALIA, EDENTATA, AND SIRENIA. 177 twin, and has been observed in the oxyhemoglobin of many species that twin in this way, where it is easily seen that the crystal is composite. If both kinds of twins formed in the same crystal, the averaging of the elasticities might be perfect. But, as has been observed in other species that form hexagonal plates (compare rats, squirrels, etc.), the growth of the composite plate by this form of twinning produces an averaging of the angles, so that prism angles that are nearly 60° (58° 30' as in this a-oxyhemoglobin) become exactly 60° in the twin. It might be possible that this crystal was an averaging of right- and left-handed forms, resulting in the more symmetrical mimetic twin. From the forms of twinning assumed, the elasticity axis, c, remains always in the same position in all of the members of the composite crystal, and hence the vertical axis, £, becomes the axis of less elasticity, and the composite remains positive. If the above view of these crystals is correct the substance of the a-oxyhemoglobin and of the /3-oxyhemoglobin may be the same unless perhaps the /?-oxyhemoglobin is a union of right-handed and left-handed crystals of the a-oxyhemoglobin. Reduced Hemoglobin of Didelphis virginiana. Monoclinic: Axial ratio a : b : t =1.963 : 1 : I; /3 = 66°. Forms observed: Prism (110), base (001). Angles: Prism angle traces on the base of 110 A 1TO=54°; prism edge to base 110-lTO A 001=/? = 66°; base to plane of symmetry or to side prism edge 001 A 110- 110=90°. (.7 FIGS. 66, 67. Dutelphit mrginiana Reduced Hemoglobin. FIGS. 68. 69. D. virginiana a-Carbon-monoxide Hemoglobin. Habit, rhombic plates with oblique sides, composed of base and prism, the crystals generally very perfect and sharp (text figures 66 and 67). They usually occur singly, but also twin with the normal to the base as the plane of twinning and the twin axis normal to the prism-base edge, the composition face being the basal pinacoid. This type of twin, " horse-type," is seen in a-oxyhemoglobin (text figure 51) and is the common twin on the base in all of these monoclinic reduced hemoglobins and oxyhemoglobins, especially when the prism angle is near 60°. These twins are often complex and the polysynthetic arrangement is very common. The crystals are readily distinguished from the a-oxyhemoglobin by their color, and by the fact that they occur singly and not in parallel growth, as is so commonly the case in the a-oxyhemoglobin. In the photographs they appear as lighter-colored, more transparent crystals than the oxyhemoglobin crystals. Pleochroism is very strong; a very pale violet, nearly colorless; b deep reddish; c deep claret-color to purple. Ex- tinction is symmetrical or nearly so on the flat basal face; on edge looking along the axis 6 it is oblique; the extinction angle is a A a = 13°, in the obtuse angle. The orientation of the elasticity axes is as follows: The axial plane is the plane of symmetry; a A a = 13° in the obtuse angle, b = 6, c A a>, and the optical character is positive. Compared with the /3-oxyhemoglobin these crystals are seen to have identical characters, and there is probably no doubt that if the /?-oxyhemoglobin crystals are mimetic twins these are also. The forms of twinning noted for the a-oxyhemoglobin would produce such mimetic forms if the twinning was repeated or polysynthetic. Such twins, with the twin axis lying in the basal pinacoid and normal to the prism-base edge, and the base as the composition face, were apparently observed in the a-CO-hemoglobin along with the type of twin already described. The close resemblance of the oxyhemoglobins and the CO-hemoglobins in this species is what might be expected from the other resemblances between these compounds. It will be noted, however, that the reduced hemoglobin varies from either in the incli- nation of the base and in the prism angle. Table 38 shows these differences plainly. TABLE 38. — Differences of the oxyhemoglobins and CO-hemoglobins in Didelphis virginiana. FIG. 70. Didelphis virginiana 0-Oxy hemoglobin . Substance. Axial ratio. Angle 3. Prism angle. Extinc- tion angle. Optical character. System. a-oxy hemoglobin a =1.7856 o 48 (43?) o / 58 30 17 Positive Monoclinic. a — 1 804 41 58 30 13 Do. Do a-reduced hemoglobin a - 1 963 66 54 0 13 Do. Do /3-oxyhemoglobin 90 60 0 0 Do. Hexagonal /?-CO-hemoglobin 90 60 0 0 Do. Do. TASMANIAN DEVIL, Sarcophilus ursinus. Plate 18. The specimen was obtained from the National Zoological Park at Washington, District of Columbia, and consisted of about 2 c.c. of oxalated blood preserved in our usual collecting tube. The blood was centrifugal- ized and the corpuscles separated and laked with ether. Preparations were made in the usual manner. The blood crystallized very readily and photographs could be taken within 2 hours of making the preparations. The blood being in good condition, the crystals were oxyhemoglobin, as determined by the spectroscope. 180 CRYSTALLOGRAPHY OF THE HEMOGLOBINS Oxy hemoglobin of Sarcophilus ursinus. Monoclinic: Axial ratio a : b : 6 =1.804 :!: 6 > a. On the base, the crystal extinguishes symmetrically, with the axis of greater elasticity bisecting the acute angle of the plate. On the clinopinacoid sections the extinction is oblique, making an angle of 9° with the edge 010-001 or with the clino-axis, a, in the obtuse angle. The plane of the optic axes is normal to the plane of symmetry and a =b. The orientation of the other elasticity axes is, 6 A a =9° in the obtuse angle, the extinc- tion angle; and c A 6 = 26°, in the obtuse angle. Traces of the brushes of an interfer- ence figure show on some of the plates that are tilted, with the orientation as above for the elasticity axes. On the clinopinacoid sections the interference figure shows when looking along a, the brushes being only slightly separated. The acute bisectrix is hence the axis of greatest elasticity, Bxa = a, and the optical character is hence negative. RAT-KANGAROO, &pyprymnus rufescens. Plates 21 and 22. The specimen was received from the National Zoological Park at Washington. The blood was putrid, but, being collected in oxalate, was liquid. The contents of the tube were centrifugalized. The separated cor- puscles were then laked with ether and again centrifugalized, and slides prepared in the usual manner. Crystallization was rapid; the crystals formed readily at room temperature and appeared to be relatively insoluble. The color of the solution was almost discharged, showing that the crystal- lization was very complete. Negatives were made within 5 hours after the slides were prepared. The crystals were oxyhemoglobin as determined by the spectroscope. OF THE MARSUPIALIA, EDENTATA, AND SIRENIA. 185 Oxy hemoglobin of sEpyprymnus rufescens. Monoclinic: Axial ratio a : b : 6 =1.4825 : 1 : 1.338; /? = 67°. Forms observed: Prism (110), clinopinacoid (010), base (001); and, in twins, positive hemiorthodome (101), orthopinacoid (100). Angles: Prism angle 110 A 110 = 112° (68°); 001 A 010=90°; 100 A 001=0 = 67°; 100 A T01=51°, from twin. Habit prismatic, elongated on the vertical axis, with the prism (110) and base (001) alone (text figure 89) or in combination with the clinopinacoid (010) (text figure 90), and frequently flattened on the clinopinacoid; the base produces an oblique termi- nation in all crystals. The prisms are sometimes long, but in the greater number of crystals are not more than 4 to 6 times as long as wide. The orthopinacoid is not de- veloped as a face and only appears in twins; hence no square crystals are seen. Twin- ning is of several types, interpenetrant twins on the prism being common (text figure 91), see plate 22, fig. 127; twins also form on the orthopinacoid (text figure 92) (gypsum type) and on the clinopinacoid similar to the carlsbad type (text figure 93). The twin on the positive hemiorthodome (T01), from which the value of t was obtained, is an interpenetrant twin, often seen in crystals with the combination (110) (010) (001). The base of one member is nearly parallel with the orthopinacoid or prism edge of the other and the acute angles are opposed with the obtuse angles pointing outward (see text figure 94). In the twins of the carlsbad type the opposed prism faces on either side of the plane of twinning appear to be developed more than the other pair in each case. The twins on the prism are not only interpenetrant, they are frequently juxtaposed, and even in this case polysynthetic (see plate 22, fig. 128). a/ a /90 91 92 Fics. 89, 90, 91, 92, 93, 94. .Epyprymnut rufescens Oxyhcmoglobin. Pleochroism is very strong; a pale yellowish-red, b rather deep red, c very deep red; the pleochroism is readily observed on account of the almost complete crystalliza- tion of the oxyhemoglobin, leaving a nearly colorless solution. Extinction on all of the usual aspects is oblique, the crystals generally presenting the clinopinacoid or prism face. In the twins on the orthopinacoid, prism, and orthodome the extinction is sym- metrical with the plane of twinning. The plane of the optic axes is the plane of symmetry, the orientation of the elasticity axes is a A a = ll° in the obtuse angle; b=&, c A 6= 12° in the obtuse angle. Only brushes of the interference figure in unsymmetrical arrange- ment were seen on such optical sections as could be observed; the optical character appeared to be negative, or Bxa—a. KANGAROO, Macropus giganteus (?). Plate 22. The specimen was received from the National Zoological Park at Washington during the summer and was kept frozen in the original collect- ing tube until examined. The blood was rather putrid and contained many small clots and amorphous matter. It had been drawn into a collecting tube supplied with oxalate, hence did not clot after the specimen was 186 CRYSTALLOGRAPHY OF THE HEMOGLOBINS placed in the tube. Ether added to lake the blood appeared to increase the precipitate of amorphous granular matter, and centrifugalizing did not entirely free the blood from this precipitate. The crystals formed readily at ordinary room temperature and did not appear to be very soluble. Pho- tographs could be made inside of 4 hours after the slides were prepared. Examination with the spectroscope showed the crystals to be oxyhemo- globin. Oxyhemoglobin of Macropus giganteus. Monoclinic: Axial ratio a : 6 =1 : 0.497; /?=87° (93°). Forms observed: Orthopinacoid (100), clinopinacoid (010), base (001); also an orthodome (T01). Some crystals apparently showed a square prism (110) in place of the two vertical pinacoids; this was not clearly made out. Angles: 100 A 001 -0=-87° (93°); 001 A 010=90°; 100 A 101 =66°. The posi- tive hemiorthodome (T01) was also observed as a plane of twinning with angle between the (! axes of the twin of 48° 30' as measured. This gives the angle 100 A T01 =65° 45'. Habit lath-shaped crystals, consisting of orthopinacoid (the principal plane) and clinopinacoid; the two forming a flattened prismatic crystal elongated along the vertical axis and terminated by the base (001) (text figure 95). They, being flattened on (100), generally present this aspect and hence appear to be terminated by a plane normal to the length, but the square end is produced by the aspect in which the crystal is usually presented, and on an edge view, looking along the 6-axis, the end is seen to be oblique. The orthodome was seen in a few crystals only. The lath-shaped crystals aggregate into sheaf- like bundles and stellate radiating groups, and also grow singly. Twins on the orthopina- coid (100), gypsum type, were occasionally seen (text figure 96) and one distinct twin on the hemiorthodome (T01) was observed with the angle as given above (text figure 97). Pleochroism is very pronounced; a pale pink, b rose-pink, c deep rose-pink. Extinc- tion is straight or nearly so in both side and edge views. The end view was not seen. The orientation of the elasticity axes is c=<5, a =6, b A a =3°, in the obtuse angle; the axial plane is normal to the plane of symmetry. No interference figure could be observed, but indications seemed to show that Bxa=c, and the optical character is hence probably positive. " J, <*, 100 101 Fioa. 95, 9G. 97. Macropia eigant*\a Oxyhemoglobin. FIOB. 98, 99, 100, 101. Pilrogalt sp. Oxyhemoglobin. ROCK-KANGAROO, Petrogale sp. Plate 23. The specimen was received in July and kept frozen in the original col- lecting tube until examined. The blood was full of a granular precipitate which did not separate on centrifugalizing. The blood had been oxalated when collected and was not ether-laked. The slides were prepared in the OF THE MARSUPIALIA, EDENTATA, AND SIRENIA. 187 usual manner, and crystals formed readily at room temperature, showing no signs of dissolving. They were oxyhemoglobin, as shown by the spec- troscope. Oxyhemoglobin of Petrogale sp. Monoclinic: Axial ratio not determined. /?— about 84° (96°). Forms observed: Orthopinacoid (100), clinopinacoid (010), prism (110), base (001). Angles: 100 A 001=/?=84°; 001 A 010=90°. Habit lath-shaped by development of the two vertical pinacoids (100) and (010) and terminated obliquely by the base (001) (text figure 98), also prismatic with the com- bination of prism (110) and base (001) (text figure 99). The crystals are capillary in some cases (probably the prism and base combination) ; and in other cases are long lath- shaped, with the orthopinacoid predominating, and then with square ends (text figure 98); or short lath-shaped, with the clinopinacoid predominating (text figure 100), and then with oblique ends. The prism seems to be nearly square, but its angle could not be obtained. Contact twins on the orthopinacoid (gypsum type) are common (text figure 101) ; twins on a pyramid of the interpenetrant type were also observed (compare text figure 97). Pleochroism is rather strong; a pale yellowish-red, 6 red, c deep cochineal-red. Extinction was apparently straight in both aspects, on (100) and on (010), the side and edge views. The orientation of the elasticity axes is a =6; b A a =6°, in the obtuse angle; c= b > a. Extinction is straight on (100) and on (010). The orientation of the elasticity axes is fl=a; b=6; ( = 6. No interference figure was seen on b; but on a, or looking along a, traces of a figure were seen, the brushes passing out of the field in open position. The aspect looking along c could not obtained, but there seems to be no doubt that Bxa—c, or that the optical character is positive. The axis of b > a. From the double refraction and the pleochroism, as well as from the absorption, which show c and b to be nearly equal, it would seem that the acute bisectrix Bxa = a, and the optical character is negative. Reduced Hemoglobin of Sus scrofa, Domesticated Variety. In the slides after standing for 24 hours there always developed numerous long prismatic crystals of reduced hemoglobin, which appear at first around the margin of the cover, and later throughout the body of the slides. They show straight extinction on most aspects, but have a decidedly monoclinic habit, being terminated obliquely in many cases. Some appeared to have square ends; others, a single plane like a basal pinacoid, but oblique. They appear to be monoclinic. They grow in tufts and in sheaf-like aggre- gates, sometimes even in feathery groups. They appear to twin on a dome or pyramid, and also on the prism. The terminal plane is usually very imperfect, due to a fibrous character which the crystals show, the ends of the fibers making a rough plane. Smaller crystals and short stout prisms show a very monoclinic aspect. Muis DEER OR CHEVROTAIN, Tragulus meminna. Plate 34. The specimen was obtained from the post mortem of an animal that died in the Philadelphia Zoological Gardens. The blood was oxalated, ether-laked, and centrifugalized; the slides were prepared in the usual manner. Crystals formed readily and did not show a tendency to dissolve on bringing them into a warm room. They were oxyhemoglobin. Later, the same slides developed crystals of reduced hemoglobin, along with those of the oxyhemoglobin; these latter being relatively enormous. Both kinds of crystals were very sharp and well defined. CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE UNGULATES. Oxy hemoglobin of Tragulus meminna. = 63°. 201 Monoclinic: Axial ratio a : b : t =1.804 : 1 : 6; /? = Forms observed: Unit prism (110), base (001). Angles: Traces of prism on the base, edges 110-001 A 110-001=59°; true angle 110 A 1TO = 64° 50' (calculated); prism edge to base, edge 110-110 A 001 = 63° (normals) =/?. Habit of the single crystals tabular on the base (text figures 142, 143), the plate bounded by the prism faces, generally symmetrical or nearly so; but most of the crystals are twinned with the prism-base edge (110-001) as twin edge and a normal to this edge in the plane of the base as the twin axis (text figures 144 and 145). In these twins the composition face is the base and along one of the prism-base edges, where they unite, there is a reentrant angle, while on the opposite edge there is an ordinary dihedral angle. In these twins (horse-type), which are common in all hemoglobins with angles that approximate 60°, the compound crystal in this species is usually elongated along the common edge, and the two crystals overlap each other at the ends of this elongated crystal forming reentrant angles in the outlines of these ends. The twinning is frequently repeated in polysynthetic order; and it is often complicated by parallel growth in one or more of the members of the twin. It does not appear to tend to produce hexagonal forms by twinning on more than one pair of the prism-base edges, however, as is com- monly the case in this kind of twinning. Twinning on the base as twin plane is also found apparently, but it is rare. This twinning seems to tend to make the angle of the plate nearer 60°. In some cases the opposite prism-base edges do not appear to be par- allel, due perhaps to a vicinal prism face in one member of the twin; this non-parallelism would tend to average the angles to near 60°. 147 FIGS. 142, 143, 144, 145. Tragulus meminna Oxyhemoglobin. FIGS. 14ti, 147. TruQulus meminna Reduced Hemoglobin. Pleochroism is strong; a is pale yellowish with a reddish tinge, 6 is a blood-red and c is still deeper red than b. Extinction seems to be symmetrical with the sides of the plate; in some cases it appeared a little oblique, but probably the plates were some- what tilted. In twins with symmetrical extinction the angle was about 10° from the prism-base edge. Looking along the symmetry axis, it was about 15° from the trace of the base. The orientation of the elasticity axes is a A a = 15°, in the obtuse angle, b=b, c A > e. RED-BACKED DEER (PROBABLY THE RED BROCKET, Cariacus rufus). Plate 35. The specimen of blood was received from the Philadelphia Zoological Gardens, and was beginning to putrefy. It was treated by oxalating and freezing, then laked with ether and centrifugalized. The slides were pre- pared in the usual manner and kept at a temperature near the freezing-point. It crystallized readily, the crystals that were the first to form being long lath-shaped rods ; these were followed by large rectangular plates, very thin, and evidently the same as the rods, but of a tabular habit. When the slides were brought into a warm room the plates were rapidly dissolved ; the rods showed more resistance to solution, but it was found necessary to examine the slides in the cold, and all photographs were taken at a temperature near the freezing-point. The crystals gave the spectrum of reduced hemoglobin. Reduced Hemoglobin of Cariacus rufus. Monoclinic: Axial ratio could not be determined, as only pinacoids were seen. Angle p appears to be about 90°. Forms observed: Base (001), clinopinacoid (010), orthopinacoid (100). Angles: Clinopinacoid to orthopinacoid, the outline of the plates, 010 A 100=90°; base to orthopinacoid, angle /?, about 90°, perhaps exactly 90°. The third angle was not observed, but must be 90° in this system. Habit lath-shaped crystals elongated on the clino-axis, and flattened on the base; these, by elongation on the ortho-axis also, became rectangular plates, which are fre- quently as long as the lath-shaped rods and perhaps 30 times as wide. The plates appear to be produced by the piling up of narrow plates all in parallel position and this produces striation on most of these plates parallel to the a axis. The rods grow in tufts, radiating or brush-like, generally united with each other on the base, or in the zone of (OOl)-(lOO). The composite plates are produced in the same way, by uniting on the base. No definite twinning was observed, but possibly the piled-up plates may be polysynthetic twins. 204 CRYSTALLOGRAPHY OP HEMOGLOBINS OF THE UNGULATES. Pleochroism was strong, as is common in reduced hemoglobin; a pale pink, nearly colorless; b purplish, deeper than a; c deep purplish-red. Absorption was in the order c > b > a. Extinction on the base was straight; on edge views the extinction angle was about 30° with the length of the rod-like section. The plane of the optic axes is the plane of symmetry; the orientation of the elasticity axes is a A a =30°, b —b, c A 6 =30°. On the base, in convergent polarized light, a single brush of a biaxial interference figure is seen, the optic axis emerging at a small angle with c or the normal to the plate; the acute bisectrix is hence evidently c, and the optical character is positive. VENEZUELA DEER, Mazama americana savannarum (?). Plate 36. The specimen of blood was received from the National Zoological Park at Washington during the summer and was kept frozen until examined. The quantity of blood was small, and it was quite thick and putrid, and full of extraneous matter. This latter was centrifugalized off as far as possible, but the specimen was not thoroughly cleansed, owing to an accident to the centrifugal machine. The slides were prepared in the usual manner, and crystals formed readily in the cold. They were not dissolved at a tempera- ture of 10° C. A spectroscopic examination of the plasma showed the presence of oxyhemoglobin, but only crystals of reduced hemoglobin were obtained, they being determined as such by the microspectroscope. Reduced Hemoglobin of Mazama americana savannarum. Monoclinic: Axial ratio not determinable as the pinacoids only are developed. The angle /? seems to be 90°. Forms observed: Base (001), clinopinacoid (010), orthopinacoid (100). Angles: Clinopinacoid to orthopinacoid, the outline of the plates 010 A 100=90°; base to orthopinacoid 001 A 100=90°=^. The third angle could not be obtained, but is necessarily 90°. Habit broad or narrow lath-shaped, flattened on the base and elongated parallel to the clino-axis (text figure 151); the lath-shaped crystals by development along the sym- metry axis b become broad plates. When the plate-like habit is assumed, the tabular crystals are seen to be composite, by par- allel growth and uniting on the base, producing strong striation parallel to the clino-axis. The crystals grow in tufts, radiat- ing from a center, and the majority of the crystals are broad a lath-shaped or tabular, with the length of the plate 2 to 3 times FIG. 151. Mazama amencana ! savannarum Reduced the width. On edge view they do not show the usual tendency Hemoglobin. to racjjate jn a brush-like manner to any very marked degree. The color is reduced hemoglobin purple; the pleochroism, as usual in hemoglobin, is very strong; a is pale rose-pink; b is strong rose-pink; c is deep rose-red. On the flat the extinction is straight, parallel to the edges of the plate or lath-shaped crystals; on edge it is oblique, about 30° measured from the length of the rod. The plane of the optic axes is the plane of symmetry; and the orientation of the elasticity axis is a A a = 30°, the extinction angle; b=&; c A 6=30°. Traces of the interference figure were seen on the flat view, on (001), but it was not definitely observed, except as to the position of the plane of the optic axes. Either the angle between the axes is large or the axis of greatest elasticity is the acute bisectrix. FALLOW DEER, Cervus dama. Plate 36. The specimen of blood was received from the New York Zoological Gardens, having been collected in a tube containing oxalate. It was clot- ted and somewhat putrid. The clots were ground in sand with ether and the mixture centrifugalized, and from the clear solution the slides were CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE UNGULATES. 205 prepared in the usual manner. Crystallization began in the protein ring shortly after covering the slides, but soon after they formed these crystals began to dissolve; due, no doubt, to the establishment of equilibrium between the concentrated solution near the protein ring and the less con- centrated solution throughout the slide. The crystals subsequently formed mainly around the edge of the cover, owing to concentration of the solution in that region, due to evaporation through the balsam seal. Crystals of oxyhemoglobin, in the form of bipyramids, and of hemoglobin, in the form of broad lath-shaped crystals, were developed side by side ; but the reduced hemoglobin crystals began to appear later than those of oxyhemoglobin. Both kinds of crystals were tested by the spectroscope. The crystals formed practically at room temperature, although the slides were kept at a tem- perature below 10°. Oxyhemoglobin of Cervus dama. Tetragonal: Axial ratio a : 6 =1 : 1.200. Forms observed: Unit pyramid (111), traces of base (001). Angles: 111 A TTl =61°, measured over the pole; the angle of the pole edges, measured in the same way, was edge 111- 1T1 A 1T1-IT1 =79° (calculated 79° 36') ; profile of pyramid look- ing along <} gave 90° between the edges. Habit pyramidal (text figure 152), the unit pyramid in very perfect development or with some faces larger, due to lying on the slide; a few crystals seemed to show the base (001). The crystals \/ 152 are very small, but well formed. In some cases they appeared like FIG. 152. Cmiu» dama oxy- skeleton crystals, due to some tendency to parallel growth appar- ently; some formed interpenetrating groups resembling twins; in one or two cases these appeared to be twins on the pole edge as twin axis. From the optical anomalies noted in some crystals, they may be some form of a mimetic twin of the orthorhombic system, with the groups producing tetragonal symmetry. The skeleton-like crystals mentioned might be such interpenetrant orthorhombic twins. Pleochroism was not very strong, but the axis of greater elasticity was the axis of less color. On the basal aspect, looking along 6, the crystals normally show single refrac- tion, but the crystals presenting this aspect were too small to show an interference figure. A few of them showed double refraction, not very strong, but extinguishing along the two equal diagonals of the square section. This optical anomaly may indicate a twinned orthorhombic structure. On the side views normal to 6, the extinction is parallel to the vertical axis t in all aspects. The double refraction is fairly strong, the vertical axis (i =e being the axis of greater elasticity. Hence w > s and the optical character is negative. Reduced Hemoglobin of Cervus dama. Probably orthorhombic, perhaps rnonoclinic; the crystals were very imperfect. They showed sometimes a roughly four-sided cross-section, with an angle of perhaps 85°, but most of them seemed to be rather lath-shaped. They were generally not ter- minated; a few showed square-cut ends, but the majority were merely shred-like masses, with more or less straight sides and splintery looking ends. They were also in spheru- litic masses and are probably parallel or radiating groups of smaller prisms; the parallel groups forming the prism-like shreddy crystals and the radiating groups the spherulites and tufts of crystals. The parallel masses show straight extinction, the spherulites extinguish parallel to the fibers and show the usual extinction cross of spherulitic masses of crystals in polarized light. The straight groups of crystals show the length of the prisms to be the direction of greater elasticity and the direction normal to this to be the 206 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE UNGULATES. direction of less elasticity. Pleochroism is strong, parallel to the length of the fibers; a is colorless, normal to the length, b and c are purple. In the spherulitic masses the pleochroism shows strongly, dividing the spherulite into sectors, the opposite sectors being of the same color; a colorless, c and & purple, as in the prisms. MDNTJAK, Cervulus munljak. Plates 36 and 37. The specimen of blood was received from the New York Zoological Gardens and was in a somewhat putrid condition. The blood, containing oxalate, was laked with ether and centrifugalized and from the clear solu- tion slides were prepared in the usual manner. The blood crystallized slowly and the crystallization was therefore carried on at temperatures near 0° C. The first crystals to form were oxyhemoglobin, short prisms with very oblique terminations; later, crystals of reduced hemoglobin formed in the shape of long square-ended rods or lath-shaped crystals, and in curving arborescent forms. The crystals of the oxyhemoglobin with- stood changes of temperature fairly well, but the crystals of reduced hemo- globin were rapidly dissolved when brought into the warm room. Oxyhemoglobin of Cervulus muntjak. Monoclinic: Axial ratio a : b : 6 =1.303 :!: 6 > a. Extinction is straight or symmetrical in all aspects. The plane of the optic axes is the basal pinacoid and the orientation of the elasticity axes is a—b, b=c, c=a. On side views of the prism or on the vertical pinacoids traces of an interference figure are seen in convergent light; on (010) the two brushes of the biaxial figure are seen in the field, but they are widely separated. On (100) two brushes show also, but pass out of the field in the diagonal position. The acute bisectrix is hence evidently a, and the crystals are optically negative. This is indicated also by the pleo- chroism, for b and c are much nearer together than & and a. DOKCAS GAZELLE, Gazella dorcas. Plate 39. This specimen was received from the Philadelphia Zoological Gardens. The blood was clotted, but in good condition. The clot was ground in sand with ether and the mixture centrif ugalized ; from the solution the slide preparations were made in the usual manner. Crystals formed readily in the dried protein ring, but were gradually dissolved as equilibrium was established in the solution after covering; and then they reformed along the cover edge, as they were dissolved from the protein ring, until the solution in the slides was homogeneous. When the condition of equilib- rium was reached in the solution, the crystals showed no sign of being dissolved and were in good condition for days. The first crystals to form are small rectangular plates, but with them are long rods; both seem to be the same, however, and both are oxyhemoglobin, as determined by the spectroscope. Oxyhemoglobin of Gazella dorcas. Orthorhombic : Axial ratio a : b : c =0.3639 : 1 : 0.4452. Forms observed: Brachypinacoid (010), base (001), unit prism (110), brachydome (Oil), and, without measurement of angles, the macrodome (101) and the macropinacoid (100). Angles: Prism angle 110 A HO =40° (normals) ; brachydome angle Oil A Oil = 48° (normals); outline of plates, 100 A 001=90°. Habit tabular on (010), the plate bounded by the other two pinacoids (100) and (001) when the crystals begin to grow (text figure 160) or by the combination of the prism (110) and the brachydome (010) in the larger crystals (text figure 161). The macropinacoid disappears as the crystals increase in size, but the base sometimes appears to persist, although it is generally replaced by the brachydome. The crystals are usually elongated along the vertical axis, so that on the brachypinacoid aspect the length is 14 210 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE UNGULATES. about double the width; but some are much more elongated, and others are reduced to nearly square tabular plates. They frequently show parallel growth, uniting on the brachypinacoid or on the macropinacoid and also grow in irregular, somewhat radiating aggregates. Pleochroism is rather strong; a is pale yellowish-red; & is deep blood-red, c is deep red, somewhat deeper than b. Extinction is straight in all aspects. The orientation of the elasticity axes is a=a, b=c, c=6. The plane of the optic axes is the base, and a is the acute bisectrix, Bxa =a. Looking along this axis of a, the interference figure is seen in convergent light, with the angle between the optic axes, 2E =40°. The optical character is negative. 10 160 162 161 163 Fias. 160, 161. Gtuella dorcae O.xyliemoglobin. FIG. 162. Cephalophus yrimmi a-Oxyhemoglobin. Flo. 163. Cephalophut grimmi /3-Oxyhemoglobin. DUICKERBOK, Cephalophus grimmi. Plate 40. The specimen was received from the National Zoological Park at Washington. The blood was oxalated, laked with ether, and centrifu- galized, and preparations were made in the usual manner. The slides were kept at a temperature of about 0° C., and soon were filled with the small pyramidal crystals, which showed a tendency to dissolve, and were hence examined and photographed at temperatures near the freezing-point. Later, the slides developed the second kind of crystals, the hexagonal plates. Both kinds of crystals were oxyhemoglobin. a-Oxyhemoglobin of Cephalophus grimmi. Tetragonal: Axial ratio a : c =1 : 0.8687. Forms observed: Unit pyramid (111), also traces of (100). Angles: Between the pyramid edges in the horizontal plane, normal to the axes = 110 A 1TO = 90°; between the pyramid edges in the vertical axial plane (calculated) = 101 A T01=9S°, observed angle of pyramid over the pole = lll ATTl=77°. Habit pyramidal (text figure 162), the crystals occurring singly or in irregular groups and in parallel growths. Twinning seems to occur on the pyramid as the plane of twinning. The color is the normal oxyhemoglobin red; pleochroism is scarcely noticeable. Double refraction is weak, but the extinction is symmetrical on the aspects normal to the vertical axis. Looking along this axis, the crystals are singly refracting in parallel polarized light and do not polarize; but in convergent light they show a faint dusky uniaxial cross. By the quartz wedge it is seen that the vertical axis is the direction of greater elasticity, hence w > s and the optical character is negative. CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE UNGULATES. 211 ^-Oxyhemoglobin of Cephalophus grimmi. These crystals develop after the pyramids of the a-oxyhemoglobin. They are in the form of very thin hexagonal plates (text figure 163), occurring both singly and also growing in groups, often with the orientation of parallel growth. They are evidently hexagonal, the angle of the plate being 120° (60° normals) and the sides square with the terminal plane. No axial ratio could be determined, as the combination of forms is simply unit prism (10TO) and base (0001). They are singly refracting when viewed on the base, but too thin to give an interference figure. On edge view they show very weak double refraction and extinguish parallel to the base. The optical character could not be determined owing to the very weak double refraction. SHEEP, Ovis aries. Plates 40-42. The fresh blood was collected in oxalate from the abattoir, and centri- fugalized to throw down the corpuscles. The plasma was drained away, the corpuscles were laked with ether, oxalate added almost to saturation, and the solution centrifugalized for 2 hours. From the clear liquid the slide preparations were made as usual. The preparation crystallized at room temperature, and the crystals showed no tendency to dissolve. Some crystals were obtained within 5 hours of making the preparations. The crystals at first formed were fine needles, but soon tabular crystals began to appear. Several other preparations were made from the same blood, and in all the crystals kept well. After about a week, crystals of reduced hemoglobin began to make their appearance, along with the crystals of oxy hemoglobin, which formed in the freshly prepared slides. These crystals of reduced hemoglobin, like the oxyhemoglobin, were not dissolved on slight increase of temperature. The slides were kept cool, at about 10° C., except when under examination. Both the oxyhemoglobin and the reduced hemoglobin were identified by the spectroscope. Oxyhemoglobin of Ovis aries. Monoclinic: Axial ratio a : b : 6 =1.140 : 1 : 0.970; /?=54°. Forms observed: Unit prism (110), positive hemiorthodome (T01), base (001), clinopinacoid (010), orthopinacoid (100). Angles: Prism angle 110 A HO, traces on the base, or angle of edges 110-001 A 110-001= 82° 30' (actual angle); orthodome to orthopinacoid T01 A 100=72°; ortho- pinacoid to base 100 A 001 = 54° = /? (normals). Habit of the first crystals to form minute needles without definite outlines, tapering to a point at either end; with these soon appear tabular crystals consisting of the base with a very short prism, tabular on the base (text figure 164). After about a day, long prismatic crystals appear consisting of the three pinacoids, elongated parallel to the vertical axis and generally flattened on the orthopinacoid. These sometimes show the prism as a bevel on the edges (text figure 165), but more often are simply the three pina- coids. These crystals twin and form networks of rods, and frequently on the orthopina- coid faces twin growths develop, producing a cross-banded effect due to the strong pleochroism. Twins are hemitrope, on the orthopinacoid (100) and on the hemiortho- dome (10T), the two occurring together and making fivelings of exactly pentagonal shape (text figure 166). These little fivelings grow on the sides of the long crystals, or singly, scattered through the slides; and they grouped themselves along the crystals of oxalate that formed in some of the slides, strung like beads along the needles of the oxalate. In this occurrence they present edge views to the observer. When seen in side view they are generally more or less perfect pentagons, divided by the contact planes into five 212 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE UNGULATES. sectors meeting at a point in the center; sometimes, by irregularity of growth, 7 individuals are seen in the group. The following more detailed description will make the arrangement plain. Suppose a fiveling, the members taken in cyclic order around from 1 to 5. Nos. 1 and 2 twin on the orthopinacoid (100); 3 is twinned to 2 on the hemiorthodome (10T) and to 4 on the orthopinacoid; 5 is twinned to 4 on the hemiortho- dome which brings its orthopinacoid in parallel position with the hemiorthodome of 1, thus completing the cycle. In each member of the twin the base forms one of the sides of the pentagon. n\ 164 166 168 Fios. 164, 165, 166. Omi arlet Oxyhemoglobin. FIGS. 167, 168. Ovit ariet Reduced Hemoglobin. These twins are often seen terminating a long prismatic crystal and, as already noted, they sprout out of the side planes of the long crystals, producing the banded appearance seen in the photographs. Pleochroism is very strong; a nearly colorless, b moderately strong red, c very deep blood-red. In the plates, the extinction is symmetrical on the base, and straight on the aspect looking along a; on the (010) aspect, the extinction angle is 30° with the prism edge. The long prismatic crystals show the same extinction as the plates. The orientation of the elasticity axes is a A a = 6°, b = b, c A <5=30°. The axial plane is the plane of symmetry, a is probably the acute bisectrix, but the interference figure was not observed. The optical character is probably negative. Reduced Hemoglobin of Ovis aries. Orthorhombic: Axial ratio a : b : t — 0.7813 :!: s and the optical character is slightly positive. On the basal aspect a very faint dusky cross in convergent light shows the uniaxial character of the crystals. ^-Oxyhemoglobin of Sciurus vulgaris, from the Putrid Blood. Orthorhombic: Axial ratio a : b : c =0.577 : 1 : c. Forms observed: Unit prism (110), base (001). Angles: Prism angle 110 A 110=60°; prism to base 110 A 001=90°. 220 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. Habit very thin tabular on the base, with the very short prism determining the outline of the rhombic plate (text figures 181 and 182). The crystals rarely occur singly, however, but in twins of the usual type on an axis in the base and normal to a prism-base edge, the composition face being the basal pinacoid. These occur on any or all of the prism-base edges, making a group that is composed of many individuals and generally of a roughly hexagonal outline (text figure 183). Hexagonal plates of normal develop- ment occur with these twins, similar to the plates of a-oxyhemoglobin; and they are probably mimetic twins of the /?-oxyhemoglobin, due to the complicated groups above described becoming developed into hexagonal plates. Twins on pyramid faces, as seen in the hexagonal a-oxyhemoglobin, were not observed. The color was the usual oxyhemoglobin red, perhaps a little darker than for the corresponding thickness in the a-crystals. Pleochroism is very slight on edge and not noticeable on the basal aspect; a deep red, b = c somewhat deeper red. Double refrac- tion on the base is not noticeable, even with the quartz wedge (b = c) ; on edge the extinc- tion is straight and the relative elasticities may be made out with the quartz wedge. On the base no interference figure of any kind could be detected in convergent light, but it is evident that the vertical axis is the axis of greatest elasticity, and that b=c; hence the acute bisectrix Bxa = a, and the optical character is negative. On comparing these two types it is evident that the characters of the /?-oxyhemo- globin are such that it would readily become hexagonal by mimetic twinning, the prism angle being exactly 60°, and the double refraction of the ^-modification is such that, but for the form of the crystal, it might be hexagonal. In the mimetic twins, produced by piling up of the rhombic plates to build a hexagonal composite plate, it might readily happen, with the very weak double refraction, that the crystal might become more dense in the direction in which the plates are piled, and hence the vertical axis, or normal to the plates, become the axis of greater density or less elasticity, when the pseudohexagonal crystal would become positive. It is hence entirely probable that the two modifications are really one and the same, the a-oxyhemoglobin being a mimetic twin of the ^-oxyhemo- globin and only pseudohexagonal. FOX-SQUIRREL, Sciurus rufiventer neglectus. Plate 46. The specimen was purchased from a collector at Orlando, Florida, and was bled in the laboratory, oxalated, ether-laked, centrifugalized, and the slide preparations made as usual. Crystals formed rapidly in the slides, and showed no tendency to dissolve. The blood crystallizes more readily than that of the related gray squirrel. The crystals were shown to be typical oxyhemoglobin by the spectroscope. Oxyhemoglobin of Sciurus rufiventer neglectus. Hexagonal: Axial ratio not determinate. Forms: Unit prism (10TO), base (0001). Angles: Prism angle 60°, prism to base 90°. Habit, thin tabular on the base, very symmetrical hexagonal plates consisting of the short prism and the basal pinacoid (text figure 184). The crystals occur singly, or in parallel growths and piled groups on the base, the smaller crystals piled concentrically on a larger crystal. In single crystals the thickness of the plate is one-tenth to one- twentieth of the width, but this is very variable. Many single perfect plates are seen, but this varies in different slides. In some cases the plates elongated on two prism faces or along the diameter of the hexagon parallel to a crystal axis, becoming somewhat orthorhombic looking; most of them are almost perfect hexagons. Twins on a first- order pyramid occur, mostly contact twins (text figure 185). The color of the plates is variable with the thickness, but pleochroism is very slight. On the base they are singly refracting, and polarize very faintly on edge; the double CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. 221 refraction is very weak. The elasticity for the ordinary ray, oj, is somewhat greater than for the extraordinary ray or « > aj in refraction indices, and the optical character is hence weakly positive. GRAY SQUIRREL, Sciurus carolinensis. Plates 46 and 47. The living animal was obtained from a collector at Newport News, Virginia, and was bled in the laboratory. The oxalated blood was laked with ether and centrifugalized, and the slide preparations made in the usual manner. Crystals formed more slowly than with the other squirrels examined; they were larger and showed more tendency to produce com- posite crystals than in the other species. They showed no tendency to dissolve, however, and are evidently quite difficultly soluble in the plasma. Examination with the microspectroscope shows that these crystals are typical oxy hemoglobin. 187 FIGS. 184, 185. Sciurut rufivcnter neglectut Oxyhemoglobin. FIGS. 186, 187. Sciurut carolinentie Oxyhemoglobin. Oxy hemoglobin of Sciurus carolinensis. Orthorhombic; pseudohexagonal: Axial ratio a : b : c =0.577 : 1 : 6. Forms observed: Unit prism (110), brachypinacoid (010), basal pinacoid (001); or, as pseudohexagonal, prism and base. Angles: Prism angle 110 A 110=60° (normals); prism to brachypinacoid 110 A 010=60° (normals), the two making a perfect hexagonal plate; prism to base 110 A 001=90°. Habit pseudohexagonal, tabular on the base, and with the prism and brachypina- coid faces in equilibrium, so that the plate is a perfect hexagon; sometimes, however, the plate is elongated on the brachy-axis, producing a distinctly orthorhombic habit (text figures 186, 187). The plates are large and perfect hexagons, but are not often simple; they produce groups by piling up on the base, more or less concentrically, and often with curving of the crystals, producing the form of the "eisen rose" of hematite, (see plate 47, fig. 277). The parallel growths on the base may, however, start from several centers, and it is very common to see a small group of this kind near one side of a large plate, not central, but in perfect orientation with the large plate. Twinning seems to be on a brachydome. In the protein ring the crystals form spherulitic masses of the radiating plates, and when these are seen on edge, or interfered with by the cover-glass, they look like lath-shaped crystals. When the piled-up plates are seen on edge, in section, they present a sheaf-shaped appearance. The color varies much with the thickness, but in the thicker crystals it shows the normal oxyhemoglobin red. Pleochroism does not show on the flat aspect, the crystal 222 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. acting like a hexagonal crystal; but on edge the pleochroism is noticeable, the color being deeper normal to the plate than parallel to it. On the base the crystal is nearly singly refracting; but by use of the quartz wedge it is seen to be very weakly doubly refracting; on edge the double refraction is easily seen, and extinction is parallel with the base. In convergent light, a uniaxial cross shows in most aspects; but on revolution the brushes open slightly and the crystal is as strongly biaxial as some of the biotite micas. The orientation of the elasticity is a = c, b=a, c=b. The macropinacoid is the plane of the optic axes and the acute bisectrix Bxa = a. The optical character is hence negative. FLYING-SQUIRREL, Sciuropterus volans. Plate 47. Blood was obtained from the living animal, oxalated, ether-laked, and centrifugalized, and the slide preparations made as usual. Crystal- lization begins as soon as the blood is laked, and proceeds with great rapid- ity, so that the preparations are soon full of minute scales or tabular crystals. To retard the formation of the crystals somewhat, and permit them to grow to a larger size, some preparations were made by diluting the blood with about 3 times its volume of the blood plasma; but, even with this dilution, the crystals begin to form immediately upon laking the corpuscles. They are always small, much smaller than in other species of squirrels, but other- wise resemble those formed in the squirrel bloods in general. They were oxyhemoglobin, as determined by the microspectroscope. Oxyhemoglobin of Sciuropterus volans. Hexagonal. No axial ratio determinable. Forms observed: Unit prism (1010), base (0001). Angles: Prism angle 60° (normals) ; crys- tals are so thin that prism to base could () not be measured with any exactness, but it appears to be 90°. Habit very thin tabular on the base (text figure 188), minute hexagonal scales or plates, with very little color, owing to their being so thin. They develop in enormous 191 numbers, the slides becoming completely filled with them. They generally occur singly or in irregular groups, but a twin on a pyramid of the second order seems to occur, interpenetrant and of the same general form as the tridymite twin (text figure 189). The crystals are very faintly colored, when seen on the flat, but on edge have the red color of oxyhemoglobin, and show pleochroism; a> deep red, « very pale red. On the flat, the crystals are singly refracting; on edge they polarize, but not strongly. The direction of the vertical axis or of the optic axis s is the direction of greater elasticity; hence u> > £, and the crystals arc negative. GROUND-SQUIRREL OR HACKEE, Tamias striatus. Plate 48. The specimen was purchased from a collector in eastern Pennsylvania. The animal was bled into oxalate, the blood laked with ether and centrif- ugalized, and slide preparations made in the usual manner. The blood crystallized readily at a temperature of 22° C. The crystals are quite insol- 190 Flos. 188. 189. Sciuropterug volans Oxyhemoglobin. Flos. 190, 191. Tamias striatu* Oxyhemoglobin. CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. 223 uble and keep well at ordinary room temperature, showing no tendency to dissolve, even in the rays of the electric arc lamp, when making the photo- micrographs. They are the usual oxyhemoglobin red, and were determined as oxyhemoglobin by the spectroscope. Oxyhemoglobin of Tamias striatus. Orthorhombic (?): Axial ratio about a : 6 : 6 =0.9246 : 1 : 0.589. Forms observed: Unit prism (110), macrodome (101), brachydome (Oil). Angles: Macrodome angle 101 A 101=65° (normals); brachydome angle Oil A 011=61° (normals); prism angle (calculated) 110 A 1TO=85°30'. The prism angle was not observed, but was calculated from the two dome angles which were measured, but not very satisfactorily; hence the uncertainty as to the exact axial ratio. Habit prismatic on the vertical axis; the first prisms that develop are very long and slender; later, stouter crystals form on which some measurements of the terminal planes can be made. The common termination is the macrodome, one face much more developed than the other, giving the crystal a very monoclinic aspect (text figure 190). It may in fact be monoclinic, but the measurements of prism edge to macrodome seemed to be symmetrical in the crystals examined, and extinction is straight in all aspects. The prisms range in ratio of length to thickness from 15 : 1 to 100 : 1, and in most of them the terminal macrodome is unsymmetrically developed. In some a brachydome appears (text figure 191) and, some days after the slides were prepared, the two domes were seen in equilibrium, in a few cases. The crystals grow in radiating tufts from the protein ring and cover edge, and also scattered irregularly through the body of the slide; but they do not appear to form twins. Pleochroism is rather pronounced; a pale yellowish-red, b pale rose-pink, c deep red. The orientation of the elasticity axes is apparently a = a, b=b, c=i; but no inter- ference figure was made out. As stated above, the extinction is straight in all aspects of the crystals that could be examined. The optical character could not be determined, but, from the pleochroism, it should be positive. PRAIRIE-DOG, Cynomys ludovicianus. Plate 48. Specimens of prairie-dogs were purchased from collectors in Ohio and in Kansas City, and the animals were bled in the laboratory. Prep- arations were made from the corpuscles, but not from the whole blood, which probably prevented the characteristic plate-like crystals, common in rodent blood, from developing. The corpuscles were oxalated, ether- laked, and centrifugalized and from the clear solution the slide prepara- tions were made as usual. Only one type of crystals developed and these were not very favorable for observing the characters. They were oxy- hemoglobin. Oxyhemoglobin of Cynomys ludovicianus. Probably orthorhombic: No axial ratio determinable. Forms observed: Evidently a unit prism, but the terminations were not perfect. Angles: No angles of the crystals could be measured. Habit of the crystals obtained was long prismatic, practically hair-like, and taper- ing gradually to an acute point; but, in the larger crystals, a high power showed that they were four-sided prisms, with a lozenge-shaped cross-section; and they probably are orthorhombic, possibly tetragonal, but certainly not hexagonal. The polarization characters showed that they must be one of these three systems. The needles grow in tufts, radiating from a center, the adjacent tufts penetrating each other and forming networks of interlacing fibers. 224 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. The needles in the dense tufts show the oxyhemoglobin color, but individual needles are very pale owing to their tenuity. Pleochroism is noticeable, the direction of the length of the needles showing more color than the normal to the length. The elasticity is greater normal to the length of the fiber and less parallel to the length. They are so thin that no characters can be made out in convergent light; but extinction is straight in all aspects; and this, with the four-sided cross-section, reduces the possible crystal systems to two, orthorhombic and tetragonal. The lozenge-shaped section indicates that the crystallization is orthorhombic. The blood was examined before we had devel- oped our methods of retarding crystallization in order to produce better crystals, and hence this blood should be further investigated. GROUND-HOG OR WOODCHUCK, Marmota monax. Plates 49 and 50. Specimens of this animal were purchased at different times from collectors in eastern and central Pennsylvania, and were bled in the lab- oratory. The blood was collected in oxalate. The first preparations were made by laking the oxalated corpuscles, and centrifugalizing, and from the clear solution preparing the slides as usual. As these preparations produced mainly long needles, that did not show the crystallographic characters definitely, and as the hexagonal plates that finally appeared were so im- perfect that better preparations seemed necessary, others were made, using the whole blood, the preparations being made as above described. In these preparations from the whole blood, the first crystallization in the dried protein ring is in the form of minute hexagonal plates; these soon become covered by the rapidly developing needles, and in part dissolve; so that the slides finally contain only masses of the needles. A preliminary trial of diluting with the blood plasma, and etherizing strongly before centrifugalizing, proving satisfactory in developing the plates, preparations were made by diluting the whole blood with an equal volume of the blood plasma and laking, and carrying out the preparation as above described. In this diluted blood, the plates developed readily and grew to large size, with only a slight development of the rods. The hexagonal plates kept well and passed by paramorphous change into reduced hemoglobin and also into metoxyhemoglobin. The crystals at first formed were, in all cases, oxyhemoglobin. Crystals form very readily in solutions of either the corpuscles, the whole blood, or the whole blood diluted with plasma; but much more rapidly, of course, in solutions of the corpuscles alone than in the less concentrated solutions. The development of the needles, or of the plates, can be controlled at will by the amount of dilution. The same principle applies to other bloods that develop needles or hair-like crystals from the whole blood. Unfortunately, however, the amount of blood in the samples received was rarely enough to try the experiment, or the plasma was not in good condition owing to putrescence. In rodents in general, dilution of the blood by the plasma or serum will probably be found advantageous. Two kinds of tabular crystals were observed in the blood of the ground-hog; the one, hexagonal plates, that are probably only pseudohexagonal and mimetic twins of the second kind, which latter are in the form of rhombic plates, belonging to the monoclinic system. These two will be described as a-oxyhemoglobin and y-oxyhemoglobin, CRYSTALLOGRAPHY OF HEMOGLOBINS OP THE RODENTIA. 225 respectively. The rods are possibly a form of the y-oxyhemoglobin with prismatic development, but they appear to be orthorhombic, and will be called /3-oxyhemoglobin. The other forms observed, which are reduced hemoglobin and metoxyhemoglobin, were simply paramorphous alterations of the normal crystals. They appeared mainly in the a-oxyhemoglobin form, and only in slides that had been kept for some days. a-Oxy hemoglobin of Marmola monax. ^^—- — v Hexagonal or pseudohexagonal: Axial ratio not deter- \J" - • - 1 --- " ^J minable, as no pyramidal forms were observed. >• ---- ?92 Forms observed: Unit prism (10TO), base (0001). FlG- 192- ^a™'\0J?onax a'°xy Angles: Prism angle 60°; prism to base 90°. Habit thin tabular; in the whole blood preparations, the first crystals to appear are very minute hexagonal plates in the protein ring; these are later dissolved with development of the needles of /?-oxyhemoglobin. In diluted blood, the typical a-oxy- hemoglobin plates are developed; they are large, well-formed, and very regular hexagonal plates (text figure 192), occurring singly or in complicated groups in parallel growth orientation, either piled on the base (plate 49, figs. 290, 291, and 292) or in arborescent forms (plate 50, fig. 297) ; also in partial orientation, which looks complete on the base, but is seen to be partial in edge view, the plates radiating from the center of the main groups as though twinned in the zone of two opposite unit-pyramid faces (plate 59, figs. 295 and 296) . Interference with the slide and cover produces in these groups on edge broad lath-shaped individuals which look rather orthorhombic. Often a single large plate may have on its basal surface several small concentric groups, all in perfect parallel growth orientation with the main large crystal. Twins are on the unit pyramid, but owing to the tendency to produce radiating groups the angle of the pyramid could not be determined with any certainty. The color of the plates varies with the thickness, but they show rather strong pleo- chroism; u> deep red, e pale reddish to colorless. On the base, the crystal is singly refract- ing; on edge, the double refraction is quite strong, and the extinction is straight parallel to the base. In convergent light, a dusky cross appears on the basal aspect, showing the uniaxial character. The vertical axis is the direction of greater elasticity, cu > s, and the optical character is negative. [3-Oxyhemoglobin of Marmota monax. Orthorhombic or monoclinic: No axial ratio is determinable. Forms observed: Apparently two vertical pinacoids and a terminal dome or some- times one plane of such a dome. Angles: The crystals were not perfect enough to measure angles with exactness; the angle of the terminal dome seemed to be about 58° (normals), and the two pinacoids at right angles. Habit ordinarily hair-like, the ends tapering to a point, without any definite plane terminations; some larger crystals were lath-shaped and showed the dome or oblique termination described above (text figure 193). The crystals grew in tufts, radiating slightly; or in groups of such tufts, sometimes radiating from a center like the spokes of a wheel; along the protein ring they shoot out normal to the surface and form a con- tinuous mass of hairs on the inside of the ring; outside of it the crystals are larger and longer and the tufts more dense. The crystals are quite elastic, bending considerably before they break. They reach a large size, the individual tufts of hairs being easily seen with the unaided eye. When the crystals are lath-shaped, the flat surface of the lath is usually presented; and on this surface the pleochroism is quite marked. The length of the lath is apparently c, the width b, and the thickness a. On this aspect above described the axes b and c 15 226 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. show. The crystal is very thin so that the a direction is very short. The pleochroism on the flat is b pale yellowish-red, c pale red; or, when very thin, 6 colorless and c pale pink. On edge view the crystal shows probably from 5 to 10 times the thickness seen on the flat, and the colors are deeper; a pale pink, c deep red. On the flat aspect in con- vergent light a pair of dusky brushes of a biaxial interference figure shows; the conjugate axis is the long dimension of the lath c, but the brushes pass out of the field on rotation of the crystal. It seems probable from this that the acute bisectrix of the optic axes Bxa=c, and the optical character is positive. Calling the flat side of the lath the macro- pinacoid, the narrow edge the brachypinacoid and the dome a brachydome, the orienta- tion of the elasticity axes is a=a, b=6, c=<<; and the axial plane is the brachypinacoid. ir-Oxyhemoglobin of Marmota monax. Monoclinic: Axial ratio a : b : 6 =1.804 : 1 : 6; /? near 90° (?). Forms observed: Unit prism (110), base (001). Angles: Prism angle 110 A HO =58° (about ) ; the angle /? was not exactly observed, but appears to be near 90°. 193 Flo. 193. Marmota monax fl-Oxyhemoglobin. FIGS. 194. 195, 196. Marmota monax y-Oxyhsmoglobm. Habit thin tabular, consisting of the basal pinacoid, bounded by the unit prism (text figures 194, 195); but the crystals rarely occur singly, they form groups radiating from a center both on edge and on the flat (see plate 50). They occurred sparingly as a second growth in preparations made from whole blood, and appeared to be more soluble than the a and /^-crystals. The prism faces at first were curved, making measurement of the prism angle impossible, but later the crystals became more perfect. Single rhombic plates were rare, but the groups were not all irregular. Twinning is the normal type for these rhombic plates ("horse-type," text figure 196) with the twin axis in the base and normal to a prism-base edge. The composition face is the base. The crystals do not seem to elongate along the common edge so much as is usual in this type of twin, but remain symmetrical rhomboidal plates. Pleochroism and absorption are not noticeable on the flat view; the color is bright oxyhcmoglobin red. Double refraction is strong on the basal aspect as also on edge; extinction on the base is symmetrical. On edge the extinction is oblique looking along 6, and straight looking along a; the extinction angle is 11° from the trace of the base. On the basal aspect, in convergent light, a biaxial interference figure is seen, with the brushes unsymmetrically placed with respect to the normal to the base. The orientation of the elasticity axes is a A A about 10°, b=b, c A a = 11°. The angle /? not being exactly determined, the angle a A <5 is somewhat uncertain. The plane of the optic axes is the plane of symmetry (010) and the acute bisectrix Bxa=a; the optical character is hence negative. CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. 227 It would appear very probable that the hexagonal crystals of o-oxyhemoglobin were simply mimetic twins of this 7-oxyhemoglobin ; the twins consisting of a number of individuals twinned on the different prism-base edges, and producing a uniaxial effect as in the artificial twin of mica. Both the monoclinic and the hexagonal plates are nega- tive; the monoclinic have their axis of greatest elasticity within 10° (about) of the normal to the plate, which becomes exactly normal, by averaging, in the multiple twins; while, in the hexagonal, the axis of greatest elasticity is normal to the plate also. The double refraction is not so strong in the hexagonal form as in the monoclinic; and this would have to be the case in such mimetic twins, as the elasticity of a is diminished by its being inclined to the plate at an angle of some 80°, which would allow the influence of b and c to be shown in the direction normal to the plate; and, of course, these latter axes would neutralize each other to produce the apparently uniaxial character. If the ^-crystals were orthorhombic this lessening of the strength of the double refraction would not be so noticeable. Reduced Hemoglobin and Metoxyhemoglobin of Marmota monax. As noted above, the hexagonal plates passed by paramorphous change into reduced hemoglobin and metoxyhemoglobin without any apparent change of form or of optical character. This could readily happen in such mimetic twins, even though the angles of the original rhomboidal plates should be slightly different from those of the 7"-oxyhemo- globin; for the composite hexagonal crystal always changes the angles of the rhomboidal plates a few degrees, if necessary, to be exactly 60°. The change seemed to be from a-oxyhemoglobin to reduced hemoglobin, and then from reduced hemoglobin to the metoxyhemoglobin. In the reduced hemoglobin the pleochroism on edge is quite strong; e nearly colorless, usually pale lilac; a> deep rose-pink. The elasticity was e > w, and the optical character was negative. The metoxyhemoglobin showed the mixed spectrum of oxyhemoglobin and methemoglobin that is often described as methemoglobin. It seemed to be the final paramorphous change, following the change to reduced hemo- globin. The metoxyhemoglobin is also quite strongly pleochroic; e is colorless or pale yellow, w is deep reddish-brown. The elasticity and optical character are as in the reduced hemoglobin. BEAVER, Castor canadensis. Plate 51. The specimen was received from the Philadelphia Zoological Gardens in a putrid condition. The usual method of preparation was employed. Crystals formed readily after the slides were covered, and were at first long needle-like rods; but soon they became lath-shaped, and then plate- like crystals began to appear. They were not very stable, many crystals disintegrated and were dissolved within 24 hours after making the prep- aration. The crystals were oxyhemoglobin. Oxyhemoglobin of Castor canadensis. Monoclinic: Axial ratio a : 6 : t =1.732 : 1 : 6; /?=78° (about). Forms observed: Unit prism (110), base (001), orthopinacoid (100). Angles: Prism angle 110 A 1TO=60° (or very nearly); orthopinacoid to base 010 A 100=78° (about) =/?; prism edge to base, edge 110-TlO A 001=90°. Habit tabular on the base, the combination being prism and base with a greater or less development of the orthopinacoid, making generally hexagonal plates or truncated lozenge-shaped plates (text figures 197, 198). The first crystals to appear are needles; when these attain dimensions to show planes they are generally seen to be twinned, and are the twin on a twin axis in the base and normal to a prism-base edge, along which the crystal appears to be elongated (text figure 199). In some cases the twin in these prismatic crystals seems to be on a unit pyramid, interpenetrant and forming an oblique cross-shape in the cross-section (text figure 200). The plates appear when these twins 228 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. are visible; some are rhomboidal plates, apparently untwinned, but most of them seem to be twinned and in the twins the orthopinacoid planes seem to be more developed (text figure 201). By this twinning being several times repeated the crystals become nearly symmetrically hexagonal in outline, and perfect hexagonal plates appear sparingly (along with the obviously twinned crystals) that are apparently mimetic twins and really hexagonal in symmetry (text figure 202). The rhomboidal plates tend to grow into groups, by piling up of the plates (plate 51, fig. 303), and, as these are nearly all hexagonal in outline, due to development of (100), these groups closely resemble the similar forms seen in the hexagonal plates of other rodents, as the squirrels for example. 199 FIGS. 197, 198. 199, 200, 201, 202. Castor canadenrit Oxyhemoglobin. The color of the crystals is a bright scarlet or blood-red. Pleochroism on the basal aspect is hardly noticeable, but probably most of the crystals examined were twinned. The colors were: a yellowish, b yellowish-red, rather a strong color; c deep blood-red. On the basal aspect the double refraction is very weak, and extinction is very hard to observe; it is, however, symmetrical. On the edge view, the double refraction is stronger, and looking along a the extinction is straight; along 6 it is 8° from the trace of the base or from the clino-axis, a. On the base in convergent light the interference figure is readily seen — a nearly uniaxial cross, which opens and closes as the crystal is revolved, showing the crystal to be biaxial. The angle of the optic axes, 27? is not above 7° or 8°. The orientation of the elasticity axes is a A a =8°, the extinction angle; b=6, c A 6=4° (about). The plane of the optic axes is the plane of symmetry, and the acute bisectrix is the axis of least elasticity, Bxa =c. The optical character is hence positive. As nearly all of the crystals examined were twinned, and as these mimetic twins tend to become uniaxial, it is possible that the above-described interference figure is due to twinning; but if so, the orientation of the optic axes is not altered nor is the optical character. MUSKRAT, Fiber zibethicus. Plates 51 and 52. The living animal was procured from a collector and bled in the lab- oratory. The blood was oxalated, laked with ether and centrifugalized ; from the clear solution slide preparations were made in the usual manner. The crystals formed readily soon after covering the slides; at first, the crystals were fine needles, but afterwards these became lath-shaped, or flat prismatic crystals appeared amongst the needles; and, at the same time, tabular crystals began to appear. They kept well, showing no sign of dis- solving. Crystallization continued after sealing the slides, until practically the entire slide was filled with crystals. The crystals are oxyhemoglobin. CRYSTALLOGRAPHY OP HEMOGLOBINS OF THE RODENTIA. 229 Oxyhemoglobin of Fiber zibethicus. Monoclinic: Axial ratio a : b : t =1.6318 : 1 : 6; /? = 68°. Forms observed: Unit prism (110), base (001). Angles: Prism angle 110 A HO =63°; prism edge to base, edge 110-lTO A 001 =68° =/?. 204 206 Flos. 203, 204, 205, 206. Fiber zibethicu» Oxyhemoglobin. Habit thin tabular on the base, the crystal consisting of the base (001) bounded by the prism (110). The first crystals to form are long needles or lath-shaped crystals with oblique ends; among these soon appear blade-like crystals of the same habit, often twinned; and, at about the same time, lozenge-shaped tabular crystals appear all through the slides. These latter (text figures 203 and 204), which are undistorted crystals, are very symmetrical rhombic plates, sometimes untwinned, sometimes twinned repeatedly. They do not seem to form mimetic twins and develop into hexagonal plates, as is so commonly the case with rodents. The twins are of the usual horse-type (text figure 205), on a twin axis normal to a prism-base edge and lying in the base. But in the blade-like crystals these appear as contact twins with the common prism-like edge parallel to the length of the blade-like crystal; the blades being elongated in the direction of two oppo- site prism faces, and consisting, therefore, of the two basal faces and two prism faces and terminated by the prism faces. This elongation produces in the untwinned crystals a triclinic appearance. In these same blade-like crystals another kind of twinning is very commonly seen, on a unit pyramid as the plane of twinning, the twin being inter- penetrant and showing an X-shaped cross-section (text figure 206). These were also observed in the plates. The twins of the plates are, as stated, of the usual horse-type, but the plates being very symmetrical the group formed by the twinning has often the outline of a truncated triangle, and sometimes is nearly triangular. By parallel growth the plates become greatly elongated in the direction of the clino-axis, forming parallel growth groups, and they also grow together on the base in groups, extending in the direc- tion of the same axis. Sometimes the rhombic plates form radiating groups by uniting on the base, the radial character showing when the plates are seen on edge. On the base, the color of the crystals is a deep scarlet, owing to the very slight pleochroism on this aspect. Pleochroism is weak on the base, but strong when the edge aspect is presented; a pale reddish-orange, b blood-red; c blood-red, somewhat deeper than 6, but the two practically equal. Double refraction is so weak on the basal aspect that the quartz wedge scarcely shows the difference between 6 and c. On the edge view, looking along c or b, however, the double refraction is quite strong. On the edge view looking along b the extinction is oblique, about 15° in the acute angle. The orientation of the optic axes is a A «S=37°, in the obtuse angle; b=b; c A a = 15°, in the acute angle. The plane of the optic axes is the plane of symmetry (010), and the acute bisec- trix is the axis of least elasticity, J5x0 = a; the optical character is hence negative. 230 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. WHITE RAT, ALBINO OF Mus norvegicus (Mus norvegicus var. albus Hatai). Plate 53. A number of specimens were examined at different times, the living animals being bled in the laboratory. The general method of preparation was to bleed the animal into oxalate, lake the whole blood with ether, and centrifugalize. From the clear solution slide preparations were made as usual. Modifications of the method above described, using corpuscles and adding variable amounts of plasma in excess of the normal, gave about the same results as the preparations of the whole blood. The blood crystallizes very readily, so much so that the crystals are usually small unless methods of preparation are used that retard crystallization. Being small, they show but little color; the crystals examined were, in each case, determined to be oxyhemoglobin by the microspectroscope. A superficial examination shows that the crystals are of several habits, and they look as though they were of different systems. Careful study shows, however, that they are all of the same crystallization, although one type, a hexagonal plate, seems to be sometimes a mimetic twin of the normal crystals. Oxyhemoglobin of Albino of Mus norvegicus. Orthorhombic : The axial ratio was calculated from the traces of the macrodome on the prism, assuming the same prism as in Mus norvegicus; it is a : 6 : 6 =0.7829 : 1 : 0.7332. Forms observed: Unit prism (110), brachydome (101). Angles: The only angles that can ordinarily be observed are the plane angles between the edges produced by a prism face intersecting the two brachydome faces, that is edges 110-011 A 110-OTl =120°. The half of this angle can also often be meas- ured, and it is 60° actual angle. In twins of the stellate shape, the edges are inclined to each other at 60°. As this dome angle on the prism is the same as in Mus norvegicus, the true dome angle Oil A Oil is assumed to be the same also, 72° 30', which makes the prism angle 110 A 1TO=76° 7'. Habit thin tabular, elongated along the vertical axis and the crystal tabular on two opposite faces of the unit prism (110), the end being formed by the faces of the brachydome (text figure 207). The tabular crystals are thus roughly six-sided with two sides longer than the other four. Some symmetrically developed crystals of the combination of prism and brachydome were seen (text figure 208), but they were always very small. Generally when the prism faces were equally developed, which occasionally happened, the dome faces were unsymmetrical, two opposite faces being larger and the other two smaller, but usually two opposite prism faces were larger, making the tabular crystal, and the other two prism faces were smaller (text figure 209). The prism is very nearly square, although evidently not quite so; but no cross- sections of it could be obtained for measurement. The ratio given a : b =0.7829 : 1 was calculated by assuming the same prism that was determined for the Norway rat crystal, which gave the same plane angle of macrodome on prism face as in this albino variety. The usual crystals are hence like vertical sections of the prism, parallel to one pair of faces; and, as the section approaches the exterior of the symmetrical crystal, the outline of the section becomes nearly four-sided; whereas a median section is nearly regularly six-sided, with four short and two long sides. This flattening of the prism produces the tabular effect, and there is, therefore, a flat view and an edge view of each crystal possible. The above descriptions refer to the flat view, but the edge view is quite analogous. The crystals twin by growing together on a prism face either on the flat or on the edge aspect, with the prism edges of the individuals of the trilling (which it usually is), at almost exactly 60° with each other. When this is on the flat aspect the crystals seem to pile up on each other at the 60° angle (text figure CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. 231 210) and there may be more than three in the combination. This kind of grouping pro- duces roughly hexagonal plates, and even fairly regular hexagonal tabular crystals, in which the composite character can, however, usually be made out. Rarely, this com- posite character almost disappears when the members of the twin are many and very thin, and the crystal then becomes pseudohexagonal; this is the normal hexagonal crystal of the rodents. When, on the other hand, the crystals twin on edge, they seem to be more interpenetrant; although in these, too, there is often the appearance of piling up. The twins of crystals on edge are in the form of six-rayed star-shaped groups (text figure 211). In some cases the two aspects of the crystals are presented in the same group, as is natural, for there is no essential difference of structure on the two kinds of prism faces, the broad face and the narrow face. The twin on the flat seems to be on a brachypyramid nearly, or quite in the zone of the prism-dome edge, but the composition face is the unit prism; in the star-shaped twins, the twin plane and composition face are a pyramid of the unit series. 207 208 Fios. 207, 208, 209. 210, 211. Mia nonegicvi aibus Oxyhemoglobin. The crystals therefore occur in five habits or forms: (1) Prismatic crystals, long or short, consisting of the symmetrically developed prism and generally unsymmetrically developed brachydome. Rather rare, but the most nearly symmetrical crystal. (2) Elongated six-sided plates formed from (1) by flattening on two opposite prism faces. The common single crystal. (3) Composite and rough hexagonal plates, twins of (2) on the brachypyramid, presenting the tabular aspect. The common crystal. (4) Six-pointed star-shaped twins, produced by twinning on the unit pyramid, presenting the narrow planes or edge aspect. Almost as common as (3). (5) More or less regular hexagonal plates, mimetic twins of the type of (3). Rather rarely observed. The color of the crystals is rather pale, owing to their very small size, but pleo- chroism is quite noticeable; a and 6 nearly equal, almost colorless in these minute crys- tals; c is reddish-orange to deeper red. The orientation of the elasticity axes is only approximately made out for a and 6, which are nearly equal, but t = 6. The peculiar development of the crystals renders it nearly impossible to get views along axes a and b; there can be little doubt, however, that the acute bisectrix Bxa—c, which would make the optical character positive. 232 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. NORWAY OR BROWN RAT, Mus norvegicus. Plate 54. The specimen of blood was received from the Wistar Institute of Anat- omy, Philadelphia. The animal was bled into oxalate, and the blood used immediately. On laking, the blood at once began to crystallize, and within a few minutes a considerable amount of the crystals of oxyhemoglobin had formed in the tube. These were separated by centrifugalization, and from the clear mother-liquor the slide preparations were made. After cover- ing the slides crystals formed rapidly, and they were quite insoluble. The color of the plasma was almost entirely discharged, showing the crystal- lization to be nearly complete. The crystals were small and thin, as in the case of the white rat. They kept well and showed no tendency to dissolve N/213 Fios. 212, 213, 214, 215, 216, 217. Mia noruegicut a-Oxyhemoglobin. upon moderate increase of temperature. Even after a month the form of the crystals in the slides had not changed materially. The crystals were oxyhemoglobin. Two forms of the oxyhemoglobin appeared: one pris- matic, and probably orthorhombic, like the white-rat oxyhemoglobin; the other isotropic and apparently isometric, but showing hexagonal out- lines. The prismatic form was the first to appear; the isotropic form developing later may be an isomer of the first form or a mimetic twin. They are distinguished as a-oxyhemoglobin and /3-oxyhemoglobin. a-Oxyhemoglobin of Mus norvegicus. Orthorhombic: Axial ratio a : b : 6 =0.7829 : 1 : 0.7332. Angles: Brachydome angle Oil A Oil =72° 30'; the prism angle was not observed, but was calculated as 76° 7' ; profile of dome edges over pole when the prism lies on its side, edges 110-011 A TTO-OTl = 120°; this is the plane angle of the dome on the prism. Habit of the first crystals to form prismatic and generally flattened on two opposite prism faces, making a six-sided tabular crystal elongated on the vertical axis, as is com- mon in the white rat (text figure 212). Some symmetrically developed prismatic crystals were observed that showed the dome termination in symmetrical development; these looked like normal orthorhombic crystals (text figure 213). But the distorted crystals, CRYSTALLOGRAPHY OP HEMOGLOBINS OF THE RODENTIA. 233 flattened on two opposite prism faces (text figure 214), have a decidedly monoclinic aspect. Nothing that was observed of the optical characters could determine that these crystals were not orthorhombic ; but no end views, looking along the length of the crystal, could be obtained, and hence they may be really monoclinic. Twins on the brachy and unit pyramids formed as has been described for the white rat; on the flat aspect, twinned on the brachypyramid, they make pseudo-hexagonal groups; and twinned on the unit pyramid, with the edge of the flattened prism presented, they make six-pointed star- shaped groups (text figures 210, 211). What appears to be a twin on the prism also occurs, in some cases producing the effect of a carpenter's miterbox, where the two crystals on edge appear on either side of one presenting the flat aspect (text figure 215). As the crystals continue to develop, short prismatic crystals, flattened on two prism faces, appear, and they produce hexagonal plates, owing to the angle of 120° of the dome profile, which is of course the same as the profile of dome to prism outline (text figure 216). These crystals show much less double refraction than the elongated crystals and are sometimes practically isotropic. When the prism is symmetrically developed and in equilibrium with the dome (text figure 217) the crystals resemble octahedra, and they appear to pass into isometric octahedra, the /3-oxyhemoglobin crystal. Pleochroism is marked in the elongated prismatic crystals, but wanting in the hexag- onal plates and in the equidimensional, octahedral-looking, prism-dome combinations. The colors are a = 6 (about), pale yellowish-red to pale red; c deeper red. No end views were seen, so that the pleochroism of a and 6 could not be differentiated. Double refrac- tion is strong in the long crystals, but very weak or entirely wanting in the equidimen- sional crystals and in the hexagonal plates. The symmetrical crystals in convergent polarized light showed traces of the brushes of an interference figure, looking along the macro-axis, the brushes passing out of the field upon rotation of the stage, showing that the observation was being made on the obtuse bisectrix of the optic axes. The orienta- tion of the elasticity axes is a =6; 6=a; c = i. No observation of the interference figure, looking along the acute bisectrix was possible, but the acute bisectrix is the axis of least elasticity, Bxa=c, and the optical character is positive. p-Oxyhemoglobin of Mus norvegicus. Isometric or pseudo-isometric. Forms observed: Octahedron (111). Angles: The angle over the pole of the octahedron 111 A III —71° (about). 218 220 FIGS. 218,219,220. M us nortitgicut ,8-Oxyhemoglobin. Habit octahedral; symmetrical isometric octahedra, or distorted octahedra formed by the crystal lying on one face and hence developing into forms with a nearly triangular to almost hexagonal profile (text figures 218, 219, 220). These hexagonal sections of the octahedron closely resemble the hexagonal-looking tabular crystals of the a-oxyhemo- globin, but may be distinguished from them by careful examination. The crystals show the oxyhemoglobin red of the white and Norway rats, rather a pale brownish or yellowish-red. There is no pleochroism nor double refraction; the crystals appear to be absolutely isotropic. 234 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. The passage of the orthorhombic crystal of a-oxyhemoglobin into the isometric or pseudo-isometric crystal of /3-oxyhemoglobin, that was described above, depends upon the pseudo-isometric character of the a-oxyhemoglobin (its angles being near the angle of the octahedron), if no change in substance occurs in the change from the dis- tinctly orthorhombic crystal to the pseudo-isometric crystal of the hexagonal or equi- dimensional type. More probably there is a change of substance and an isomer or polymer is formed (or may already exist in the solution) and then the hexagonal or the equidimen- sional form is a mixed crystal containing both a-oxyhemoglobin and /3-oxyhemoglobin. The influence of the /3-oxyhemoglobin on the a-oxyhemoglobin, or the concentration of the solution, determines the conversion of the a-oxyhemoglobin into the other isomer (or polymer) /3-oxyhemoglobin. BLACK RAT, M us rattus. Plates 54 and 55. Specimens of the blood of the black rat were obtained from the Wistar Institute of Anatomy, of Philadelphia. The animal was bled into oxalate and the blood used immediately. The corpuscles, separated by centrif- ugalizing, were laked with ether, oxalated, and the solution again centrif- ugalized. From the clear solution thus obtained the slides were prepared. The blood crystallized very readily; in fact, it is probable that better prep- arations would have been obtained if the whole blood had been used. The crystals do not form so readily as those of the white rat or the Norway rat, but they are quite permanent, show no signs of dissolving on slight increase of temperature, and they keep for weeks in the slides. They are not nearly so insoluble as the crystals of the white or Norway rats, however, and upon an increase of temperature, up to a temperature of 25° C., they begin to dissolve, so that they can not be satisfactorily studied in warm weather. This character is in sharp distinction from the insolubility of the oxyhem- oglobin crystals of the white and Norway rats, which are permanent at temperatures up to 35° C. The crystallization is not so complete as in the case of the other rats mentioned, so that the fluid remains of a strong red color, showing much oxyhemoglobin still in solution. Oxyhemoglobin of Mus rattus. Orthorhombic: Axial ratio 0:6: 6=0.7829 : 1 : 0.5864. Forms observed: Unit prism (110), macrodome (101). Angles: No cross-sections of the prism could be observed. The only angle that can be determined is the plane angle of the brachydome on the prism face, edges 110- 011 A 1 10-01 1 =130° 26', average of nine measurements. Assuming the same prism for this rat that was determined for the Norway rat from the true dome angle and the plane angle of the dome on the prism, the axial ratio was calculated. This makes the macrodome of the black rat (101), the macrodome (405) on the axial ratio of the Norway rat, the average measured angle for the edges, 130° 26', agreeing exactly with the calculated value. Habit tabular on two faces of the prism, the crystal consisting of the prism (110) and the brachydome (Oil). The prism is flattened on two opposite faces, as is common in the rats (text figure 221), and the dome termination may be of four equally developed dome faces, or two large and two small dome faces, or even of two equally developed faces on one end and one large and one small face on the other end. In some crystals two dome faces appear at one end of the prism and only one at the other end, making a five-sided plate (text figure 222). When two dome faces on the same side of the crystal are developed (one at each end), the plate becomes unsymmetrically four-sided (text CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. 235 figure 223). By shortening of the prism the tabular crystal becomes hexagonal in outline (text figure 224) ; the crystals do not elongate into the long tabular crystals so common in the Norway and albino rats. Twinning on the flat and on edge, as seen in the white rat, was observed, but it did not occur so frequently as in the case of that species. The twinning is upon a pyramid in each case, as is common in the rats. The twins on edge were of two individuals only, as a rule, and did not form the six-pointed star like Norway and white rat twins (text figure 225). On the flat, the twin consists of two individuals also (text figure 226), and does not result in the formation of a hexagonal plate, as in the case of the Norway and white rat crystals. The difference in the twins is no doubt due to the fact that in the twin on the brachypyramid the prism edge of one individual is parallel to the prism-dome trace of the other; but in the corresponding twins of the white rat and the Norway rat crystals the angle of the twin of the prism edges in the three members is 120°, while in the black rat (and Alexandrine rat) the angle of the prism edges in the two members of the twin is about 130° 25', being for the Alexandrine rat 130° 19' and for the black rat 130° 26'. Three crystals of the Norway or white rat could twin at the angle of 120° to make a regular hexagonal plate; but three crystals of the black or Alexandrine rat so twinned could not produce a regular hexagonal plate. 224 FIGS. 221. 222, 223, 224, 225, 226. M u» ratlut Oxyhemoglobin. Pleochroism is fairly strong, but from the positions of the crystals presented a and b can not be directly observed. The colors of a and b are evidently close together, rang- ing from pale yellowish-red to deeper red, according to the thickness of the crystal. The color of c is always much deeper; even in the thinner plates it is a deep red. Double refraction is strong, extinction is straight in all aspects presented. The orientation of a and b could not be observed; it is probably the same as in the Norway rat, a =b, b =a; the axis of least elasticity c = deep red. The vertical axis (=) is the direction of greatest elasticity; all directions normal to this (o») are of equal and less elasticity. Double refraction is strong; extinction is parallel to the vertical axis, or symmetrical in all of the usual aspects. Looking along the vertical axis, in convergent polarized light, the uniaxial cross is seen. The refractive indices are w > E and hence the optical character is negative. CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. 243 ^-Oxy hemoglobin of Hydrochcerus capyvara. Orthorhombic : Axial ratio not determinable. Forms: Unit prism (110)? terminations were wanting. Angles: No angles could be measured. Habit prismatic, the crystals appear to be a prism, much elongated; but perhaps the planes may be two pinacoids. When examined, a few hours after the crystals had begun to form, the terminations were imperfect and not measurable. Later they were lost by resolution. Pleochroism was weak; c deep red, a (or b) somewhat paler. Extinction is parallel to the length of the crystal in all aspects that were examined. On the side view, in some cases, a biaxial figure was seen, with the plane of the optic axes including the vertical axis, which is evidently c; this must, therefore, have been seen when looking along a and the optical character is negative, as in the case of the tetragonal crystals of a-oxyhemoglobin. The crystallization is evidently orthorhombic, but the crystallographic constants can not be determined beyond those already stated. DOMESTIC RABBIT, Lepus cuniculus. Plates 59-61. The living animal was purchased and bled into oxalate in the labora- tory. The corpuscles were separated from the plasma by centrifugalizing; and the preparations were made from the corpuscles by laking with ether and centrifugalizing for 3 hours. The slide preparations were made from the clear centrifugalized blood as usual. Crystallization begins in the protein ring soon after the slides are covered, and it proceeds rapidly at room temperature. As the solution under the cover comes to an equilib- rium, these first crystals dissolve and disappear from the slides. Upon putting the slides in the cold at near 0° C. a second crop of crystals appears, some of which are like the first crop and some are of a different type. These are distinguished as a-oxyhemoglobin and /2-oxyhemoglobin. The a-crystals are less soluble and may be examined at near room temperature; but the /3-crystals are much more soluble, and dissolve rapidly when the tempera- ture is raised a few degrees above 0° C. They had to be examined and photographed in a room temperature near the freezing-point. Another preparation, made from the same blood, was evidently not evaporated quite to the same point as the first before applying the cover; for, while two types of crystals developed, both tended to dissolve upon increasing the temperature a few degrees above 0° C., and they therefore had to be exam- ined at about this temperature. So long as the preparations were kept at about the freezing-point the crystals continued in excellent condition. a-Oxyhemoglobin of Lepus cuniculus. Monoclinic: Axial ratio a : b : c =0.643 : 1 : 0.797; /? = 85°. Forms observed: Unit prism (110), clinoprism (320), clinodome (Oil), clinopina- coid (010), orthopinacoid (100). Angles: Unit prism 110 A 1TO=65° 30' (normals); clinoprism 320 A 320=88° (normals); clinodome Oil A Oil =77°; prism edge to dome edge, in the plane of sym- metry = 85° (normals) =/?, or 95° actual angle. Two habits of crystals develop: (a) the first crystals to appear are usually pris- matic, consisting of the two pinacoids and the clinodome, elongated vertically and flat- tened on (100) (text figure 247), and with or without the unit prism (110) (text figure 248) ; (b) the second type, which is much more symmetrically developed, consists of the clinoprism (320) in combination with the clinodome and the clinopinacoid (text figure 249). Type (a) crystals are elongated vertically and striated on the orthopinacoid; 244 CRYSTALLOGRAPHY OF HEMOGLOBINS OF THE RODENTIA. by the disappearance of this plane and the development of the clinoprism, while, at the same time, the clinopinacoid becomes larger, they pass into the second type of crystals. Type (b) crystals are tabular on the clinopinacoid, elongated vertically, and generally smooth, not striated, as in the type (a) crystals. Both kinds of crystals of the a-oxyhemo- globin are much smaller than the crystals of the ,3-oxyhemoglobin. The (b) type of crystals form parallel growths and also seem to twin on the orthopinacoid; the twinning was observed in polarized light. The color is the usual oxyhemoglobin red, but the crystals are quite pleochroic; a colorless or pale yellowish; b rose-pink to pale red; c deep red. Double refraction is moderately strong on most aspects; in all sections in the zone of 100-001 the extinction is straight or symmetrical; on the plane of symmetry, looking along b, the extinction is oblique; 15°, in the obtuse angle, from the prism edge. On this aspect in some crystals, a biaxial interference figure was seen with the above orientation, the plane of the optic axes being inclined to the vertical axis 6 at 15°. The orientation of the elas- ticity axes is a A (5 = 15°, in the obtuse angle; b A a = 10°, in the acute angle; c=&. The plane of the optic axes is hence normal to the plane of symmetry. The angle between the optic axes was not accurately measured; the separation was considerable, however. The acute bisectrix emerges normal to the plane of symmetry; it is c and hence the optical character is positive. 247 N/ a/ 248 249 251 FIGS. 247,248,249. Ltput cuniculus a-Oxs-hemoglobin. FIGS. 250,251. Lepui cuniculue B-Oxyhemoglobin. ^-Oxyhemoglobin of Lepus cuniculus. Orthorhombic : Axial ratio a : b : c =0.5317 : 1 : c. Forms observed: Unit prism (110), base (001). Angles: Unit prism angle 110 A 110=56° (normals); prism to base 110 A 001 = 90°. Many oblique sections of the prism in the position of a brachydome were produced by the slide and cover; these had the angle of the prism (210), but were oblique sections, as could be shown by their optical properties. This angle of the prism (210) is 30°. Habit tabular on the base; or, in case of the crystals that were interfered with by the slide and cover, flattened on a brachydome. The tabular crystal consists of the very short prism cut by the base, and in some cases traces of a macroclome were seen (text figures 250 and 251). These crystals are many times the size of those of the a-oxy- hemoglobin. The oblique sections of the crystals are particularly common, but they are not always at the same angle, nor in the zone of the brachydomes. Their angle generally runs near 30°. These /^-crystals are much more soluble than the a-crystals as a rule, and they are corroded so rapidly that, in spite of the fact that they were photo- graphed at a room temperature of about 0° C., they show the effect of solution in etching figures, which appear on their surfaces in many of the photographs. The crystals were somewhat more of a scarlet-red color than the a-crystals, but the absorption spectrum was the same, that of oxyhemoglobin. The difference in color is due to the difference in the pleochroic colors. The pleochroism is a pale yellow-red to nearly colorless; b pale scarlet-red; c deep red. The orientation of the elasticity axes is 0=6, b=o, C = -OHb. Castor canadensis 1.732 :1:4 (58* 0) 120 0 78 aAo= 8" Positive Do. OHb. Fiber zibet hieus 1.6318: 1:4 (60* 0) 117 0 68 cAa=15° Negative Do. OHb. Mus norvegicus albino 0.7829 • 1 • 0.7332 (63* 0) 76 7 90 0° Positive Orthorhombic OHb. Mus norvegicus 0.7829 : 1 : 0.7332 76 7 90 0° Do. Do. a-OHb. Do 1:1:1 90 0 90 Isotropic Isometric /J-OHb. Mus rattus 0.7829 : 1 : 0.5864 76 7 90 0° Positive Orthorhombic OHb. Mus alexandrinus 0.7829 : 1 : 0.5880 76 7 90 0° Do. Do. OHb. 0.5543 : 1 : t 58 0 56 bAa = 20° Do. Monoclinic a-OHb. Do 0.8170: 1: t 78 30 90 0° Do. Orthorhombic ,3-OHb. Cavia cutleri, domesticated variety 90 0° Do. Do. OHb. 1 : 1.8184 90 0 90 0° Negative Tetragonal a-OHb. Do . . 90 0° Do. Orthorhombic /3-OHb. Lepus cuniculus . . 0.643 : 1 : 0.797 65 30 85 aA6 = 15° Positive Monoclinic a-OHb. Do 0.5317:1:6 56 0 90 0° Do. Orthorhombic ,:i-OHb. Lepus europceus 0.6588 : 1 : 0.8069 66 0 85 a A 4= 15° Negative? Monoclinic a-OHb. * True angle of traces of prism on base. CHAPTER XIV. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE OTARIID^E, PHOCID-ffi, MUSTELID^E, PROCYONID^E, AND URSID-ffi. Under Carnivora zoologists distinguish two suborders, (1) Carnivora vera and (2) Pinnipedia. The distinction is mainly in the structure of the limbs, which in the Carnivora vera are normal for terrestrial animals and in the Pinnipedia are modified for aquatic progression. In the arrangement of species here adopted the Pinnipedia are considered first, and then the Carnivora vera, beginning with the species which, from their hemoglobin crystals, appear to be most nearly related to the Pinnipedia, namely the Mustelidce, Procyonidce, and Ursidce. The Pinnipedia are divided into the eared seals, Otariidce, including the sea-lions and sea-bears, of which one species, the California sea-lion, was examined ; the walruses, Trichechidce, of which we had no representa- tive; and the Phocidce or earless seals, of which the harbor seal was exam- ined. These are all evidently descendants of some terrestrial mammals, and from the resemblances between the skulls of the eared seals and the bears of the Carnivora vera, Mivart (Proc. Zool. Soc., 1885, p. 497) has suggested that there exists a true genetic relationship between the two groups. Mivart states that while the sea-bears may be thus related to the bears, both being derived from bear-like carnivores, the true seals may on the other hand be genetically related to the sea-otters. The true bears are a modern group, and a common bear-like ancestor for them and for the eared seals is entirely possible. It will be seen from a comparison of the hemoglobin crystals of the sea-lion and of the bears that they have a certain very remarkable character in common, namely, a habit of twinning that is very unusual, and is identical in the two groups, but which produces differ- ent-looking crystals, owing to the development of the planes of the crystals being different in the two groups. This is not the only point of resemblance between the two groups, as will be shown. The Mustelidce and Ursidce are evidently closely related, as indicated by their hemoglobin crystals ; the Procyonidce, however, so far as our investi- gations have gone, do not show close relationship to the other groups above mentioned. Of the Mustelidce the common ferret, Mustela putorius (domes- ticated variety) ; the skunk, Mephitis mephitica putida; the badger, Taxi- dea americana, and the otter, Lutra canadensis, were examined. They show strong resemblance to each other and to the bears and seals, with the exception of the skunk, which more nearly resembles the Procyonidce from what data we were able to obtain in regard to its hemoglobin crystals; but in the case of this species the data were incomplete. 247 248 CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE Of the family Procyonidoe, the species from which crystals were exam- ined were the kinkajou, Cercoleptes caudivolvulus, and the cacomistle, Bassariscus astuta. The blood of the raccoon was experimented with early in our work, but no satisfactory crystals were obtained. They no doubt can be produced by the use of a suitably modified process of preparation, such as we have resorted to in our more recent work. The family Ursidce is represented by three species, the black bear, Ursus americanus; the polar bear, Ursus maritimus, and the sloth bear, Melursus ursinus. Their crystals all closely resemble each other, and are characterized by the peculiar cyclic trillings already alluded to as being found in this group and in the Otariidce. All belong to the same class of crystals, monoclinic hemimorphic or monoclinic sphenoidal, which has thus far been seen only in this family and in the Otariidce and Phocidce, but may very possibly occur in the otters and ferrets also, although they have been determined as monoclinic hemihedral, or domatic (clinohedral group of Dana). PHOCIDiE. HARBOR SEAL, Phoca vitulina. Plate 62. Specimens of blood of the harbor seal were received from the Phila- delphia Zoological Gardens, from the National Zoological Park at Washing- ton, District of Columbia, and from the Zoological Garden at Detroit, Mich- igan. In each case the blood was not quite fresh, but all were in fairly good condition and were not putrid, except in the case of the specimen from Detroit, which was slightly putrid. The blood from Washington was frozen; all of the specimens were treated in the same manner with this exception. The bloods were oxalated, ether-laked, and centrifugalized, generally from 2 to 3 hours; and from the clear solution thus obtained the slide prepara- tions were made. The crystals formed slowly at room temperature, and rather more rapidly at a temperature near freezing, but the color of the solution remained a deep red, showing that much of the oxyhemoglobin was still in solution. The crystals were kept at temperatures near the freezing-point, but when brought into the warm room did not appear to dissolve, and even on the stage of the photomicrographic apparatus they did not lose their form. The solution was very deeply colored and of about the color of the crystals, which were oxyhemoglobin. Oxyhemoglobin of Phoca vitulina. Monoclinic hemiruorphic, or monoclinic sphenoidal (tartaric acid type) : Axial ratio 0:6: (5 = 1.2131:1 : 1.1970; /? = 75°. Forms observed: Unit tetartopyramid (Til), unit hemiprism (1TO), orthopinacoid (100), base (001). Angles: Unit prism to unit pyramid edges on base, edges 1TO-001 A llT-001 =101°, or traces of unit prism edges lTO-001 A TTO-001 =79°, and the traces of the unit pyramid on the base give the same angle. The angle ,3 on sections parallel to (010) or 100 A 001 = 75°; and the angle of the trace of the unit pyramid on the same section or 101 A 001 =52°. Habit tabular on the base, the crystal consisting of the basal pinacoid bounded by the positive unit pyramid at the positive extremity of the ortho-axis and by the unit prism at the negative extremity of this axis (text figures 256 and 257). In many cases the orthopinacoid developed, but it was not always present. Sometimes this orthopina- OTARIID^E, PHOCID.E, MUSTELID^, PROCYONID^E, AND URSID^E. 249 coid was so strongly developed as to produce a prismatic habit on the ortho-axis, the crystal being 5 to 10 times as long on this axis as on the clino-axis. The plates were generally rather thin ; edge views showed a ratio of length on the ortho-axis to the thick- ness of the plate of 10 : 1 or more, but in some cases this became 5:1. The crystals grow singly or in radiating groups, showing a tendency to twinning on the clinodome; but this plane was not developed and there were no definite twins made out. Parallel growth by piling up on the base and the group extending along the ortho-axis was a very common habit in the larger crystals. Pleochroism is hardly noticeable on the flat aspect of the plates, but quite strong on edge views. The colors are: a pale red, b and c nearly equal and deep red. The orientation of the elasticity axes is a = i, b=6, c A a = 15°, in the obtuse angle. The plane of the optic axes is the plane of symmetry. On the base, in convergent polarized light, one brush of a biaxial interference figure is seen and traces of the other. The acute bisectrix of the optic axes is the axis of greatest elasticity, Bxa=*a and the optical char- acter is hence negative. FIOB. 256, 257. 1'hucu vitulina Oxyhemoglobin. Flos. 258, 259, 260. Otaria gttletpii Oxyhemoglobin (first orientation). CALIFORNIA SEA-LION, Otaria gillespii. Plates 63 and 64. Specimens of the blood of the sea-lion were received from the National Zoological Park at Washington and from the New York Zoological Park. In both cases the blood was rather stale, and in one case it was clotted. The specimens were oxalated and laked with ether, and cleared by centrif- ugalizing for several hours. From the clear blood slide preparations were made in the usual manner. The blood crystallized at room temperature, and the crystals did not readily dissolve on slight increase of temperature. Bacteria in the preparations destroyed the crystals after some days. A preparation of CO-hemoglobin kept well and developed very fine crystals. From these the most satisfactory measurements were obtained. The crys- tals grew to much larger size than was the case with the oxyhemoglobin crystals in the other preparations, and the planes of the crystals were per- fectly developed. The angles, as far as they could be measured in the oxy- hemoglobin crystals, corresponded to the good measurements obtained from the CO-hemoglobin crystals, and the optical characters appeared to be identical. The crystallographic and optical constants are hence derived from the CO-hemoglobin with greater exactness. 250 CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE Oxyhemoglobin of Otaria gillespii. Monoclinic hemimorphic or monoclinic sphenoidal (tartaric-acid type) : Axial ratio a : b : 6 = 0.7883 : 1 : 1.7314; /?= 74°. Forms observed: Pyramid of unit series, not measured, may be called unit pyramid (111), unit prism (1TO), orthopinacoid (100), clinodome (Oil), base (001). Angles: Traces of unit pyramid on base, edges llT-001 A Tl 1-001 =76° 30', or edges llT-001 A lTO-001 =103° 30'; traces of unit prism on base at opposite pole, edges 110-001 A TTO-001 =76° 30'. The angle of the clinodome was not accurately determined, but was very near 60°. The angle of the orthopinacoid to base 100 A 001 = 73° 30' to 74° =0. Habit tabular on the base and elongated on the ortho-axis, the crystal consists of the base bounded by the orthopinacoid and at the positive end of the ortho-axis the unit pyramid, while at the negative end of this axis is found the unit prism and the clinodome (text figures 258 and 259). The crystals show a tendency to grow together in radiating groups uniting at the negative end of the ortho-axis and also in parallel growth on the base, piling upon each other in perfect orientation. These groups in parallel growth also expand along the clino-axis, frequently with the prism and clinodome planes in common, but the pyramid planes showing the parallel growth (1559). Twins on the clinodome are common, the angle being nearly 60°; they form in trillings, very frequently radiating from a common axis, the common clinodome edge (text figure 260). The hemimorphic character is found in the smallest crystals as well as in the larger ones, and there is not much change of shape of the crystals as they grow from small to large size. Pleochroism is strong; a nearly colorless, b rather strong red, c deep red. Double refraction is fairly strong, extinction is straight or nearly so in all ordinary aspects. The orientation of the elasticity axes is a=a or veiy nearly, b=6, c A 6 = about 16° 30' in the obtuse angle or the angle =90°-/?; hence the axis c is nearly or quite normal to the base (001). The plane of the optic axes is the clinopinacoid and the acute bisectrix is the axis of greatest elasticity; Bxa = a and the optical character is negative. The axial angle 2£=35° to 40°. CO-hemoglobin of Otaria gillespii. Monoclinic hemimorphic or monoclinic sphenoidal (tartaric acid type) : Axial ratio a : b : c =0.7883 : 1 : 1.7314; /3 = 74°. Forms observed: Unit pyramid (111), unit prism (1TO), clinodome (Oil), ortho- pinacoid (100), base (001). Angles: Traces of unit pyramid or of unit prism on base 76° 30' actual angle, or 103° 30' normals as measured at the ends of the ortho-axis, making the edges of these two unit forms on the base llT-001 A 110-001 = 103° 30' as recorded for the oxyhemoglo- bin; clinodome angle Oil A OTT = 60° to 61° (taken as 60° in calculation of the axial ratio); orthopinacoid to base 100 A 001 =74°=/3. The pyramid called unit on the base, angle 111 A 001, was not obtained. Habit tabular on the base and elongated on the ortho-axis, exactly as described for the oxyhemoglobin (text figures 258, 259). The crystals grow in groups and in regular growths as there described, but are much more perfect. Twins and trillings are very common on the clinodome as described under the oxyhemoglobin; the angle of the trilling is generally 120°, but some were 121°, 118°, perhaps not quite in symmetrical position (text figure 260). These twins show a cyclic arrangement of the unit pyramid planes, the three planes of the unit pyramid on one side of the trilling being corresponding planes. The crystals are strongly pleochroic; a colorless, b old rose, c deep crimson. Double refraction is rather strong, except on edge views looking along the axis of greatest elas- ticity. Extinction is straight with the basal edges and symmetrical on the base, as though orthorhombic. The orientation of the elasticity axes is the same as in the oxyhemoglobin, a=a, b=6, c A OTTh Do 1.8019:1:0.7883 1.S019: 1 : 07883 76 30 74 0 cAi— 16° Do Do f'OTTh Mustelidse: Mustela putorius, var .... 1.2799:1:1.1105 76 0 68 0 Do. Monoclinic OHb Mephitis mephitica putida a : k = 1 : 0.4877 90 0 0° c Aa— 15° domatic OHb Taxidea americana 1.0355- 1 • 1 0125 88 0 54 32 a A t — 20° 28' OPTh Lutra canadensis 1 2131 • 1 • 0 6794 79 0 72 0 b Aa— 15° Dn nwh Procyonidae : Cercoleptes caudivolvulus Bassariscus astuta 0.6556 : 1 : 0.4663 0 7399 • 1 • 0 3939 66 30 73 o 90 0 90 0 CAi= 3° 0° 0° Do. domatic Orthorhombic Do OHb. riTTh Ursidse: Ursus americanus 1.2239: 1 • 1.1429 78 30 75 5 c Aa— 19° OHh Ursus maritimus 1 2088 • 1 'k 79 12J 73 2 C Aa — 20° Do hemimorphic Dn OTTK Melursus ursinus 1.2857 • 1 • 1 498 75 35 68 40 c Aa — 20° Do Do OHh CHAPTER XV. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE CANID^E— DOGS, WOLVES, AND FOXES. Twelve members of the family Canidoe were studied, representing 10 distinct species, and two varieties or crosses. Some of the species were represented by several specimens, so that their crystals could be studied under different methods of preparation. As a group, the dogs possess hemoglobins of a rather insoluble character, the crystals form readily and they do not readily dissolve. All members of the family furnished oxy- hemoglobin crystals which closely resembled each other, so that the differ- ences between species were not readily made out. All the crystals, without exception were orthorhombic, and the optical character was negative in each case. The axial ratios were so nearly alike, especially the ratio of a : c, which was determinable in each case, that, in spite of the fact that the zoologists place the 10 species examined in three different genera, they show much less difference among themselves than is common with the species of a single genus. Six species and two varieties of the genus Cam's were studied, also three species of the genus Vulpes, and the gray fox Urocyon. Of the genus Cam's, the species examined included the common dog, Cam's familiaris; the chow dog, a variety of the same species; a cross between the coyote and a collie dog; the gray wolf, Cam's lupus mexicanus; the coyote, Cam's latrans; the jackal, Canis aureus; the dingo, Cam's dingo; and Azara's wild dog, Canis azarce. This list of dogs includes animals from Europe, Asia, and Australia, besides those from North and South America. In spite of this wide range, however, the species examined show a remarkable resemblance in their crystals. The four foxes studied were the Swiss fox, Vulpes vulpes, the American red fox, Vulpes fulvus; the Arctic or blue fox, Vulpes lagopus, and the gray fox, Urocyon cinereoargenteus. The general type of crystal, common to all species of this family, is a more or less elongated prism, with a diamond-shaped cross-section, and usually strongly striated longitudinally, terminated by a rather flat dome. The striation of the prism is due to the tendency of the crystals to form in needles and aggregate into bundles of crystals; and this makes the dome terminations rather small, so that the angles, on which the crystallographic constants are based, are only determined with some difficulty. The prism angle is still more difficult to observe, as cross-sections are hard to find that are sufficiently symmetrical for trustworthy measurements; and all examinations of angles must be made with a moderately high-power objec- tive. On account of these difficulties of measurement, the complete axial 265 266 CRYSTALLOGRAPHY OF THE HEMOGLOBINS ratio was not always obtained ; but, as noted above, the dome angle, giving the ratio of a : 6, was made out in each case. It is very constant in the entire group, as will be seen from table 44, page 279 ; and the common dog and the chow dog, domesticated varieties, include the extremes of varia- tion noted in this ratio. Different strains of the common dog vary among themselves in such a way as to lead to the conclusion that they are a poly- phyletic group; some individuals seem to approach the wolf, others the jackal, etc. But this part of the subject has not been worked out with sufficient detail to warrant any final conclusions. In all cases but one the material examined was oxyhemoglobin ; and as the material in this one case was metoxyhemoglobin, into which the oxyhemoglobin was in several other cases seen to pass by paramorphous change, it may safely be said that all of the crystals examined were strictly comparable. It is well known that crosses between the dogs are readily obtained; and, from the close resemblance of the crystals in the species examined, it would seem probable that any one of these species could cross with any other. CANIDJB. DOG, Cants familiaris. Plate 72. Specimens of dog blood were obtained from living animals in the laboratory. Preparations were made from the whole blood defibrinated by beating; from blood kept liquid by oxalating; and also from mixtures of whole blood and the blood plasma. The blood, either defibrinated or oxalated, was laked with ether and centrifugalized ; and from the clear solution thus obtained, with or without the addition of plasma, the slide preparations were made. Two forms of crystallization were observed in the oxyhemoglobin; the first, distinguished as a-oxyhemoglobin, is the normal form and crystallizes in the orthorhombic system; the second, which is designated as /2-oxyhemoglobin, is monoclinic. The a-oxyhemoglobin is readily obtained by any of the methods of preparation above mentioned and is very insoluble, the crystals continuing to form until the color is practically discharged from the solution, and the slide is filled with a mass of needles in most cases. When the preparation is thick, the crystals become so massed together in the slides that the preparations are useless for crys- tallographic investigation (see plate 72, fig. 429). The /3-oxyhemoglobin crystals were only occasionally observed (in perhaps one out of a dozen slides), and appeared to develop more readily in the blood to which no oxalate had been added. Aside from this, no difference was noted in preparations made with and without oxalate. All crystals became brownish on standing under the cover for some days, and passed by paramorphism into the metoxyhemoglobin, but without any change in their angles. a-Oxyhemoglobin of Canis familiaris. Orthorhombic: Axial ratio a : b : 6 =0.6745 : 1 : 0.2863; a : c = l : 0.4245. Forms observed: Unit prism (110), macrodome (101), base (001). Angles: Prism angle 110 A 110=68° (normals); macrodome angle 101 A 101=46° (normals) (also measured on some specimens as 44°). OF THE DOGS, WOLVES, AND FOXES. 267 Habit prismatic, elongated parallel to the vertical axis, and the prism terminated by the flat dome (101) (text figure 293) or sometimes by the base (text figure 294). The first crystals to form are usually hair-like, but stouter crystals later develop along the protein ring and the cover edge. These stouter crystals may also appear in the body of the slide, and may even be doubly terminated. They are often seen to be composite, by parallel growth in the zone of the vertical axis; and are strongly striated in this direction, because of the parallel growth. Cross-sections are rare, and almost never appear in measurable form until the slides are several days old. The ratio of length to thickness varies from 500 : 1 in the hair-like crystals, down to 20 : 1 in some of the stouter crystals. Twinning was not observed, but a parallel growth, in which the crystals grow together upon the brachypinacoid, is very commonly seen. In such groups two prisms are seen in perfectly parallel orientation, and united side by side on the brachy- pinacoid. This is a character quite common in all of these rod-like orthorhombic crystals in other species also. Pleochroism is marked; a nearly colorless, 6 rather pale red, c deep red. Double refraction is strong; and extinction is straight in all side views and symmetrical on cross-sections of the prism. The orientation of the optic axis a=a, b=6, c=6. No interference figure was observed; but, from the relative elasticities in different directions, it would appear probable that the optical character was negative and the acute bisectrix Bxa=a. (3-Oxyhemoglobin of Cam's familiaris. Monoclinic: Axial ratio not determined, ,3=78°. Forms observed: Unit prism (110), clinopinacoid (010), orthopinacoid (100), basal pinacoid (001). Angles: Orthopinacoid to base 100 A 001=78°=/?; the prism angle appears to be acute on a or a > 6, but no cross-sections were found from which this angle of the prism could be obtained. Habit (a) prismatic on the vertical axis, the crystal consisting of the unit prism, clinopinacoid, and base (text figure 295); the crystals rather large and well-formed; also (b) tabular on the clinopinacoid, the crystal consisting of the three £ pinacoids only, with the base and P**1 orthopinacoid in equilibrium; thus making a rhomboidal plate (text figure 296). Both kinds of crys- tals, types (a) and (b) , were found very sparingly, in a preparation of defibrinated blood without oxalate; the plate-like crystals of type (b) were seen still more sparingly in a preparation in which oxalate was used. Often preparations of defi- brinated blood failed to develop these crystals. Pleochroism is very strong; a pale yellowish-red, b rose-pink, c deep blood-red. Double refraction is strong, and the extinction, meas- ured from the prism edge, is 15°. The orientation of the optic axes is as follows: a A a =27° in the obtuse angle; b=b; c A c = 15°, in the acute angle. Looking at the crystal, normal to the orthopinacoid, in convergent light, one brush of the interference figure is seen, showing that the axis of greatest elasticity is the acute bisectrix Bxa=a, and the optical character is hence negative. The plane of the optic axes is the plane of symmetry, and the axis of mean elasticity is the ortho-axis. The crystals twin on a prism, and are frequently seen so twinned, in such a position that the extinction is symmetrical to the 293 295 297 294 FIGS. 293, 294. Canis familiaria a-Oxyhemoglobin. FIGS. 295, 296. 297. Canis familiaris |S-Oxyheraoglobin. 268 CRYSTALLOGRAPHY OP THE HEMOGLOBINS twin plane (text figure 297) . In such twins, the extinction angle is, of course, less than that recorded above, about 7° or 8°. CHOW DOG, Cam's familiaris var. Plate 73 . The specimen of blood was received from the Philadelphia Zoological Gardens, and was in a clotted and rather putrid condition. The specimen was ground in sand and etherized and then centrifugalized for several hours; and from the clear solution thus obtained the slide preparations were made as usual. Crystals form rapidly and readily at room temperature, and show no sign of dissolving. Within 3 hours after the slide preparations were made, satisfactory photographs were procured. The crystals were oxyhemoglobin, and resemble those of the common domestic dogs very closely; appearing, however, to differ slightly in angles. Oxyhemoglobin of Cam's familiaris var. Orthorhombic : Axial ratio a : b : , deep red. Ex- tinction is straight in all side views and the crystals are singly refracting on the base. In convergent light a faint uniaxial figure is seen on the base. The axis of greatest elasticity is the vertical axis c. Hence w > £ and the optical character is negative. Hemoglobin of Scalops aquaticus. Hexagonal, only observed in paramorphs after the oxyhemoglobin. The forms and angles are hence identical in the two substances. The reduced hemoglobin para- morph is produced by bacterial action. The bacteria enter at the basal depressions and frequently penetrate the crystal from end to end, which then becomes like a short hex- agonal bead with a central perforation. They work through the substance of the crystal and completely honeycomb it, but usually leave a shell of unaltered oxyhemoglobin on the exterior, including the pyramidal planes, but not the base, which is completely eaten away. In some cases the crystals were thus completely converted to reduced hemo- globin and the channels made by the bacteria were even repaired and filled up by re- crystallized hemoglobin, making quite perfect crystals. Pleochroism was very strong, a=e, nearly colorless, pale lilac; c —to, deep purplish- red. The double refraction is strong and extinction straight. The axis of greatest elasticity is the vertical axis, w > s; hence the optical character is negative, the same as in the oxyhemoglobin. CHIROPTERA. FOX-BAT OR FLYING-FOX, Pteropus medius. Plate 94. The specimen was received from the Philadelphia Zoological Gardens, and was in a putrid condition. It was oxalated, a little ether added, and preparations made as usual. The blood crystallized readily, and the crys- tals did not appear to dissolve at first, but after a few hours they began to break down and by the next day had disappeared from the slides. The photographs were taken inside of 4 hours after the preparations were made. The crystals were oxyhemoglobin. The examination of the crystals was incomplete, owing to their disappearing from the slides so rapidly; and hence the crystallographic constants were imperfectly determined. Oxyhemoglobin of Pteropus medius. Monoclinic (or perhaps triclinic): Axial ratio a : b : c =1 : 6 : 1.2808; /? = 56°30'. Forms observed : Unit prism (110), orthodome (T01), clinopinacoid (010), base (001). Angles: The prism angle was not obtained. Hemiorthodome to prism edge or orthopinacoid T01 A 100=50°; hemiorthodome to base 101 A 001 =73° 30'; ortho- pinacoid to base (or prism-edge to base) 100 A 001 =56° 30' =/?. 302 CRYSTALLOGRAPHY OF THE HEMOGLOBINS Habit generally tabular on the clinopinacoid (text figures 363 and 364) ; also short prismatic to tabular on the base. The planes seem to be irregularly developed, and may represent triclinic symmetry. The cover-edge crystals are elongated, apparently on the vertical axis, and generally flattened on the plane of symmetry; they grow crowded together, but as irregular aggregates, not apparently twinned. Pleochroism was only observed on the clinopinacoid aspect, and is a pale yellowish, nearly colorless; c deep red. Double refraction is strong; extinction is nearly or quite parallel to the prism edges. The plane of the optic axes appears to be the plane of sym- metry, or parallel to the plane taken as the clinopinacoid. The optical character can not be determined because of insufficient data. 363 365 366 |c FIGS. 363, 364. Pteropus mediut Oxyhemoglobin. FIGS. 365, 366. Vetpertilio jutna Oxyhemoglobin. BROWN BAT, Vespertilio fuscus. Plate 95. The specimen was bled in the laboratory. The few drops of blood ob- tained were caught in oxalate, and ether-laked. The quantity was not enough to centrifugalize. The slide preparations were made with the laked blood in the usual manner. Crystallization proceeded rapidly, and the ciystals appeared to be rather insoluble, keeping well and showing no tendency to dissolve. Crystallization was quite complete, but little color remaining in the solution. The crystals were shown to be oxyhemoglobin by the microspectroscope. Oxyhemoglobin of Vespertilio fuscus. Monoclinic: Axial ratio not determinable; /? = 81°. Forms observed: The three pinacoids only, orthopinacoid (100), clinopinacoid (010), base (001). Angles: Base to orthopinacoid, 001 A 100=S1°=/?; clinopinacoid, 010 A 100=90°. Habit tabular on the base, and elongated on the clino-axis, producing broad lath- shaped crystals (text figures 365 and 366), with a ratio of length to width on the base of about 8 : 1 to 5 : 1. The tabular crystals are thin, the thickness is one-twentieth of the length or less. They usually grow singly or sometimes in parallel growth on the base. In some cases the plates pile up on -the base into parallel groups or bundles of crystals. No evidence of twinning was observed. Pleochroism moderate; a pale yellowish-red, b rather deep red, c very deep red. Double refraction fairly strong, extinction straight on the basal aspect; on the clinopina- coid aspect the extinction is 16°, measured from the basal edge. The orientation of the elasticity axes is a A 6=25°, in the obtuse angle; b=b; c A a = 16°, in the acute angle. On the base, in convergent light, a single brush of a biaxial interference figure is seen, which passes out of the field upon rotation. As the normal to the base is 16° from n it would appear that the acute bisectrix Bxa = t, and the optical character is hence positive. OF THE INSECTIVORA AND CHIROPTERA. 303 TABLE 47. — Crystallographic characters of the hemoglobins of the Insectivora and of the Chiroptera examined. Name of species. Axial ratio. Prism angle. Angle p. E&tinction angle. Optical character. System. Sub- stance. Insectivora: Scalops aquaticus . Do 1 : 3.2931 1 : 3.2931 o 60 60 o / 90 0 90 0 0° 0° Negative Do. Hexagonal Paramorphous, OHb. Hb. Megachiroptera : Pteropus medius . . 1:6:1.2808 56 30 0° (nearly) after OHb Monoclinic (or per- OHb. Microchiroptera : Vespertilio fuscus. . 81 0 CAa=16° Positive haps triclinic) Monoclinic OHb. CHAPTER XVIII. CRYSTALLOGRAPHY OF THE HEMOGLOBINS OF THE PRIMATES— LEMURS, BABOONS, AND MAN. The specimens of blood of the Primates received were not very repre- sentative of the order. They comprised one lemuroid, Lemur catta, the ring-tailed lemur; 6 species of the Cercopithecidce, all members of the genus Papio (baboons) ; and the blood of man, Homo sapiens. The 6 species of baboons were the yellow baboon, Papio babuin; the drill, Papio leuco- phceus; the Guinea baboon, Papio sphinx; the long-armed baboon, Papio langheldi; the chacma, Papio porcarius, and the Anubis baboon, Papio anubis. In the case of several of the baboons, the supply of blood was suffi- cient to allow several methods of preparation to be used, and the crystals obtained were satisfactory for study. Three kinds of oxyhemoglobin crys- tals were observed in baboon blood, which are distinguished as a-, /?-, and y-oxyhemoglobin, respectively. Two of these types were observed in human blood. All species of the baboons did not develop these three kinds of oxyhemoglobin crystals, but when all three kinds were not observed it was due to the condition of the blood, or to lack of sufficient material. In comparing the hemoglobins of the species of Primates, the corresponding kind of oxyhemoglobin should be used. TABLE 48. — The three kinds of oxyhemoglobin observed in baboons and in man, with their optical characters. Name of species. a-oxyhemoglobin, orthorhombic. (3-oxyhemoglobin, monoclinic. V-oxyhemoglobin, orthorhombic. Negative 1.6801:1:6 Positive 0.5317 : 1 : 6 Negative. Do Papio sphinx Do. 1.8418:1:6 Negative 0.3346 : 1 : 6 Negative. a • 1 • 0 543 Do. 1 655 : 1 : 6 Positive e Do. 1.732 :1:6 Do. 1.737 :1:6 Do. 0.3268 : 1 : A Negative. Negative Optical character not determined. The a-oxyhemoglobin of the baboons and of man showed only the pinacoidal planes, so that the axial ratio could not be determined; but, in the monoclinic /3-oxyhemoglobin crystals, the prism angle gave the ratio of a : b. The optical characters were determined in practically every case. The crystals of y-oxyhemoglobin were not observed so frequently as those of the a- and /3-oxyhemoglobin; in these y-oxyhemoglobin crystals, also, the prism was developed and gave the axial ratio of a : b. Table 48 shows the distribution of these three kinds of oxyhemoglobin in the baboons and in man, as they were observed in our experiments. 20 305 306 CRYSTALLOGRAPHY OF THE HEMOGLOBINS From table 48 it will be seen that the a-oxyhemoglobin was observed in all species except in Papio anubis, and in this species the blood was not fresh. The /3-oxyhemoglobin was observed in all species except the drill and man; when it did not develop, it was probably a question of its solu- bility. The y-oxyhemoglobin was observed in three species of baboons and in man, this last a rather doubtful observation, as the angles of the crystals could not be definitely measured. As the orthorhombic forms, a- and y-oxyhemoglobin, are both optically negative, it is possible that they are the same substance; this does not seem likely from the way that they developed, but there are no data to prove that thej^ are the same or differ- ent substances. The y-oxyhemoglobin of the yellow baboon, the guinea baboon, and the anubis, the three species in which this form yielded a par- tial axial ratio, show ratios of Papio babuin =0.5317 : 1 : c, Papio sphinx = 0.3346 : 1 : c, and Papio anubis = 0.3268 : 1 : t. This apparent discrepancy disappears if the prism in Papio babuin is regarded as (230) ; then the ratio becomes for Papio babuin = 0.3545 : 1 : 6. It will be noticed that while the two orthorhombic forms of the oxyhemoglobin are, as stated above, both negative, the monoclinic form is always (with the exception of this form in Papio sphinx) optically positive. The orthorhombic crystals of the Lemur catta are optically negative. PRIMATES. RING-TAILED LEMUR, Lemur catta. Plate 95. The specimen was obtained from the Philadelphia Zoological Garden, and was in the form of clots, slightly putrid. The clots were ground in sand, with a little ether, diluted with a little normal saline solution, and centri- fugalized for 2 hours. The specimen had been collected in oxalate, hence no addition of this salt was necessary. From the clear solution obtained after the centrifugalizing, the slide preparations were made in the usual manner. The blood crystallized very slowly ; after 2 hours only very small crystals began to appear, and these were very poorly formed and only irreg- ular aggregates of rods. After standing in the cold for some time, tabular crystals appeared; but these dissolved rapidly when the slides were brought into a warm room. The tabular crystals improved in character, and became quite large and perfect in 4 days. They dissolved rapidly when the tem- perature was raised only slightly, and all examinations and photographs had to be made in a room temperature near the freezing-point. The rods at first formed were so poorly developed that their crystallographic char- acters could not be made out. The plates developed later are probably the same substance as the rods, and were well-formed crystals. They, as well as the rods, were found to be oxyhemoglobin by the spectroscope. Fine hair-like rods were seen with the tabular crystals ; these were evidently of the same crystallization, but were not of sufficiently definite form to determine the crystallographic constants with certainty. Oxyhemoglobin of Lemur catta. Orthorhombic, pseudo-hexagonal by twinning: Axial ratio a :6: violet. •jr-Oxyhemoglobin of Papio anubis. Orthorhombic: Axial ratio a : b : 6 =0.3268 : 1 : t. Forms observed: Unit prism (110), base (001). Angles: Prism angle 110 A 1TO=36° 12' average; prism to base 110 A 001=90°. These crystals appeared as a "second crop" about three days after the first preparations were made. Habit thin tabular, consisting of the very oblique prism cut by the base (text figures 406 and 407), and with a thickness of plate of one-fifteenth to one-twentieth of the length of the 6-axis. The crystals grow in tufts, often radiating, and occasionally are seen isolated; they are very thin and show scarcely any difference in the color from the solution when viewing a single crystal on the basal aspect. They do not appear to twin. They developed usually around the edge of the protein ring. On account of the thinness of the plates, pleochroism is not very noticeable on the basal aspect, on edge view it is stronger; the colors are shades of the OHb red, with a the palest color and c the deepest. Double refraction is easily noted and extinction is straight on the edge view and symmetrical on the base. The orientation of the optic axes is fl = 6; b = o; c = r siniple crystals. 21. Oxyhemoglohin and Methemoglobin of the Shad, slimvintc li:i>al aspect of regular growth. 22. Oxyhemoglobin and Methemogloliin cif the Shad, showing edge aspect cit a crystal ol oxyhemo- gliibin inclosed by methemoglobin in regular growth and twinned. 23. Oxyhemoglobin and Methemoglobin of (he Sliail. sliowing the two substances crystallized separately and also in regular growth. 24. Reduced Hemoglobin and Methemoglobin of the Shad, showing separate crystals, not in regular growth; also prismatic cleavage of reduced hemoglobin. PLATE 5 30 -.">. Metoxyhempglobin of the Carp (Ci/jinnux car/iin), showing sheaf-like aggregates. 26. Same, showing parallel growth of plates, in polarized light. 27. Same, showing irregular aggregates, produced hy piling up of plates. 28. Same, showing parallel growth of tabular crystals. 29. Reduced Hemoglobin of the Carp, showing long prismatic crystals with acute macrodorne (401), and also tabular crystals. 30. Same, showing prismatic crystals growing in sheaf-shaped tufts. PLATE 6 32. 33-36 36 Reduced Hemoglobin of the Carp (Ci//iri>iux riir/iin), showing smaller tabular crystals as they appear when they first begin to develop. Same, showing larger tabular crystals aggregated in [larallel growth. Oxyhemoglobin of the Necturus (Ncctxritx niiiriilntns), showing normally developed crystals, some twinned. PLATE? 41 42 37. Oxyhemoglobin of the Necturus (Nectunix maculatus) , showing long prismatic type of crystal. 38. Same, showing short prismatic development. 39. a-Oxyhemoglobin of the Python (Pijtlion iiinliirus), showing habit (l>>, the tabular crystal in flat view, and oblique section of crystal. 40. u-Oxy hemoglobin of the Python, habit (b), showing parallel growth on base. 41, 42. a-Oxyhemoglobin of the Python, habit (b), showing flat and edge views. 43 « ' PLATE 8 i ' ' V ' ' v ., \ v, -& i * -x K < , ^'' >*' 0 \V >^ 46 48 43, 44. /9-Oxyhemoglobin of the Python (I'l/llmn innliiriix). slmuinu sin:ill |>ynuni>hil crystals as crop on «-(>xy!iciiii'i;]i'lH[i crystals. 45,46. Oxyhemoglobin of the Ostrich (Strulliin c(inielus), showing small rhombic tabular crystals. 47,48. Oxyhemoglobin of the Cassowary (( 'iifiimrnis galeatua), showing prismatic crystals (dark), with crystals of ammonium oxalate (white). PLATE 9 50 51 53 49. Oxyhemoglobin of the Goose (.!««)• utisrr), showing clusters of tabular crystals, twinned in zone of pyramid. 50. Same, showing aggregates like figure -t'.i. hut with more individuals, producing rosette-shaped grnup. 51, 52. Same, tabular crystals seen on the basal aspect. 53. Oxyhemoglobin of the Trumpeter Swan (Olnr buccinator), showing first-formed simple crystals. 54. Same, showing large irregular aggregate of later growth. PLATE 10 59 60 55. Oxyhemoglobin of the Trumpeter Swan (l>lur hurritiittur), showing large simple crystal. 56. Same, showing arborescent aggregates. 57, 58. Oxyhemoglobin of the Whistling Swan (Olur <'iiliiiiilii/iiiiix~i. showing largr iwinnrd crystals in flat ami edge aspects. 59, GO. Same, showing large composite crystals as seen on basal aspect. PLATE 11 65 61-63. Oxyhemoglobin of the Chicken (Gullus donicstica), showing tabular composite crystals. 64. Same, showing single twinned crystal. 65, 66. Same, showing composite crystals in groups showing basal and edge aspects. 66 PLATE 12 68 i\ 7 ^ '- 70 4 * ; '< 71 , 67, 68. Oxyhemosloliin of the Quail (Colinus virgininnus), showing same compnsitu tabular erystal in two different stages of development. 69. Same, showing single and composite tabular crystals. 70, 71. Oxyhemoglobin of the Guinea-fowl (Nurnida meleagris), showing small simple crystals. 72. Same, larger crystals, showing twin on pyramid. PLATE 13 74 73. Oxyhemoglubin of the Pigeon (Columba livia), showing comparatively simple twinned aggregate, in the basal aspect. 74, 75. Same, showing more complex aggregates, some on edge or inclined. 76. Metoxyhemoglobin and Oxyhemoglohin of the Pigeon, showing disintegration of oxyhemoglobin crystals as metoxyhemoglobin crystals develop. 77, 78. Metoxyhemoglobin and Reduced Hemoglobin of the Pigeon, showing the needle-like crystals of reduced hemoglobin growing in tufts from crystals of metoxyhemoglobin. PLATE 14 80 84 7'J. Oxyhemoglobin of the Crow (Corpus americanus) , showing single, nearly equidimensional crystal attacned to cover by false plane near (100). 80-82. Same, showing groups of crystals presenting various orientations. 83. Reduced Hemoglobin of the Crow, showing group of elongated crystals. 84. Same, showing usual tabular crystal. PLATE 15 89 90 8.5. Oxyhemoglobin and Reduced Hemoglobin of the Crow ((.'nrrux u»iei'i<'-v Srv^^ f€* ^ * M s I ™ \ '1 •<. 125 121. Oxyhemogloliin of the Vulpiin- Pluilanscr (Tricltu.iiirus rul/teculn), showing tabular habit. 122. Same, showing short prismatic lialiit. 123. Same, showing long prismatic crystals and edge views of plates growing from cover edge. 124. Same, showing stout crystals in region of cover edge. 125. Oxyhemoglobin of the Rat-kangaroo (.K/ii//iryi>iiiiis nifi-scens), showing smaller crystals, many in twinned position. 126. Same, showing medium-sized crystals, some twinned on the orthodome. PLATE 22 131 127. Oxyhemoglnbin of the Rat-kangaroo (.£'/»//» •(/'"» <<•••' rufescens), law rnstals griming from cover edge and some showing interpenetrant twin on prism in the flattened crystals. 12s. Sanir, showing gypsum t\|.c of t \\iii. These large crystals are flattened lietween cover and slide. 129, 180. Oxyhemoglobin of the Kangaroo (.Mm-i-ii/ms i/ii/niiteii.-i'i, showiiig lath-shaped crystals growing singly and aggregated into sheaf-like bundles. 131. Same, showing stellate group of crystals along cover edije. 132. Same, showing larger lath-shaped crystals of parallel growth. PLATE 23 ' Tiff*-' ^ i • . .- »k*-i*»- -s*--1 nfvtSfK.- -T 136 137 138 133. Oxyhemoglobin of the Rock-kangaroo (Petrngalr s\>.), showing short lath-shaped crystals. 134, 135. Same, showing long and short lath-shaped crystals and capillary crystals. 136. Same, large crystals of long, lath-shaped type'-, showing parallel growth. 137. Oxyhemoglobin of the Ant-eater (M i/rmecoplmgri f), showing capillary crystals growing from pro- tein ring and large lath-shaped crystals. 138. Same, showing large lath-shaped crystals growing from cover edge. PLATE 24 139 143 144 139. a-Oxyhemoglobin of the Horse (Equus caballus), showing group "' longer prismatic crystals (preparation without oxalate). 140. Same, showing long prismatic crystals growing from protein ring (preparation without oxalate). 141. Same, showing shorter prismatic crystals near protein ring I preparation with oxalate). 142. Same, showing twin on pyramid (preparation with oxalate). Some crystals of the /y-nxyhemo- glohin are seen near e(>. Same, showing elongated horse-type twin. PLATE 27 ? -- 157 58 ' ,61 * 160 162 ^^^^^^^^^^v^^ 157. a-Oxyhemoglobin of the Mule (Equus asinus c? X Equus caballus ?), showing short prismatic crystals. 158. Same, showing long prismatic crystals growing from protein ring. .59, 160. Same, showing large stout prismatic crystals growing along cover edge. 161. /3-Oxyhemoglobin of the Mule, showing equidimensional development of crystals, mostly un- twinned. 162. Same, showing elongated horse-type twins. 159 PLATE 28 / 163 164 168 163. fl-Oxyhemoglobin of the Mule (A'i/w/i.s ]M>tamitii iitii/iliibiiix), showing groups of large twinned crystals. 183. Same, showing single large twinned group of horse-t \ pi- twin. 184. Same, showing twin aggregates ami ladder-like form produced by twinning. 18S, 186. Oxyhemoglobin of the Peccary (l)/i-nli/l<:t Inliinliix}. showing dodecahedron-like romliination of short diametral prism with unit pyramid. PLATE 32 188 189 O I W 191 192 • ,V_ A • 1.S7, 1S.S. Oxyhemoglohin uf the Colhiml IVccary (Dimljilfx Injm-ii), *\u>\\\\\g small pyramidal crystals. ISO, ]9(). Oxyhemoglobin of the Pin (,s'i/.v acmfn), sliowiiiic sinitlc crystals oonsisliiii;1 of unit prism and 1X7, is ISO, brachydome, growing in protein ring. 191. Same, showing large crystals in various orientations. I'.I'J. Same, showing group of crystals and aggregates from protein ring. • ~«v ~ ff- t&ff -4'j' I ;j 'f Sfi $ti*W£ - • ' • \e -;x : 193 • . . . », ^ 196 197 198 I'.):1.. ( Nxlirinoiil.il.in of the 1'ig (N».s s,-rnj'ii}. shciuing small cryslals from protein ring. I!l4-19(i. Reduced Hemogloliin ,,| the I'ig, showing long prismatic crystals, some terminated l>y hase. growing in sheaf-like groups and sometimes I \\iiined. 1(17. Same, showing thick and thin prismatic crystals at liasal termination. I'.ts. Same, showing feathery groups of crystals. PLATE 34 204 I'.Hi. Oxyhemoglobin of the Muis Deer (Tnujitlux mcminna), showing single laluilur crystals. '200, 201. Same, showing horse-typt- twin. 202. Reduced Hemoglobin of the Muis Deer, showing part of very large crystal. 203, 204. Oxyhemoglobin of the Elk (Cervus canadensis), showing simple pyramidal form of crystals. PLATE 35 205^ . - % i * * • ^ r b ^f 206 207 V ' 208 209 -'!!:>, •jiic,. 207, 208. 209. 210. - / :.,-< > Oxyhemoglobin of the Elk (('CITH.S cuiuidciixix). sliowiug Hat pyramid in tlnvr ;r side elevations. Reduced Hemogloliin of the Red Brocket (Cnrinciix nifnx), showing composite of parallel growth; also narrow lath-shaped crystals. Same, showing simple tabular crystals and lath-shaped crystals. Same, showing rods growing in sheaf-like tufts. 210 s. plan and lar crystals PLATE 36 :• 1 I, V | ' 'v^tr 3 * • y 214 .^'--v • " -- - ^«^ i ^ i 211. Reduced Hemoglobin of the Venezuela Deer (Muza/na l:iU. 238. /3-Oxyhemoglobin of the Duickerbok, showing lic.\ai;ciii;il plutes on flat ami on edge, also several groups of a-oxyhemoglobin crystals in parallel growth. 239.'Oxyhemoglobin of the Sheep (Ovis aries), showing first-formed needle-like crystals and small tabular crystals. 240. Same, showing long prismatic crystals that develop after 24 hours. PLATE 41 241 m^wmm ^tilp^ ^•* ^| 242 245 241. Oxyhemo?lobin of the Sheep (Ovis aries), showing network produced by twinning and pentagon twins. 242. Same, showing isolated pentagon twins in side and edge views. 243. Same, showing composite groups produced by twinning. 244. Same, showing cross-banded effect on pinacoid produced by twinning. 245. Same, showing isolated pentagon twins strung like beads on needles of oxalate. 246. Same, showing pentagon twins seen in polarized light. 246 PLATE 42 ll;l ' 7- :/ 251 252 247,248. Reduced Hemoglobin of the Sheep (Ovis aries), showing tabular crystals in various orifiitatnins. 249-251. Oxyhemofflobin of the Bharal (Oi'is nahiira), showing lon^c jirisniMtic crystals, many flattened on two opposite prism faces, thus producing a triclinic aspect. 252. Same, showing flattened prisms shortened to almost rhombic plates. PLATE 43 V * \V •• ; * ' ': 258 .iil. Oxyhemoglobin of the Bharal «>rix raoAuro), showing nt-t work of |>risin> ]iri»incr.i I>\- 2.">t. SLUIIC, .sliowiiif; liarreil eft'cot pnjiluccil mi prisms l>y tvvinniiifr. , 2.">ti. SMIIIC, sliciuini; isolated pentason twins in various as]»cts. , 258. Same, slioxviii;; pL-ntafion twins frrowiiif; on ami capiiiiiL; prisms. PLATE 44 ' "" *'/ 259 \\fr. ~,^ &&$ \ . 7 m -, J[ ;V*"-d 261 263 264 259. Oxy hemoglobin of the Bullock (Bos taurus), showing small, first-formed crystals ooiisi-iiinst of unit prism and brachydome. 'JiiO. Same', showini; larper prismatic crystals with iiiifi|ii:illy developed dome faces. 'Jlil. Same, short stout crystals, some showing bracln prism in combination with unit prism. -I')-. Sami'. showing group of crystals growing attached to an oxalate crystal. '_'!>:;. ( Nyheiiiogloliin of tlie Bison (Has l>ixi»i). shouing irregular aggregate of thin lath-shaped crystals in protein ring. 264. Same, showing long crystals growing in tufts from protein ring. * «r * =; *i tr ' PLATE 45 ** k T^a " * . If , "":f-", 265 f' I 1 r , 269 I 270 265-269. a-Oxyhemoglobin of the European Red Npiirrpl (Scinriix vulijurix), showing lifxasronul tubular crystals, many in parallel growth. 270. /9-OxyhemoffIobin of the European Red Squirrel, showiiiff composite crystal*. PLATE 46 (&**«. renter ««<«*«.), show,,,, single crystals and ,. showing pseudohexagona] si PLATE 47 278 V *% ; -H&V, o*; j:. ^<&* >% ^V>/ c tSkflS^- v •:, : • • . V i < • •• : - 279 %&& ^,- ^^3^" ^;^f ^-.-^-VCj^-"1.^ • ^^^-•/c^r1''-' t-..».-.- , r^^ - i •v.'it x ST'i r , 5"' • * x x ^"W^ fe ^?f ;v K V'^^i.-^ et;! ** • «* -^ Wk^jJ . ^>/ i^*^ 28° -; { :, 4*^' - .4-WT > -£» /_ 282 27S. 279-2S2. Oxylit'nioitlobin of the (iray Sijiiirrel (ficiurux curiilinciixix), shi)\\inir "cii-fii i-nsr" sphenflitic masses ol crystals in protein rinsr. Same, lar^e crystals showinj; |iarallel srrowth. Oxyheinoi;loliin of the Flying S(]ViirreI (Stiwopterus ru/unx). shuwiiis small, simple hexagonal crystals characteristic of the species. PLATE 48 283 284 287 283, 284. '_'s.~>. L'SI, 'JsT. 2SS. 288 Oxyhemoglobin of the Ground-sqmrrel (Tamias sti-fatus), showing irregular pinups nf crystals growing in protein rins; Siune showing long rrysl:ils grottiiiir in ru'lialiir,' gn>n|is I'miii protein riii'r. Oxyhemoglobin of the Prairie Dog ( ''//'"""//••' Inilnririiiniini, showing ilivergcnt tufts of hair- like crystals, growing from cover edge. Same, showing divergent groups of larger ucicular crystals, seen in polarized light. PLATE 49 289. /3-Oxyhemoglobin of the Ground-hog (Marmnta nwnax), showing radiating tufts of hair-like crystals. 290. a-Oxyhemoglobin and /3-oxyhemoglobin of same, showing tufts of jS-oxyhcmuglnhin and small hexagonal plates of first-formed a-oxyhemoglobin crystals. 291. Same, showing single large crystal with groups of smaller crystals on it in parallel growth. 292, 293. Same, showing large simple and composite crystals, the parallel growth preserving general hex- agonal outline. 294. Same, showing irregular aggre'gate of hexagonal plates, all in parallel growth. PLATE 50 300 \ 295. a-Oxyhemoglobin of the Clround-hog (Marmnta mnnax), showing two groups nf hexagonal plates in partial orientation, looking complete as seen on liase. and one group seen in side view, showing radiating character due to partial orientation. 296. Same, showing larger group in this partial orientation, as seen on base and on edge. In these two figures the lath-shaped section of plates is illustrated. -!I7. Same, a very complex group in edge view, showing; arborescent form of group. 2!IS. j-Oxyhrmoglobin, showing arborescent and feathery forms of groups of crystals. 299. Same, showing simpler group of crystals on base and on edge. 300. Same, showing twinned group of crystals in polarized light, In 299 and 300 the imperfect curved outlines of crystals are shown. PLATE 51 303 304 305 306 301. Oxyhemoglobin of the Beaver (Castnr canailcnsis), showing rhomhoi.lal and hexagonal tabular crystals, on base and edge. 302. Same, showing four and six-sided tabular crystals in different aspects and small hexagonal plates of mimetic twin. 303. Same, large hexagonal plates, showing twinning and parallel growth. 304. Same, elongated crystals showing twinning. 305, 306. Oxyhemoglobin of the Muskrat (Fiber zibethicus), showing needle-like first-formed crystals with elongated horse-type twins of lath-shaped crystals. PLATE 52 311 307-309. OxyhemoRlobin of the Muskrat (Filter ziltethicus), showing lozenge-shape. I tabular crystals m different orientations and some horsr-typr twins. 310-312. Same, showing different kinds of crystal aggregates in parallel growth, arborescent irronpin" and sheaf-like forms being produced. PLATE 53 313 315 *3fiT>IEAl - « WFK* I * ^ 316 317 :U:;-:;i."i. ( Kyln-nioulobin of the White I!;il (Alliinn of Mi/x inn ny/i-u*), showing eloii.naUMi six-M.lcil ].l;iti- prinliicfil liy Huttciiinu' of priMii; niiiiihlv ln'xai;onal groups due to t\vinninic on lir;icli\- pynuiiiil ;iiid interpenetran! twins mi unit pyramids. :;iii. Sunir. shouina: star-shape' 1 twins on unit pynuniil. :I17. S:inic, slicnviii.i; hfxasronal composites with higher nuiL'tiificatiou. • \]^. ^;i!m'. ^lio\\iiur star-shapeil twin witli liinliiT magnification. PLATE 54 fi •324 . 322. 323, 324. o-Oxyhemoglobin of the Norway Rut (M us norn iiim.-.), showing symmetrical and flattened prisms and hexagonal plates produced by shortening of prism. Star-shaped twin on unit pyramid seen at two places in lower part of h'eld. Same, showing especially nearly hexagonal plates produced l>y shortening of llattrned prism. 0-Oxyhemoglobin of the Norway Rat, showing symmetrical and flattened octahedra in dillVrent a-pects. with higher magnification. «-( Ixyhemoglobin of the Norway Rat. showing symmetrical and unsymmetrifal prismatic crystals and pseudo-hexagonal jilates, with some hexagonal plates of /3-oxyhemoglobin due to flat ten- ing of octahedron. Oxyhemoglohin of Black Rat (Max rtittux), showing flattened prism terminated liy dome, \\ilh dome faces sometimes unequally developed and producing four-, live-, and six-sided plairs. PLATE 55 Oxyhemoglobin of the Black Rat (Mus rnttus), showing twin on the flat, consisting of t\\i> individ- uals and not producing a hexagonal plate as in White Rat twin. .'i'-'ii. Same, showing thicker crystals and olilique termination of dome faces. • >-7. Oxyhemoglobin of the Alexandrine Rat (Mux alc.randri>ius), showing unsymmetrical flattened prisms and a twin on flat aspect to lower left of field. 3'JX. Same, showing four-, five-, and six-sided tabular crystals, due to unsymmetrical development of dome faces. 329. Same, showing star-shaped twins. 330. Same, showing larger crystals. PLATE 56 fe 331 332 ' '.t'.n. a-Oxyhemoglobin of the Porcujiinc (Kr. and r. ;!4'2. Same, showing large crystals with twins of types u and /<. PLATE 58 344 i «p" « U k - *' " •- ^^ /*• 5 o ^^u V I ^1 if •' ~^" - jfe^ • r» ""^ * ? " ,vjW ^ T ? c" "<5- -, • •"* •-- -' Itft •' •"• - ' 345 346 :!4i;. Oxyhemoglobin of the Guinea-pig (Ca«(acuyh;ini (Hydrochierim ni/iy/ivim), showing \>\ raniidal er\stals In various orientations. M4s. ^-Oxyhemoglobin of the Capybara, showing lonj; prismatie crystals. PLATE 59 349 I 350 V 352 354 34!l. a-Oxyhemoglobin of (lie ('apybaru (Hi/ill nc/iarux <-/i/>iii-ur\y hemo- globin crystals in ordinary light. PLATE 61 362 ^> 365 366 :>61. /9-Oxyhemoglobin of the I!al>l>it i/.i/n/.s ciiiiiciiltm), showing oblique sections of crystals. iUi'J. Same, showing ol)lic|uc sections; crystals are [ileoclirdic in ordinary lis'i'- :i(i:j, M(i4. S;inie, sliowiiif; groups of kirire cryst:ils in nl>lii|iie section. 304 sliows ilprided pleochroisni. 365. o-Oxyhemoglobin of tin- Helgiun Hare (Lf/n/K CHI-H/HI nx}, showing piisniatic crystals of second crop, firouini; in rndiatiii}: groups from protein ring. A few sliow single oblique termination. 366. Same, showing doulily terminateil prismatic crystals from protein ring. PLATE 62 371 372 367,368. a-Oxyhemoglobin of the Belgian Hare (Li'/mx r»Mi/«r/i.s->, showing large prismatic crystals uf second crop. 369. Oxyhemoglobin of the Harbor Seal (Phoca ritulina), showing first-fonneil, small tabular crystals. Their hemimorphic character is easily observed. 370. Same, showing larger tabular crystals, some exhibiting parallel growth. Hemimorphism is evi- dent in large central crystal. 371. Same, showing crystals elongated into prisms by development of orthopinacoid. 372. Same, showing simple symmetrical crystals consisting of base (001). unit prism (110), and unit pyramid (111). PLATE 63 \ c 374 376 377 378 373. Oxyhemoglobin of ( 'alit'ornia Sea-lion (Oturin gilli-s/iii), showing small, first-formed crystals, mostly simple an. Same, showinft Sea-lion twin in edge view, consisting in tliis case of two iniliviilnals. 7ti. Same, showing parallel growth and an edge view of Sea-lion twin in parallel growth. 77. Same, showing flat view of crystals in parallel growth. Cross-barring is due to twinning of a number of individuals in parallel position. !7s. Same, showing parallel growth in twins seen on edge, producing comb-like appearance and cross- barring when seen on the flat. PLATE 64 • 383 -. 379. C'O-Hemoglobin of the California Sea-lion «>ttu-iu i/iltfx/in), showing small, tirst-l'onneil crystals, some twinned. 380. Same, showing the crystals seen on edge and in section. 381. Same, large crystals showing parallel growth. 382. Same, showing Sea-lion twin in edge view, consisting of three nearly symmetrically developed indi- viduals. 3S3. Same, showing parallel growth. •i*t Same, showing single large crystal in symmetrical position. Small crystal below to the left shows profile view looking along ortho-axis. PLATE 65 386 389 x"\ 390 38.5. Oxyhemoglobin of the Skunk (Mephitis inepliilii-'i /mliilu), showing long jirismatic crystals grow- ing in radiating groups. 386. Same, showing long prismatic crystals and brush-like tuft. :is7. Same, showing irregular aggregate of shurter prismatic crystals growing in proti-in ring. 388. Same, showing large groups growing in parallel orientation. 389, 390. Uxyhemoglobin of the Ferret (Mustela put >riun), showing tabular crystals and twins. PLATE 66 395 396 391. Oxyhemoglobin of the Ferret (Mustela jiutorius), showing small, first-formed somatic crystals in protein ring. 392. Same, showing larger tabular crystals in various orientations, growing in protein ring. 393-395. Oxyhemoglobin of the Otter (Littra canadensis), showing tabular crystals in different positions. many in twin position. Sphenoidal or hemimorphic character may be seen in ci-vsial on edge near middle of Held in 395. 396. Same, snowing thicker tabular crystals in basal and edge views; basal aspect shows parallel growth. PLATE 67 %7 I - w • ' Wi. L^ &%^e$&A '^-JU^- . " M ^v '• ,-^^S \fe^- •• -J 8 •^E^SSPr' i ^^m* ^KH. A^ ;Wa>:d | yx'^ ^ ^^^ip VJSJ- -. V ^ &* # 402 M07. ()xyhciiiiif;li>bin of the Badger (Tax idea americana), type a, showing small, first-formed tulmlur cry-ilals, five-sided, and consisting of unit prism and base with one face nf hemiorthodome. Uadger l\vin shown in many of these crystals. ;-!98. Same, type a, showing tabular crystals more symmetrically developed and four-sided. 399. Same, type a, showing more prismatic development of crystal consisting of prism, base, and hemior- thodome; also penetration twins. 411(1. Same, showing crystals of type o in short prismatic form and long prisms of type /« growing from cry si a Is of oxalate. 401. Same, showing long prisms of type b growing in a tuft. 40'J. Same, showing radiating group of type b crystals growing from an oxalate crystal. 403 - PLATE 68 404 V 407 408 403,404. Oxyheinogloliin of the Kinknjou (Cerrtcs caiidirnlriiliix), showing prismatic crystals of type a in different aspects. 405. Oxyhemogloliin of the Cacomyxl (Bassariscus nxlutu}, showing longer type of prismatic crystal, consisting of unit prism and brachydome. 406. Same, shorter type of crystal showing same combination as 405. 407, 40S. Same larger crystals of type of 405, hut showing brachypiiuicoid in addition to unit prism and brachydome. Some are in twin position, and in 408 cross-sections show brachypinacoid. PLATE 69 $ • 'J, A-- -v\ S 409 410 - - -- \ 412 , am, «? -i i •' 1^ S»H, 414 40!l. Oxyhemoglobin of the Black Bear (L'rsus omericanus), showing small. livst-i'iinniMl crystals in trilliiiKs, the bear-type twin. 410. Same, lar^f, single crystals slio\vin<; jiarallcl growth. Combination is liasc cut at one eml of orthii-axis by unit i>rism aiul at opposite end by unit pyramiil. 411. Same, showing larger crystals in same combination as 411) with clinopinacoid cutting unit pyramiil at one end of axis. Small section of crystals above, to the right slums hrminiorphic form. 412. Same, showing crystals in various orientations and in section. 413, 414. Same, showing aggregation of crystals in parallel growths and radiating groups along cover edge. PLATE 70 419 420 41."). Oxyhemoglobin of the Bl:ick Bear (I'rxux americanus), showing large cnstals along nner edge. mostly in section. •116. Same, showing two large groups of crystals along cover edge in |i:inillel orientation, diviileil liy large crystals in oblique section. 417, 41S. Oxyhemoglobin of the Polar Bear (I.' runs inari/iiinix), showing small. first-fornn-il ci\>l:ils (\\iimeil in bear-type twin, growing in protein ring. 419. Same, single crystals showing hemimorphisrn ami bear-type twins, near COMT edge. 420. Same, larger tabular crystals along cover edge, showing parallel growl h. 42! \\ PLATE 71 '• .-., ff- ,\ \ , V**" -J • "' — -a "i *" \» .— j»- "•«'.*-'. i .*>:(! b^._ JJ-, - - — : is . - • .. •'•..,'• ••' jk *-. .-.. • • :s 422 423 424 425 426 421, 4'22. Oxyheinofslubin of the Sloth Bear (Mi'Iiirmix urxiinix). showinji small, tirsl-i'onnrcl siM^li' crvslals and l»'ar-l vpi' twins. Coinliinalion is liasc cut by the unit prism at one c'in! of ortho-axis and by unit pyramid at the other end, and \vilh or without orthopiuaeoid. 4'j:;. Samr, larjii'i- crystals, most, of them showing orthopiuaeoid. 424, 42."). Same, larger second-crop crystals from along cover eilsre, a few showini: bear-type I \\in 426. Same, group of larger crystals from near cover edge, showing parallel growth ami cross-sections of crystals. PLATE 72 I /*l?\n*, ;">$ -. fcAJ&4S$y'* 431 432 427. Oxyhemoglobin of the Sloth Bear (Mcliir..-inillrl ^KI\\ th :ili>M<; ortho-axis. The white crvst:ils arc oxalatc. 4L'S. Same, •ilinwinjj irregular groups in parallel growl li orientation. 4120. a-Oxyhemoglobill of the Dog (Cards familiaris), showing mass of ca|iillarv rrvstals proilui'ril in thick slides. 4MO. Same, sliouinj; striated prisinutii' crystals growing from protein rins;. l, 4:>'J. Same, showing divi'i'irent groups of long prismatic crystals. PLATE 73 -- "', 437 4:;:.. 4:;ii. 4:17. 438. 438 Oxyhemoglobin of the fho\v Dog (Canis familiaris var. ), showing capillary and long |irisni:ilic crystals doubly terminated. Same, showing shorter prismatic crystals in divergent tufts and overgrown by s|ilienelitic groups of smaller prisms. Same, showing radiating cluster of larger prisms growing from protein ring. Same, showing large crystals growing from protein ring. Oxyhemoglobm of the cross between Collie (Canix fninilittris) and Coyote (I'units liilninx), showing capillary and thicker prisms, several being the group of two and others more c iposite. Same, showing groups of crystals, some probably in twin orientation. PLATE 74 443 l:'.'.i. Metoxyhempglobin of the Wolf (Can/x lujiux iiic.rii-niin^}, ^ll(l\vinic ladialini; Crimps of crystals. 440. Same, showing lirush-like ends on some crystals. 441. Oxyhemoglobin of the Coyote (<'unix lutninx). sliowin;; mass of oa|iillary crystals. 4412. Same, showing short capillary crystals ami single shorter composite crystal. 44M. Same, showing radiating groups of crystals. 141. Same, showing mass of short needle-like crystals. PLATE 75 445 X 446 ^*^/ < 449 450 445. Oxyhemoglobin of the Jackal (Cants owm/.--), showing divergent and radiating aggregate- "I capillary crystals. Flexibility of crystals shown by curvatures. 440. Same, showing capillary and shorter prismatic crystals. 447. Same, showing shorter prismatic crystals along protein ring. 1 Is. Samr. showing part of the protein ring that lias developed into doubly terminated, composite crystals. 449. Oxyhemoglobin of the Dingo (Cunis ilh-iin}, showing short prismatic doubly terminated crystals, some in twin position as they occur through body" of slide. 450. Same, showing short composite prisms that develop near protein ring. PLATE 76 451 452 453 455 456 4.11. Oxyhemoglobin of the Dingo {(.'nnis ilimjui, showing sioi 4.VJ. Same, showing medium thick prisms along protein ring. !.">:). Oxyhcmoirloliiii of Azara's Dog (('emit /cimc), showing divergent tufts of lonn capillary cr\Mals. •154-4.">ti. Same, showing masses of crystals aggregateil in approximately parallel grow I h along protein ring or cover edge. PLATE 77 459 460 461 461 •457. Oxyhemoglobin of tin- Red Fox ( I '»//«'* /»/r».s-i, showing radiating groups of linn prismatic crvsials. •45S. Same, showing radiating groups of Immlles of crystals, probably in t\vin orii-nlation. t."i!l. Same, showing mass of long prismatic crystals. l(i(). Same, sliowing bunilles of slightly divergent crystals. 4til. Same, showing approximate parallelism of a large group of the crystals. 4lil2. Same, showing groups of crystals in approximately parallel growth. PLATE 78 467 468 lii"', Itil. Oxyhemoglobin «( the Swiss Fox ( 1 ul/irx rnl/ics), shouing long stri:iU' if% 469 111 469. Oxyhemoglobin of the Gray Fox (Urocynn i-nn-itn-iirijenteus), showing sphonelitic group* uf tliiu prismatic crystals, an occurrence rarely seen. 470. Same, showing large masses of crystals in parallel growth, a characteristic aggregate in this species. 471, 472. Same, showing radiating tufts that develop in retarded crystallizations. 473, 474. Same, showing shorter thicker crystals developed in undiluted preparations. These crystals formed in protein ring, and in 474 one of the small rare sphenelitic groups is seen. PLATE 80 - • . • ^ 475^ n;r ; 476 'X*£ "'^ *' '3$ v' t< 'Sr^ • "' ' -^SL v - 'S '-*t-' ^- V'^^*^ ^ .,' FV^.fT^ - ,&,,^ ^,- H ^ •-i, 4* ' e 479 480 475. Oxyhemoglobin of tlie Lion (t'elis leu), showing type o crystals ciccurriiif: singly and cxliil>itiiiK panillfl growth. Sliort prismatic crystals in this plate are rc-dnci'il liemoglobin ul' lirst crop. 476. Same, showing an irregular II.HK resale of type a crystals. 477-4SO. Same, showing crystals of type « and type l> along cover edge. Characteristic form of type l> crystals may l>e seen to lower left of Held in figure 4X0. All figures show reduced hemoglobin crystals. PLATE 81 485 486 4S1. Oxyhemoglobin of the Lion (Felis leo), showing group of type a, rhombic crystals, some exhibiting parallel growth. 482. Oxyhemoglobin and Reduced Hemoglobin of the Lion, showing rhombic plates of Oxyhemoglobin and brush-like aggregates of hemoglobin needles of second crop. 483. Same, showing large crystals of Oxyhemoglobin showing parallel growth, embedded in tuft of crystals of reduced hemoglobin of second crop. 484. Oxyhemoglobin of Lion, showing large type a crystal with smaller crystals on it in parallel growth. 485. Oxyhemoglobin and Keduced Hemoglobin of Lion, showing large type a Oxyhemoglobin crystals on Hat and on edge, and containing small embedded crystals of reduced hemoglobin. Needle-like crystals to left are reduced hemoglobin. 486. Reduced Hemoglobin of Lion, of the short prismatic type; pleochroism very distinct. PLATE 82 488 492 <0 %* 4S7. Reduced Hemogloliin of the Lion (Felis leo), showing smaller, more normally developed first -formed crystals of third crop in various orientations. 488. Same, showing larger crystals of third crop. Traces of needles of second-crop hen.oglobin ervstids may be seen in crystals at top of figure, penetrating the large crystals. 489. Same, showing one of these large, third-crop crystals penetrated liy needles of second-crop crystals. 490. Same, various sections of these luri^e, third-crop crystals, showing traces of penetration by needles of second-crop reduced hemoglobin. 491. Reduced Hemoglobin of the Tiger (Ft'lix tlgris), showing small, first-crop crystals. 492. Same, showing larger crystals of first crop, some distorted by growth or by contact with slide or cover. PLATE 83 497 493. Reduced Hemoglobin of the Tiger (Felis tigris), showing various aspects of medium-sized cr\>t:il- Angle of macrodome may be seen in crystal to left of center of field. 494, 495. Same, showing different orientations of medium-sized crystals. 496. Same, showing large crystals. 497, 49S. Same, showing large crystals of second crop, along with small crystals of first crop. PLATE 84 499 501 502 & i 503 )'.)!>. «.-< >xyhemoglobin of the Jaguar (Felis mica), two groups of tabular crystals showing parallel growth. 500. Same, showing large group of crystals all in parallel growth orientation. Hod-like crystals are reduced hemoglobin. 501. Same, showing three groups of tabular crystals, each in parallel growth orientation. 502. Same, showing large group in parallel growth orientation. 503. /3-Oxyhemoglobin of the Jaguar, showing dodecaheriral crystals. 504. Same, showing octahedral crystals. PLATE 85 PHI fc « & V " \ / 509 505. 3-Oxyhemoglobin of the Jaguar (Felis onca), showing octahedral crystals. 506. Reduced Hemoglobin of the Jaguar, showing long, square, four-sided prisms ending in a brush of fibers. 507, 50S. a-Oxyhemoglobin of the Mountain Lion (Felis concolur), showing type a crystal, consisting of unit prism and brachydome. 509. Same, showing type b crystal. 510. Same, showing type 6 crystal, some passing into /?-Oxyhemoglobin. PLATE 86 514 516 oil. Oxyhemoglobin of the Mountain Lion (Felis concolor), showing type c crystal. 512. ^-Oxyhemoglobin anil o-Oxyhemoglobin of the Mountain Lion, showing type 6 crystal of a-Oxyhemo- globin twinned and passing into /3-Oxyhemoglobin octahedron. 513. a-CO-Hemoglobin of the Mountain Lion, snowing type c crystal. 514. CO-IIemoglobin of the Mountain Lion, showing tabular type c crystals of a-C'O-Hemoglobin and crystals of /M'O-Hemoglobin that, have grown on a-CO-Hemoglobin crystals and gradually absorbed them. 515. Reduced Hemoglobin of the Leopard-cat (Felis bengalensis), showing small, first-formed crystals. 516. Same, showing symmetrical crystal consisting of unit prism and macrodome. PLATE 87 519 320 521 522 517, 51S. Reduced Hemoglobin of the Leopanl-cal (/ciii/(ili:iixi.<<), showing single crystals in \arious orientations. In 51S a number of different sections of tlie prism are shown. 519. Same, showing two skeleton groups in parallel growl li orientation. 520. Same, showing group of large crystals growing from a square cross-seel ion of a prism as a nucleus. Cross-section of a prism seen near bottom of figure. 521. Same, showing group similar to 520. with single large prism ami oblique section of another prism that resembles an acute rhombohedron. 522. Same, showing single large crystal with outgrowth of .smaller crystals. PLATE 88 *1 527 525, 523. Reduced Hemoglobin of the Ocelot (Felis pttrdalis), showing small, first-formed crystals, growing singly or twinned. 524. Same, showing large crystals covered with radiating tufts of smaller cnstals. 526. Same, large crystals, many in cross-section, showing maerppinacoid. 527. Reduced Hemoglobin of the Cat (Felix domestica), .slimving lirst .K'liuiic crystals to form and parallel growth aggregates of reduced hemoglobin rods. These groups are later uhsorU'd as symmetrical crystals develop. 528. Same, showing network of long prismatic crystals of second crop. •- ' PLATE 89 529 %$$ ; 1 ' 531 532 533 529. Reduced Hemoglobin of the Cat (Felis dnmesticn), showing rough parallel growl h aggregates like those of first-formed crystals. .530. Same, showing network of large prismatic crystals. 531. Same, showing single crystal lying on face- o|: prism. ._>.'!'-!. Same, showing smaller, later-crop crystals, growing from cross-section of large crystal. .">:;:;. Same, showing smaller crystals growing in radiating form from large crystals. 534. Same, showing dome symmetrically and unsymmetrically developed; and o\yhemoglol>in of Wild Cat (Lynx rufus). PLATE 90 Jm vv - ^ ^ES*1' tr-C£* VrV; , *\*sm *. .-^>w 536 540 A \ .">::.">. Reduced Hemoglobin of the Cat (Fclis dnmcsticn), showing short prismatic type of crystal. '}'•',('<. Same, showing cross-section of prism. 5M7. Same, showing group of short type of crystals growing «>n an n|.li.|iic cross-section of prism; with one dome face developed making a monocfinic-lookine crystal. .VIS. Same, showing radiating groups of smaller crystals growing on larger' prisms. 539. Same, showing prismatic type of crystal. 540. Same, showing tabular type of crystal. PLATE 91 541 543 546 541, 542. Oxyhemoglobin of the Wild Cat (Lynx rufiis), showing tabular type of crystal, the base bounded by unit prism and two pinacoids. 543. Same, showing large ]irismutio crystals covered with a secondary growth. Unsymmetrical dome termination seen in large crystal below middle of plate. 544. Same, showing larger prismatic crystals covered by growth of smaller crystal*. 545, 546. Reduced Hemoglobin of the Wild Cat, showing long prismatic type of crystal and (in 544) form- ing network. Many of these are iu twin positions. PLATE 92 I 551^ 547. Reduced Hemoglobin of the \Vild (';U {l.i/n.r riijuxi. showing long prismatic crystals, sonic with unsymmetrieal ends, some with an overgrowth of radiating smaller crystals. The \\\n crys- tals to upper left are in twinned position. 548. Same, showing larger long prismatic crystals with attached overgrowth of smaller crystals. 549. Same, showing short type of prismatic crystal. 550. Same, showing twin on braehydome in upper left and immediately below it a parallel growth showing group extending in direction of macro-axis. Crystals on either side of middle indi- vidual have unsymmetrieal development of braehydome. but this is arranged symmetrically with respect to middle individual, one right-handed and the oilier left-handed. 551, 552. Oxyhemoglobin of the Lynx (L'/n.i' riinmli'iixifi var.). showing thin tabular crystals, somr in parallel growth orientation and some in divergent tufts. PLATE 93 556 Jl 558 553. Reduced Hemoglobin of the Lynx (Li/n.r i:iimi:/i:nxix vur.), showing type n crystals, consisting of btacliv- prism and macrodome; in some the prism is very short. 554. Same, showing typ.' n crystals, some showing macropinacoid in addition to brachypris-in and marro- dome. Distorted crystal with unei|iially developed dome faces seen on left. 555. Same, showing type b crystal with long type n prisms growing out from it. Several smaller tvpe h crystals are seen in this figure. 55(1. Same, showing parallel growth in groups of type a crystals. 557. Same, showing large type 6 crystal \\itli a parallel growth group of typr .">! I, ."i(jO. Oxyhemoglobin of the Mole (Sctdnps wjuaiicus), showing small barrel-shaped crystals, some in twinned position. 561, 562. Same, showing large crystals along with crystals of first crop. 563, 564. Oxyhemoglobin of the Fox-bat (Pteropus medius), showing small tabular crystals. PLATE 95 V ^S •> :^/ 566 568 5(j5, ")(i(j. Oxyhemoglobin ut the Urown Bat (Vespertttiofuscus), showing broad lath-shapi'd cry.slals, tabu- lar on base. ."il)7. Oxyhemoglobin of the Ring-tailed Lemur (Lemur cnl/n), showing lirst crystals to form imperfect prisms consisling of bundles of fibers crossing each other at definite angles and also small imperfect tabular crystals. 568. Same, showing hexagonal plate produced by twinning. Other crystals are seen growing on ha-e of main plate, not all in exact orientation. 569. Same, showing mimetic hexagonal tabular crystals. 570. Same, showing rosette-shaped groups of crystals. . - •' PLATE 96 571 WlPJU, _.. s $*35? •-' • *$s. • *'^ j^yx-^'-: ^' y.>;< ^\ 573 574 575 576 571. a-Oxyhemoglobin of the Yellow Baboon (l}api» bahuin). showiiii; small tuhulur crystals along protc'in ring. 572. Same, showing thicker tabular, nearly cubical crystals. .573, ,574. /?-Oxy hemoglobin of (he Yellow Baboon, showing tabular and prismatic types of crystals. 575, 57(i. Oxyhemoglobin of the Drill (Papin leucophceus) , showing long roil-like crystals growing in diver- gent tufts and irregular aggregates. PLATE 97 578 1 ! ^ i -: ),v - - UA ':;--i.-- • -,-••. . ' • v ? r 580 581 582 577, 57S. y-Oxyhemoglobin of the Guinea Baboon (Papio a/iliinf). showing lung (liai>ionil-.-.]i:i|>ccl inhnljir crystals, growing in divergent tufts and piling up in approximately parallel growth. 579. a-Oxyhernoglobin and /3-Oxyhemoglobin of the Long-anncd Baboon (Papio liini/lu-ltli}, shuwing short lath-shaped crystals of a-Oxyhemoglobin and rhombic tabular crystals of 8-Oxvhemo- globin. 5SO. a-Oxyhemoglobin of the Long-armed Bal>oon, showing thicker tabular crystals. .">.sl. Same, showing twin on brachyilomc. 582. 0-Oxyhemoglobill of the Long-armed Baboon, showing thick tabular' and prismatic crystals. Different depth of shading is due to pleochroiMii. \ PLATE 98 586 587 * 588 583. /3-O.\yhemoglobin of the Long-armed Baboon (Papio langheldi), showing tabular and prismatic types of crystals. 584. Same, showing tabular and prismatic types of crystals. Horse-type twin on edge is seen near lower part of field. 585. Same, showing horse-type twin on right of field. 586. Same, showing long and short prismatic crystals. .riS7. a-Oxyhemoglobin of the Chacnia (Pajiin jmrcarius), showing tabular crystals. Difference in tint is due to pleochroism. 588. Same, showing partial parallel growth and fan-shaped appearance of group when seen on edge. PLATE 99 589; L ^~~-' 591 594 589. a-Oxyhemoglobin of the Chaema (I'li/iin /nirciiriiix), showing Ihick tabular crystals in various orienta- tions and illustrating pleochroism as crystals are viewed in different posh inn-. 590. Same, showing parallel growth and fan-shaped aggregates. 591. j3-Oxyhemoglobin of the (.'haenia, showing side view of horse-type twin. 592. Same, showing parallel growth in direction of ortho-axis. 593. «-<>xy hemoglobin and /9-Oxyhemoglobin of the Chacma. showing liorsi-type twins of <-( Ixyhemoglobin. 594. a-Oxyhemoglobin and ,9-Oxyhemoglobin of the Chacma, showing parallel growth and hor-r-t \ pe twinning. PLATE 100 595 596 598 599 600 595. /3-Oxyhemoglobin of the Anubis Baboon (Pa/n'o anubis), showing tabular crystals with unequal devel- opment of ortbopinacoid fact's. 596. Same, showing tabular crystals in horse-type twins, cm cilue ami mi Hal. 597. Same, showing single tabular crystals. 59$. Same, showing prismatic type of crystal. 599. Same, showing large crystals with tufted groups of thin crystals of y-Oxyhemoglobin. 600. /?-Oxyhemoglobin ami >--Oxyhemoglobin of the Anubis. LIBRARY UH Ifliu K