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Full text of "The chemical constitution of the proteins"

==s Monographs on Biochemistry 




TH E 



CHEMICAL CONSTITUTION 



FHR PROTEINS 

PART i 

ANALYSIS 

BY 

R. H. A. PLIMMER, D,Sc. 



I 



MONOGRAPHS ON BIOCHEMISTRY 

EDITED BY 

R. H. A. PLIMMER, D.Sc. 

AND 

F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S. 



MONOGRAPHS ON BIOCHEMISTRY. 

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THE NATURE OF ENZYME ACTION. By 
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THE CHEMICAL CONSTITUTION OF THE 

PROTEINS. By R. H. A. PLIMMER, D.Sc. 
Third Edition, Part I. Analysis. 
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THE VEGETABLE PROTEINS. By THOMAS B. 
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THE SIMPLE CARBOHYDRATES AND THE 
GLUCOSIDES. By E. FRANKLAND ARMSTRONG, 
D.Sc., Ph.D. Second Edition. 55. 6d. net. 

THE FATS. By J. B. LEATHES, F.R.S., M.A., M.B., 
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ALCOHOLIC FERMENTATION. By A. HARDEN, 
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THE PHYSIOLOGY OF PROTEIN META- 
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THE DEVELOPMENT AND PRESENT POSI- 
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THE POLYSACCHARIDES. By ARTHUR R. LING, 
F.I.C. 

COLLOIDS. By W. B. HARDY, M.A., F.R.S. 

PHYSICAL METHODS USED IN BIOLOGICAL 
CHEMISTRY. By S. G. WALPOLE, D.Sc. 

PROTAMINES AND HISTONES. By A. KOSSEL, 
Ph.DX 

LECITHIN AND ALLIED SUBSTANCES. By H. 
MACLEAN, M.D., D.Sc. 

THE ORNAMENTAL PLANT PIGMENTS. By 
A. G. PERKIN, F.R.S. 

CHLOROPHYLL AND HEMOGLOBIN. By H. 
J. PAGE, B.Sc. 



LONGMANS, GREEN AND CO., 

LONDON, NEW YORK, BOMBAY, CALCUTTA, AND MADRAS. 



MPhy 
P 



THE 



CHEMICAL CONSTITUTION 



OF 



THE PROTEINS 



v^ 1 ^J* 

KT Hf Af 



BY 



(1ST) 



PLIMMER, D.Sc. 



UNIVERSITY READER IN PHYSIOLOGICAL CHEMISTRY, UNIVERSITY COLLEGE, LONDON 



IN THREE PARTS 
PART I 

ANALYSIS 



s 





WITH DIAGRAMS 



THIRD EDITION 



LONGMANS, GREEN AND CO. 

39 PATERNOSTER ROW, LONDON 

FOURTH AVENUE & 30TH STREET, NEW YORK 
BOMBAY, CALCUTTA, AND MADRAS 

1917 



Bebicatefc 

TO 

EMIL FISCHER 

THE MASTER OF 
ORGANIC CHEMISTRY IN ITS RELATION TO BIOLOGY 



GENERAL PREFACE. 

THE subject of Physiological Chemistry, or Biochemistry, is 
enlarging its borders to such an extent at the present time, 
that no single text-book upon the subject, without being 
cumbrous, can adequately deal with it as a whole, so as to 
give both a general and detailed account of its present 
position. It is, moreover, difficult, in the case of the larger 
text-books, to keep abreast of so rapidly growing a science 
by means of new editions, and such volumes are therefore 
issued when much of their contents has become obsolete. 

For this reason, an attempt is being made to place this 
branch of science in a more accessible position by issuing 
a series of monographs upon the various chapters of .the 
subject, each independent of and yet dependent upon the 
others, so that from time to time, as new material and the 
demand therefor necessitate, a new edition of each mono- 
graph can be issued without re-issuing the whole series. In 
this way, both the expenses of publication and the expense 
to the purchaser will be diminished, and by a moderate 
outlay it will be possible to obtain a full account of any 
particular subject as nearly current as possible. 

The editors of these monographs have kept two objects 
in view : firstly, that each author should be himself working 
at the subject with which he deals ; and, secondly, that a 
Bibliography, as complete as possible, should be included, 
in order to avoid cross-references, which are apt to be 
wrongly cited, and in order that each monograph may yield 
full and independent information of the work which has been 
done upon the subject. 

It has been decided as a general scheme that the volumes 
first issued shall deal with the pure chemistry of physiological 



viii GENERAL PREFACE 

products and with certain general aspects of the subject. 
Subsequent monographs will be devoted to such questions 
as the chemistry of special tissues and particular aspects of 
metabolism. So the series, if continued, will proceed from 
physiological chemistry to what may be now more properly 
termed chemical physiology. This will depend upon the 
success which the first series achieves, and upon the divisions 
of the subject which may be of interest at the time. 

R. H. A. P. 
F. G. H. 



PREFACE TO THIRD EDITION. 

WORK upon the chemistry of the proteins is always in progress, 
and a large amount of new matter has required incorporation 
in this edition. At the same time a thorough revision has been 
made, and the former omission of references in the text to the 
papers quoted in the bibliography has been remedied. 

The new matter in this edition is mainly connected with the 
analysis of proteins. It has therefore been thought advantage- 
ous to divide this part of the monograph into two : Part I. 
Analysis. Part II. The Amino Acids. New or improved 
analytical methods are constantly being devised, whilst the 
description of the amino acids is a more definite part of the 
subject matter. 

The division into two parts will be a saving of labour to 
author, publisher and printer, and the expense to the purchaser 
will be lessened. The editors mentioned these considerations 
at the time the monographs were first issued. 

R. H. A. P. 

April, 1917. 



CONTENTS OF PART I. 

PAGE 

INTRODUCTION i 

I. HYDROLYSIS 9 

II. THE ISOLATION AND ESTIMATION OF THE UNITS - 15 

A. THE MONO-AMINO ACIDS - 1 7 

TYROSINE - 17 

(1) Isolation and Gravimetric Estimation - 17 

(2) Colorimetric Estimation - 20 

(3) Estimation by Bromination - 2 2 

CYSTINE - - 24 

(1) Isolation and Gravimetric Estimation - 24 

(2) Separation of Cystine and Tyrosine - 25 
TRYPTOPHAN - 27 

(1) Isolation and Gravimetric Estimation - -27 

(2) Colorimetric Estimations - 28 

(3) Estimation by Bromination - 32 
THE OTHER MONO-AMINO ACIDS - - 33 

(1) Isolation of Glutamic Acid as Hydrochloride - 33 
(ia) Isolation of Glutamic Acid as Zinc Salt - - 34 
(ib) Isolation of Glutamic and Aspartic Acids as Silver Salts 34 
(ic) Isolation of Glutamic and Aspartic Acids as Calcium Salts 34 

(2) Esterification - - 35 

(3) Isolation of Glycine as Ester Hydrochloride - - 36 

(4) Extraction of the Esters of the Amino Acids - - 37 

(5) Fractional Distillation of the Esters in Vacua - 40 

(6) The Isolation of the Individual Mono-amino Acids - 42 

(a) Proline - 43 

(b) Glycine, Alanine, Valine, Leucine, and Isoleucine - - 44 
i. Separation of Valine from Leucine and Isoleucine - - 45 
ii. Valine - 46 
iii. Leucine and Isoleucine - 47 
iv. Separation of Valine and Alanine - - 48 
v. Separation of Glycine and Alanine - 5 1 

(c) Phenylalanine - 52 



xii CONTENTS OF PART I. 

PAGE 

(d) Aspartic Acid 52 

(e) GlutamicAcid - 53 
(/) Serine - 53 
(g) The Distillation Residue 54 
(h) The Isolation of Oxyproline - 54 

B. THE DI-AMINO ACIDS - - - 55 

I. Hydrolysis and Estimation of Protein - 55 

II. Removal of Sulphuric Acid. Estimation of Ammonia 

and Humin Nitrogen - 55 

III. Precipitation of Arginine and Histidine - 56 

IV. Estimation and Isolation of Histidine - - 5 7 

V. Estimation and Isolation of Arginine - - 59 

VI. Estimation and Isolation of Lysine - - 60 
Colorimetric Estimation of Histidine - 61 

THE RESULTS OF THE ANALYSIS A - - 63 

THE RESULTS OF THE ANALYSIS B - - 69 

Protamines - - ?o 

Histones - 72 

Albumins and Globulins - 73 

The Vegetable Proteins - 74 

Phosphoproteins - - 76 
The Scleroproteins - .77 

Various Proteins - - - - 81 

Derivatives of Proteins - - - - - 82 
ANALYSIS OF PROTEINS BY THE DISTRIBUTION OF THE VARIOUS 

KINDS OF NITROGEN - - - 85 

A. Distribution of the Nitrogen in Three Groups - 87 

B. Distribution of the Nitrogen in Seven Groups - - 89 

I. Estimation of Amino Nitrogen - 89 

II. Estimation of the Different Groups of Amino Acids - 97 

ANALYTICAL DATA - lll 

I. Composition of Proteins in Amino Acids - -in 

II. Distribution of Nitrogen in Three Groups - - - 131 

III. Distribution of Nitrogen in Seven Groups - - - 132 

IV. Distribution of Nitrogen in Seven Groups, in Foodstuffs 134 

V. Effect of Carbohydrate on the Distribution of Nitrogen in 

Seven Groups - . - 135 
TABLE : Milligrams of Amino Nitrogen corresponding to i c.c. 

of Nitrogen Gas . . - 136 

BIBLIOGRAPHY - I ^ 

INDEX 



THE CHEMICAL CONSTITUTION OF THE 
PROTEINS. 

PART I. 
INTRODUCTION. 

THE proteins, of which we know some forty or fifty natural ones 
occurring in both animals and plants, are divided according to their 
origin, solubility, coagulability on heating and other physical char- 
acteristics into the following groups : 

I. Protamines, e.g., salmine, sturine, clupeine, scombrine, cyclop- 

terine, cyprinine. 

II. Histones, e.g., thymus histone, Lota histone, Gadus histone, 
histone from blood corpuscles. 

III. Albumins, e.g., ovalbumin, conalbumin, serum albumin, various 

vegetable albumins. 

IV. Globulins, e.g., serum globulin, fibrinogen and its derivative 

fibrin, myosinogen and its derivative myosin occurring in 
the muscles of animals ; legumin, conglutin, amandin occur- 
ring in plants, and some crystalline vegetable globulins, e.g., 
edestin, excelsin. 
V. Glutelins, e.g., glutenin in wheat, oryzenin in rice, soluble in 

very dilute alkali. 
VI. Gliadins, e.g., wheat-gliadin, hordein, zein, occurring in cereals 

and soluble in 70-80 per cent, alcohol. 
VII. Phosphoproteins, e.g., caseinogen, vitellin, ichthulin. 
VIII. Scleroproteins, e.g., keratin from hair, horn, feathers, egg- 
membrane. Collagen, gelatin, elastin. Silk-fibroin, silk- 
gelatin. 
IX. Conjugated Proteins : 

(a) Nucleoproteins : nucleic acid in combination with protein, 

generally I., II., III. 

(&) Chromoproteins : chromogenic substance in combination 
with protein, e.g., haemoglobin. 



PT. I. 



2 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

(c) Glucoproteins : carbohydrate in combination with protein, 

e.g. t mucin, ovomucoid. 
X. Derivatives of Proteins : 

(a) Metaproteins, e.g., acid-albumin, alkali-globulin. 

(b) Proteoses, e.g., caseose, albumose, globulose. 

(c) Peptones, e.g., fibrinpeptone. 

(d) Polypeptides, e.g., glycyl-alanine, leucyl-glutamic acid, 

a tetrapeptide (2 glycine + I alanine + I tyrosine). 
Except the protamines, the histones, and the derivatives of the 
proteins, all the proteins contain carbon, hydrogen, nitrogen, sulphur 
and oxygen, and they possess the following elementary composition : 

C 51-55 percent. 

H 7 

N 15-19 

S 0-4-2 -5 

O 20-30 
from which a formula such as 

r H N S O 

^726 " 1174^ 1943^214> 

which is that of globin, the basis of haemoglobin, can be calculated. 

The phosphoproteins and the nucleoproteins contain also the 
element phosphorus ; in the former, probably combined directly with 
one of the constituents of the protein molecule ; in the latter, combined 
with a pyrimidine or purine base or a carbohydrate ; these substances, 
together with phosphorus, constitute nucleic acid. 

Investigations upon their chemical constitution have been carried on 
now for nearly a century, but it is only since 1901 that, by the work 
of Emil Fischer and his pupils, any clear view has really been obtained 
of their actual constitution. The main result of these investigations 
is that the protein molecule is built up of a number of amino acids, 
belonging to four different series, and of which the following have 
been definitely determined : 

A. Monoaminomonocarboxylic acids. 

1. Glycine, C 2 H 5 NO 2 , or amino-acetic acid. 

CH 2 . (NH 2 ) . COOH 

2. Alanine, C 3 H 7 NO 2 , or a-aminopropionic acid. 

CH 3 . CH(NH 2 ) . COOH 

3. Valine, C 5 H n NO 2 , or a-aminoisovalerianic acid. 

CH 3 \ 

)CH . CH(NH 2 ) . COOH 



INTRODUCTION 

4. Leucine, C 6 H 13 NO 2 , or o-aminoisocaproic acid. 

CH 3 v 

\CH . CH 2 . CH(NH 2 ) . COOH 

5. Isoleucine, C 6 H 13 NO 2 , or o-amino-/3-methyl-)8-ethyl-propionic acid. 

CH,\ 

\CH . CH(NH 2 ) . COOH 

6. Phenylalanine, C 9 H U NO 2 , or 0-phenyl-a-aminopropionic acid. 

C 6 H 5 .CH 2 .CH(NH,).COOH 

7. Tyrosine, C 9 H n NO 3 , or /3-parahydroxyphenyl-o-aminopropionic acid. 

HO . C 6 H 4 . CH 2 . CH(NH 2 ) . COOH 

8. Serine, C 3 H 7 NO 3 , or )3-hydroxy-o-aminopropionic acid. 

CH 2 (OH) . CH(NH 2 ) . COOH 

g. Cystine, C 6 H 12 N 2 O 4 S 2 , or dicysteine, or di-(-thio-a-aminopropionic acid). 
HOOC . CH(NH 2 ) . CH 2 . S S . CH 2 . CH(NH 2 ) . COOH 

B. Monoaminodicarboxylic acids. 

10. Aspartic acid, C 4 H 7 NO 4 , or aminosuccinic acid. 

HOOC . CH 2 . CH(NH 9 ) . COOH 

11. Glutamic acid, C 5 H 9 NO 4 , or o-aminoglutaric acid. 

HOOC . CH 2 . CH 2 . CH(NH 2 ) . COOH 

C. Diaminomonocarboxylic acids. 

12. Arginine, C 6 H 14 N 4 O 2 , or a-amino-S-guanidinevalerianic acid. 

\NH . CH 2 . CH 2 . CH 2 . CH(NH 2 ) COOH 

13. Lysine, C 6 H 14 N 2 O 2 , or a, e-diaminocaproic acid. 

H 2 N . CH 2 . CH 2 . CH 2 . CH 2 . CH(NH 2 ) . COOH 

D. Heterocyclic compounds. 

14. Histidine, C 6 H 9 N 3 O 2 , or j8-imidazole-a-aminopropionic acid. 

CH 

</ \ 

N NH 

CH = C CH 2 .CH(NH 2 ).COOH 

15. Proline, C 5 H 9 NO 2 , or o-pyrrolidine carboxylic acid. 

CHo CH, 



CH 2 CH. 



COOH 



16. Oxyproline, C 5 H 9 NO 3 , or 7-hydroxy-o-pyrrolidine carboxylic acid. 
HO.CH CH 2 

CH., CH.COOH 



4 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

17. Tryptophan, CjjH^N^Og, or 0-indole-a-aminopropionic acid. 
C CH . CH(NH 2 ) . COOH 

/ \ 
C 6 H 4 CH 

\ / 
NH 

E. Ammonia. 

These seventeen amino acids, together with ammonia, which is 
present in the form of acid amide groups, are the basis of the com- 
position of the protein molecule. They are generally referred to as 
" Bau-steine," the bricks, or foundation stones ; but as the English 
translation of the German word is not entirely expressive of its mean- 
ing, it is preferable to use the term unit or element for these compounds 
in their relation to the proteins. 

Most proteins contain all these amino acids or units in various 
proportions, but some proteins, such as gelatin, contain only fourteen 
or fifteen, and some, such as salmine, are built up of three or four units, 
these units being di-amino acids and histidine. Full data of the pro- 
portions, so far as is known, are given in the tables on pp. 111-130, 
and further reference to details is made on pp. 63-84. 

Our purely chemical knowledge of the proteins has led to a greater 
knowledge of the digestion of proteins. Proteins are completely con- 
verted into their constituent units in the alimentary canal of animals ; 
in this form they reach the bloodstream and circulate to the various 
organs of the body. Each organ rebuilds its tissue from the circulat- 
ing amino acids, as well as giving rise to them during its catabolism. 
In plants, too, protein is transferred from one region to another in the 
form of amino acids. We think of and work with proteins in terms 
of amino acids. All considerations and deductions as to the dietetic 
value of proteins can no longer be based upon their total nitrogen 
content, but must be based upon the amounts of the various amino 
acids in their molecule. Nitrogen equivalence is not amino acid equi- 
valence. Proteins devoid of certain units are of no value for the 
maintenance of life ; proteins deficient in certain amino acids are of 
little or of no value for ensuring the growth of young animals. These 
particulars are given in Cathcart's Physiology of Protein Metabolism, 
second edition. The analysis of proteins with special reference to 
those units of metabolic value is therefore of the greatest importance. 

The long list of amino acids is sufficient evidence of the complexity 
of the protein molecule ; and, as yet, it seems to be incomplete, for 
several other products have been described : 



INTRODUCTION 5 

1. Aminobutyric Acid. The presence of aminobutyric acid, which 
would complete the series of monoaminomonocarboxylic acids, was as- 
sumed by Schiitzenberger [1879]. A small amount of a substance of 
the empirical formula C 4 H 9 NO 2 was isolated by Foreman [191 3, 2] from 
the products of hydrolysis of caseinogen. Foreman considered it to 
be a-aminoisobutyric acid, but his evidence is not quite sufficient for 
the identification of the product. Abderhalden [Abderhalden and 
Weil, 1913, 3] subsequently stated that an amino acid of this composi- 
tion had been obtained from a great number of proteins but that its 
constitution was not established. 

2. Norleucine or a-amino-n-caproic Acid. In addition to the two 
isomers, leucine and isoleucine, of the empirical formula C 6 H 13 NO 2 , 
another isomer, norleucine, has been shown to be present in the protein 
of the nervous tissue of the brain [Abderhalden and Weil, 1912, 2 ; 
1913, 1,2, 3]. It is apparently identical with glycoleucine described 
by Thudichum [1901]. This unit is most likely present in other pro- 
teins also. Further evidence of its more general occurrence is given 
by the results of Skraup and Witt [1907], who obtained ^-valerianic 
acid, which can only arise from a normal caproic acid, by the action 
of alkaline hypobromite on caseinogen and of Heckel [1908], who by 
the same method obtained w-valerianic acid from the leucine fraction 
of amino acids prepared from caseinogen ; also of Samec [1908], who 
obtained ^-caproic acid by the reduction of the leucine fraction of amino 
acids prepared from elastin. 

3. Skraup [1904] added several new units to the list. Two of 
these, diaminoadipic acid and diaminoglutaric acid, were subsequently 
[1905, 2] shown to be alanine and a mixture of glycine and alanine. 
The other compounds were hydroxyaminosuccinic acid, C 4 H 7 NO 5 , di- 
hydroxydiaminosuberic acid, C 8 H 16 N 2 O 6 , caseanic acid, C 9 H 16 O 7 N 2 , and 
caseinic acid, C 12 H 16 O 5 N 2 . These substances were not found by Skraup 
[1905, i] in gelatin, but another compound, C 12 H 25 N 5 O 10 , was obtained. 

Adensamer and Hoernes [1905], who used the same method as 
Skraup, could only isolate small quantities of caseanic and caseinic 
acids from egg-albumin. All these substances are most probably 
mixtures. Caseinic acid resembled in many ways the substance 
diaminotrioxydodecanic acid. 

4. The two substances, hydroxyaminosuberic acid, C 8 H 15 O 5 N, and 
hydroxydiaminosebacic acid, C 10 H 20 O 5 N 2 , described by Wohlgemuth 
[1904, 1905] in the protein of the liver are, like the above, probably 
mixtures. 



6 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

5. Diaminotrioxydodecanic acid, C 12 H 26 O 5 N 2 , was isolated by 
Fischer and Abderhalden [1904] from some tyrosine prepared from 
caseinogen. Other workers have subsequently searched for this com- 
pound, but have never isolated it. Abderhalden and Weil [1912, 2] 
stated that it had been obtained in very varying yields, which made it 
appear probable that it was a secondary product of hydrolysis and that 
its constitution appeared to be different to that implied by its name. 

6. A substance of the formula C U H 15 O 5 N was isolated by Torquati 
[1913, I, 2] from the seedlings and green pods of the vetch, ViciaFaba. 
This compound was investigated by Guggenheim [1913], who ascer- 
tained that its elementary composition corresponded more closely to 
the formula C 9 H U O 4 N, and that its constitution was 3, 4, dihydroxy- 
phenylalanine, 

OH 




CH 2 . CH(NH 2 ) . COOH 

In properties it resembled the synthetical dl-dihydroxyphenylalanine 
described by Funk [1911] and also by Stephen and Weizmann [1914]. 
Like adrenaline this substance possesses a pyrocatechin nucleus. (See 
Barger's Simpler Natural Bases.} 

7. Gortner [1911] mentions the presence in wool of an aromatic 
phenolic compound, which gives Millon's reaction but is not tyrosine. 
If it should prove to be dihydroxyphenylalanine, this compound may 
prove also to be present in other proteins and to be a unit of general 
occurrence. 

8. Abderhalden and Kempe [1907, i], in preparing tryptophan by 
the tryptic digestion of caseinogen, observed the presence of another 
compound of the formula C n H 12 N 2 O 3 . It was found more frequently 
when the digestion was prolonged and the subsequent preparation was 
slow ; it seems to be an oxidation product of tryptophan. It does not 
give the usual reactions for tryptophan and is converted by hydrochloric 
acid into a quinoline derivative. It is known as oxy tryptophan. 

9. 3, %-Diiodotyrosine is present in the protein contained in corals 
and other sea animals : it is formed when proteins are treated with the 
halogen : it is not included in the above list since it is of such rare 
occurrence and presumably a derivative of tyrosine. 

10. Glucosamine. The presence of glucosamine in the protein 
molecule is a disputed question ; there is no doubt that a carbohydrate 



INTRODUCTION 7 



containing nitrogen is contained in the glucoproteins in their prosthetic 
group, but it is doubtful if it be present in the protein part of the mole- 
cule, although a carbohydrate has been obtained from carefully purified 
proteins containing no prosthetic group, such as crystallised egg-albu- 
min and serum-albumin. The fact that the yield of carbohydrate from 
such a protein becomes smaller the more often it is recry stall ised, 
suggests that the presumably pure protein still contained an impurity ; 
this impurity would be a glucoprotein, which is found in both egg 
white and serum from which the crystallised proteins are separated, 
and this would give rise to the carbohydrate. Glucosamine is therefore 
excluded from the above list. 

The composition of the protein molecule has been determined by 
the method of hydrolysis. As the result of hydrolysis a complex 
mixture of all, or nearly all, the previously mentioned units is ob- 
tained. These have been isolated by various methods based upon 
the fractional crystallisation of the compounds themselves, or of their 
copper, silver and other salts. Only when one or more of the amino 
acids occurred in somewhat large amounts was their isolation and 
characterisation effected ; their amount seldom reached a value higher 
than 20 per cent, of the total quantity, and the remainder was 
represented by uncrystallisable syrups of unknown nature. The 
products, termed leuceines, acids of the series C M H 2 -iNO2, and 
glucoproteines, acids of the series C W H 2 MN 2 O 4 , in gelatin, glucopro- 
teines-o. and tyroleucine in albumin, described by Schiitzenberger 
[1879] and by Lepierre [1903] have been shown by Hugounenq and 
Morel [1906, 1907] and Galimard, Lacomme and Morel [1906] to be 
mixtures of now definitely known substances. 

A great advance was made when Drechsel discovered that the 
protein molecule contained di-amino acids as well as mono-amino acids, 
and to Kossel and Kutscher we owe our chief knowledge concerning 
their isolation and estimation. Emil Fischer, in 1901, by his study 
of the amino acids and their derivatives, introduced a new method of 
isolating and separating the mono-amino acids, which depended upon 
the fractional distillation in vacuo of their esters, and which is now 
commonly known as the ester method. This method, though not yet 
really quantitative, has enabled us to obtain a knowledge of some 70 
per cent, of the total products resulting by hydrolysis, and it has shown 
us that phenylalanine, serine and alanine, which were only known to 
occur in a few, are present in all proteins, and that phenylalanine 
in its distribution is the principal aromatic constituent, for it often 



8 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

exceeds in amount that of tyrosine and occurs when this latter is 
absent. Further, it has demonstrated the presence of two new com- 
pounds, proline and oxyproline. 

New units have thus been discovered with each improvement in, 
and development of, the methods of analysis of the proteins. The 
exact constitution of these units had also to be determined. This 
portion of the subject has been attended with entire success ; we 
now know the chemical constitution of every clearly defined unit 
in the protein molecule. Except glycine, all the amino acids con- 
tained in proteins are optically active and this property must also 
be considered. 

The final problem in the chemical constitution of the proteins 
the synthesis remains. This problem is still in its infancy. After 
numerous attempts by the earlier investigators its foundation was 
laid by Emil Fischer, who has synthesised a compound which, if 
it had been found in nature, would have been described as a protein. 
The difficulties in this part of the subject are very considerable. Not 
only is the amino acid required, but also its natural optical isomer is 
required. Many of the amino acids can only be readily obtained by 
decomposition of the protein, and, even if they be prepared by synthe- 
sis, much time and expense is involved. The proper conjunction of 
the amino acids is then necessary ; the results of analysis give no 
clue as to whether the arrangement is a, b, c, d, e or b, c, d> a, e, or 
d, e, b, a, c> etc. Some idea of the arrangement of the units in the 
protein molecule has been obtained by the isolation of several poly- 
peptides, as the combinations together of the amino acids are termed, 
from proteins. Their isolation is so difficult that there must be many 
years of incessant labour before a real natural protein will be actually 
produced in the laboratory. 

The study of the Chemical Constitution of the Proteins can there- 
fore be divided into three sections : 

I. The Chemical Composition of the Protein Molecule. Analysis. 

II. The Chemical Constitution of the Units. The Amino Acids. 

III. The Synthesis of the Proteins. The Polypeptides. 



HYDROLYSIS 



I. HYDROLYSIS. 

The complex problem of the composition of the protein molecule 
has been solved by the method of hydrolysis, though other methods 
such as fusion with alkali, oxidation with permanganate, chromic acid, 
etc., action of halogens have been employed. Hydrolysis has been 
effected by (i) boiling with acids, (2) boiling with alkalies, (3) the 
action of the various proteoclastic enzymes which occur in animals 
and plants. Proteins were first hydrolysed by acids in 1820 by 
Braconnot, who used dilute sulphuric acid; between 1850 and 1875 
hydrochloric acid was most frequently used as the hydrolysing agent 
by Ritthausen, Hlasiwetz and Habermann, and others; from 1870 
to 1880 Schiitzenberger employed baryta water under pressure. The 
action of vegetable enzymes on proteins was studied chiefly by 
Schulze and his co-workers, that of animal enzymes by Kiihne, 
Kossel, Kutscher, Drechsel, and numerous other investigators. 

(a) Hydrolysis with Hydrochloric Acid. 

(i) Concentrated Acid. 

Hydrolysis of proteins by boiling with concentrated hydrochloric 
acid for several hours produces a dark brown solution of the products. 
Hlasiwetz and Habermann [1871, 1873] carried out the hydrolysis in 
the presence of stannous chloride in order that the solution should 
remain colourless. This addition is not necessary, as was shown by 
Cohn [1896-97] and was not made by E. Fischer in his researches. 

The hydrolysis of protein is usually carried out by heating it with 
three times its quantity of concentrated hydrochloric acid of specific 
gravity 1*19. The protein is placed in a round flask and covered with 
the acid ; on shaking and warming slightly it gradually passes into 
solution. The solution, which at first may become violet in colour, 
gradually turns brown ; it is then boiled under a reflux condenser for 
six to twenty-four hours, depending on the particular protein. The 
solution has finally a dark brown colour and during the hydrolysis a 
portion of the hydrochloric acid is evolved as gas. It is filtered from 
humin substances (secondary products arising from carbohydrate and 
tryptophan and other units, see p. 65) and fatty material by passage 
through a Buchner funnel covered with linen, and these are well washed 
with water. The products are isolated from this solution. 



io THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(ii) Dilute Acid. 

Hydrolysis may be equally well affected by boiling the protein 
with from 1020 parts of 20 per cent, hydrochloric acid for twenty- 
four to forty-eight hours. [Van Slyke, 1911, 4.] 

Henriques and Gjaldbak [1910] have found that complete hydro- 
lysis is produced by heating the protein with 3N hydrochloric acid 
in an autoclave at 150 for one and a half hours. These observers 
made no statement as to the quantity of acid they used. Their ex- 
periments were confirmed by Van Slyke [1912, 2] who hydrolysed I '5 
grams of protein with 50 c.c. of 3N acid, i.e., with thirty-three times 
the quantity. Van Slyke showed further that complete hydrolysis was 
effected either by heating at 100 for forty-eight hours with 20 per 
cent, acid or at 150 with 3N acid. Henriques and Gjaldbak [1910] 
found that the hydrolysis of egg albumin was not complete by 
heating for twelve hours with concentrated acid, but was complete on 
heating at 150 for 1-5 hours in an autoclave with 3N acid. There 
was no advantage in heating at 1 50 for a longer period of time ; at 
1 80 in an autoclave decomposition of the amino acids occurred with 
the formation of ammonia. 

(iii) Alcoholic Hydrochloric Acid. 

Proteins are also hydrolysed by boiling with alcohol saturated with 
hydrogen chloride. Pribram [1911] first drew attention to this fact, 
showing that hydrolysis occurred with carefully dried protein and 
carefully dried reagents ; the hydrolysis was more complete if the alco- 
hol contained 3-5 per cent, of water. Similar results were previously 
published by Pfannl [1910]. They are not in accordance with those 
of Abderhalden and Hanslian [1912], who showed that no appreciable 
hydrolysis took place if moisture was carefully excluded during all 
operations. Weizmann and Agashe [1913] have isolated small quan- 
tities of amino acids, but consider that the main result of hydrolysis 
with alcoholic hydrochloric acid is the formation of complex products 
intermediate between protein and amino acid. Herzig and Land- 
steiner [1914] estimated the amount of esterification in the case of 
serum proteins which were heated with various strengths of alcoholic 
hydrochloric acid. Their results also show that hydrolysis of the 
protein takes place, but only to a small extent. 



HYDROLYSIS 1 1 

(b) Hydrolysis with Sulphuric Acid. 

The protein is hydrolysed by boiling for fifteen to twenty-four 
hours with six times its quantity of 25-33 per cent, sulphuric 
acid, or with a mixture of three times its weight of concentrated 
sulphuric acid and six times its weight of water under a reflux con- 
denser. Generally, the mixture is heated for one to one and a half 
hours on a water-bath until frothing has ceased and then in an oil- 
bath at 105 for the necessary length of time. 

(c) Comparison of the Hydrolysis with Hydrochloric and 
Sulphuric Acids. 

Several observers have maintained that the results obtained by 
hydrolysing with concentrated hydrochloric acid and with 25-33 per 
cent, sulphuric acid are different, but investigations by Abderhalden 
and Funk [1907] and by Skraup and Turk [1909] have proved that 
complete hydrolysis is effected by both acids, if the boiling be con- 
tinued for a sufficiently long time. 

(d) Hydrolysis with Hydrofluoric Acid. 

Hugounenq and Morel [1908; 1909, I ; 1909, 2], who have em- 
ployed hydrofluoric acid, found that the results depend on the strength 
of the acid ; the stronger the acid the greater is the amount of com- 
plex polypeptides. Many hours' boiling with dilute acid are required 
to effect complete hydrolysis. 

(e) Hydrolysis with Alkalies. 

Proteins are not generally hydrolysed by boiling with alkali. 
Alkali hydrolysis is only used for special purposes. The amino acids 
produced are completely racemised, whereas acids cause only partial 
racemisation. Arginine is destroyed t by boiling with alkali and con- 
verted into ornithine and ammonia. Cystine is also decomposed by 
boiling with alkali. 

(/) Comparison of the Hydrolysis with Acids and Alkalies. 

Abderhalden, Medigreceanu and Pincussohn ,[1909] compared 
the hydrolysis by acids and alkalies. Alkalies produce the most com- 
plete hydrolysis. Abderhalden and Brahm [1909] found that a body, 
formed from silk, and resistant to hydrolysis by acid, could only be 
completely hydrolysed by alkali. 



12 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

(g) Determination of the Completion of Hydrolysis. 

The completion of the hydrolysis has usually been ascertained by 
performing the biuret test on the solution and on any residue remain- 
ing when the time of hydrolysis is completed. Further hydrolysis is 
required if the biuret test is positive. Many proteins are hydrolysed 
with great slowness and, as has been pointed out by Osborne and 
Jones [1910, 3], boiling with acids for a much longer time than stated 
above is necessary ; in the case of caseinogen, Osborne and Guest 
[1911] found that hydrolysis with concentrated hydrochloric acid was 
only complete after three to five days. 

The biuret test is not altogether a satisfactory way of determining 
the completion of hydrolysis. It is necessary to determine whether 
an increase of ammonia and amino nitrogen occurs on a further period 
of six or eight hours' boiling. This increase can be estimated either 
by Van Slyke's amino nitrogen method or by Sorensen's method of 
titration with formalin. 

(i) Van Slykes Amino Nitrogen Method. 

The hydrolysis is carried out in a tared flask under a reflux con- 
denser ; after six or eight hours the heating is discontinued. A portion 
of I or 2 c.c. is removed with a pipette and diluted to 10 c.c. The 
amino nitrogen is estimated in this solution as described on pp. 89-96 ; 
the reaction is allowed to proceed for five minutes in the stationary 
apparatus and subsequently shaken for one minute. The flask in 
which the hydrolysis is carried out is weighed, and the boiling con- 
tinued for another six or eight hours. The flask is again weighed to 
ascertain if the solution has become more concentrated ; water may 
be added to replace the loss or a correction made for the decrease in 
volume. The hydrolysis is continued until the amino nitrogen is 
constant. 

(ii) Sorenseris Titration Method with Formalin. 

Sorensen [1907] proved that amino acids could be accurately 
estimated in solution by titration with alkali after they had been treated 
with neutralised formalin. The reaction of the amino acid with 
formalin results in the formation of a methylene compound, the neutral 
amino acid becoming a free carboxylic acid : 

CH 2 . NH 2 CH 2 . N = CH 3 

| + OHCH =| + H 2 

COOH COOH 



HYDROLYSIS 13 

Some modification of the method is necessary for the titration of 
amino acids when they are present in dark brown solutions such as 
result from the hydrolysis of proteins. Sorensen and Jessen-Hansen 
[1907] have given the following procedure: The reaction of the solu- 
tion must be approximately decinormal ; 25 c.c. of such a solution is 
used and placed in a 50 c.c. measuring flask. If the solution is not 
of this concentration in acid, 20 c.c. are taken and treated with '5N 
hydrochloric acid or sodium hydrate as required to produce the proper 
acidity. In the case of a solid substance 1-3 grams are dissolved in 
25 cc. of *i N hydrochloric acid. Four c.c. of 2N barium chloride solu- 
tion (244 grams per litre) are added and then gradually about 20 c.c. of 
*33N silver nitrate solution (56-7 grams per litre), which is best dropped 
into the solution from a small measuring cylinder. After each addition 
of silver nitrate the mixture is frequently and thoroughly shaken. The 
foam settles on standing for a short time and the flask is then filled with 
water free from carbon dioxide ; if great accuracy is required four drops 
more of water are put in to allow for the volume of the silver chloride. 
The solution is filtered through an 1 1 cm. filter paper, taking care to 
bring as much as possible of the precipitate upon the filter. The 
filtrate, which is at first cloudy, is again poured carefully on the filter. 
The filtrate is now quite clear, and 15 or 30 c.c. are titrated with forma- 
lin using phenolphthalein as indicator. The reagents required are (i) 
a solution of 0*5 gram of phenolphthalein in 50 c.c. alcohol + 50 c.c. 
water ; (2) a neutral formalin solution which must be prepared fresh 
for every series of experiments. It is prepared by adding I c.c. of 
phenolphthalein solution to 50 c.c. of commercial formalin (30-40 per 
cent.) and adding *2N baryta or sodium hydrate solution until it has a 
faint pink colour. In order to' be sure of the end point in the titration 
a control colour is prepared in the same volume of liquid as is used in 
the actual experiment. E.g. y 10 c.c. of the formalin solution are added 
to 20 c.c. of boiled distilled water : 5 c.c. of *2N baryta or sodium 
hydrate are added and the solution titrated with -2N hydrochloric 
acid, which is dropped in until the solution has a faint pink colour. 
A drop of *2N baryta or soda is added to produce a distinct red 
colour. Twenty c.c. of the solution l to be estimated are treated with 
10 c.c. of the formalin solution and then with -2N baryta or soda until 
there is a red colour ; a few more c.c. of alkali are added. The solu- 
tion is titrated with -2N hydrochloric acid until the colour is paler 

1 Henriques and Gjaldbak [1910] neutralise the solution to litmus before titrating in the 
presence of formalin. 



I 4 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

than that of the control and finally *2N alkali is added until the 
colour matches that of the control. To the control solution two drops 
of *2N alkali are now added so as to produce a deep red colour, and 
alkali is added to the solution until it attains the same deep red colour. 
The difference between the alkali and acid used in the solution and in 
the control (from 0-0-2 c.c.) gives the titration figure. 

(h) Hydrolysis with Enzymes. 

Hydrolysis by the action of proteoclastic enzymes is never com- 
plete. The earlier investigators always observed that a complex 
body antipeptone resistant to the further action of trypsin was 
formed. Fischer and Abderhalden [1903, I, 2] have confirmed this 
observation and have found that the resistant body contains all the 
phenylalanine and proline present in the protein molecule ; even by 
the combined action of pepsin and trypsin, although phenylalanine 
and proline are formed under these conditions, a body resistant to 
hydrolysis by trypsin still remained. Almost complete hydrolysis 
may be effected by trypsin and the enzymes in the small intestine 
if a sufficiently long time be given for the digestion. 

Henriques and Gjaldbak [1910] estimated the amount of unhydro- 
lysed protein, and found that in a prolonged digestion some 5-10 
per cent, of the amino nitrogen still remained in combination. 

Hydrolysis by enzymes, though of use in the discovery of new 
units in the protein molecule, is not serviceable for a complete analysis 
of the decomposition products. . 



ISOLATION AND ESTIMATION OF UNITS 15 



II. THE ISOLATION AND ESTIMATION OF THE UNITS. 

The units composing the protein molecule belong to four different 
classes of organic compounds, but are divided into two main groups for 
the purpose of isolation and estimation : 

A. The mono-amino acids, including proline and oxyproline. 
Tyrosine and cystine differ from the other mono-amino acids by 

their extremely slight solubility in neutral aqueous solutions. They 
are therefore easily obtained after hydrolysis by acids by neutralising 
and concentrating the solution, when they crystallise out. 

B. The di-amino acids, including histidine. The three compounds 
in this group were formerly called the hexone bases on account of 
their basic properties and the fact that each of them contains six 
carbon atoms. 

The remaining unit, tryptophan, is almost completely destroyed 
by hydrolysis by acids ; it is usually isolated after hydrolysis by trypsin. 

Ammonia (amide nitrogen) is estimated by hydrolysing the protein 
with concentrated hydrochloric acid, removing the great excess of acid 
by evaporation under reduced pressure, adding excess of magnesia, 
distilling off the ammonia in vacua and collecting it in excess of 
standard acid. This operation is usually carried out in the determina- 
tion of the distribution of the total nitrogen amongst the two main 
groups (p. 97). The estimation of ammonia is frequently combined 
with the estimation of the di-amino acids (p. 55). 

The separation and estimation of the two main groups of amino 
acids is generally not carried out in one experiment, but only when 
the amount of protein available is small, as very different quantities 
of material are required. Thus, the di-amino acids can be determined 
in 25-50 grams of protein with considerable accuracy, whereas the 
mono-amino acids can only be determined with fair accuracy when 
250-500 grams of protein can be used. The two processes, of 
which the details are given under the two sections, may be combined 
as follows : 

The protein is hydrolysed by boiling for fifteen to twenty-four hours 
with six times its quantity of 25-30 per cent, sulphuric acid. The 
solution is neutralised with baryta and the filtrate and washings from 
the barium sulphate are evaporated down to a small volume. Tyrosine 
(and cystine) crystallise out. The filtrate is diluted with water and 
sulphuric acid added till the content of acid is 5 per cent. The 



16 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

di-amino acids are then precipitated with phosphotungstic acid (pp. 60, 
98) ; from this precipitate they are obtained by decomposition with 
baryta and separated by means of their silver compounds as described 
in section B (p. 55). The filtrate is freed from phosphotungstic acid 
and sulphuric acid with baryta, excess of which is removed with carbon 
dioxide and sulphuric acid, and then treated for the other mono-amino 
acids as described in section A (p. 33). 

On the whole it is not advisable to combine the two processes, 
since the phosphotungstic acid precipitation does not effect a perfect 
separation of the two groups and considerable loss occurs. 



ISOLATION AND ESTIMATION OF TYROSINE 17 

A. THE MONO-AMINO ACIDS. 
Tyrosine. 

(l) Isolation and Gravimetric Estimation. 

Hydrolysis by sulphuric acid possesses one great advantage over 
that by hydrochloric acid, as it can be subsequently completely and 
easily removed by baryta. 

The protein is hydrolysed by boiling with five to six "times its 
quantity of 25 per cent, sulphuric acid for twelve to fifteen hours ; the 
solution, after filtration, is diluted with twice its volume of water and 
neutralised with barium carbonate, or a strong solution of baryta, the 
excess of which is then quantitatively removed by dilute sulphuric 
acid. The solution, together with the water used in thoroughly wash- 
ing the precipitate of barium sulphate, is then evaporated down, 
until the compound crystallises out. It is filtered off, the filtrate is 
concentrated, and further crops of crystals are removed until the 
mother liquor no longer gives Millon's reaction for tyrosine. The 
amount of cystine in most proteins is so small that the product 
generally consists only of tyrosine. It is purified by recrystallisation 
from water, decolorisation of the solution being effected by charcoal. 
The yield of tyrosine so obtained is the measure of its amount in the 
protein. 

On account of the insolubility of tyrosine and the difficulty of 
filtering and completely washing the barium sulphate precipitate in 
order to abstract from it the whole of the tyrosine, Abderhalden and 
Teruuchi [1906], in the case of silk, hydrolysed the protein with 
hydrochloric acid, the greater part of which was then removed by 
evaporating several times in vacuo after diluting the concentrated 
solution with water ; the. remainder of the hydrochloric acid was then 
estimated in a small aliquot portion, and the main bulk neutralised 
with the calculated amount of caustic soda. The tyrosine then cry- 
stallised out and was purified by recrystallisation from water. 

Abderhalden [1912] shortened the process by hydrolysing the silk 
by boiling for three hours with three times its quantity of concentrated 
hydrochloric acid, evaporating the solution to drynegs repeatedly in 
vacuo to remove the hydrochloric acid, dissolving the residue in water 
and passing in ammonia gas, or dissolving in ammonia, again evaporat- 
ing in vacuo to dryness and treating the residue with cold water. 

PT. I. 2 



1 8 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

Tyrosine remained. It is better to dissolve the residue in hot water, 
decolorise with charcoal, and allow the tyrosine to crystallise out. 

When large quantities of protein are under investigation, the 
removal of the hydrochloric acid, after evaporation in vacuo, is effected 
by treating the solution with cuprous oxide until it is green in colour, 
filtering off and washing the cuprous chloride, and removing dissolved 
copper by hydrogen sulphide. A current of air is then passed 
through the solution to remove the hydrogen sulphide, and the re- 
mainder of the hydrochloric acid is either neutralised with the calcu- 
lated quantity of soda or is removed by treating with silver carbonate. 
The solution on concentration deposits the tyrosine. 

Levene and van Slyke [1908, 2] prefer the use of hydrochloric 
acid to that of sulphuric acid for separating tyrosine on account of the 
difficulty of completely extracting it from the barium sulphate precipi- 
tate and of obtaining it in a state of purity. Their procedure is the 
following : The protein is hydrolysed with concentrated hydrochloric 
acid ; the solution is concentrated and saturated with gaseous hydro- 
chloric acid. Glutamic acid hydrochloride separates out. The filtrate 
and washings from this precipitate are concentrated in vacuo to remove 
the greater part of the hydrochloric acid. The solution is then 
diluted to 7 litres (for 400 grams protein) and boiled with lead oxide 
till its reaction is alkaline. 1 The lead oxide is prepared by precipita- 
tion with baryta, washed by decantation and preserved in the form of 
a paste. The precipitate of lead oxychloride is filtered off when the 
solution has cooled. It retains the resinous matters and a nearly 
colourless filtrate results. The remainder of tr>e chlorine, which is 
estimated in an aliquot portion, is removed by means of the calculated 
quantity of silver sulphate, the excess of lead by adding sulphuric acid 
and passing in hydrogen sulphide, and of sulphuric acid by baryta. 
On concentrating the solution to one-seventh almost pure tyrosine 
separates out ; it is filtered off, washed, dried and weighed. A portion 
of the other amino acids can be obtained by further concentration, 
and treated for leucine and valine (see p. 45). The di-amino acids are 
then precipitated (pp. 60, 98) and the filtrate is treated for the other 
monoamino acids. 

If the solution be highly concentrated a mixture of tyrosine and 
leucine may separate out. This mixture may be separated by treat- 
ing with glacial acetic acid. Leucine is soluble, tyrosine is insoluble 
[Habermann and Ehrenfeld, 1902]. 

1 Levene and Van Slyke [1910] state that excess of lead oxide should be avoided so as to 
prevent the formation of the insoluble lead salt of tyrosine. 



ISOLATION AND ESTIMATION OF TYROSINE 19 

The data for tyrosine by the gravimetric method by various workers 
are in some cases quite concordant, but there are instances in which 
the data are very different, e.g., Abderhalden and Langstein [1910] give 
4 -6 per cent, for the amount of tyrosine in caseinogen, whereas Osborne 
and Guest [1911] were only able to isolate 3-4 per cent. The same 
quantity was obtained by Totani [1916]. Totani was able to isolate 
up to 4 -I per cent, by the further treatment of the liquid with 
mercuric sulphate. Osborne and Clapp [1907, 8] have emphasised the 
fact that from zein it is extremely difficult to crystallise the whole of 
the tyrosine, and Abderhalden and Fuchs [1913] also make a similar 
statement. They state further that better yields are obtained by 
evaporating the solutions in vacua, which prevents access of acid and 
alkali vapour which keeps the tyrosine in solution and prevents it from 
crystallising. Sometimes the basic di-amino acids such as lysine form 
a combination with tyrosine preventing crystallisation. It may be 
generally considered that all these data are minimal. 

In order to obtain tyrosine E. K. Marshall, jun. [1913], pointed out 
that it was made more quickly and with less manipulation if it were 
prepared by the tryptic digestion of caseinogen. Pig's pancreas was 
finely minced, mixed with an equal weight of water, and allowed to 
stand in the presence of chloroform for two days, whereupon the mix- 
ture was incubated at 37 for twenty-four hours, cooled and filtered. 
100-150 grams of caseinogen are added per litre of filtrate, the solu- 
tion is made slightly alkaline with ammonia and digested at 37 for 
three to seven days. Tyrosine separates out and is filtered off after 
cooling. The solid matter is extracted with 1000, 500, and 250 
c.c. of boiling water and these extracts are evaporated to 250 c.c. 
The tyrosine which crystallises out is obtained quite pure by one re- 
crystallisation. The yield is given as 5 grams from 100 grams of 
caseinogen and I -2 grams from I litre of pancreas extract. 

This method of preparation of tyrosine has been used for many 
years by the author by digesting caseinogen dissolved in alkali with 
various preparations of trypsin. The yields were never so high as the 
3 -8 per cent, obtained by Marshall ; on the average about 2-5 per cent. 



20 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(2) Color imetric Estimation. 

Folin and Denis [1912, 2] introduced a method for the quantita- 
tive estimation of tyrosine in proteins depending upon the blue colour 
which tyrosine gives with a phosphotungstic-phosphomolybdic reagent 
[1912, i]. 

The estimation is carried out as follows : 

A weighed quantity about I gram of dried protein ishydrolysed 
by boiling under a reflux condenser for twelve hours with 25 c.c. of 
20 per cent, hydrochloric acid. The solution is transferred to a 100 
c.c. measuring flask and made up to volume. One or two c.c. of this 
solution are placed in a 100 c.c. flask, 5 c.c. of the reagent * are added, 
and after five minutes 25 c.c. of a saturated solution of sodium car- 
bonate. The volume is made up to 100 c.c. with water. At the same 
time 5 c.c. (= i mgm.) of a standard tyrosine solution 2 are placed 
in another 100 c.c. measuring flask and treated in the same way. The 
maximum blue colour develops in half an hour. After this time the 
colours of the two solutions are matched in a Duboscq colorimeter, 
the standard being set at 20 mm. The solutions, if not clear, are 
filtered before making the comparisons. 

Folin and Denis' results, compared with the gravimetric data, 
were : 



Wool (sheep) . 
Hair (human 
Horn (cow) 
Gelatin 
Ovomucoid 

Globulin (flax seed) . 
Glycinin (soy bean) . 
Phaseolin (white bean) 
Globulin (squash seed) 
Corylin (hazel nut) 
Edestin (hemp seed) . 
Amandin (almond) 
Globulin (cotton seed) 



Colori- 


Gravi- 


metric, 


metric, 


per cent. 


per cent, 


6-0 


2' 4 


4'3 


3-0 


6-5 


4-6 


trace 





5 '4 





3*3 





4-0 


1-9 


4'5 


2'2 


4'9 


i*4 


4-0 





5*2 


2'I 


47 


1*1 


47 


23 



Caseinogen 
Vitellin 
Conalbumin 
Ovalbumin 
Lactalbumin 
Globulin (ca 
Vignin (cow pea) 
Legumin (pea) 
Glutelin (maize) 
Zein (maize) 
Hordein (barley) 
Gliadin (wheat) . 
Glutenin (wheat) 





Colori- 


Gravi- 




metric, 


metric, 




per cent. 


per cent. 




6-5 


4'5 




5'2 


3'4 




4'9 









1-8 




4'9 


0-9 


bean 


4'3 




) 


4-6 


2-3 




4'5 


2'8 


. 


5*5 


3-6 




47 


1-7 


. 


3 '3 


2-4 




5*8 


4'3 



In all cases the values by the colorimetric method are higher than 
those by the gravimetric. These higher values for tyrosine were shown 
by Folin and Denis not to be due to the reaction taking place with 

1 100 grams of sodium tungstate, 20 grams of phosphomolybdic acid and 50 c.c. of 85 
per cent, phosphoric acid are added to 750 c.c. of water. The solution is boiled for two 
hours under a reflux condenser, cooled and diluted to 1000 c.c. 2 c.c. of this reagent will 
give the maximum colour with i mgm. of tyrosine. 

2 Pure tyrosine dissolved in -iN HC1 so that 5 c.c. contain i mgm. of tyrosine. 



ISOLATION AND ESTIMATION OF TYROSINE 21 

other amino acids, all of which were tested except tryptophan, oxy- 
tryptophan and oxyproline, nor to the formation of phenols during 
hydrolysis. They therefore concluded that, in the absence of any 
amino acid at present unknown, these values more correctly represent 
the amounts of tyrosine in proteins than the gravimetric values. 

Abderhalden and^uchs [1913] subsequently maintained that the 
values for tyrosine by the gravimetric method were more exact than 
those by the colorimetric method. They showed that tyrosine added 
to gelatin could be almost entirely Recovered from the mixture of the 
products of hydrolysis, and that a reaction was given by oxytryptophan 
and by tryptophan. These reactions are not of a similar colour in- 
tensity as the reaction with tyrosine. 

Folin and Denis [1913] conceded that their figures may be a little 
too high, but do not consider that the presence of tryptophan, though 
it may react after boiling with acids, will cause the great differences 
observed especially as the higher results are of a similar magnitude with 
caseinogen and with zein. 

Abderhalden [1913] stated that oxyproline gave a blue colour re- 
action with the reagent, but seeing that gelatin with 3 per cent, of 
oxyproline only shows a trace of colour and that other proteins prob- 
ably contain still smaller amounts of oxyproline, the error due to 
oxyproline, together with that due to oxytryptophan and tryptophan, 
is not sufficient to account for the differences. We must therefore 
conclude that the data by the colorimetric method more nearly repre- 
sent the actual amount of tyrosine in proteins than the data by the 
gravimetric method. 



22 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(3) Estimation by Bromination. 

It was shown by J. H. Millar [1903] that tyrosine was readily 
brominated and converted into dibromotyrosine, and that the amount 
of tyrosine in a simple mixture of amino acids could be accurately 
estimated by means of this reaction. 

A. J. Brown and E. T. Millar [1906] using this reaction showed 
that the tyre sine in proteins was completely liberated at a very early 
stage in the hydrolysis by trypsin. Their data gave the tyrosine con- 
tent of edestin as 4-06 per cent, a figure which is considerably higher 
than that obtained by direct isolation (2-1 per cent). They made no 
estimations of tyrosine in other proteins. 

Plimmer and Eaves [1913] studied this reaction more closely with 
a view to the estimation of tyrosine in proteins. It was not found 
possible to apply the reaction to the products of hydrolysis of proteins 
by acids, since tryptophan, though almost entirely decomposed by 
boiling with acids, gave rise to products which absorbed bromine, but 
if the hydrolysis of the protein were effected by the action of trypsin, 
and if the amount of bromine absorbed were determined after about 
six hours, values for tyrosine were obtained which agreed closely with 
those by the gravimetric method. If the amount of bromine absorbed 
was determined after twenty-four to twenty-eight hours' hydrolysis, 
the values for tyrosine were very similar to those by the colorimetric 
method of Folin and Denis. 

The procedure was to digest a known weight of protein in faintly 
alkaline solution with trypsin, to remove measured volumes of the 
digest after the stated period, and to treat this volume with a 5 per 
cent, solution of phosphotungstic acid in 5 per cent, sulphuric acid to 
remove histidine, which absorbs bromine. A measured volume of the 
filtrate was treated with a known volume of *2N sodium bromate solu- 
tion and 10-20 c.c. of 20 per cent, sodium bromide solution. After 
half an hour the excess of bromine was determined by titration with 
thiosulphate solution, using starch and sodium iodide as indicator. 
At the same time the same volume of trypsin solution was digested 
by itself and treated in a similar way so as to deduct the amount of 
tyrosine in this solution. 

The values of tyrosine by bromination are given in the following 
table and are compared with the gravimetric and colorimetric : 



ISOLATION AND ESTIMATION OF TYROSINE 23 





Bromination after 
6 hours' digestion, 
per cent. 


Gravimetric, 
per cent. 


Bromination after 
24-28 hours' 
digestion. 


Colorimetric, 
per cent. 


Caseinogen 


5'3 


4'5 


6-3 


6'5 


" Peptone Roche " 


1O'2 




IO'2 




Silk fibroin 


9'5 l 


9 to 10-5 








Conglutin 


i'3 


2'I 


3'i 





Legumin 


27 


2'8 


4*2 


4'5 


Edestin 


17 


2'I 


3'6 


5'2 


Vignin 


3'4 


2- 3 


6-0 


4-6 


Globulin (squash seed 


3'2 


3*i 


5'8 


4*9 


Amandin 


2-9 


I'l 


5'i 


47 


Glycinin 


IT 


1-9 


3'5 


4-0 


Excelsin 


2'5 


3'i 


4-6 





l By acid hydrolysis. 



2 5-2 after forty-eight hours. 



The bromination method thus does not decide whether the gravi- 
metric value or the colorimetric value represents the tyrosine content 
of protein the more accurately and consequently is of little value for 
determining the tyrosine content of proteins. It may prove of use to 
ascertain the amount of tyrosine in the impure tyrosine which is iso- 
lated from proteins, if this impure tyrosine is shown to be free from 
tryptophan. The gravimetric values may then be found to be higher 
than those given by the various workers, who have usually only 
weighed pure recrystallised tyrosine. 



24 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

Cystine. 

(i) Isolation and Gravimetric Estimation. 

With few exceptions cystine has not been isolated from the pro- 
ducts of hydrolysis of proteins other than the keratins, and in these 
cases usually mixed with tyrosine from which it required separation. 
Morner [1901-2], who first isolated cystine from the products of 
hydrolysis, heated the protein- hair, keratin from horn, egg-shells, 
etc. with five times its quantity of 13 per cent, hydrochloric acid 
under a reflux condenser on a water-bath for six to seven days. The 
solution was then decolorised with charcoal and evaporated in vacuo, 
and the residue dissolved in 60-70 per cent, alcohol. On neutralisa- 
tion with soda a mixture of cystine and tyrosine separated out. 

Embden [1900] hydro lysed horn by boiling under a reflux with 
three times its quantity of concentrated hydrochloric acid. The liquid 
was neutralised to amphoteric reaction and allowed to stand for not 
more than twenty-four hours. The black pigment (melanin) was 
filtered off and the filtrate after acidifying was boiled with charcoal. 
The pale yellow filtrate gave on standing in a cool place a large 
precipitate of tyrosine and cystine. Further quantities were obtained 
in the same way on concentrating the mother liquor. 

Some alterations in this procedure were made by Friedmann [1902]. 
The hydrolysed protein was nearly neutralised with a concentrated 
solution of caustic soda, decolorised by boiling with large amounts 
of charcoal, and allowed to cool. Cystine and tyrosine crystallised out. 

A simple method of preparing cystine from wool was described by 
Folin [1910]. The wool is boiled with concentrated hydrochloric acid 
in the proportion of 100 grams of wool to 200 c.c. of acid for three to 
five hours. The hot solution is then neutralised to congo red by adding 
sodium acetate in the form of crystals. Almost the whole of the cystine 
separates out on standing. After several hours the precipitate is dis- 
solved in 3-5 per cent, hydrochloric acid, the solution is boiled with 
charcoal, and the hot colourless solution is neutralised as before by 
adding a hot concentrated solution of sodium acetate. Cystine 
separates out in the characteristic hexagonal plates as the solution 
cools. No data of the yield were given. 

The mother liquor on dilution and on standing deposits tyrosine. 
This is most readily purified by dissolving in hydrochloric acid, de- 
colorising the solution with charcoal, and then neutralising exactly 
with ammonia, when almost pure tyrosine separates out. 



ISOLATION AND ESTIMATION OF CYSTINE 25 

(2) Separation of Cystine and Tyrosine. 

The mixture of cystine and tyrosine was separated by Morner by 
fractional crystallisation from ammonia ; if much tyrosine was present 
it separated out first, but if cystine exceeded tyrosine in quantity this 
compound crystallised out first ; the remainder was only separated 
with difficulty. 

The method adopted by Embden and followed by Friedmann was 
the solution of the tyrosine from the mixture, suspended in water, by 
the addition of very dilute nitric acid. The separation could be 
checked by microscopic observation of the crystals, the needle-shaped 
crystals of tyrosine dissolving whilst the plates of cystine remained. 
After washing free from acid with water, the cystine was recrystallised 
from ammonia. 

Separation of the mixture was effected by Abderhalden [1903] 
and by Abderhalden and Pregl [1905, 2] by dissolving in ammonia 
and adding glacial acetic acid keeping the reaction alkaline ; tyrosine 
was precipitated : on acidifying with glacial acetic acid the cystine 
separated. 

The separation of cystine from hydrolysed Bence-Jones protein by 
Hopkins and Savory [1911] was carried out by precipitation of a 
tryptic digest with mercuric sulphate in acid solution (see under 
tryptophan). Before the second precipitation the solution was con- 
centrated after removal of the acid ; cystine crystallised out. 

The precipitation of cystine by mercuric sulphate in 5 per cent, 
sulphuric acid solution in which the mercury compound of tyrosine is 
soluble was used by Osborne and Clapp in the cases of squash seed 
globulin [1907, 6] , and wheat gliadin [1906] respectively. 

Winterstein [1901] showed that cystine was precipitated by phos- 
photungstic acid, but the observation seems to have been overlooked. 
Cystine and tyrosine can be separated by means of this reagent. The 
mixture is dissolved in 5 per cent sulphuric acid and treated with 
excess of phosphotungstic acid solution. Cystine phosphotungstate 
generally separates out in a crystalline condition. From this precipi- 
tate the cystine can be obtained by the usual method (see under 
di-amino acids), but a large excess of baryta must be avoided as 
cystine is readily decomposed by alkalies. Cystine crystallises out on 
neutralising the filtrate from the barium phosphotungstate. Winter- 
stein decomposed the phosphotungstate with hydrochloric acid. The 
precipitate is made into a paste with water, placed in a separating 



26 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

funnel and treated with small quantities of concentrated hydrochloric 
acid. Ether is added and the mixture well shaken. At first an 
emulsion is formed, but on adding more ether and acid and thoroughly 
shaking a clear ethereal solution of phosphotungstic acid settles to the 
bottom ; if this does not occur, decomposition is not complete and 
more acid must be added and the shaking repeated. The middle layer 
containing the cystine hydrochloride is separated ; the other layers are 
treated once more in the same way and the middle layers are combined. 
Ether is removed from the acid solution by warming on the water- 
bath and the cystine is separated by exactly neutralising with soda. 

The separation of cystine and tyrosine has been examined by 
Plimmer [1913]. Neither the phosphotungstic acid method nor the 
mercuric sulphate method was found to be satisfactory, as loss of 
both substances occurred. In the case of phosphotungstic acid some 
tyrosine was carried down with the precipitate and was only removed 
by prolonged washing, and some of the cystine was decomposed in 
recovering it from its phosphotungstate. In the case of mercuric 
sulphate, the cystine is not completely precipitated. A quantitative 
separation of the two compounds was effected by means of absolute 
alcohol saturated with hydrogen chloride. Tyrosine is converted into 
its ethyl ester hydrochloride; cystine is not acted upon. The mixture 
of the two compounds is covered with the alcoholic hydrochloric acid 
and warmed upon the water-bath to dissolve the tyrosine. An equal 
volume of absolute alcohol is now added to precipitate any cystine 
which may dissolve. The undissolved cystine is filtered off and 
washed with absolute alcohol till it is free from acid and recrystallised 
from ammonia in the usual way. The filtrate containing the tyrosine 
ester is diluted with two volumes of water and boiled for eight hours, 
water being added when necessary. The ester is thus hydrolysed and 
on neutralising the tyrosine is precipitated. 

The usefulness of this method is shown by the treatment of pre- 
sumably pure tyrosine prepared from wool by Folin's method for ob- 
taining cystine ; one specimen of 5 grams contained 0*05 gram of 
cystine, another specimen of 4 grams yielded O'2 gram of cystine. 



ISOLATION AND ESTIMATION OF TRYPTOPHAN 27 

Tryptophan. 

(i) Isolation and Gravimetric Estimation. 

Tryptophan is not obtained by the hydrolysis of proteins by acids 
owing to its decomposition and is prepared by the action of trypsin. 
By the method of Hopkins and Cole [1901, 2] the protein is digested 
in alkaline solution by trypsin, until the solution gives a maximal 
coloration when tested with bromine water ; the solution is acidified, 
boiled and filtered. The clear solution (better after concentrating in 
vacuo and filtering off tyrosine, which crystallises out) is acidified with 
sulphuric acid until it contains 5 P er cent., and then 10 per cent, 
mercuric sulphate in 5 per cent, sulphuric acid solution is added as 
long as a precipitate, which contains tryptophan, cystine and tyrosine, 
is formed. The yellow precipitate is freed from tyrosine by washing 
with 5 per cent, sulphuric acid in which the tyrosine compound is 
soluble, that is, until the washings no longer react with Millon's 
reagent. The precipitate is decomposed by sulphuretted hydrogen, 
and the solution containing cystine and tryptophan after removing the 
hydrogen sulphide is again acidified with sulphuric acid to 5 per cent, 
and fractionally precipitated with the mercuric sulphate reagent. The 
cystine is thrown down first, filtered off, and then the tryptophan is 
precipitated. The precipitate is again decomposed by hydrogen sul- 
phide, and the solution, freed from sulphuric acid, is evaporated down, 
alcohol being continually added to hasten the evaporation and prevent 
decomposition of the tryptophan, which crystallises out. It is purified 
by recrystallisation from water. The yield represents its amount in 
the protein. 

Neuberg and Popowsky [1907] introduced a few alterations in the 
procedure, such as evaporation to one quarter and filtration from tyro- 
sine before precipitating with mercuric sulphate ; in decomposing the 
precipitate with hydrogen sulphide they make the solution faintly 
alkaline with baryta and treat with the gas for twenty-four hours, 
warming several times. The removal of sulphuric acid in the last 
operation is carried out with lead carbonate in ammoniacal solution, the 
lead being subsequently removed with hydrogen sulphide. 

Abderhalden and Kempe [1907, i] made very little alteration in 
the procedure of Hopkins and Cole beyond evaporation of the solutions 
in vacuo and removal of hydrogen sulphide with -a current of carbon 
dioxide. The yield of tryptophan obtained by these workers (0-53 
per cent.) is less than the yield mentioned by Hopkins and Cole in the 
case of caseinogen (1*5 per cent). Usually about I per cent, is ob- 
tained from caseinogen. 



28 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

(2) Colorimetric Estimations. 

Attempts have been made by several investigators to estimate the 
amount of tryptophan in proteins by colorimetric methods. 

i. Levene and Rouiller [1906-07] suggested a procedure depend- 
ing upon the bromine absorption of tryptophan. The protein was 
digested by trypsin and the tryptophan precipitated by mercuric 
sulphate as described above. This precipitate was decomposed by 
hydrogen sulphide in 1-2 per cent, sulphuric acid solution and the 
filtrate from the mercuric sulphide made up to a definite volume. To 
15 c.c. of this solution 2 c.c. of amyl alcohol were added and then 
gradually bromine water with vigorous shaking. The end point was 
reached when the purple colour of the amyl alcohol disappeared and 
became yellow. 

The absorption of bromine is influenced by the presence of tyrosine 
and cystine, which are precipitated by mercuric sulphate with trypto- 
phan. The amount of tyrosine thrown down is negligible, if the 
mercuric sulphate reagent is added only as long as the solution gives 
a reaction for tryptophan with bromine water and if the precipitate is 
washed until the washings give no reaction for tyrosine. A correction 
is made for the absorption by cystine, the amount of cystine in the 
solution being determined by a sulphur estimation. T}ie bromine 
water used is standardised against solutions of tryptophan and cystine. 

Up to the present no values of the amount of tryptophan in pro- 
teins have appeared, the further work of these authors [1907] being 
concerned with the hydrolysis of the protein by baryta and the nature 
of the bromine derivatives of tryptophan. They found that tryptophan 
was not completely separated from protein by the hydrolysis with 
baryta unless the hydrolysis was prolonged and that under these con- 
ditions the colour reaction was not satisfactory. By a short hydrolysis 
polypeptides containing tryptophan are present, which also absorb 
bromine and affect the estimation. At the stage when a violet colour 
is produced by bromine water they consider that a mixture of mono- 
and dibromo-tryptophan is present and that the di bromide is present 
when excess of bromine has been added. 

2. Proteins containing tryptophan give a reddish-violet to blue 
colour reaction with glyoxylic acid and sulphuric acid the Adamkie- 
wicz-Hopkins reaction. It is positive for a concentration of tryptophan 
of I in 100,000. This reaction has been made use of by Fasal [1912] 
for estimating tryptophan in proteins and is carried out in the follow- 



ISOLATION AND ESTIMATION OF TRYPTOPHAN 29 

ing way : Solutions of tryptophan of a concentration of I in 1000, 
I in 2000, I in 3000 and so on up to I in 10,000 are prepared and 
also I in 20,000 up to I in 50,000. To I c.c. of these solutions in 
test tubes of equal size and bore are added 2 c.c. of glyoxylic acid 
solution 1 and 6 c.c. of concentrated sulphuric acid. A series of 
coloured solutions from deep blue to red-violet result. A known 
weight (about O'l gram) of previously purified and dried protein is 
placed in another test tube and treated with 2 c.c. of glyoxylic acid 
and 6 c.c. of concentrated sulphuric acid. The colour so produced is 
compared with the other colours. The one showing the closest com- 
parison is then matched with the one from the protein in a Duboscq 
colorimeter, the cups of which are entirely made of glass. Readings 
are taken from both above and below in the usual manner. Usually 
the comparison was made with the tryptophan solution I in 4000, 
i.e., i c.c. = 0*00025 gram tryptophan. If the colorimeter readings 
were for example 22 : 18 (standard) the protein contains i -23 times as 
much tryptophan as the solution, i.e., O'l gram protein contains 
o '00025 * 1*23 gram tryptophan or 0*3 per cent. 

In most cases the colours could be matched fairly well, but in some 
cases the comparison was difficult owing to brown shades in the pro- 
tein solution. This could be sometimes overcome by taking different 
amounts of protein. In these cases Fasal [1913] found that a com- 
parison was possible if the colours were compared in the apparatus 
with colour filters described by Sorensen. 2 Numerous estimations 
were made by Fasal ; they are given in the following table in which 
the figures are the percentage of tryptophan : 

Caseinogen 0*65 Epidermis 0^30 

Edestin 0*38 Cutis o o 

Lactalbumin . . . .3*07 Psoriasis scales 0^41 

. 1*00 Carcinoma (epithelial) . . i*7 
. 0*17 ,, (liver) . . . -i*7 

+ ,, (mouse) . . . .1*6 

. o Sarcoma (rat) 1-4 

+ Carcinoma (breast) . . .. . o 



Liver tissue 
Horn (ox) . 
Nail (human) 
Hair (human) 
Wool (sheep) 



The different kinds of keratin, for which the method was particu- 
larly devised on account of their insolubility by pepsin and trypsin, 
show very striking differences. The value for caseinogen is higher 
than that found by the gravimetric method by Abderhalden and 
Kempe, but lower than that found by Hopkins and Cole. The large 

1 Prepared by reducing a saturated aqueous oxalic acid solution with 3 per cent, sodium 
amalgam. 

* Biochemische Zeitschrift, 1909, 21, 201. 



30 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

amount of tryptophan in lactalbumin is most noticeable. Greater 
amounts of tryptophan were present in carcinoma than in the normal 
tissue, but tryptophan was absent in carcinoma of the breast. 

3. Tryptophan is decomposed by putrefactive bacteria with the 
formation of indole. An investigation by Herzfeld [1913, i] into the 
formation of indole by the action of alkali (the highest yields being 
given by boiling with 1000 c.c. of 9 per cent, caustic soda and I gram 
of copper sulphate), showed that the estimation of tryptophan under 
these conditions was not practicable. Sanders and May [1912-13] 
have also tried to estimate tryptophan by the formation of indole. 
The pancreatic digest of protein was inoculated with faecal bacteria, 
distilled, and the indole in the distillate treated with nitrous acid. 
At the same time an indole solution was treated in a similar way and 
the colours compared. Caseinogen gave an amount of indole cor- 
responding "to I -6 percent, of tryptophan. 

4. Herzfeld [19 1 3, 2] determined the tryptophan content of several 
proteins by the colorimetric and spectrophotometric comparison of 
the colour given by tryptophan with /-dimethylaminobenzaldehyde 
and concentrated hydrochloric acid. A blue colour is given by this 
reagent with one part of tryptophan in 1 ,000,000. As a standard 
colour a solution of I gram of ignited copper sulphate dissolved in 
100 c.c. of water is used, I c.c. of this solution being treated with 20 c.c. 
of ammonia and diluted to 100 c.c. This blue solution has the same 
colour as 0*0001 gram of tryptophan in its reaction with /-dimethyl- 
aminobenzaldehyde and hydrochloric acid. The two are carefully 
compared before use. About I gram of the purified and dried protein 
is accurately weighed out and dissolved in 500 c.c. of O'5 per cent, 
sodium carbonate solution to which 0-5 gram of pancreatin is added. 
The solution is kept at 37 in the presence , of chloroform and xylene. 
50 c.c. are then taken and treated with 10 c.c. of /-dimethylamirio- 
benzaldehyde solution 1 and 40 c.c. of concentrated hydrochloric acid. 
After thirty minutes' standing the blue colours are compared. The 
tryptophan content of the pancreatin is also determined and the 
amount deducted from the digest of protein with pancreatin. The 
colorimetric values agreed closely with the spectrophotometric. 

The tryptophan content of several proteins was determined : 

1 20 grams /-dimethylaminobenzaldehyde dissolved in 500 c.c. cone. HC1 and 500 c.c. 
water. 



ISOLATION AND ESTIMATION OF TRYPTOPHAN 31 



Albumin (blood) 
Albumin (milk) 
Fibrin (blood) 
Caseinogen 
Elastin 
Albumin (egg) 
Keratin 
Witte-Pepton 
Bread (white) l 
Milk . 






0-95 P er 
0-91 
1-05 
0-51 

O'22 
0-52 
0-03 
1-25 
0'03 
0'02 


cent. 



Caseoglutin 

Conglutin (yellow lupin) 

Edestin (oil seeds) 

Globulin (squash seed) 

Glutencasein (wheat gluten 

Legumin (pea) . 

Plant-fibrin 

Plant-gelatin 

Tyroalbumin 

Vitellin (squash seed) 



0-08 per cent. 

o'o6 

0-58 

o 61 

0-07 

0-83 

0-04 

0-42 

0*05 

0-30 



1 69-9 per cent, dry substance. 



These values are not in agreement with those found by Fasal in 
the cases of milk albumin, caseinogen, edestin and keratin (horn). 

In all cases the time of digestion of the protein was presumably 
twenty-four hours, and in this time the whole of the tryptophan is not 
likely to be liberated from the protein. A maximal colour reaction 
for tryptophan with bromine water as stated by Hopkins and Cole is 
only obtained after several days, and the rate of digestion of proteins 
is not likely to be the same ; keratin is not dissolved by trypsin and 
hence the low value. 

The tryptophan content of the kidneys in normal and pathological 
conditions has been determined by this method by Kurchin [1914]; 
normal kidneys contained slightly more than pathological. 



32 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

(3) Estimation by Bromination. 

The bromination of tryptophan with a view to its estimation in 
solution has been studied by Homer [1915]. Solutions of tryptophan 
were treated with excess of (a) nascent bromine liberated from 10 
c.c. of a sodium bromate solution (15*1 grams per 1000 c.c.) and 10 
c.c. of sodium bromide solution (51*5 grams per 1000 c.c.) by 5 c.c. 
of concentrated hydrochloric acid ; (ft) saturated bromine water. 
After varying periods of time, from thirty minutes to eight hours, the 
excess was titrated with -iN thiosulphate solution after adding 
potassium iodide. Under these conditions tryptophan was found to 
absorb eight atoms of bromine. The first reaction is identical with 
that used by Plimmer and Eaves in their estimation of tyrosine; these 
workers found that tryptophan absorbed six atoms of bromine. 

In carrying out the estimation in proteins the protein was hydro- 
lysed by boiling 100 grams with 350 grams barium hydrate dissolved 
in 2500 c.c. of water for 20 to 120 hours. In the absence of metallic 
salts tryptophan was found to be stable to the action of baryta (see 
above, 3). The baryta was removed with sulphuric acid and the 
solution acidified to 5 per cent. On the addition of the mercuric sul- 
phate reagent the tryptophan is precipitated. The precipitate was 
filtered off after forty-eight hours and washed until free from tyrosine ; it 
was then suspended in 2 per cent, sulphuric acid and decomposed with 
hydrogen sulphide. The filtrate from the mercuric sulphide was freed 
-from hydrogen sulphide and treated with phosphotungstic acid to 
remove polypeptides. The filtrate containing the tryptophan was 
treated with baryta to remove phosphotungstic acid, and with sul- 
phuric acid to remove baryta, and made up to a definite volume (usually 
1000 c.c.). 50 c.c. of this solution were treated with sodium bromate 
and bromide solutions, and at the same time a tryptophan solution was 
treated in the same way. The excess of bromine was titrated with 
'iN thiosulphate. From the bromine absorption of the tryptophan 
the amount of tryptophan in the solution was calculated and hence the 
amount in the protein. 

The tryptophan content of caseinogen was determined in this way 
and was found to be from 0-99-1 -59 per cent. No other determina- 
tions were made. 



ISOLATION OF GLUTAMIC ACID 33 

THE OTHER MONO-AMINO ACIDS. 

The isolation and estimation of these units is usually not carried 
out separately, except occasionally for glutamic acid and glycine. 
The process is one which yields the mono-amino acids in sequence. 

The method generally adopted in isolating the other mono-amino 
acids is that first employed by E. Fischer [1901]. It has been modi- 
fied in certain details by his pupils, particularly Abderhalden [1910] 
and other workers have contributed to its improvement. 

Hydrolysis is more conveniently effected by concentrated hydro- 
chloric acid than by dilute sulphuric acid, and is carried out as 
mentioned on p. 9. 

(i) Isolation of Glutamic Acid as Hydrochloride. 

The method of isolation of glutamic acid as hydrochloride was 
introduced by Hlasiwetz and Habermann [1873]. 

The solution of amino acids in 25 per cent, hydrochloric acid is 
concentrated in vacua to a half or a third of the original volume, and 
glutamic acid, if present in any large amount, is removed as its hydro- 
chloride by saturating the solution with dry gaseous hydrochloric acid. 
After allowing to stand at o for some days, glutamic acid hydro- 
chloride crystallises out. This occurs in the case of caseinogen and 
certain vegetable proteins, which contain from 10-40 per cent, of this 
amino acid. The glutamic acid hydrochloride is filtered off after add- 
ing an equal volume of ice-cold alcohol, washed with alcoholic hydro- 
chloric acid, redissolved in water, and boiled with baryta to remove 
ammonia. The barium is removed with sulphuric acid and the gluta- 
mic acid is again precipitated as hydrochloride by saturating the 
solution with gaseous hydrogen chloride. It is usually quite pure. 
The mother liquor on further concentration may give further crops of 
glutamic acid hydrochloride. These are treated in the same way as 
the first crop. The whole of the glutamic acid in the protein is not 
always precipitated as hydrochloride ; the remainder is then isolated 
later. The precipitation of glutamic acid hydrochloride is, however, so 
nearly complete that the yield is considered quantitative. Many com- 
parative determinations of the glutamic acid content of various pro- 
teins have been made by Abderhalden and his co-workers, and a series 
of values in numerous vegetable proteins and a few animal proteins 
have been published by Osborne and Gilbert [1906]. Abderhalden 
PT. i. 3 



34 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

[1912] states that glutamic acid is most rapidly obtained from the 
hydrochloride by dissolving in water, passing in ammonia, evaporat- 
ing to dryness, and recrystallising from water. The remaining glutamic 
acid is obtained from the mother liquor by adding alcohol. 

(ia) Isolation of Glutamic Acid as Zinc Salt. 

In view of the sometimes incomplete separation of glutamic acid as hydrochloride, 
especially in the cases of the proteins containing less than 10 per cent, of this unit and in 
view of its incomplete isolation as ester at a later stage, the observation of Kutscher [1903] 
that glutamic acid forms a very insoluble zinc salt might be made use of to isolate it. The 
mixture of amino acids, after removal of the greater part of the hydrochloric acid by 
evaporation in vacua, is diluted with water and boiled with excess of zinc oxide and allowed 
to cool. The precipitate consisting of the excess of zinc oxide and zinc glutamate is dis- 
solved in hot dilute acetic acid and treated with hydrogen sulphide. The filtrate on 
evaporation yields glutamic acid. 

A disadvantage of this method is the fact that leucine also forms a zinc salt which is 
soluble with difficulty. The separation of glutamic acid and leucine may be effected by the 
method of Osborne and Liddle [1910, 2], i.e., neutralising the acidity of the glutamic acid 
and crystallising out the leucine. The glutamic acid is then obtained on acidifying and 
crystallising, or as hydrochloride. 

(ib) Isolation of Glutamic and Aspartic Acids as Silver Salts. 

Glutamic acid and aspartic acid form silver salts which are not easily soluble in water 
[Habermann, 1875]. Siegfried [1891] was able to isolate aspartic acid as silver salt and 
Kutscher [1903] has shown that the solubility of the silver salts of these amino acids in 
water is very slight. Kutscher devised a method for isolating these units depending upon 
the insolubility of the silver salts. The mixture of amino acids after hydrolysis with 
sulphuric acid and removal of the di-amino acids, or similarly after hydrolysis with hydro- 
chloric and removal of di-amino acids, is evaporated in vacua to remove as much as possible 
of the hydrochloric acid ; its removal is completed as silver chloride. The solution is 
treated with silver nitrate or better another soluble silver salt and baryta water carefully 
added, avoiding excess which decomposes the silver salts. This may be tested by with- 
drawing a drop of the solution and placing it in contact with a drop of ammoniacal silver 
nitrate ; a turbidity indicates that more baryta is required. The silver salts are decom- 
posed and the mixture is separated, either by preparing glutamic acid hydrochloride, or by 
preparing the zinc salts ; the zinc salt of aspartic acid is easily soluble in water. 

(ic) Isolation of Glutamic and Aspartic Acids as Calcium Salts. 

Foreman [1914, i] has found that the calcium salts of these dibasic amino acids 
are insoluble in alcohol and has described a method for isolating them based upon 
this property. 

The hydrochloric acid solution is evaporated to a syrup in vacua ; the syrup is dis- 
solved in 200-400 c.c. of water per 20-40 grams of protein and 0-5 gram of slaked 
calcium oxide per gram of protein is added. The mixture is shaken well and filtered from 
excess of lime and humin substances. The filtrate is evaporated in vacua at 40-45 to a 
volume of 3*5-4 c.c. per gram of protein ; ammonia is thus removed. The temperature 
at which evaporation is carried out should not exceed 45 so as to prevent the transforma- 
tion of glutamic acid into pyrrolidone carboxylic acid. Rectified spirit is now added in 
small quantities at a time with vigorous shaking so as to prevent clumping of the pre- 



ESTERIFICATION 35 

cipitate ; alcohol is added so long as precipitation occurs, about i litre being required. 
The precipitate is filtered off and washed thoroughly with alcohol. If a small precipitate 
forms as the wash alcohol comes in contact with the filtrate, it is neglected : it does not 
contain glutamic acid and aspartic acid, but may consist of the calcium salt of tyrosine. 
The precipitate is dissolved in about 300 c.c. of water and treated with oxalic acid to re- 
move the calcium completely. The filtrate from calcium oxalate is treated with silver 
sulphate solution to remove traces of chloride and pigmented substance ; the pigmented 
substance dissolves if the solution be heated, but is removed later. Excess of silver is re- 
moved from the solution with hydrogen sulphide and the volume reduced to about one 
half. By now adding a solution containing 5-10 grams of phosphotungstic acid the 
colouring matter is precipitated, and the excess of this reagent is thrown down by baryta, 
which is added until the blue colour, if formed, disappears ; excess of baryta is removed by 
sulphuric acid and the filtrate evaporated to about 50 c.c. The solution is transferred to a 
weighed dish with a stirring rod, further evaporated to 20 or 25 c.c. for 40 grams of protein 
and allowed to cool. The acids crystallise out, are stirred and dried further in a desiccator 
over calcium chloride. 

The mass does not consist of a mixture of the pure amino acids ; it is therefore stirred 
up with cold glacial acetic acid ; the insoluble residue is filtered off, again stirred with 
glacial acetic acid, filtered off and washed ; the residue is dried and weighed. The acetic 
acid solution dries in vacuo over potash to a gum which contains pyrrolidone carboxylic 
acid, which may be estimated by determining the amino nitrogen before and after hydro- 
lysis with concentrated hydrochloric acid. 

The residue of glutamic acid and aspartic acid is separated by preparing the copper 
salts and crystallising out copper aspartate. The glutamic 'acid is isolated from the filtrate 
as hydrochloride ; or glutamic acid may be isolated first as hydrochloride and the aspartic 
acid from the filtrate. By determining the carbon content of the mixture of the acids, the 
proportion of aspartic acid in the mixture is given by multiplying the yield by 

40*82 - per cent, of carbon 

40-82 - 36-09 
the remainder is glutamic acid. 

The method has only been carried out with caseinogen ; the yield of aspartic acid was 
1*8 per cent, and of glutamic acid about 16-2 per cent. On adding the amount of glutamic 
acid corresponding to the amino nitrogen determination of the pyrrolidone carboxylic acid 
the yield was 2i'8 in one experiment and 24-1 in another. This is considerably higher than 
the yield of 15-6 by the hydrochloride method ; the yield of glutamic acid actually isolated 
was in good agreement with the above. 

(2) Esterification. 

The filtrate from the glutamic acid hydrochloride, to which the 
mother liquors from the recrystallisations are added, is concentrated 
in vacuo at a low temperature to a thick syrup ; this is dissolved in 
absolute alcohol (3 litres to I kilo, protein), and the amino acids are 
esterified by saturating the alcohol with dry gaseous hydrochloric acid 
at the ordinary temperature and then warming on the water-bath for 
half an hour. In the process of esterification a large amount of water 
is formed, which prevents its completion ; the alcohol is therefore 
evaporated off in vacuo at a temperature below 50 and the result- 
ing syrup again dissolved in absolute alcohol and saturated with 

3* 



36 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

dry gaseous hydrochloric acid. In some cases it may be necessary to 
repeat this operation once more. 

The esterification, according to Osborne and Jones [1910, 2], is 
more advantageously effected by the method of Phelps and Tillotson. 
The concentrated solution of amino acid hydrochlorides is dissolved 
in alcoholic hydrochloric acid and zinc chloride is added as a catalyst. 
The solution is maintained at a temperature of 100 and the vapours 
of absolute alcohol containing some hydrochloric acid are passed into 
the solution. The water arising during the process is removed by the 
alcohol vapours as fast as it is formed, and complete esterification 
results in a shorter time. 

Since the completion of the esterification of the amino acids necessitates at least two 
evaporations and two treatments with absolute alcohol and hydrogen chloride, Foreman 
[1913 and unpublished a ] proposed that the esterification be effected with the dry lead or 
copper salts of the amino acids. The solution of amino acids is evaporated in vacuo and 
steam is passed into the remainder to remove hydrochloric acid as completely as possible. 
It is diluted to about 2000 c.c. (with 320 grams protein) and heated with 100 grams of pre- 
cipitated lead hydroxide. The undissolved matter, which is dark in colour and contains 
humin, is filtered off. The filtrate is boiled for forty-five minutes with 400-500 grams of 
litharge. It is best to treat first with precipitated lead hydroxide, as under these conditions 
the humin separates in a flocculent state. The excess of litharge is filtered off and washed, 
and the solution is evaporated to about 500 c.c. On cooling it sets to a semi-solid mass, 
but it is stirred up on a water-bath until it becomes too viscous and dried in a steam oven. 
The dry mass, so obtained, can be powdered, but this is not necessary. The lead salts are 
suspended in about 1500 c.c. of absolute alcohol and dry hydrogen chloride passed in until 
the liquid is saturated. The solution is allowed to cool and again saturated with hydrogen 
chloride. The lead chloride is filtered off and washed with absolute alcohol ; the filtrate 
and washings are evaporated to half their volume at 40 and 15 mm. The excess of hydro- 
chloric acid is removed by slowly adding at o alcohol saturated with dry ammonia until 
the liquid is only faintly acid, thus avoiding liberation of the esters from their salts. The 
ammonium chloride, thus formed, is filtered off and washed with absolute alcohol and the 
alcohol evaporated off in vacuo at 40 and 15 mm. (the further treatment is described 
on p. 40). 

(3) Isolation of Glycine as Ester Hydrochloride. 

At this stage, glycine, if it occurs in the protein, e.g., in gelatin, in 
any considerable amount, is separated as glycine ester hydrochloride 
by concentrating in vacuo at 40 to two-thirds and seeding the solu- 
tion with a crystal of this compound and allowing to stand for twenty- 
four hours at o. The precipitate is filtered off while the liquid is kept 
cold and is washed with ice-cold alcohol ; the mother liquor, on further 
concentration in vacuo and saturation again with hydrochloric acid, 
may give another crop of glycine ester hydrochloride, which is treated 

1 Mr. Foreman has kindly informed me of the full details of his process, which gives as 
good, or even better, yields than the direct esterification process. 



ESTERIFICATION 37 

in same way. The glycine ester hydrochloride is dried in vacua over 
lime and sulphuric acid, and is purified by recrystallisation from ab- 
solute alcohol, charcoal being used to decolorise the solution. Almost 
the whole of the glycine may be isolated in this way [Fischer, 1902]. 
The filtrate containing the esters of the hydrochlorides of the other 
amino acids and the filtrate from the recrystallised glycine ester hydro- 
chloride are combined and concentrated to a syrup in vacuo at 40, 
the process of esterification is best repeated again, and any further 
quantities of glycine ester hydrochloride separated. 

(4) Extraction of the Esters of the Amino Acids. 

The esters are liberated from their hydrochlorides in the solution 
which has been concentrated in vacuo at 40 to a syrup by one of 
the following methods : 

(a) About one-third to one-half the volume of water is added to 
dissolve the syrup, and, if I kilo, of protein has been used, the solution 
is divided into two or four portions for convenience and to ensure the 
subsequent thorough cooling ; to each portion two or three volumes of 
ether are added, and the mixture is thoroughly cooled in a freezing 
mixture of ice and salt; strong caustic soda (33 per cent.) is now 
added till the free hydrochloric acid is neutralised ; this can be tested 
by adding a small quantity of a saturated solution of potassium 
carbonate. The feebly basic esters of aspartic and glutamic acids, 
which are very sensitive to free alkali, are thus liberated and are dis- 
solved by the ether, which is quickly poured off and replenished by a 
fresh quantity. Caustic soda and solid potassium carbonate added in 
small portions at a time set free the other esters from their hydro- 
chlorides ; these are dissolved by the ether, which is frequently renewed 
throughout the process and thoroughly mixed with the mass of salt and 
potassium carbonate ; sufficient caustic soda must be added to com- 
bine with the whole of the hydrochloric acid, and as much potassium 
carbonate as is necessary to form finally a pasty mass in order that 
the esters, which are very easily soluble in water, are salted out and 
dissolved by the ether. A large amount of ether is required for this 
extraction, which is continued until the ether separates in a colour- 
less state, and an essential condition is that, throughout the process 
of extraction the various portions be kept thoroughly cold by shaking 
in the freezing mixture. 

The several ethereal extracts are each dried by shaking for about 
five minutes with potassium carbonate ; they are then combined to- 



38 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

gether and allowed to stand for twelve hours with anhydrous sodium 
sulphate. 

The ether is next evaporated off, preferably in small quantities at 
a time and in vacua at the ordinary temperature ; in this way the 
lower boiling esters do not distil with the ether and the danger of 
decomposing them by a higher temperature is avoided. A brown oil, 
consisting of the esters of the amino acids, results ; this is fractionally 
distilled in vacuo. 

A considerable amount of the lower boiling esters nevertheless 
distils with the ether, especially those of alanine and glycine. Leucine 
ester has also been found in the distillate. They are recovered by 
shaking out the ether with dilute hydrochloric acid and evaporating 
the acid aqueous solution to dryness, when they remain as amino acid 
hydrochlorides. The glycine is separated as ester hydrochloride and 
the alanine, and leucine if present, are separated as esters by fractional 
distillation in vacuo (see p. 40). They may also be obtained by evaporat- 
ing their solution with water, removing the hydrochloric acid with lead 
oxide and silver carbonate, the excess of these by hydrogen sulphide 
and fractional crystallisation. Leucine, if present, separates out first, 
and subsequently the alanine. A mixture of small quantities of glycine 
and alanine generally remains which is too small for further separation ; 
it may be treated with picric acid (see p. 51). 

By this method of extracting 'the esters from their hydrochlorides, 
neither that of tyrosine, which remains behind combined with alkali, 
nor those of the di-amino acids, which are soluble with difficulty in 
ether, are obtained. This is advantageous for the^ subsequent process 
of separation, but the method has the disadvantage that the whole 
quantity of esters is not taken up by the ether. Extraction with 
chloroform after ether increases the amount of ester ; it separates the 
pasty mass of potassium carbonate into particles which can be washed 
with the solvent ; this extract contains the ester of tyrosine. 

The main loss seems to be caused by the destruction of the esters 
by the alkali. In order to avoid this loss, the mass of carbonate is 
treated with excess of hydrochloric acid and evaporated down, the 
potassium chloride being filtered off as it separates out ; the residue 
is extracted with alcohol and the above process of esterification is re- 
peated. It is simpler to suspend the mass of carbonate in absolute 
alcohol and to saturate the solution with gaseous hydrochloric acid. 
The salts remain insoluble and are filtered off and the alcoholic solution 
is treated as has been described. 



ESTERIFICATION 39 

() In order to separate the amino acids as completely as possible, 
Fischer introduced another method of liberating the esters from their 
hydrochlorides, i.e., treatment with sodium ethylate. The hydrochloric 
acid is removed as completely as possible by evaporation in vacua and 
the mixture of ester hydrochlorides is dissolved in five times its 
quantity of absolute alcohol. The amount of chlorine is estimated in 
a small portion of this, and to the remainder the calculated quantity 
of sodium dissolved in absolute alcohol and freshly prepared, so as to 
make a 3 per cent, solution, is added. The sodium chloride formed is 
filtered off. Its separation is greatly facilitated by the addition of 
ether and cooling to o. The alcohol is removed by evaporation in 
vacuo. A small quantity of the lower boiling esters of the amino acids 
passes into the distillate with the alcohol, but is recovered by acidify- 
ing with hydrochloric acid and evaporating when the amino acid 
hydrochlorides are obtained. A dark-brown oil again results, which 
is fractionally distilled in vacuo. 

Although this method prevents loss by the action of alkali, the 
yield of the higher boiling fractions is not so great on account of the 
more complex nature of the mixture of esters. The residue which 
does not distil contains the tyrosine, the di-amino acids, and other 
substances. 

(c) Instead of employing caustic soda and potassium carbonate for 
the liberation and salting out of the esters, Levene[i9O5] proposed the 
use of barium oxide, for which he claims the following advantages : 

(i) On account of its small solubility in water, a large excess of 
alkali which causes saponification of the esters is avoided. 

(ii) In neutralising the free acid the rise in temperature is less than 
with caustic soda. 

(iii) When the second esterification is required it is more easily 
removed. 

The procedure described by Levene and Van Slyke [1909, 2] is 
as follows : 

The concentrated solution of the ester hydrochlorides is poured 
into a porcelain or enamelled vessel of capacity of I litre for every 125- 
150 grams protein, and the flask is washed out with ice-cold baryta 
solution. The vessel is placed in a freezing mixture. When the con- 
tents are cold excess of crystallised baryta is added, and the mixture 
is thoroughly stirred with a wooden or porcelain spatula. The peculiar 
sticky mass in a few minutes becomes liquid and the solution becomes 
alkaline in reaction. Several volumes of ether are then poured over 



40 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

the solution. The ether is then poured off and a fresh quantity added. 
More baryta is added and the stirring continued. The ether is re- 
plenished and more baryta added several times. During the process 
the mixture becomes cloudy and pasty, but finally a light dry mass of 
baryta is left. 

The ethereal solutions are treated* as described in the (a) method. 

For the second esterification process the residue is stirred up several 
times with water, filtered through asbestos, and washed with water till 
no more organic matter is extracted. Most of the baryta remains un- 
dissolved. The solution is freed from that which has dissolved by the 
equivalent quantity of sulphuric acid, acidified with hydrochloric acid, 
and esterified in the usual manner. 

(d) Pribram [1910] found that the esters may be liberated very 
conveniently by the action of ammonia. His preliminary experiment 
with glycine gave a yield of 69 per cent, and an equally good yield of 
esters was obtained from gelatin. Abderhalden has also found this 
method very serviceable. 

The concentrated solution of ester hydrochlorides is mixed with 
ether and surrounded with a freezing mixture. Ammonia, dried by 
passing through three towers of caustic soda and lime, is then passed 
into the solution. The ammonium chloride which is formed is filtered 
off and washed with absolute alcohol. The ether is distilled off and 
the esters fractionally distilled in vacuo. 

(e) Zelinsky, Annenkoff and Kulikoff [1911] suggested a still simpler procedure 
for obtaining the esters. The concentrated solution of the ester hydrochlorides is mixed 
with lead oxide (200 grams to 100 grams protein) and the mass is directly distilled in 
vacuo. Abderhalden and Weil [1912, 3] do not recommend this method on account of the 
great loss which occurs during extraction. 

(/) Foreman (see p. 36) dissolves the syrup of ester hydrochlorides in 1000-1500 c.c. of 
dry chloroform and filters from ammonium chloride. 300-350 grams of anhydrous baryta are 
added in small quantities at a time and the mixture well shaken after each addition. The 
liquid is tested with silver nitrate and nitric acid and if a precipitate forms it is allowed to 
stand until this no longer occurs. The excess of baryta is filtered off and washed with 
dry chloroform, the chloroform is evaporated off in vacuo and the residue dissolved in ether ; 
only a gum-like substance remains undissolved. The ether is removed by distillation in 
vacuo and the residue of ester distilled in vacuo. The baryta was found to contain some 
amino acids ; it was decomposed with sulphuric acid and the solution treated with lead 
hydroxide in the same way as the original solution. 

(5) Fractional Distillation of the Esters in Vacuo. 

The fractional distillation of the brown oil, which is obtained by 
either of these methods, is carried out firstly at a pressure of 10-12 
mm. produced by a water pump, and then at a pressure of 0*5 mm. 



FRACTIONAL DISTILLATION OF ESTERS IN VACUO 41 

produced by a Geryck vacuum pump, as described by Fischer and 
Harries [1902]. In order to preserve the high vacuum in this process 
liquid air is used for condensing the alcohol vapour arising from 
the decomposition of the esters ; carbonic acid has been used by 
other investigators, and Levene and Van Slyke [1908, i] employed 
sulphuric acid, contained in cotton wool and cooled by a freezing 
mixture, as an absorbent for this purpose. A small quantity of 
ester also passes over with the alcohol ; this cannot be recovered if 
Levene and Van Slyke's modification be used. The accompanying 
figure shows the arrangement for the distillation at low pressure. 
At 10-12 mm. pressure the receiver should be cooled in a freezing 
mixture. 




FIG. i. 

From Berichte der Deutschen Chemischen C^esellschaft, 35, 2160. 

The temperatures at which the various fractions are collected are 
those of the vapours of the esters at 10 mm. pressure, and those of 
the water-bath at 100 and of an oil-bath, which replaces the water- 
bath for the higher temperatures up to 1 60, at 0-5 mm. pressure. 

Formerly the lower boiling fractions were again distilled in vacuo 
so as to obtain a further fractionation, but each fraction, even then, 
did not generally contain a single ester of an amino acid. A second 
fractionation is therefore no longer carried out. 

In the case of the higher boiling fractions a second e fractionation is 
not necessary, since the esters contained in them can be separated by 
their varying solubility in water, ether and petroleum ether. Accord- 



42 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



ing to Osborne and Jones [1910, 2] the distillation of the esters boil- 
ing above 110 can be dispensed with. An equally good separation 
of this residual mixture can be effected if it be carried out in the same 
way as is described for the esters after they have been distilled. 

The following table shows the fractions which are usually collected, 
and the amino acid esters which they may contain : 



Temperature. 


Pressure. 


Esters of 


Fraction I. To 60 (vapour) .... 

II. 60-90 (vapour), 100 (water-bath) . 
III. 100 (water-bath) .... 
IV. 100-160 (oil-bath) . 


10 mm. 

10 mm. 
0-5 mm. 
0*5 mm. 


Glycine, alanine, leucine, 
proline. 
Valine, leucine, proline. 
Leucine, proline. 
Phenylalanine, glutamic acid, 
aspartic acid, serine. 



Sometimes no fractionation of the esters boiling up to 100 at 0*5 
mm. is made, the whole mixture being simply divided into esters 
volatile below 100 at 0-5 mm. and a residue, which is not distilled. 

(6) The Isolation of the Individual Mono- Amino Acids. 

The separation and characterisation of each constituent contained 
in an ester fraction has now to be carried out. A somewhat special 
process has to be adopted for each individual product. 

Fractions I. ,1 7. , ///. 

The esters contained in these fractions are immediately reconverted 
into the amino acids. This is effected by boiling the fractions with 
5-10 volumes of water under a reflux condenser for six to seven hours, 
until the alkaline reaction has disappeared. 

If leucine be present in considerable amount, as may be the case in 
fraction III., the solution on cooling may deposit crystals of this sub- 
stance. These are filtered off, washed with water, dried and weighed. 
Identification of the product is carried out as described below. 

The three solutions are then evaporated to dryness in vacuo in 
weighed flasks and the amount of residue ascertained for each fraction. 

Levene and Van Slyke [1908, 2] have found that leucine ester, 
like phenylalanine ester, is readily dissolved by ether from water. The 
ester fractions may therefore, before hydrolysis, be mixed with 3 
volumes of water and extracted with ether, and the ether extract 
washed three times with water. The aqueous solution is then 
saponified by boiling and the extract, after removal of the ether, is 
treated in the same way. 



ISOLATION OF PROLINE 43 

(a) Proline. 

Proline is the only product contained in these fractions which is 
soluble in alcohol ; it is also much more easily soluble in water than 
the other products. 

The dry residues are therefore extracted several times with boiling 
absolute alcohol ; these extracts on cooling frequently become turbid, 
and on standing deposit other amino acids which, though insoluble in 
alcohol, are dissolved when proline is present. They are filtered off 
and returned to the portion insoluble in alcohol. 

The combined alcoholic extracts of the three fractions are again 
evaporated to dryness in vacuo and the residue is treated several times 
with cold absolute alcohol. A considerable amount is undissolved ; 
as before this is returned to the insoluble portion. 

The alcoholic solution is again evaporated to dryness in vacuo and 
extracted with cold absolute alcohol, and this operation is continued 
until all the insoluble amino acids are removed. 

The final alcoholic solution is evaporated to dryness and the residue 
weighed. As thus obtained, the proline is a mixture of the optically 
active and the racemic forms. These are separated by conversion into 
their copper salts by boiling with freshly precipitated copper oxide. 
The resulting dark-blue solution is evaporated to dryness, and the re- 
sidue is treated with absolute alcohol which dissolves the copper salt 
of the optically active proline. This solution on concentration yields 
the greater part of the compound in a crystalline state, but the remain- 
der is amorphous. The copper salt of the racemic proline, which is 
insoluble in alcohol, is purified by crystallisation from water. 

The identity of the compounds is established by a determination 
of the water of crystallisation and of the copper. The racemic copper 
salt contains 2 molecules of water of crystallisation, and in this state 
it is dark blue in colour ; in the anhydrous state its colour is violet. 
Further characterisation is obtained by preparing proline from it. The 
copper salt is dissolved in water and decomposed with hydrogen sul- 
phide. The filtrate is concentrated to a small volume and precipitated 
with alcohol. The product, crystallised from alcohol, is obtained in flat 
needles, /-proline has a sweet taste, melts at 206-209 and has a 
rotation of [~ a l D = - 77*4. The phenylhydantoin of /-proline is the 

L J 20 

most suitable derivative for still further characterisation. 

The amount of proline in the protein is given by the yield of the 
two copper salts obtained in a pure state. The actual proline content 



44 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

of the protein is considerably larger than the figures given in the tables 
(pp. 111-130). Abderhalden and Kautzsch [1912] consider that the 
yields of both forms of proline should be always separately given. 
The yield if it be based on the yield of copper salts is not a proper 
value. 

The real proline content can be accurately determined, as Van 
Slyke [1911, 3] has shown by his nitrous acid method (p. 89), by a 
determination of the total and amino-nitrogen of the product soluble 
in absolute alcohol. The alcoholic solution is made up to a definite 
volume and aliquot portions are taken for these estimations. The 
difference gives the amount of nitrogen present as proline from which 
the amount of proline can be calculated. In the case of caseinogen 
the proline content was found to be 6*7 per cent., a figure which is 
twice that found by Abderhalden. It agrees with that found by 
Engeland by his method of methylation (p. 69). Abderhalden and 
Kautzsch [1912] believe that the proline content determined by the 
Van Slyke method is not accurate, as the solution may contain other 
nitrogenous products which do not evolve nitrogen on treatment with 
nitrous acid. 

(b) Glycine, Alanine, Valine, Leucine, and Isoleucine. 

These five amino acids are present together in varying proportions 
in the residues which are insoluble in absolute alcohol. Their 
separation is only effected with great difficulty, and the procedure 
depends very largely upon which amino acids are present in the 
several fractions. 

In each case the residue is dissolved in water and, if necessary, the 
solution is decolorised by boiling with charcoal. The aqueous solu- 
tions are concentrated and fractionally crystallised ; the final mother 
liquor is evaporated to dryness. Each fraction is dried and weighed. 
Indications of the constituents of each fraction may be obtained : 

1. By elementary analysis of the carbon, hydrogen and nitrogen 
content. 

2. By determining the melting-point; the substance must be 
rapidly heated. Glycine melts at 240, alanine about 297, leucine 
about 300, and valine about 315. 

3. By the taste. Glycine and alanine have a sweet taste ; valine 
is less sweet and it leaves a bitter after-taste ; leucine is insipid and 
slightly bitter. 

The following fractions may be obtained by the fractional crys- 



ISOLATION OF VALINE, LEUCINE AND ISOLEUCINE 45 

tallisation of the amino acids obtained from the esters after removal 
of the proline : 

1. Valine + leucines. 

2. Valine + alanine. 

3. Alanine. This is purified by recrystallisation from dilute alcohol. 

4. Alanine + glycine. 

If the esters be separated by distillation into three fractions, the 
residues from fraction I. will contain chiefly glycine and alanine ; from 
fraction II. valine, leucine and isoleucine; from fraction III. leucine 
and isoleucine. 

i. Separation of Valine from Leucine and Isoleucine. 

These three compounds are the most difficult to separate from each 
other. Their separation has really only been accomplished by chance. 

In those cases where the isolation of the individual substance has 
succeeded it has been effected by the fractional crystallisation of the 
amino acids themselves and of their copper salts and by the different 
solubility of the compounds in methyl alcohol. Valine is soluble in 
methyl alcohol, isoleucine is insoluble in the cold solvent, but soluble 
in the hot. On cooling the solution, however, the presence of valine 
prevents its separation. The copper salt of leucine is very insoluble, 
but the mixed copper salts are relatively soluble. Leucine and iso- 
leucine were first separated by F. Ehrlich [1904] by the different 
solubility of their copper salts in methyl alcohol ; that of leucine is 
insoluble. The separation is most tedious and not at all satisfactory. 

In order to give us more information about the constituents of this 
fraction, Levene and Van Slyke [1909, I, 2] worked out a method 
which depends upon the precipitation of leucine and isoleucine as their 
lead salts from ammoniacal solution and the subsequent separation of 
these two amino acids by means of the different solubility of their 
copper salts in methyl alcohol. The details are as follows : 

The mixture is carefully analysed and the amount of leucine + 
isoleucine is calculated from the carbon content : 



Leucine and isoleucine contain 54-92 per cent. C. j difference = 3 . 68 per cent> 
Valine contains . . . 51-24 ,, ,, J 

Per cent, carbon - 51*24 = cent . of leucine isomers in the mixture . 
3-68 

The mixture is pulverised and suspended in 7 parts of water and 
the water is raised to the boiling-point : for each gram of substance 
I -5 c.c. of concentrated ammonia solution is then added. The flask 



46 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

is stoppered and shaken so as to dissolve the amino acids ; if necessary 
the solution may be again heated. 

4 c.c. of I -I M lead acetate solution (sp. gr. 1*254 at 20) for 
each gram of leucine and isoleucine are slowly added to the hot solu- 
tion, which is thoroughly stirred during the addition. The solution 
is then cooled in ice-water and after one to two hours is filtered 
through a Buchner funnel, or a Gooch crucible, according to the 
amount of precipitate. The solid matter is pressed down so as to 
remove the mother liquor as completely as possible, and it is washed 
firstly with 90 per cent, alcohol, then with ether, and dried in vacua 
over sulphuric acid. 

It is curious that the presence of valine facilitates the separation of 
the lead salts of the leucine isomers. 

If the proportion of isomers : valine be less than 2 : I the pre- 
cipitation is not so complete. In these cases less lead acetate solution 
(3 7 c.c.) should be taken and the filtrate concentrated in vacuo till the 
percentage of valine reaches 10. Ammonia is again added and the 
precipitate treated as before. It is preferable to treat the filtrate once 
more in the above manner after the valine has been separated. 

ii. Valine. 

The filtrate is freed from lead by means of hydrogen sulphide, and 
the solution, filtered from lead sulphide, is evaporated to dryness. The 
dry residue is treated with an alcohol-ether mixture (3 : i) to extract 
acetic acid and ammonium acetate. The small amount of valine 
which dissolves is recovered by again evaporating to dryness and 
extracting with alcohol and ether. 

Pure valine generally remains ; it is identified by recrystallisation 
from water, elementary analysis, and rotation in 20 per cent, hydro- 
chloric acid of [a] D = + 28'8; a rotation of 26-28 is generally found 
as racemisation occurs in the process. 

If analysis shows leucine to be present the above treatment must 
be repeated. 



ISOLATION OF VALINE, LEUCINE AND ISOLEUCINE 47 

iii. Leucine and Isoleucine. 

The purity of the lead salt, as obtained above, is tested by analysis. 
This is performed by dissolving about 0*3 gram in 5 c.c. of normal 
nitric acid in a 100 c.c. beaker and precipitating the lead with 5 c.c. of 
normal sulphuric acid followed by 50 c.c. of absolute alcohol. The 
lead sulphate, which settles rapidly in a granular form, is collected 
after about fifteen minutes in a Gooch crucible and washed with 95 
per cent, alcohol acidified with sulphuric acid. The crucible is placed 
in another crucible, heated gently until the alcohol is driven off, and 
then with the full heat of the Bunsen burner for ten minutes. 

If the lead content be too high, due to contamination with the lead 
salt of valine, the mixture is purified by dissolving it, after thorough 
pulverisation, in 5 parts of hot water + J part of glacial acetic acid 
and reprecipitating by adding 0*5 c.c. of concentrated ammonia solu- 
tion for every gram of salt. The precipitate is collected and treated 
as above under the conditions where the amount of valine present is 
small. 

The last portions of the mixed leucines are recovered by repeating 
the entire process. 

The mixed lead salts are dissolved in 15-20 parts of hot water + 
part of glacial acetic acid, and hydrogen sulphide is passed into the 
solution. The filtrate from the lead sulphide is evaporated in vacua to 
dryness, and the residue is washed with a mixture of equal parts of 
alcohol and ether to remove acetic acid. 

Since these isomers have been shown not to be racemised by heating 
with acids, the composition of the mixture can be ascertained by de- 
termining the rotation in 20 per cent, hydrochloric acid : 

d-isoleucine has [a] 10 = 



/.leucine [op '" ' 23 

hence percentage of d-isoleucine = 100 x 1 

/-leucine = 100 x 37'4 ~ g . 

From the weight of the mixed amino acids and these data the 
amount of each isomer can be calculated. 

The mixed isomers are converted into their copper salts by boiling 
with excess of copper oxide, which is thoroughly boiled out with water 
to remove the last traces of the copper salt of leucine which is pale 
blue in colour and very insoluble. 

The solution of the copper salts is evaporated to dryness in vacua. 



48 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

The dry and finely pulverised copper salts are then shaken in a shaking 
machine with 94 per cent, methyl alcohol. The insoluble copper salt 
of leucine is filtered off and washed with solvent. The soluble copper 
salt of isoleucine may be contaminated with some of the copper salt 
of leucine. It is therefore decomposed with hydrogen sulphide, re- 
converted into copper salt and again extracted with methyl alcohol. 
Both the leucine and isoleucine are obtained in the usual way from 
the copper salt and recrystallised from water. 

They are identified by elementary analysis, rotation and estimation 
of copper in their copper salts. 

iv. Separation of Valine and Alanine. 

Levene and Van Slyke [1913] described a method for separat- 
ing these two amino acids. It depends (i) upon the greater insolu- 
bility of alanine phosphotungstate than valine phosphotungstate in a 
solution containing 10 per cent, of sulphuric acid and 20 per cent, of 
phosphotungstic acid ; (2) the greater insolubility of valine in 80 per 
cent, acetone. Alanine combines with 14 parts of phosphotungstic 
acid. The solubility of alanine phosphotungstate in the above solution 
is 0*1 5 gram per 100 c.c. ; that of valine phosphotungstate is i '2 gram. 
A mixture of the two compounds can be separated by crystallisation 
from this solution. Glycine behaves like alanine; its phosphotung- 
state has the solubility of O'2 gram per 100 c.c. If leucine be present 
in the mixture it must be first separated as lead salt (above). 

In carrying out the separation the mixture should preferably con- 
tain not more than 50 per cent, of valine, and in precipitating the 
alanine the volume of liquid should be as small as possible. There 
are two alternatives : (a) if the alanine phosphotungstate is not re- 
crystallised, the volume should be 100 c.c. for every gram of valine 
present; (&) if the alanine phosphotungstate be recrystallised, the 
volume need only be 30-40 c.c. per gram of valine; one recrys- 
tallisation from the same volume yields pure alanine phosphotungstate. 

The most satisfactory separation is conducted by dissolving the 
mixture of valine and alanine in a hot sulphuric acid solution contain- 
ing 10 grams per 100 c.c. in the proportion of 30-40 c.c. per gram 
of valine present. This amount is given by an elementary analysis 
of the mixture. Phosphotungstic acid l in the ratio of 14 : i according 

1 The phosphotungstic acid is purified by Winterstein's method by dissolving in a 
small volume of water and extracting with ether. An oily solution heavier than water 



ISOLATION OF VALINE AND ALANINE 49 

to the maximum amount of alanine in the mixture and I gram excess 
for every 5 c.c. of solution is dissolved in the hot solution. A greater 
excess does not interfere with the separation, but it requires removal 
subsequently. The solution is kept at o, or in ice water in an ice 
box for at least twenty-four hours. 1 The alanine phosphotungstate 
separates in large transparent crystals forming a solid layer on the 
bottom and sides of the vessel. The supernatant solution is decanted 
and the crystals are redissolved in the same volume of 10 per cent, 
sulphuric acid as that originally used, I gram of phosphotungstic acid 
per 4 or 5 c.c. of liquid are added, and the solution kept at o for 
twenty-four hours. The alanine phosphotungstate is filtered off by 
suction and washed with 10 per cent, sulphuric acid containing 20 per 
cent, of phosphotungstic acid. 

The alanine in the phosphotungstate is determined and recovered 
by dissolving the salt in hot water in which it gives a somewhat 
turbid solution and making up to a known volume containing 50-100 
mgm. of alanine per 10 c.c. The nitrogen in this solution is esti- 
mated by Van Slyke's method (p. 89) using 10 c.c., or 2 c.c. if 
the micro-apparatus be used. It can also be estimated by Kjeldahl's 
method, but this is not so convenient on account of the violent bump- 
ing caused by the separation of tungstic acid. This estimation gives 
a more accurate value for the alanine than its isolation, as loss occurs 
during the removal of the phosphotungstic acid ; the actual amount 
isolated is from 90-95 per cent, of that found by the nitrogen esti- 
mation. A correction for the solubility of alanine phosphotungstate 
'15 gram in 100 c.c. solution can be applied. 

The remainder of the solution is washed into a beaker, heated to 
boiling, and treated with excess of pure 20 per cent, lead acetate solu- 
tion ; z excess of lead acetate is ascertained by removing a drop and 
testing with dilute sulphuric acid. Lead sulphate and phosphotung- 
state are filtered off and thoroughly washed. The filtrate and wash- 
ings are concentrated to about 50 c.c. per gram of alanine, an equal 
volume of 95 per cent, alcohol added, and the mixture heated on the 
water-bath for about one hour. This removes the remainder of the 

results. It is washed several times with water and the ether removed on the water-bath. 
The product is not hygroscopic and forms a colourless solution. It should leave no residue 
after precipitation with pure lead acetate and evaporation of the filtrate. 

1 Working at room temperature, about 75 per cent, of the alanine is precipitated if one 
half of the volumes given are used. 

2 This should leave no residue after removal of the lead with hydrogen sulphide and 
evaporating to dryness. 

PT. I. 4 



50 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

lead sulphate. The excess of lead is removed with hydrogen sulphide, 
the lead sulphide washed with water containing hydrogen sulphide and 
the liquid is evaporated in vacua to a small volume. It is transferred 
to a Jena glass dish, evaporated until all liquid disappears and the 
alanine dried by placing in a vacuum desiccator over sulphuric acid 
and caustic potash. The substance becomes discoloured if it be dried 
on the water-bath. The product is nearly ash-free alanine, but it may 
contain glycine, if glycine was present in the original mixture. The 
rest of the alanine in the mixture is obtained after isolating the valine. 

The phosphotungstic acid filtrates and washings containing the 
valine is made up to a known volume and the amount of valine esti- 
mated by a nitrogen determination by Van Slyke's method, using 10 
per cent, sulphuric acid instead of water in a blank determination. 
The reagents are removed with lead acetate as described above under 
alanine, and the solution evaporated till valine begins to crystallise ; 
2-3 volumes of 80 per cent, acetone are added and the mixture 
washed into a flask with 80 per cent, acetone ; the flask is stoppered 
to prevent evaporation of the acetone and allowed to stand for twelve 
hours. Valine crystallises out ; it is filtered off and washed with 80 
per cent, acetone. Its yield is 80-85 P er cent, of the amount found 
in the mixture by analysis. 

The filtrate is evaporated to dryness and the above separation re- 
peated. It is then practically quantitative. 



ISOLATION OF GLYCINE AND ALANINE 51 

v. Separation of Glycine and Alanine. 

There are two methods of separating these compounds : 

(a) By reconverting the mixture into esters, separating the glycine 
as ester hydrochloride, and distilling the alanine ester, which 
is then decomposed by boiling with water and the alanine 
obtained by crystallisation. 

(&} By precipitating the glycine as picrate [Levene, 1906 ; Levene 
and Van Slyke, 1912]. 

Glycine picrate, which was first described by Levene, was shown 
by Levene and Van Slyke to have the composition of 2 molecules of 
glycine and I molecule of picric acid. The pure compound on heat- 
ing softens at 200 and melts at 202 ; it is soluble to the extent of 
'35 gram (= '14 gram glycine) in 20 c.c. of water. In preparing it, 
it is not advisable to use a large excess of picric acid, as this increases 
the solubility of the picrate though the solubility is lessened in the 
presence of alanine. 

The separation of glycine and alanine is effected by dissolving the 
mixture in 3-4 parts of hot water and then adding an amount of 
picric acid exceeding that required to combine with the glycine (1-5 
grams per I gram glycine) but not exceeding the amount required to 
combine with the whole of the amino nitrogen in the mixture if this 
be calculated as glycine. The solution is cooled to o ; glycine picrate 
crystallises out completely in about one hour. It is filtered off and 
washed with a small quantity of water, followed by 95 per cent, 
alcohol. Its purity is controlled by a melting-point determination and 
amino nitrogen content. The filtrate is acidified with a known excess 
of normal sulphuric acid and freed from picric acid by shaking out with 
the ether. The sulphuric acid is removed by adding an equivalent of 
standard baryta solution and the filtrate from the barium sulphate is 
evaporated to dryness. The residue consists of alanine, generally of 
over 90 per cent, purity, the impurity being glycine. Pure alanine 
results on recrystallisation and a separation of the remainder can be 
effected by repeating the process. 

Glycine is identified by the melting-point and analysis of its ester 
hydrochloride and picrate ; its amount in the protein is given by the 
yield. 

Alanine is identified by elementary analysis and its rotation in 
hydrochloric acid solution. The amount in the protein is also given 
by the yield. 

4* 



52 THE CHEMICAL CONSTITUTION OF THE PROTEINS 
Fraction IV. or Non-distilled Higher Boiling Esters. 

(c) Phenylalanine. 

The ester of phenylalanine differs from the esters of aspartic acid, 
glutamic acid and serine, which are present with it in this fraction, by 
being only slightly soluble in water. 

The mixed esters are dissolved in 5 volumes of water. If a 
large amount of phenylalanine be present, it may separate in the form 
of oily drops. The aqueous solution is extracted with an equal volume 
of ether. The ether extract is then washed several times with water 
to remove the last traces of any of the other esters which may have 
been dissolved by the ether. The ether is removed by distillation and 
the ester is hydrolysed by evaporation with concentrated hydrochloric 
acid. The resulting phenylalanine hydrochloride is purified by crys- 
tallisation from hydrochloric acid, and can be identified by an estima- 
tion of its content in chlorine. 

The free amino acid is obtained from the hydrochloride by treating 
with sodium acetate, or ammonia, and precipitating from hot aqueous 
solijtion with alcohol. A determination of the melting-point of 283 
and rotation of [a] D = - 35'i in aqueous solution characterises the 
compound. 

(d) Aspartic Acid. 

The esters in the aqueous solution from which the phenylalanine 
ester has been extracted with ether are saponified by boiling with 
baryta ; the solution of baryta is prepared by/ dissolving twice the 
quantity of barium hydrate to that of ester in sufficient hot water, 
filtering and allowing to cool. The clear solution is poured off from 
the crystals and to it is added the solution of esters. Hydrolysis is 
then effected by heating for two hours on the water-bath. The solu- 
tion is allowed to stand for several days to allow the barium salt of 
racemic aspartic acid to crystallise out. 

The barium aspartate is decomposed with sulphuric acid, the 
barium sulphate filtered off, and the excess of sulphuric acid quantita- 
tively removed with baryta. Pure aspartic acid crystallises out from 
the solution on evaporation. It is identified by analysis and by the 
analysis of its copper salt. 

The remainder of the aspartic acid is isolated after removal of 
glutamic acid as hydrochloride (e) as follows : 

The solution is evaporated in vacuo to remove as much hydrochloric 



ISOLATION OF SERINE 53 

acid as possible. The residue is dissolved in water and boiled with 
yellow lead oxide until a test portion of the cold solution no longer 
gives a reaction for chlorine. 1 The filtered solution is freed from lead 
by hydrogen sulphide and the filtrate from lead sulphide is evaporated 
to a small volume, when aspartic acid crystallises out. The mother 
liquor contains principally serine, but more aspartic acid and other 
products are also present. 

Note. Osborne and Liddle [1910, 2] have observed that an intermediate fraction between 
fractions III. and IV. containing aspartic ester and leucine ester and possibly also phenyl- 
alanine ester may distil. The separation of leucine and aspartic acid (or glutamic acid) 
is impossible by fractional crystallisation. The fraction should be treated as described and 
the leucine separated from the aspartic acid by neutralising with soda and crystallising. 
Leucine results. On acidifying the filtrate and again crystallising the aspartic acid is 
obtained. 

(e) Glutamic Acid. 

The filtrate from the barium aspartate is exactly freed from barium 
by sulphuric acid and the solution is evaporated to dryness in vacuo. 
The residue is dissolved in water, the solution decolorised, if neces- 
sary, by boiling with charcoal and the glutamic acid is precipitated 
as hydrochloride by passing in dry gaseous hydrogen chloride. A 
further quantity of glutamic acid hydrochloride may be obtained from 
the mother liquor by concentration and similar treatment. Practically 
the whole of the glutamic acid present in the protein is thus obtained 
as hydrochloride. The larger portion is separated directly, before 
the mixture of amino acids is esterified. 

Glutamic acid is obtained from the hydrochloride by treatment with 
the calculated quantity of caustic soda to combine with the hydro- 
chloric acid and by crystallisation from water, in which it is soluble, 
when pure, with some difficulty. Elementary analysis of the free 
acid, or of its hydrochloride, determines its identity and its weight 
gives the amount in the protein. 

(/) Serine. 

It is most difficult to isolate serine and obtain it in a pure state. 
The solution from which the active aspartic acid has crystallised 
out is neutralised, if acid, with caustic soda and concentrated. Serine 
crystallises out in crusts of monoclinic crystals, and "is identified by its 
melting-point of 240 and elementary analysis. 

1 Levene and Van Slyke [1910] point out that, if excess of lead oxide be used, the 
insoluble lead salt of aspartic acid is formed with consequent loss of this amino acid. 



54 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

Its ^-naphthalene sulphonyl-derivative, 

/CH 2 OH /CHoOH 

C 10 H 7 S0 2 C1 + H a N . CH^ = HC1 + C 10 H 7 SO 2 . NH . CH< 

\COOH \COOH 

which is prepared by shaking in alkaline solution with /3-naphthalene 
sulphonyl-chloride, serves for the isolation of the remainder from the 
filtrate. This compound is very suitable for its characterisation. 
(M.P. = 2i4corr.) 

(g) The Distillation Residue. 

If the higher boiling esters are distilled a dark reddish-brown and 
thick syrupy residue remains in the distilling flask and sometimes 
crystals are observed clinging to the walls of the vessel. The mass 
consists chiefly of the anhydrides of the amino acids. Leucinimide 
may be extracted by boiling it with ethyl acetate. The residue is 
generally so small in amount that it is not further examined, but if this 
be necessary the mass is boiled with baryta for sixteen hours, the 
baryta removed and the process of separation repeated. 

(h) The Isolation of Oxyproline. 

Only in a few cases has this compound been isolated from the 
products of hydrolysis of proteins, since its separation is extremely 
laborious. Its ester is not extracted by ether, and it consequently 
remains behind in the mass of carbonate ; if its isolation be required 
a second esterification is generally not performed. 

The mass of carbonate is treated as previously described (p. 38). 
The aqueous solution is evaporated in vacua td^ remove hydrochloric 
acid as completely as possible. The organic matter is dissolved in 
water so that its content is about I per cent. ; sulphuric acid is added 
and the di-amino acids are precipitated with phosphotungstic acid 
(see p. 60). The excess of reagents are removed from the filtrate 
with baryta, the solution is concentrated in vacuo to a small volume 
and hydrochloric acid is removed with silver sulphate. The silver and 
sulphuric acid are precipitated in the usual way, the solution is eva- 
porated in vacuo to a small volume and allowed to stand in a desiccator 
over sulphuric acid. Oxyproline slowly crystallises out. 

Oxyproline is more easy to obtain when the esters have been 
separated with sodium ethylate. It is then present in the distillation 
residue ; this is treated in a similar way. It is identified by its melt- 
ing-point of 270, rotation of [a] D =- - 81-04 in aqueous solution 
and by conversion into its /9- naphthalene sulphonyl-derivative. 



ISOLATION AND ESTIMATION OF DI-AMINO ACIDS 55 

B. THE DI-AMINO ACIDS. 

The isolation and estimation of the three compounds arginine, his- 
tidine, lysine are carried out by the method described by Kossel and 
Kutscher [1900-1] which was slightly modified by Kossel and Patten 
[1903]. Further modifications and improvements have been added by 
Steudel [1903], Kossel and Pringle [1906] and Osborne, Leavenworth 
and Brautlecht [1908]. The method is based upon the earlier work of 
Drechsel, Hedin, and Kossel, and depends upon the precipitation of 
arginine and histidine as their silver salts, their separation by difference 
in solubility in water and in strongly alkaline solution, and the pre- 
cipitation of lysine from the filtrate by phosphotungstic acid, and then 
by picric acid. 

The method has been described in full by Weiss [1907] and by 
Steudel [1910] and is carried out as follows : 

I. Hydrolysis and Estimation of Protein. 

About 25-50 grams of protein are hydrolysed by boiling with 
dilute sulphuric as stated on p. 1 1. The exact amount of protein is 
then estimated by making the volume up to I litre with water and 
determining the nitrogen in 5 or 10 c.c. by Kjeldahl's method; from 
this figure the amount of protein can be calculated, if the amount of 
nitrogen in it be known. 

II. Removal of Sulphuric Acid. Estimation of Ammonia and 

Humin Nitrogen. 

The acid solution is heated to boiling and treated with a hot 
concentrated solution of baryta until the reaction is only faintly acid 
and almost the whole of the sulphuric acid is precipitated as barium 
sulphate, which is filtered off by suction and thoroughly washed 
with boiling water, by stirring up and raising to the boiling-point. 
This should be repeated twice or until the filtrate gives no precipitate 
with phosphotungstic acid. The filtrate and washings are evaporated 
down best in vacuo at 70 and again made up to I litre. A deter- 
mination of the nitrogen in 5 or 10 c.c. of this solution gives by differ- 
ence the amount of nitrogen contained in the melanin, which is 
carried down by the barium sulphate. It is known as " humin nitro- 
gen I. " 

In this liquid two determinations are made of the amount of 



56 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

nitrogen present as ammonia, by distilling portions of 100 c.c. with 
magnesium oxide. 

The ammonia is removed from the remainder by evaporating with 
magnesia, or better barium carbonate, on the water-bath. 

The two portions, freed from ammonia, are combined, and made 
alkaline with baryta, or barium carbonate. 

The separate solutions are now combined, the precipitate of barium 
carbonate and barium sulphate is filtered off and washed by boiling 
with water three times ; the excess of barium is removed from the 
filtrate by dilute sulphuric acid and the precipitate again filtered off 
and washed out. Filtrate and washings are combined together, 
evaporated down and made up to I litre and a Kjeldahl nitrogen de- 
termination again made. Allowing for the nitrogen given off as 
ammonia, the difference between this and the previous estimation 
gives the humin nitrogen II. contained in the alkaline barium magnesia 
precipitate. 

III. Precipitation of Arginine and Histidine. 

The solution, which now contains a small quantity of sulphuric 
acid, is placed in a 5 litre flask, and treated with a hot saturated solu- 
tion of silver sulphate, 1 which is slowly added, until the solution con- 
tains sufficient to give a yellow-brown, not a white or pale yellow 
precipitate, on removing a drop and testing it with baryta water in a 
watch-glass. If, during the process, there be any undissolved silver 
sulphate at the bottom of the flask, it is dissolved by adding more 
water before a fresh quantity is added, in order that a yellow-brown 
precipitate be given in the test drop with baryta. As soon as sufficient 
silver is present to combine with all the arginine and histidine, the 
solution is allowed to cool to 40 and is saturated with finely 
powdered baryta, i.e., until some remains undissolved after repeated 
shaking. The precipitate, which is thus formed and which consists 
of the silver salts of arginine and histidine, is filtered off and stirred 
up together with the filter paper in a mortar with baryta, when it is 
again filtered off and washed with baryta water. The lysine in the 
filtrate is separated according to VI. 

The precipitate of the silver salts of arginine and histidine is 
suspended in water containing sulphuric acid and decomposed with 
hydrogen sulphide. The filtrate from the silver sulphide and barium 

1 Osborne prefers to use silver nitrate. 



ISOLATION AND ESTIMATION OF DI-AMINO ACIDS 57 

sulphate, which is thoroughly extracted in the usual manner with 
boiling water, is evaporated down to remove the hydrogen sulphide 
and again made up to I litre ; a Kjeldahl nitrogen determination in 
20 c.c. now gives the amount of nitrogen in the substances precipi- 
tated by silver and baryta. 

IV. Estimation and Isolation of Histidine. 

(a) The solution is freed from sulphuric acid by neutralising 
to litmus with baryta and adding barium nitrate as long as a preci- 
pitate is formed ; the barium sulphate is filtered off and washed. 

The solution is concentrated to 300 c.c., acidified with nitric acid, 
if necessary, and treated with silver nitrate, as before, till a test drop 
gives a yellow-brown colour with baryta ; when this occurs it is exactly 
neutralised to litmus with baryta and 5 c.c. of a cold saturated solu- 
tion of baryta are added. If 10 c.c. of the filtered solution when tested 
with a drop of baryta give a precipitate which indicates that the silver 
salt of histidine is not completely thrown down, 2 c.c. of saturated 
baryta solution are added to the main bulk, and this test is repeated 
until a test portion remains clear. The precipitate of the silver salt of 
histidine is then filtered off. 

Instead of adding excess of baryta, Kossel and Pringle direct that 
a suspension of barium carbonate be added to the neutral solution, the 
solution warmed on the water-bath and then raised to the boiling- 
point. After cooling, the histidine silver salt is filtered off and washed 
with baryta till free from nitric acid. The filtrate and washings are 
treated as in V. for arginine. 

The precipitate of the silver salt of histidine is suspended and 
heated in water to which sulphuric acid is added until the reaction is 
acid and decomposed with hydrogen sulphide. Excess of hydrogen 
sulphide is removed by boiling and the silver sulphide is filtered off 
and washed. The solution and washings are concentrated and made 
up to 250 c.c. A nitrogen estimation in 20-25 c.c. by Kjeldahl's 
method gives the amount of histidine. 

The histidine is isolated from the remainder of the solution as 
hydrochloride or as picrolonate : 

I. As Hydrochloride. The solution is made alkaline with baryta, 
the barium sulphate formed is filtered off, excess of baryta is removed 
by carbon dioxide, and the whole is evaporated to dryness. The resi- 
due is extracted with boiling water and to the solution, filtered from 
barium carbonate, hydrochloric acid is added. Histidine dichloride, 



58 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

C 6 H 9 N 3 O 2 . 2 H Cl, is obtained on evaporating down. The yield is 7 5 -80 
per cent, of the histidine estimated by the Kjeldahl determination. 

2. As Picrolonate. The excess of sulphuric acid is removed by 
treating the hot solution with excess of baryta, and excess of the 
latter is removed by carbon dioxide ; it is evaporated down and filtered 
from barium sulphate and carbonate, which are thoroughly washed. 
The filtrate and washings are evaporated to about 10 c.c., if necessary 
after the addition of a drop of sulphuric acid to remove the last traces 
of barium. The necessary quantity of picrolonic acid (calculated from 
the above Kjeldahl determination and dissolved in a small quantity of 
alcohol) is added ; the precipitate of histidine picrolonate is filtered off 
after three days, washed with water, dried and weighed. The amount 
of histidine can be calculated from the formula C 6 H 9 N 3 O 2 . C 10 H 8 N 4 O 5 ; 
it corresponds very closely with the amount calculated from the 
Kjeldahl estimation. 

(b) Osborne, Leavenworth and Brautlecht find that it is better to 
remove the greater portion of the histidine by precipitation with 
mercuric sulphate. The solution is concentrated to about 250 c.c., 
sulphuric acid is added till the solution contains 5 per cent, of this 
acid, and it is treated with a slight excess of mercuric sulphate solution. 
The precipitate of histidine mercury sulphate is allowed to stand for 
twelve to twenty- four hours when it is filtered off, washed with 5 per 
cent, sulphuric acid, suspended in water and decomposed with hydrogen 
sulphide. The filtrate and washings from the mercuric sulphide which 
contain the histidine are neutralised with baryta and barium nitrate 
added until barium sulphate is no longer precipitated. The barium 
sulphate is filtered off and thoroughly washed. The histidine is then 
thrown down as silver compound and estimated as under (a). The 
filtrate from the mercury precipitate is freed from mercury, neutralised 
and treated as described under (a) so as to throw down the small 
quantity of histidine not precipitated by mercuric sulphate. 



ISOLATION AND ESTIMATION OF DI-AMINO ACIDS 59 

V. Estimation and Isolation of Arginine. 

The filtrate containing the arginine is saturated with baryta ; the 
precipitate of the silver salt of arginine, so obtained, is filtered off, and 
precipitate and filter paper are stirred up in a mortar with baryta, 
filtered off, and the process repeated till the precipitate is free from 
nitric acid. It is then suspended in water containing a slight excess 
of sulphuric acid and decomposed with hydrogen sulphide. The 
filtrate and washings from the precipitate of silver sulphide and barium 
sulphate are evaporated down and made up to 500 c.c., or I litre. The 
amount of arginine is estimated from the amount of nitrogen deter- 
mined in 25-50 c.c. of this solution by Kjeldahl's method. 

The arginine is isolated from the remainder of the solution as 
nitrate, copper nitrate double salt, or as picrolonate. 

1. As Nitrate. The solution is freed from sulphuric acid by baryta, 
the excess of which is removed by carbon dioxide, and evaporated 
down. The last traces of baryta are then removed with a drop of 
sulphuric acid, the solution is neutralised with nitric acid and evapor- 
ated to dryness. Arginine nitrate, C 6 H U N 4 O 2 . HNO 3 + -JH 2 O is ob- 
tained as a dry white crystalline mass. The double salt with copper 
nitrate is then prepared from the nitrate by boiling with copper oxide ; 
a yield of 85-90 per cent, is obtained. 

2. As Picrolonate. The solution is freed from sulphuric acid as 
described above, and evaporated down to about 10 c.c. The necessary 
quantity of picrolonic acid (calculated from the nitrogen determination 
and dissolved in a small volume of hot alcohol) is then added ; the 
yellow crystals of picrolonate are filtered off after a few days, washed 
with a small quantity of water, and dried at 110. The yield of 
arginine calculated from the picrolonate, C 6 H 14 N 4 O 2 . C 10 H 8 N 4 O 5 , which 
loses its one molecule of water of crystallisation at 110, is almost 
quantitative, since the picrolonate has the very slight solubility of I 
part in 1 1 24 parts of water. 



60 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

VI. Estimation and Isolation of Lysine. 

The lysine is contained in the filtrate from the precipitate of the 
silver salts of arginine and histidine. 

The solution is acidified with sulphuric acid and freed from silver 
by hydrogen sulphide ; the filtrate and washings from the precipitate 
of silver sulphide and barium sulphate, which is treated in the usual 
manner, are evaporated down to 500 c.c. Sulphuric acid is then added 
until the content is 5 per cent, and the lysine is precipitated by not 
too large an excess of phosphotungstic acid. 1 This is added until a 
portion of the clear liquid on the further addition of the reagent re- 
mains clear for ten seconds. After twenty-four hours the precipitate 
of lysine phosphotungstate is filtered off by suction and washed with 
5 per cent, sulphuric acid by stirring up in a mortar. After making 
up the filtrate and washings to a definite volume an estimation of the 
substances not precipitated may be made in an aliquot part by the 
Kjeldahl method. 

The lysine phosphotungstate is made into a uniform suspension 
with water and poured into boiling water. A hot saturated solution 
of baryta is added until the solution is strongly alkaline and contains 
excess of baryta. The precipitate of barium phosphotungstate, which 
is formed, is filtered off and boiled out several times with baryta and 
then with water. The alkaline solution is freed from baryta by means 
of carbon dioxide, concentrated, filtered, and evaporated on the water- 
bath nearly to dryness. Water is then added, the barium carbonate 
filtered off and washed, and the solution once more evaporated, after 
which it is made up to a definite volume and the lysine estimated in 
an aliquot portion by Kjeldahl's method. 

The lysine is separated from the remainder of the solution as 
picrate. The solution is evaporated down in a porcelain basin to dry- 
ness, and a small quantity of alcohol is added to the sticky residue. 
It is then treated with a saturated solution of picric acid in alcohol 
until no further precipitation of picrate occurs. After twenty-four 
hours this precipitate is filtered off and washed with a small quantity 
of absolute alcohol; it is then recrystallised by solution in boiling 
water, filtering if necessary, and evaporating to a small volume, when 
lysine picrate, C 6 H 4 N 2 O 2 . C 6 H 2 (NO 2 ) 3 OH, crystallises in needles on 

1 Schulze and Winterstein [1902] found that phenylalanine under certain conditions is 
precipitated by phosphotungstic acid and they have been able to isolate phenylalanine in 
this way. 



ISOLATION AND ESTIMATION OF DI-AMINO ACIDS 61 

cooling ; these are filtered off, washed with alcohol, dried, and 
weighed. 

The last portions of the lysine in the mother liquor from the picrate 
can be obtained by acidifying with sulphuric acid, extracting the picric 
acid with ether, precipitating as phosphotungstate, and repeating the 
above process for obtaining lysine picrate. 

Colorimetric Estimation of Histidine. 

Weiss and Ssobolew [1913] attempted to estimate histidine colori- 
metrically by its reaction with diazobenzene sulphonic acid. With 
pure solutions of histidine their attempts were successful ; the esti- 
mation was apparently not so successful in solutions containing other 
amino acids, as no data were given. In all cases, tyrosine which gives 
a very similar reaction, must be absent from the solution. 

In performing the reaction a fresh solution of diazobenzene sul- 
phonic acid is necessary. The authors prepared a standard reagent 
made up by mixing I part of solution A with 2 parts of solution B. 

The composition of these is : 

A = 4 grams of sulphanilic acid, 40 c.c. concentrated hydrochloric 
acid, water to 400 c.c. At room temperature solution takes place in 
twenty-four hours. 

B = o'5 per cent, solution of sodium nitrite in water. 

The solution is made alkaline with a 10 per cent, solution of 
anhydrous sodium carbonate. 

A standard solution of I in 10,000 of histidine monochloride is 
also required 

The standard colour for comparison is made by thoroughly mixing 
I '5 c.c. of the diazo reagent (lA + 2B) with 10 c.c. of the standard 
histidine solution and adding 3 c.c. of the soda solution. The colour 
produced is in five to ten minutes of a pure red shade, optimal, and 
stable. 

The unknown solution is successively diluted say I in 5, I in 20, 
i in 40, i in 80, I in 160. To 10 c.c. are added 1*5 c.c. of diazo 
reagent and 3 c.c. of soda as above. 1 

The dilution at which a colour corresponding with the standard 
colour is noted. As soon as the colour intensity decreases, the optimal 
dilution is passed. For example the matching of colours may be be- 
tween i in 80 and I in 160. Intermediate dilutions of I in 100 and 

1 No colour appears with very concentrated solutions of histidine, so that absence of 
colour does not imply absence of histidine, unless no colour appears on further dilution. 



62 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

I in 1 20 are made until there is the best matching of the colours. 
Finally, a stage will be reached at which I in 100 may be the best 
match and I in 101 will give a colour which is slightly less intense. 
The final matching may be performed in a Duboscq colorimeter. 

With solutions containing other amino acids, just as with concen- 
trated solutions of histidine, no colour appears unless more reagent 
is added This is due to the combination of other amino acids with 
the reagent. In order therefore to estimate histidine under these con- 
ditions more reagent is necessary and in order to avoid excess, which 
interferes by producing a dirty-green colour, a fixed volume of 20 c.c. 
was decided upon. This volume allows of an alteration of the amount 
of diazo reagent of from 1*5 to 7 c - c -> "> IO c - c - solution + 4*5 c.c. 
reagent + 3 c.c. soda +2*5 c.c. water. The standard was also diluted 
to 20 c.c. with 5 '5 c.c. water. Two variables are thus introduced in 
effecting an estimation (a) the necessary dilution of the histidine solu- 
tion, (fr) the necessary amount of reagent. 

The first testing is carried out with the reagent k, a series being 
prepared represented as & ', 6 ' 5 , k* . . . 2 , 1<5 , where the index denotes 
the amount of reagent. Testing is then done with dilutions of the 

solutions, the series being represented by , etc., where the denomi- 

4 5 

nator denotes the dilution. In practice all the preliminary experiments 
are not necessary, since as soon as the quantity of reagent is found, 
those trials with amounts greater than this quantity can be omitted. 
With greater dilution generally less reagent is required. The series 
experimentally tested may be for example 
I. k\ 6 ' 5 , k* . . . 8 , 2 - 5 , 2 , k l ' b optimal reaction with 5 . 

TT k_ R k^ A? k^ k k^ 

2'T' 2 2 y 2~2~ 2' 

in. i 4 , , -, , -, k . 

333333 3 

Wfv ft f& K ft K 
> > > j 3) 

44444 4 

7,3 7,2-5 7,2 T.1'5 

V. -, , -, _ . All colours below standard. 

5555 

The estimation is then made with the solution represented by ,*'.*., 

4 

with a dilution of I in 4 and with 3 c.c. of reagent. 



RESULTS OF ANALYSIS 63 

THE RESULTS OF THE ANALYSIS. 
A. 

Inspection of the results of analysis, which are tabulated on pages 
111-130, shows that there is a considerable deficit in the sum of the 
units composing the protein molecule. 

The best analyses are those of the protamines, of the silk-fibroin of 
spider's silk, and the gliadin of wheat ; in these, some 80-90 per cent, 
of the protein is accounted for ; in most cases, the sum of the figures 
only reaches 50-70 per cent, and in the other cases complete analyses 
do not exist. The deficiencies are due almost entirely to losses in- 
curred in isolating and purifying the amino acids. 

A careful inquiry where the loss occurs has been made by Osborne 
in conjunction with Leaven worth and Brautlecht [1908] and in con- 
junction with Jones [1910, I, 3], and by Abderhalden [1910], Abder- 
halden and Weil [1911, 2 ; 1912, i], who have estimated the nitrogen 
at each stage in the process for isolating the mono-amino acids. 

Osborne, Leavenworth and Brautlecht have proved that the loss 
does not fall upon the di-amino acids. They are convinced that the 
method for their isolation and estimation is extremely satisfactory, for 
they have been able to recover from 80-90 per cent, of the di-amino 
acids in a pure state. They consider that no other di-amino acid than 
the three hexone bases is present in most proteins ; Fischer and 
Abderhalden's diaminotrioxydodecanic acid in caseinogen may be an 
exception ; this protein seems to be the most complex in the number 
of units which it contains. 

The loss therefore occurs in the isolation and estimation of the 
mono-amino acids. Fischer pointed out, when he first described his 
ester method, that the values were not to be regarded as quantitative. 
The fact that all the figures given are those of the amount of theflure 
dry compound isolated is sufficient evidence that the total quantity of 
products is not accounted for. 

The sources of loss in the several steps of the long process have re- 
ceived special attention from Osborne and Jones [1910, I, 3], who are 
confirmed in their observations by Abderhalden and Weil [1911, 2 ; 
1912, i]. 

I. Hydrolysis. In many cases the hydrolysis of the proteins may 
not have been complete. Some proteins are very difficult to bring 
into solution in the concentrated hydrochloric acid, and portions may 



64 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

adhere to the sides of the flask and may therefore not be hydrolysed. 
Even if there be apparent total solution a small amount may escape 
hydrolysis by becoming enclosed in the " humin " which is formed to 
a greater or lesser extent. The insoluble material should be filtered 
off, washed, and tested with the biuret reaction. The absence of the 
biuret reaction is not an absolute criterion that hydrolysis is complete, 
for many polypeptides do not show it and are very resistant to 
hydrolysis. The residue, if large, should therefore be hydrolysed 
again. Complete hydrolysis should be tested for by Van Slyke's 
amino-nitrogen method (p. 89). Osborne has found that hydrolysis 
is sometimes only complete after boiling for two to five days. 

Henriques and Gjaldbak [1910] made an examination, by the 
formal titration method of Sorensen, of the completeness of hydrolysis 
of proteins by the action of enzymes and by boiling with acids. Hy- 
drolysis by enzymes was seldom complete, but complete hydrolysis 
was effected by boiling for twelve hours with 20 per cent, hydrochloric 
acid, except in the case of certain proteins. They found that com- 
plete hydrolysis occurred on heating the protein with 3N hydrochloric 
acid for one and a half hours in an autoclave at 1 50. The completion 
of hydrolysis could only be ascertained by testing the solution for in- 
crease of amino groups whilst at the same time the amount of ammonia 
must be minimal, i.e., arising only from amide groups and not by 
deamination of the amino acids. Andersen and Roed-Muller [1915] 
on account of the fact that more ammonia is slowly formed in a pro- 
longed hydrolysis are inclined to attribute its origin to uramido groups 
in the protein molecule. The rate of evolution of x ammonia was found 
by Skraup and Hardt-Stremayr [1908] to be rapid at first and quite 
slow subsequently, the greater part being evolved at the beginning of 
the hydrolysis. Van Slyke [1912, 2] maintained that the maximum 
amino nitrogen was evolved whether the hydrolysis was effected in an 
autoclave at 150 or by boiling at 1 00 for forty-eight hours ; there 
was less tendency for deamination at 100 than at 150. 

Pittom [1914] also found that ammonia is rapidly liberated in the 
early stages of hydrolysis. Amino-nitrogen is liberated in the same 
way during hydrolysis by acid, or by enzymes. Caseinogen differs 
from egg-albumin in its hydrolysis. More amino acids are formed from 
caseinogen than from egg-albumin in the earlier stages ; the reverse 
holds good at later stages ; at a still later stage the rate of amino acid 
formation is the same as in the early stage. There seems to be a defi- 
nite point at which complex polypeptides are broken down into simpler 



RESULTS OF ANALYSIS 65 

compounds. Some of the simpler polypeptides are not precipitated 
by phosphotungstic acid. 

Sulphur-containing substances and sometimes sulphur have been 
found in the reflux condenser, and the smell of iodoform has been 
noticed in the hydrolysis of spongin. 

2. Formation of Humin. Nearly all proteins on hydrolysis yield an 
insoluble brownish-black residue, -humin or melanin. Humins are 
formed by boiling carbohydrates with concentrated mineral acids, and 
if nitrogenous matter be present, the humin contains nitrogen. 
Samuely [1902] suggested that the humin formed from proteins was 
due to a secondary reaction between amino acids and carbohydrates, 
and obtained melanins containing nitrogen on boiling various amino 
acids with hydrochloric acid in the presence of carbohydrate. Maillard 
[1912] similarly obtained melanin-like substances on heating glucose and 
other carbohydrates with glycine and alanine ; the reaction was most 
rapid with xylose and arabinose, and alanine was the most reactive of 
the amino acids. These products were shown by Maillard [1913] to 
yield cyclic bases on heating, and he suggested that cellulose and pro- 
tein were the origin of the pyridine and allied bases found in coal-tar. 
Pyridine and other bases were obtained by Pictet and Chou [1916] by 
hydrolysing caseinogen in the presence of formaldehyde, and Maillard 
[1916] maintained that his results were substantially in agreement 
with those of Pictet and Chou. Gortner and Blish [1915], knowing 
that zein, which contains neither tryptophan nor carbohydrate and only 
a small quantity of histidine, gave very little humin, heated zein wfth 
acid in the presence of tryptophan and carbohydrate and obtained 
86 '6 per cent, of the nitrogen of the tryptophan in the form of humin; 
with histidine in the place of tryptophan 0*5 per cent, of its nitrogen was 
contained in the humin. Tryptophan thus is largely concerned in the 
formation of humin. Grindley and Slater [1915] found that other 
amino acids were concerned in the formation of humin. A detailed 
investigation upon the amino acids taking part in the formation of 
humin was made by Roxas [1916]. Alanine, leucine, phenylalanine 
and glutamic acid are not factors in humin formation ; proline may be 
a factor under certain conditions in the formation of humin. Trypto- 
phan gave up 71 per cent, of its nitrogen, tyrosine 15, cystine 3*1, 
arginine 2-3, lysine 2*6, histidine 1*8. Generally, fructose and xylose 
were more reactive than glucose, and the three hexone bases reacted 
more readily in weak acid solutions than in strong. The amino nitro- 
gen of arginine, histidine, and tryptophan was lost in the formation of 
humin ; tyrosine and cystine did not react with loss of nitrogen. 
PT. I. 5 



66 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



Roxas suggested that the mechanism of the reaction was : 
(i) with histidine: 



H-C 




R-CH 

] 


H- 
H 


-c 

I 


s 





N 



CH 



NH 



CH 



COOH 



COOH 
(2) with arginine : 

f 

H N - C N CH CH CH, 



H 





H- 



II 
c 
it 



i 

H-j-N CH COOH 
H 



(3) with tryptophan : 

C CH CH-COOH 




C-CHg-CH-COOH 



or 



NIB HCR 




NH 



These possible condensation products can explain the formation of 
pyridine from melaniri or humin : 



R-I-CH C - J N 




-NH 
I 
CH 

- f 

COOH 

Gortner [1916] showed that the aldehydes, furfural, benzaldehyde 
and formaldehyde reacted on boiling with hydrochloric acid in a way 
similar to carbohydrates, and in presence of amino acids the humin 
nitrogen was large. The humin was mostly insoluble in acid except 
in the case of tryosine and formaldehyde. It appears that the reaction 
consists in the formation of furfural from carbohydrate and that humin 
arises by the condensation or decomposition of furfural together with 
nitrogen from amino acids if these be present in the solution. 

The loss of products is considerably greater than the quantity of 
" humin," which generally amounts to 1-2 percent, of the protein. A 
loss, which cannot be estimated, is represented by the soluble brown 
pigment which colours the solution. 



RESULTS OF ANALYSIS 67 

3. Separation of Glutamic acid Hydrochloride. The quantity of 
glutamic acid precipitated as hydrochloride never represents the total 
quantity present in the protein. The amount precipitated depends 
very largely on conditions. The precipitate usually contains am- 
monium chloride ; this is removed by boiling with baryta, and if the 
baryta be removed with carbon dioxide the barium carbonate may 
contain barium glutamate, which is very insoluble, and consequently 
there is loss of this constituent. The remainder of the glutamic acid 
is recovered with the esters. 

4. Esterification. The loss in this process is not great, especially 
if it be repeated. Loss is chiefly due to hydrolysis of the esters when 
the water is removed by evaporation in vacuo. Phelps and Tillotson's 
method seems to be preferable. 

5. Separation of Glycine Ester Hydrochloride. The glycine is never 
completely isolated as ester hydrochloride ; the remainder is obtained 
as ester. 

6. Extraction of Esters. Loss always occurs in this part of the pro- 
cess, but is covered when the process is repeated. The loss is largely 
mechanical and cannot be avoided. A small quantity of organic 
matter is retained by the sodium sulphate used for drying the esters. 

7. Distillation of Esters. (a) In distilling off the ether, especially 
if its volume be large, a considerable quantity of esters distils at the 
same time. Two receivers should be used and the esters extracted 
from the distillate. 

(#) Decomposition of the esters occurs during the distillation, and 
a more or less large residue represents the loss. The products can 
be recovered, if necessary. 

Abderhalden and Weil have found that in the process up to this 
stage 30 per cent, of glutamic acid, 40-45 per cent, of aspartic acid, 
37-5 per cent, of glycine, 30 per cent, of alanine, and 20 per cent, 
of leucine are lost ; some of the glutamic acid is lost by conversion 
into pyrrolidone carboxylic acid. In the case of a mixture, of the 
glycine 50 per cent, was recovered, of the alanine 57 per cent, of the 
leucine 66 per cent., of the glutamic acid 58 per cent, and of the 
aspartic acid 40 per cent. The esters were liberated by treatment 
with caustic soda and sodium carbonate, alcoholic soda or ammonia. 
The best yields were obtained with alcoholic soda. The loss of higher 
boiling esters is greatly diminished if the distillation of fraction IV. be 
omitted. Osborne and Jones found that the separation of the esters 
in this fraction is not more troublesome than when they are distilled. 

5* 



68 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

8. Separation of the Individual Amino Acids. The greatest loss 
occurs here as none of the methods of isolating the compounds are 
perfect. 

(a) Proline. The quantity extracted by alcohol is much greater 
than that obtained in a pure crystalline state and reckoned as pro- 
line. An estimation by Van Slyke's method may give the actual 
amount, though Abderhalden and Kautzsch think that this value is 
not really satisfactory. Abderhalden and Kautzsch [1912] regard the 
proline content determined gravimetrically as being very much below 
the actual amount. 

(fr) Valine, Leucine, Isoleucine. The figures given represent the 
quantity of substance isolated in a pure state. The leucine figures 
in the tables are really those for leucine + isoleucine. 

(c) Glycine and Alanine. Nearly all the glycine can be obtained, 
but the actual amount of alanine is much greater. 

(d) Glutamic Acid. Probably the figures given for this amino acid 
most nearly approach the real content of the protein in this constituent. 

(e) Aspartic Acid and Serine. The figures are much too low, as 
the method of separation is extremely unsatisfactory. 

9. Oxy proline. The amount of this substance in the protein is 
greater than the quantities which have been isolated. Its method of 
isolation is so laborious that data are only available for a few proteins. 

10. Tyrosine and Cystine. The data for tyrosine most probably 
represent the content of the protein in this unit very closely. 

The data for cystine are unsatisfactory ; in many cases the data 
are calculated from the sulphur content of the jSrotein ; in the other 
cases they are those from pure isolated cystine. More satisfactory 
data are given by Van Slyke's amino method (p. 85). 

11. Tryptophan. A large amount of protein is required for the 
isolation and estimation of tryptophan ; it is on this account most 
probably that so few data exist. The isolation is easier the larger the 
quantity of material. Tryptophan might be isolated first in most cases 
and the remainder of the products isolated subsequently. 

Osborne, allowing for all losses, calculates that from 4 1-82 per cent, 
can actually be recovered. In the cases of zein and gliadin 86 and 68 
per cent, respectively are known ; in the case of vignin only 5 per cent, 
more has to be accounted for. The losses from the esters are com- 
puted to be 50 per cent, of the alanine, serine, aspartic acid ; 30 per 
cent, ot the valine, proline, glutamic acid, phenylalanine ; 20 per cent, of 
the leucine : only 50 per cent, of the cystine and tryptophan may be 
accounted for. 



RESULTS OF ANALYSIS 69 

Methylation of Amino Acids. 

A method which may prove of some service in separating these 
various mixtures was described by Engeland [1909]. The amino acids 
are methylated in alkaline solution with methyl iodide and converted 
into their betaines : 

/CH 3 

CH 2 .NH 2 CH 2 .Nf-CH 3 

+ 3 CH 3 I + 3 NaOH = 3 NaI + 3 H 2 O +11 \CH 3 
COOH CO O 

These products are separated by means of their double salts with 
mercuric chloride, gold chloride, and platinum chloride. In the case 
of caseinogen, Engeland isolated (i) 1-methyl-hygric acid, which is de- 
rived from proline ; a yield corresponding to 67 per cent, of proline 
was obtained ; (2) trimethyl leucine, (3) trimethyl valine, (4) betaine, 
(5) trimethyl-alanine. Subsequently, Engeland [1910] prepared the 
trimethyl derivative of phenylalanine and dimethyl glutamic acid, and 
[1914] isolated methyl-hygric acid from the products of hydrolysis of 
spongin. The procedure adopted was very laborious and seems* to 
offer no advantages over the ester method. Its chief use may be in 
the separation of those mixtures which result by the ester method and 
for which the methods at present in use are not so satisfactory. 



B. 

The data in our possession show definitely that the various proteins 
are composed of the same units ; in some cases certain are missing 
and in other cases one or more units are present in very much larger 
amount. These differences on the whole confirm our classification of 
the proteins on physical properties. No great differences are notice- 
able between the members of any single group except in the case of the 
scleroproteins. Although two proteins in any group may contain the 
same amount of any unit we cannot say that they,are identical. Even 
if they contained the same amount of all the units they might still be 
different, for the arrangement of the units in the molecule may not 
be the same. The analytical data are given on pp. 111-130. The 
following brief particulars may be noted : 



70 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

Protamines. 

The first work upon this group of proteins was carried out by 
Miescher [1874], who described the basic substance protamine 
present in the sperm of salmon. Piccard [1874] made similar in- 
vestigations. Kossel extended the work of Miescher by examining 
the basic proteins in the sperm of other fishes. He termed the 
whole group protamines, the individual members being called 
salmine, sturine, clupeine, etc., according to the name of the fish from 
which they were prepared. 

The first analyses of salmine and sturine [Kossel, 1896-97] were 
qualitative and showed the presence of arginine and histidine with 
quite small amounts of mono-amino acids. Lysine was found later 
in sturine [Kossel, 1898], and it was thought that the protamines 
contained the three bases as their principal constituents, but Kossel 
[1898-99] with improved methods of analysis showed that arginine 
only was present in salmine and clupeine, whereas sturine contained 
all three hexone bases. Subsequent work by Kossel [1898-99, 
1903], Morkowin [1899], Kossel and Dakin [1903-4, 1904, 1905] was 
devoted to the identification of the mono-amino acids. Quantitative 
analyses of the di-amino acids were made by Kossel and Kutscher 
[1900-1], and Kossel and Dakin [1904] published a complete analysis 
of salmine. Their result does not conform to that of Abderhalden 
[1904] and the difference is to be attributed to the material analysed ; 
Abderhalden probably used salmine prepared from unripe sperm and 
not sufficiently purified ; Kossel and Dakin used carefully purified 
salmine from ripe sperm. 

A large number of protamines have been analysed by Kossel 
[1910, 1913] and the nature of the mono-amino acids determined 
in some of them by Kossel and Edlbacher [1913]. Some of these 
were previously investigated by Ulpiani [1902], by Dezani [1908], 
and by Taylor [1908-9]. Maleniick [1908] prepared sturine from the 
Russian sturgeon, but did not analyse its constituents. His method 
of preparation was criticised by Kossel [1910]. Kossel [1913] did 
not give the actual quantity of the units in these protamines, but 
determined the percentage of the total nitrogen present as arginine- 
nitrogen, histidine-nitrogen, mono-amino acid-nitrogen. The arginine- 
nitrogen on the average was about 80 per cent, of the total nitrogen, 
the mono-amino acid-nitrogen about 10 per cent. Lysine was absent 
and histidine present only in percine. 

The analyses show that the protamines are built up almost ex- 



RESULTS OF ANALYSIS 71 

clusively of di-amino acids, especially arginine, salmine containing over 
80 per cent. Only small amounts of mono-amino acids are present in 
them. The mono-amino acids are alanine, aminovalerianic acid, serine, 
proline, tryptophan and tyrosine. It has not been determined whether 
the aminovalerianic acid is identical with valine from other proteins, 
though this is probable. 

The chief features are the presence of arginine and the absence of 
lysine and histidine in salmine, clupeine, cyclopterine and other 
protamines. Sturine only contains arginine, histidine and lysine ; 
cyprinine contains arginine and lysine ; percine contains arginine and 
histidine. The general composition of the protamines seems to be 
a^m or (ahl\m or (ah\m where a represents arginine, h histidine, 
/ lysine, and m mono-amino acid [Kossel, 1913]. 

As to the origin of the di-amino acids in the protamine of fish 
sperm the observations of Miescher suggested their formation from the 
muscular tissue of the fish. The salmon does not feed during its life 
in the river and loses weight at the expense of the increase in size of 
its roe. Kossel [1905] made calculations as to this possibility: the 
roe of a salmon contains 27 grams of protamine with 22-8 grams of 
arginine ; if the muscular tissue contain 7*1 per cent, of arginine, 321 
grams of it would be required : more than this quantity of muscle is 
decomposed during the life of the salmon in the river, so that, as there 
is no evidence for its synthesis, its origin is by transference from 
the muscular tissue. Actual analyses of the protein of salmon muscle 
and salmon tissue have been made by Weiss [1907] and they confirm 
the calculations. In the transference of the arginine from the muscle to 
the roe it seems that it is rendered stable by combination with nucleic 
acid or some other grouping and thus transported, for under the 
ordinary conditions of decomposition of tissues arginine is itself de- 
composed. The transfer appears to occur not as the transfer of a 
single unit, but as the transfer of a complex containing several units. 
Such a complex is a histone which contains more mono-amino acid 
units than a protamine. 

In general, Kossel regards the protamines as the simplest proteins, 
the more complex or ordinary proteins containing a protamine nucleus 
(i.e., of di-amino acids) to which is attached the mono-amino acid units, 
the histones being intermediate substances. This theory is supported 
by the results of analysis of other proteins, all of which contain 
arginine and other di-amino acids. The isolation from proteins of 
complexes containing only di-amino acids will be the only proof of a 
protamine nucleus in a protein molecule. 



72 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

Histones. 

Histones are found in the unripe sperm of the salmon [Miescher, 
1874], mackerel [Bang, 1899], and other fishes [Ehrstrom, 1901] ; also 
in the ripe sperm of the sea urchin [Matthews, 1897]. The first 
representative of the group was the histone, to which the name was 
first applied, prepared by Kossel [1883-84] from the red blood cor- 
puscles of the goose. Lilienfeld [1894] prepared a similar substance 
from the thymus, and Schulz [1898] found that globin of haemoglobin 
belonged to this group. Histones are characterised by being precipi- 
tated by ammonia in the presence of ammonium salts. Their other 
properties are described by Bang [1899]. They contain from 15-91- 
1979 per cent, of nitrogen. 

Histones are supposed to be intermediate compounds between 
protamines and other proteins, and this supposition is confirmed by 
the results of analysis. They are distinguished from protamines in 
their smaller content of di-amino acids, namely, about 25 per cent. 
Only in the case of thymus-histone has an estimation been made of 
the mono-amino acids. 

The protein constituent globin of haemoglobin has always been 
regarded as a histone, but the presence of only 20 per cent, of di-amino 
acids is against this supposition. Further, the principal di-amino acid 
is histidine, whereas in the other histones it is arginine. It should be 
noted that haemoglobin contains a considerably greater amount of 
histidine than the other proteins. The high content in histidine 
appears to be a peculiarity of the haemoglobins ;/it may be connected 
with the origin of the red blood corpuscles from nucleated corpuscles 
since the glyoxaline ring contained in histidine is also contained in 
the purine bases, which are present in nucleic acid. Comparative 
data of the amount of histidine in the haemoglobin of different animals 
by Abderhalden and Medigreceanu [1909] are at present only avail- 
able for the red-blood corpuscles; those of the horse contain 5-3 per 
cent., of the hen 2-8 per cent, of the duck 2-5 per cent., of the goose 
3-6 per cent. 

An examination of the mono-ammo acids in haemoglobin was 
undertaken by Proscher [1899] before Fischer had described his ester 
method. The analysis by Fischer and Abderhalden [1902] was 
repeated by Abderhalden [1903] whose data were considerably higher 
than those found by Fischer and Abderhalden ; the mixture of amino 
acids was esterified three times. Abderhalden and Baumann [1907] 
analysed the haemoglobin of dog's blood. 



RESULTS OF ANALYSIS . 73 

Albumins and Globulins. 

Albumins contain no glycine, whereas globulins contain this amino 
acid ; they show no other striking differences. Their differentiation 
on physical grounds is thus scarcely borne out by analysis, and their 
interconversion, which has been described, may be possible. 

The most recent results for crystallised egg-albumin by Osborne, 
Jones and Leaven worth [1909] confirm the earlier ones by Abderhalden 
and Pregl [1905, 2]. They show that workers in different parts 
obtain very similar results with Fischer's ester method. The values 
by Hugounenq and Morel [1906] and by Hugounenq and Galimard 
[1906] are for coagulated egg-white. 

Chapman and Petrie [1909] determined that egg-white contains 2' 4 
per cent, of arginine, 3 '2 per cent, of lysine, and 07 per cent, of 
histidine, data which were required for experimental work on nutrition. 

The comparative data by Abderhalden and Slavu [1909], both of 
serum albumin and serum globulin, with regard to their content in 
glycine, tyrosine and glutamic acid would incline one to believe that 
both the serum albumins and the serum globulins of different origin 
were of the same composition. They show no very great difference 
from fibrin. 

The composition of lactalbumin has been determined by Abder- 
halden and Pribram [1907] and by Osborne, Van Slyke, Leaven- 
worth and Vinograd [1915]. The very high lysine content of this 
protein accounts for its value in nutrition. It would appear that 
the globulin in milk contains glycine, a mixture of the coagulable pro- 
teins having been analysed by Abderhalden and Hunter [1906, i]. 
Abderhalden and Schittenhelm [1906] found 1-3 per cent, of tyrosine 
and I per cent, of glutamic acid in the albumin of human milk. 

Hopkins and Savory's thorough investigation of the Bence-Jones 
protein [1911], in which they showed that its peculiar physical pro- 
perties were due to the conditions under which it was examined, and 
that its chemical composition differed so distinctly from that of the 
proteoses, brings this protein into the class of coagulable proteins as a 
globulin. The Bence-Jones protein is characterised chemically by a 
high content of the aromatic amino acids; the combined values for 
phenylalanine and tyrosine are higher than those 'for any other blood 
or tissue protein. Both physically and chemically this protein seems 
to stand in a class by itself. Abderhalden and Rostoski [1905, 2] had 
previously analysed and examined this protein. Their figures are 
very similar to those of Hopkins and Savory. 



74 THE CHEMICAL CONSTITUTION OF THE PROTEINS 
The Vegetable Proteins. 

The vegetable proteins show no great difference from the animal 
proteins in regard to the number of amino acids which they contain in 
their molecule. The most noticeable features are their high content in 
glutamic acid and in arginine. Their ammonia content is also high. 
This is probably connected with the large amount of the dibasic 
glutamic acid and is in harmony with the occurrence of asparagine 
and glutamine in growing seedlings. 

Albumins. 

Only two vegetable albumins have so far been analysed, the 
legumelin of the pea and the leucosin of wheat. The resemblance in 
their composition extends not only to the general proportions of the 
amino acids, but also to the quantity isolated. Leucosin occurs in 
the embryo of wheat ; it is not possible to locate legumelin in any 
particular part of the seed, but, from analogy, it may be supposed that 
both these proteins are constituents of the physiologically active tissues 
rather than a constituent of the reserve food-stuff for the embryo. 
Legumelin is quite different in composition from legumin and vicilin, 
two other proteins contained in the pea. These albumins show a 
resemblance in their composition to the animal albumins. 

Globulins. 

All the globulins, which can be prepared in a crystalline state, 
have a very similar composition. Excelsin contains the greatest amount 
of arginine and edestin of glutamic acid. These proteins form the 
best source of arginine. Their content in glutamic acid is about half 
the content of the gliadins in this amino acid. It is unfortunate that 
the analysis of edestin by Osborne and Liddle [1910, i] is incomplete. 
Their figures are higher than those of Abderhalden [1902, 1903] and 
they were unable to isolate oxyproline. 

No great difference is to be noted between the crystalline globulins 
and the other vegetable globulins, except in the proportion of arginine 
which is distinctly less in most of the non-crystalline ones. 

The legumins of the pea and vetch show no real difference in 
their physical properties and elementary composition, but the analysis 
shows that differences do exist, especially in the data for lysine and 
histidine. Analysis has also shown that vignin differs from the other 
legumins. 

The vicilin of the pea contains less sulphur (o*i-O'2 per cent.) than 



RESULTS OF ANALYSIS 75 

any other protein ; its analysis shows that it is distinct from legumin. 
It contains no glycine and more glutamic acid than legumin. A 
similar protein does not exist in the vetch. 

Amandin contains 19 per cent, of nitrogen; the high content in 
arginine and ammonia serve to explain this high figure. The proteins 
of the peanut are rich in lysine, and in this respect are of great value 
for improving a diet containing protein which is deficient in this unit. 

The high figure for valine in Foreman's analysis of the protein of 
linseed is remarkable. 

Gliadins and Glutelins. 

There is a very marked difference in the composition of the two 
proteins found in cereals. Those amino acids, which are absent in the 
alcohol-soluble protein, are present in the other protein, which is 
soluble in dilute alkali. The mixture (gluten) of these proteins in the 
grain, therefore, gives all the amino acids present in other proteins. 

The gliadins are very like one another in composition. They are 
distinguished from other proteins by their high content in glutamic 
acid, proline and ammonia, their low content in arginine and histidine ; 
lysine is absent from zein, but is present in wheat gliadin in very small 
amount. On account of the high content in proline and ammonia 
Osborne suggested the name of prolamines for this group ; the group 
name gliadins has been preferred by the British workers. 

Wheat-gliadin and rye-gliadin show no great differences, and it 
seems probable that they are the same protein. Zein of maize differs 
from them in containing no glycine and no tryptophan and also in con- 
taining more leucine and less glutamic acid. The high tyrosine con- 
tent found by Kutscher does not seem to be correct. Kafir, largely 
grown in U.S.A., contains kafirin as its chief protein, which resembles 
zein, but contains lysine and tryptophan. Hordein contains more 
proline than any other protein. 

Wheat-glutenin and the other glutelins seem to contain all the 
amino acids which have been isolated. The analysis of rice by the 
Japanese workers is for the whole grain and not for the isolated pro- 
tein ; they found that the husk of the grain also contained protein, 
and that the proportions of the constituent amino acids were different. 
Data for the di-amino acids in oryzenin have been published by 
Osborne, Van Slyke, Leavenworth and Vinograd [1915]. 



76 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

Phosphoproteins. 

There is no striking peculiarity noticeable in the analyses of the 
phosphoproteins. If we disregard the small quantity of glycine found 
in caseinogen by some workers and in vitellin by Abderhalden and 
Hunter, who used the commercial product in their investigation, we 
must note the absence of this amino acid in the phosphoproteins. It 
is also absent from the albumins. Glycine is apparently, from the 
results obtained by numerous workers, the only amino acid which can 
be synthesised by the animal body from other products ; if these phos- 
phoproteins, especially vitellin, really contain no glycine a further proof 
is given of its synthesis by animals. Abderhalden and Kempe [ 1 907, 2] 
in their experiments on the synthesis of amino acids in the chick 
could not detect any differences in the amounts of glycine, tyrosine 
and glutamic acid at different periods of development. 

Caseinogen. 

Caseinogen has been hydrolysed more frequently than any other 
protein and was the protein first analysed by E. Fischer [1901] by the 
ester method. Fischer did not state the yields of the amino acids, but 
they were given later by Abderhalden [1905]. No data are given for 
isoleucine. Some samples of caseinogen seem to contain glycine ; in 
others this unit is not found. 

The analysis of caseinogen by Osborne and Guest [1911, i] is the 
most recent, and the data given are the highest which have been ob- 
served by them and other American workers. On account of the im- 
portance of this protein in nutrition it is very necessary to have as 
thorough an analysis as possible. The latest analysis has increased 
our knowledge of the constituent amino acids by about 1 5 per cent. 
Some 30 per cent, of the protein still remains unaccounted for. Fore- 
man's unpublished figures indicate that some of the amino acids are 
present in larger amounts than other workers have found. 

The caseinogens of cow's, goat's, and human milk appear to have 
the same composition. 

Vitellin, etc. 

Vitellin, which has been analysed by four sets of investigators, has 
given very different results. The values of Hugounenq [1906] and of 
Levene and Alsberg [1906] do not correspond with the values of 
Abderhalden and Hunter [1906, 2] or of Osborne and Jones [1909, i]. 
The values by the latter workers are the most recent and are probably 
the most accurate. 

The phosphoproteins in the eggs of fish (ichthulin) and the frog 
have been analysed by Hugounenq [1904] and by Galimard [1904]. 



RESULTS OF ANALYSIS 77 

Scleroproteins. 

The scleroproteins, which in their physical properties comprise a 
heterogeneous collection of proteins, give on hydrolysis, as would be 
expected, results which support their classification. 

Silk. 

Of the proteins in this group those of silk have been most 
thoroughly investigated. Silk is a mixture of two proteins silk- 
fibroin and silk-gelatin ; the latter is extracted from raw silk by boiling 
out with water under pressure when it loses 15-20 per cent, in weight ; 
the insoluble portion which has the structure of the original silk forms 
the silk-fibroin. 

Both silk-fibroin and silk-gelatin were analysed by Fischer and 
Skita [1901, 1902] when the ester method was first introduced. Silk- 
fibroin is composed of practically only three amino acids, glycine, 
alanine and tyrosine, and is probably the simplest protein known. It 
contains more tyrosine than any other protein except that of the cara- 
pace of the tortoise and is the best source of tyrosine. Silk-fibroin 
differs very markedly in composition from silk-gelatin ; this substance 
contains more serine than any other protein. 

The composition of silk-fibroin and silk-gelatin from different 
sources is under investigation by Abderhalden and his pupils. The 
present data show that the composition of the various silk-fibroins 
is fairly similar, although many differences can be noted. The New 
Chwang, Schantung and Chefoo varieties are rather peculiar in 
leaving a somewhat large residue after hydrolysis, which seems to be 
connected with the food-stuff of the silk-worms. The New Chwang 
and Schantung worms are fed on oak leaves. The Canton and Bengal 
silks are most like the Italian ; Indian Tussore silk contains a 
considerably smaller amount of glycine. African Tussore silk is like 
other Tussore silks and the silk of Bombyx mori from Africa resembled 
that of Bombyx mori from other countries. The silk of Anaphe had 
also a similar composition. In fact, the silk of caterpillars, spiders, 
and of Pinna nobilis have an almost identical composition, with 
tyrosine, glycine and alanine as the chief constituents [Abderhalden, 
1911]. 

No striking differences are to be noted in the various silk-gelatins. 

The silk of other arthropods has also been examined. The silk- 
fibroin from spider's silk, except for its high content in glutamic acid, 



78 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

closely resembles that of the silk- worm. The silks examined by 
Suzuki, Yoshimura and Inouye [1909] were distinctly different. The 
material spun by Oeceticus of the family Psychidae in order to unite 
the bits of wood together with which it builds its house contained no 
tyrosine, but otherwise resembled silk-fibroin. The absence of tyrosine 
brings out a resemblance to ovokeratin. 



Origin of the Amino Acids in Silk. 

Abderhalden and Dean [1909] and Abderhalden and Weichardt 
[1909] have tried to ascertain whether the amino acids composing the 
silk are elaborated at the moment of spinning or whether they are 
selected out of the protein material by the spinning gland. Since the 
composition of the moth and cocoon together is about the same as the 
silk-worm, it seems most likely that the spinning gland selects the 
constituents in making the silk and does not synthesise them from 
other products. 

Pigorini's experiments [1915] with Bombyx mori indicate that 
glycine is assimilated if it be added to the food in small quantities ; 
in large quantities it had a toxic action, probably due to the formation 
of ammonia and other decomposition products. This fact supports the 
hypothesis that silk-formation is mainly a protective measure of the 
silk-worms, the object being to remove free amino acids from the 
organism. 

Inouye [1912] showed that a great change in chemical composition 
of the animal takes place in the preparation of the cocoon by the 
caterpillar, but there is very little change between the pupa and the 
moth. No nitrogen is given off in the gaseous state, but fat is stored 
up in the pupa stage and consumed by the pupa and moth. In all 
stages the amount of mono-amino acids present is greater than that 
of di-amino acids ; mono-amino acids are found almost entirely in the 
cocoon. Protein is hydrolysed by a proteoclastic enzyme and its loss 
is balanced by the increase in amino acids, which are partially con- 
verted into ammonia. Inouye [1910] determined the amount of food 
(mulberry leaves) consumed by the silk-worm. 1000 silk-worms ate 
12579-6 grams of fresh leaves whose dry matter was 4056*6 grams ; 
1324 grams or nearly 33 per cent, of the mulberry leaves were assimi- 
lated. The silk-worms were hatched on July 1 2th, attained maturity, 



RESULTS OF ANALYSIS 79 

and commenced to spin on August 6th. In other terms, a silk-worm 
during the period of its life as caterpillar, consumes 12*6 grams of 
fresh mulberry leaf or 4-0 grams of dry leaf, of which 1-3 grams is 
assimilated, in a period of twenty-five days. The amino acids in 
mulberry leaves have been estimated by Katayama [1916]. There is 
a marked difference in the proportions of the different units (see p. 123). 
Of the three chief constituents of silk, alanine is present in large amount 
in the mulberry leaf, but there is very little glycine ; tyrosine is evi- 
dently present only in small amounts as no yield is mentioned. These 
data suggest that glycine may be synthesised de novo and that tyrosine 
arises from phenylalanine. 

Gelatin, Spongin, Elastin. 

Gelatin contains no tryptophan, cystine, or tyrosine, but it contains 
more glycine than any other protein, except elastin. It also con- 
tains a large amount of proline and oxyproline. Levene and Beatty's 
analysis [1906] was not made by the ester method. This protein 
appears to have no similarity to silk-gelatin, which contains so much 
serine. 

Spongin resembles gelatin in its high content of glycine, but differs 
in its content of glutamic acid. 

Elastin seems to be made up almost entirely of glycine and leucine ; 
like gelatin, it contains no tyrosine, but it differs from gelatin in con- 
taining more phenylalanine. No di-amino acids could be isolated by 
Bergh [1898] and Hedin [1898], but a minute quantity of arginine 
was obtained by Kossel and Kutscher [1898]. 

Keratins. 

The keratins, except for certain of the proteins usually in- 
cluded in this sub-group, are remarkable for containing more cystine 
than any of the other proteins ; in human hair cystine exists to the 
extent of about 14 per cent., in other keratins its amount varies from 
2-8 per cent. Tyrosine is also present in fair quantities, and the 
amounts of leucine and of glutamic acid are high. 

The amount of cystine isolated from the keratins is generally far 
below the actual quantity present. The cystine content has been 
ascertained by the determination of the total sulphur content of the 
hydrolysed protein and of the loosely-bound sulphur (see Part II.) 
[Morner, 1901-2], or by the determination of the sulphur content of 



8o THE CHEMICAL CONSTITUTION OF THE PROTEINS 

the cystine fraction dissolved in ammonia [Buchtala, 1907]. More 
cystine is present in the hair than in the horn of the same animal, as 
the following figures show : 



Human hair 13-9 per cent. (Morner) 

14-0 , (Buchtala) 



,, (white) 11*6 , 
nails 5-2 



Ox hair 7-3 per cent. (Buchtala) 



horn 6*8 

hoof 5-4 

Horse hair 8'o 

,, hoof 3-2 

Pig's bristles 7*2 

hoof 2'2 



(Morner) 
(Buchtala) 



Human hair thus contains nearly double the amount of cystine 
that is found in the hair of other animals. White hair has less 
cystine than brown hair. 

Horn and hair differ further in respect to their content in glycine 
and phenylalanine. Hair contains very little or no phenylalanine and 
from 3-4 per cent, of glycine (white hair 9 per cent.) : horn contains 
very little glycine and from 2-3 per cent, of phenylalanine. 

The keratins of sheep's wool and of goose feathers correspond in 
most respects with the keratin of hair. 

Comparative analyses by Abderhalden and Fuchs [1908] show that 
the horn of older animals contains slightly less glutamic acid than that 
of younger : ox hoof one year old contained 18 per cent., four years 
old 17; ox horn one year old 14, four years old 13. The data for 
the horny material of the epidermis of a fish, a tortoise, a snake, an 
armadillo and an elephant form an interesting series in comparative 
physiological chemistry. The high tyrosine content of tortoise shell, 
armadillo scales and snake's scales is particularly noticeable and it is 
high also in whalebone and elephant hide. , <Tortoise shell has a high 
glycine content, like gelatin and elastin, and does not appear to con- 
tain glutamic acid. Tortoise shell has the highest tyrosine content of 
any protein. 

The analysis of the egg-membrane of hen's eggs was made with 
material from 25,000 eggs ; only a qualitative analysis with 17 grams 
of the egg-membrane of the eggs of Testudo graeca was possible. No 
tyrosine is present in these proteins, but the egg-membrane of the 
eggs of Selachians contains tyrosine and only traces of cystine. 
Koilin, the horny material in birds' gizzards, has also a low cystine 
content. The egg-membranes and koilin are not considered by 
Hofmann and Pregl [1907] to belong to the sub-group of keratins. 



RESULTS OF ANALYSIS 81 

Various Proteins. 

The only analyses which we possess of glucoproteins pseudomu- 
cin and paramucin were carried out before the ester method had be- 
come of general use. Otori [1904, I, 2] has given some analytical 
figures, but Pregl [1908, 2] with the small quantity of material available 
could only perform a qualitative analysis. 

A large variety of proteins which cannot be included in any of the 
above groups have been examined, including micro-organisms and the 
muscle of Egyptian mummies. 

The qualitative data given by Abderhalden and Rona [1905] for 
Aspergillus niger are of special interest, as this mould was grown on 
different nutrient solutions and in each case the same amino acids 
were synthesised. Emmerling [1909] found the usual amino acids in 
the phosphorescent infusoria ; Tamura[i9i3, 1,2 ; 1914, I, 2] examined 
the amino acids in the bacilli of tubercle and diphtheria, Mycobacter- 
ium and a water bacillus; Omeliansky and Sieber [1913] analysed 
Azotobacter chroococcum. The protein of yeast has been investigated 
by Pringsheim [1913], Neuberg [1915], and Meisenheimer [1915]. 
The bacteria had a high content in phenylalanine, whilst the results 
for yeast are most variable. 

The data for the muscle of the Egyptian mummy are interesting 
as showing that the muscle substance is preserved from decomposition 
by the process of embalming. Autolysis of the muscle occurs, as 
amino acids could be extracted from the tissue by water. 

The analysis of tumours by the ester method may perhaps throw 
some light on cancer. Several tumours have been analysed, and they 
all gave figures approximating to those in the table (p. 129). More 
useful information was published by Drummond [ 1 91 6], see pp. 109, 133. 

By analysing the membrane enveloping the fat particles of milk 
Abderhalden and Voltz [1909] have been able to show that the protein 
is not caseinogen, but that it is most probably a mixture of proteins, 
as is generally believed. 

The analyses of chicken muscle, fish muscle, scallop muscle and ox 
muscle by Osborne and his associates are of extreme importance for 
the study of the nutritional value of these food-stuffs as compared with 
one another and with other, especially the vegetable, proteins. The 
several muscles show a very close resemblance to the vegetable globul- 
ins, but they contain less arginine and more lysine than these proteins. 
The high content in lysine is particularly noticeable ; scallop muscle 
contains the least amount; in the other muscles it is about 7-5 per 
PT. I. 6 



82 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



cent. Fish muscle contains the least amount of glutamic acid ; the 
amounts in the other muscles are very close. The muscles are alike in 
respect of tyrosine, aspartic acid, arginine, lysine and histidine, and 
also phenylalanine, which is slightly more abundant in scallop muscle. 
Glycine is present in considerable amount in the free state in scallop 
muscle ; otherwise, it is present only in ox muscle to any extent ; 
since syntonin contains very little glycine, it is probably derived from 
the connective tissue in the ox muscle. It is curious that, as we 
pass from the lower forms of life to the higher, the amounts of glycine, 
alanine, leucine and proline increase. 

The amount of protein in the nervous system seems to be small, as 
the total quantity of amino acid isolated by Abderhalden and Weil 
[1912, 2 ; 1913, i] does not exceed 10 per cent. Mention may again 
be made of the presence of norleucine amongst these amino acids. 

The crystalline protein from the juice of A ntiaris toxicaria examined 
by Kotake and Knoop [1911] is a remarkable protein probably a 
complex polypeptide with IO'6 per cent, of cystine. Further data 
will no doubt decide the nature of this protein. Only a small quantity 
of material has been so far available. 

Derivatives of Proteins. 

Complete analyses of the proteoses products intermediate between 
the proteins and amino acids have been undertaken by Levene, and 
by Levene in conjunction with Van Slyke and Birchard, and by Skraup 
and his pupils. 

In the case of the gelatoses and gelatin peptone, Levene [1902-3, 
1904] found that the gelatoses contained 17-20 percent, of glycine 
and gelatin peptone 17-4 per cent, as compared with 16-5 per cent, in 
gelatin itself. Skraup and Hummelberger [i 908] gave the composition 
of gelatoses precipitated by half, two-thirds and complete saturation 
with ammonium sulphate and of gelatin peptone as follows : 





Gelatoses by 
half and two- 
thirds Saturation. 


Gelatoses 
by Complete 
Saturation. 


Gelatin 
Peptone. 


Glutamic acid 


27 5-8 


15 II 


20*8 16 


Glycine . 


io'2 10*3 


67 10-5 


7-2 4-1 


Histidine 
Arginine 


} 5 ' 8 


} 


0-4 0-3 
6-3 4-0 


Lysine . 


1-8 


2-3 


3-8 4-8 



The gelatoses contain more glycine than gelatin which contains 
9'6 per cent, but the peptone less. Levene found that amino acids 



RESULTS OF ANALYSIS 



were separated off in the production of peptone which accounts for the 
different figures for gelatin peptone. 

The qualitative differences observed by the older workers, Kiihne, 
Chittenden, Neumeister, and the analytical differences in the case of 
Witte's peptone observed by Pick have not been found. The 
generally accepted view that heteroalbumose contains more hexone 
bases than protoalbumose is not confirmed. We may note that 
heteroalbumose contains more glutamic acid than protoalbumose, and 
that it contains I per cent, more histidine. 

Skraup and his pupils have made numerous analyses of various 
intermediate products. Skraup and Krause [1910, I, 2] analysed the 
derivatives of caseinogen. Proteose I. contained more glutamic acid 
and more tyrosine than caseinogen ; proteose II. contained less glutamic 
acid but more tyrosine, and the peptone contained more glutamic acid 
and no tyrosine. 

Skraup with Zwerger [1905] and with Witt [1906] examined the 
substances termed kyrins by Siegfried and came to the conclusion that 
they are mixtures. They found that gelatin-kyrin contained 50 per 
cent, of di-amino acids and casein-kyrin 80 per cent., the former being 
composed of I molecule of arginine, I molecule of histidine, I mole- 
cule of glutamic acid and 2 molecules of glycine, the latter of I mole- 
cule of arginine, 2 molecules of lysine and I molecule of glutamic 
acid. 

Skraup with Lampel [1909], with Hummelberger [1909], and with 
Wober [1909] gave the following data for the protein and for those pro- 
ducts formed by the action of alkali, which were first prepared by 
Paal : 





Histidine. 


Arginine. 


Lysine. 


Tyrosine. 


Proline. 


Phenyl- 
alanine. 


Glutamic 
Acid. 


Amino 
Acids. 


Serum globulin 


17 


3'7 


4'3 


3-i 


3-o 


3-6 


4'4 


18-5 


Protalbic acid . 




O 


3*9 


4*4 


3*2 


I'O 




20' 4 


Lysalbic acid . 


17 





4*4 




2*9 


27 


1*9 


21-2 


Peptone . 




o 


4'4 


I'2 


2-3 


i-8 





13-5 


Ovalbumin 


i'5 


2-9 


3*9 


2'4 


i-5 


5-8 


3'2 


7-9 


Protalbic acid . 


2'3 


0-4 


3'3 


3*4 


2'0 


I2'O 


1-8 


147 


Lysalbic acid . 


0-3 


0*2 


5'3 


2-6 


I'O 


5'2 


I'O 


7-0 


Peptone . 


0-6 


o-3 


4-0 


i-i 


0-3 




1-6 


3-2 


Edestin . 


2'2 


14-2 


17 


2*1 


17 


2'4 


6-3 


in 


Substance A . 


2'2 




0-8 


1*4 


17 


O'2 




9'4 


Substance B . 


3-6 


13*0 


0-9 




07 


0-4 


10-9 


8'5 



84 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



Even the elementary analyses of the substances differed according 
to Gupta [1909] : 





C. 


H. 


N. 


S. 


Ovalbumin .... 


52*7 


7'2 


15*3 


i'3 


Protalbic acid 


55'4 


7'2 


*4'3 


2-4 


Lysalbic acid 


52-9 


7-0 


14-9 


I'2 


Peptone .... 


46-2 


6-6 


10-3 


irg 



The analysis of plastein also does not answer the much discussed 
question whether it represents a synthetical product or a further pro- 
duct of digestion. The figures so nearly resemble those for Witte's 
peptone that one would be inclined to the view that plastein is still a 
mixture, which is precipitated under the conditions of the experiment. 

In general, the analyses of the proteoses show that they contain all 
the amino acids originally present in the protein ; if a splitting of the 
large molecule had occurred in such a way that four or five amino 
acids only were present in each product, the synthetical problem would 
be easier ; we have still no substantial clue as to the order in which 
the units are combined in the molecule (see Part III.). 

Dennstedt and Hassler [1906] believe that in the formation of 
proteoses the process of oxidation occurs at the same time as 
hydrolysis. 



DISTRIBUTION OF NITROGEN 85 



ANALYSIS OF PROTEINS BY THE DISTRIBUTION OF 
THE VARIOUS KINDS OF NITROGEN. 

Since a complete analysis of a protein is still an impossibility, 
owing to the unsatisfactory methods for isolating and estimating the 
several mono-amino acids, the proteins cannot yet be differentiated by 
means of their chemical composition. 

It has been proved by Osborne, Leavenworth and Brautlecht [1908] 
that the methods for estimating the ammonia content and the di-amino 
acid (the three hexone bases) content of a protein are almost perfect. 
We can therefore differentiate proteins by their content in these four 
products. 

A further differentiation of the units composing the protein mole- 
cule into those containing amino groups and those containing nitrogen 
in heterocyclic combination was made by Van Slyke [1910, 1911], 

This subdivision is possible, since nitrous acid reacts only with 
amino groups with liberation of nitrogen : 

CH 2 .NH 2 CH 2 OH 

+ HN0 2 =1 + N 2 + H 2 

COOH COOH 

the amount of nitrogen evolved being double that contained in the 
amino acid. 

We can ascertain the following particulars : 



I. Amide nitrogen (ammonia). 

(Di-amino nitrogen fa cystine \ contain only amino N. Sulphur content gives cystine. Lysine by 

I b lysine j difference 
xx. , and 

I /in non- Arginine evolves half its 

V Cystine nitrogen I c arginine arginine contains f of its N.I amino N as NHg by boiling 

\^ histidine histidine f of its N |, form. with alkali. Histidine 

by difference. 



III. 



Mono-amino nitrogen 



.Non-amino nitrogen 
b 



glycine, phenylalanine, aspartic acid, tryptophan 

alanine, tyrosine, glutamic acid. 

valine, 

leucine, 

isoleucine. 

proline 

oxyproline 



tryptophan () 

i.e., seven data out of the possible eighteen. 

An eighth value is obtainable by determining the mono-aminodi- 
carboxylic acid content as was shown by Andersen and Roed-Mliller 
[1915]. This determination depends upon the fact that in a mixture 
of mono-amino acids, the mono-aminomonocarboxylic acids have a 



86 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

neutral reaction whereas the monoaminodicarboxylic acids have an 
acid reaction ; by neutralising the solution the dicarboxylic acids react 
with i molecule of sodium hydroxide and on incineration leave an 
equivalent quantity of sodium carbonate, which can be titrated (see 
pp. 104-106). Only a few determinations have so far been made by 
Andersen and Roed-Miiller, but they agree with the figure calculated 
from the amounts of aspartic and glutamic acids isolated from the 
products of hydrolysis : 

Per cent, of Total Calculated from data by 

Nitrogen. 

Gliadin . . . 24-3 24*0 Osborne and Guest 

Zein .... 17-9 16-6 Osborne and Liddle 

Caseinogen . . . 17-7 10-4 Osborne and Guest 

In the case of caseinogen the value is distinctly higher, but it may 
represent the amount of dicarboxylic acid in this protein more closely 
than the calculated figure, more especially as in this case the complete 
isolation of glutamic acid is more difficult than in vegetable proteins. 

If we regard the data for tyrosine as almost accurate, we have still 
one more value of service for the chemical differentiation of the pro- 
teins. It is unfortunate that we cannot yet measure the tryptophan 
content of a protein, especially as this unit is so readily detected by 
means of its colour reactions. 



DISTRIBUTION OF NITROGEN 87 



A. DISTRIBUTION OF THE NITROGEN IN THREE 

GROUPS. 

The differentiation of proteins by the estimation of the various 
groups of the units was first attempted in Hofmeister's laboratory by 
Hausmann [1899, 1900], who estimated amide nitrogen, di-amino 
nitrogen and mono-amino nitrogen. The protein (i gram) was 
hydrolysed with 20 c.c. of concentrated hydrochloric acid by boiling 
for five hours under a reflux condenser ; the solution was diluted and 
distilled with excess of magnesia, and the ammonia, which was 
liberated, was collected in excess of standard acid ; the solution was 
then acidified with acid and precipitated with phosphotungstic acid ; 
after twenty-four hours the precipitate was filtered off, washed with 
phosphotungstic acid, dissolved in alkali and the nitrogen estimated 
in an aliquot portion by Kjeldahl's method ; the filtrate was made 
up to a definite volume and nitrogen estimated in an aliquot portion. 

Numerous objections to the accuracy of the data were raised. 
Henderson [1899] maintained that the amount of amide nitrogen varied 
according to the strength of the acid employed in the hydrolysis and 
the time of hydrolysis; Kutscher [1900], and also Chittenden and 
Eustis [1900], showed that the precipitation of the di-amino acids was 
not complete, and Schulze and Winterstein [1901, 2] found that certain 
mono-amino acids, e.g. t phenylalanine, were precipitated by phospho- 
tungstic acid under certain conditions. Hart [1901] preferred barium 
carbonate to magnesia for distilling off the ammonia. 

Osborne and Harris [1903], Gumbel [1904], and also Rothera 
[1904], critically examined the various objections. The amount of 
amide nitrogen was not found to vary as Henderson stated ; if similar 
conditions are always maintained in the precipitation with phospho- 
tungstic acid most valuable comparative results can be obtained ; the 
errors of incomplete precipitation of the di-amino acids and precipita- 
tion of mono-amino acids almost compensate each other. 

Adopting Climbers suggestion of distilling off the ammonia in 
vacuo at 40 and Osborne and Harris' procedure, the process may be 
carried out as follows : 

I. About i gram of protein is boiled with about 100 c.c. of 20 
per cent, hydrochloric acid in a 500 c.c. round bottom flask under a 
reflux condenser until the solution no longer gives the biuret reaction, 
usually from seven to ten hours. It is then evaporated in vacuo at 



88 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

40 to a volume of 2-3 c.c. ; the greater portion of the hydrochloric 
acid is thus removed. 

2. About 300 c.c. of water is then placed in the flask and a cream 
of magnesia, which has been freed from every trace of ammonia by 
long boiling, is added until in slight, but distinct, excess. The mix- 
ture is distilled in vacuo at 40 and the distillate collected in excess of 
standard acid ; about half the liquid should be distilled. Titration of 
the standard acid gives the amount of amide nitrogen. 

3. The remainder of the solution is filtered through a nitrogen-free 
paper and the residue, thus collected, washed thoroughly with water. 
The nitrogen in this precipitate is estimated by Kjeldahl's method 
and is the " humin " nitrogen. 

4. The filtered solution is concentrated to 100 c.c. and cooled to 
20 ; 5 grams of sulphuric acid and then 30 c.c. of a solution con- 
taining 20 grams of phosphotungstic acid and 5 grams of sulphuric 
acid per 100 c.c. are added. 

5. The precipitate is filtered off after twenty- four hours and washed 
with a solution containing 2 '5 grams of phosphotungstic acid and 5 
grams of sulphuric acid per 100 c.c. The washing is effected by rins- 
ing the precipitate from the filter into a beaker and returning to the 
paper three successive times, each portion of the wash solution being 
allowed to run out completely before the next is applied. About 200 
c.c. of washings are generally obtained. 

6. The precipitate is transferred to a 600 c.c. Jena glass flask and 
the nitrogen estimated in it by Kjeldahl's method, digesting it with 
35 c.c. of concentrated sulphuric acid for seven or eight hours. Potas- 
sium permanganate crystals may be added three or four times. If the 
phosphotungstic acid precipitate be small, less sulphuric acid may be 
used, but sufficient must be taken to prevent bumping. 

7. The remaining nitrogen, belonging to the mono-amino acids, 
is found by subtracting the sum of the nitrogen found in the preced- 
ing operations from the total nitrogen contained in the protein. 

The data given in the table on page 131 show that there are con- 
siderable differences in the amounts of the various kinds of nitrogen 
in proteins. 



DISTRIBUTION OF NITROGEN 89 



B. DISTRIBUTION OF THE NITROGEN IN SEVEN 

GROUPS. 

I. Estimation of Amino Nitrogen. 

The action of nitrous acid upon amino acids and amides as a method for estimating 
these compounds was introduced by Sachsse and Kormann [1875]. These investigators 
employed an apparatus consisting of a small cylinder, furnished with a rubber stopper, 
through which two tap funnels and an exit tube for the evolved gas passed. Potassium 
nitrite was placed in the cylinder, dilute sulphuric acid in one of the tap funnels, and the 
solution of the substance (o'6-i'o gram) in the other. The exit tube was placed under an 
eudiometer filled with ferrous sulphate solution to absorb the nitric oxide. Air was expelled 
from the apparatus by the decomposition of some of the nitrite with the acid. As soon as 
the expulsion was complete, the amide solution and more acid solution were allowed to 
enter into the cylinder and the gas collected. The ferrous sulphate solution freed the 
mixture from nitric oxide and more was added, if necessary. Carbon dioxide was then re- 
moved with potash and the remaining gas was measured. 

This method was exhaustively tested in the Guinness Research laboratory by Horace 
Brown and J. H. Millar [1903]. They found that there were several serious sources of error 
in the original method before they could apply it to their own subject of investigation. 
These were due (i) to residual air contained in the apparatus, or in the liquids ; (2) to the 
difficulty of absorbing the excess of nitric oxide with ferrous sulphate. Carbon dioxide 
was used to remove the air and the evolved nitrogen, and difficulty was experienced in ob- 
taining a steady and constant stream of this gas. Pure carbon dioxide was prepared by 
the action of 30 per cent, sulphuric acid on normal sodium carbonate solution. A constant 
evolution of gas was not obtained, when the two solutions were allowed to drop separately 
into a flask, owing to the supersaturation of the liquid with the gas. A steady stream of 
carbon dioxide was obtained by allowing the liquids to mix in a piece of wide glass tubing, 
which was drawn out at its lower extremity so that only a small aperture remained and 
closed at its upper end by a small cork through which tubes connected with reservoirs of 
acid and carbonate passed ; two small apertures in its sides allowed for the escape of the 
gas. This wide tube was placed in the vessel used for the generation of the carbon dioxide. 
The mixture of acid and carbonate solutions accumulated to a height of about 3 cm. in the 
tube, and then dropped into the flask, which could be emptied by an attached syphon tube. 
The carbon dioxide entered the apparatus through a special trap. Air contained in the 
apparatus was removed by a stream of carbon dioxide and steam, which was made to enter 
through this trap. All the solutions used were made with air-free water, charged with 
carbon dioxide ; the same water was used for washing purposes. 

The carbon dioxide was removed by the potash solution contained in a Lunge nitro- 
meter, with which the exit tube of the apparatus was connected. The excess of nitric oxide 
was removed by means of oxygen, prepared by the electrolytic decomposition of water, and 
passed into the nitrometer, and the excess of oxygen by passing the gas in the nitrometer 
into a double Hempel pipette containing pyrogallol dissolved in 60 per cent, potash solution. 

Allowing one to two hours for the reaction of the nitrous acid upon the amino acids, 
satisfactory results were obtained with glycine, alanine, phenylalanine, leucine, aspartic 
acid, glutamic acid and asparagine. The results with tyrosine were not satisfactory, but 
they found that it reacted quantitatively after bromination. 

The estimation of the amino nitrogen was originally performed by 
Van Slyke [1910, I, 2 ; 191 1, I, 2, 4 ] in an apparatus very similar to 
that used by Sachsse and Kormann and by Horace Brown and Millar. 



90 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

The whole apparatus was filled with nitric oxide, and this gas was used 
for washing the evolved nitrogen into the eudiometer ; the excess 
of nitric oxide was then removed with permanganate contained in a 
Hempel pipette. 




FIG. 2. 
From J. Biol. Chem., 1912, 12, 278. 

A modified form of apparatus was devised by Klein [1911] and 
this form of apparatus, greatly improved, was adopted by Van Slyke 
[1912, i]. It has entirely displaced the older form of apparatus, and 
is shown in figs. 2 and 3, the latter figure showing all the parts of the 
apparatus assembled for use upon a fixed stand. The parts are more 
frequently assembled on a stand which is movable. 

The apparatus consists of the deaminising bulb D of 40-45 c.c. 
capacity for the interaction of the nitrous acid and the solution of the 
amino substance. It has a mark at 20 c.c. Connected with this 
vessel, are 



DISTRIBUTION OF NITROGEN 91 

A, a vessel of 35 c.c. capacity with a mark at 7 c.c. and tap a for 
introducing solution into D, or receiving solution from D. 




FIG. 3. 
From Plimmer's Practical Organic and Biochemistry. 

B, a 10 c.c. burette for containing the solution of the ammo 
substance. 

A tap d, for emptying the deaminising vessel D. 



92 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

There is above D a small bulb which .keeps the reacting solution 
from splashing into the capillary. 

The deaminising bulb D is connected through a three-way tap c 
with the gas burette F, which is of 150 c.c. capacity ; its upper and 
narrower portion holding 40 c.c. is graduated in tenths ; its lower and 
wider portion in 10 c.c. divisions. This large burette will hold all 
the gas which is liberated whilst the apparatus is being used. The 
gas, which is to be finally measured, should only fill a portion of the 
narrow accurately graduated part. It is filled with water. The gas 
burette is connected with the Hempel pipette of special shape, 
which contains an alkaline solution of potassium permanganate. 
Though the nitric oxide reduces the permanganate, the manganese 
dioxide formed is in such a fine state of division that it does not in- 
terfere with the manipulation. Several determinations can be made 
with the same solution. Deposition of manganese dioxide in the 
capillary tube is prevented by allowing water from the gas burette, in- 
stead of permanganate, to remain in this tube. Any carbon dioxide 
evolved is absorbed by the alkali. 

The glass connecting tubes are strong walled and of 3 mm. 
internal diameter ; the bore of tap a is also of 3 mm. and tap d should 
have the same wide bore. The connection between D and B should 
be at least 8 mm. inner diameter, so as to allow free circulation. 

The nitric oxide is obtained from an excess of sodium nitrite solu- 
tion which is decomposed by glacial acetic acid ; less nitric oxide is 
evolved by its use than by the use of mineral acid. The amino sub- 
stance may be dissolved in semi-normal acid, 50 pejp cent, acetic acid, 
or normal alkali. 

Only a small correction for the reagents is necessary ; commercial 
nitrite gave 0*2 c.c. of nitrogen for five minutes' reaction, 0*3 c.c. for 
thirty minutes, and 0-5 c.c. for two hours. 

The estimation is performed in the following way : 

(i) Displacement of the Air in the Apparatus. The gas burette 
F is filled with water as far as the tap r, the' air being allowed to 
escape through c. One bulb of the Hempel pipette is filled with 
alkaline permanganate solution 1 through a small funnel, the tap / 
being arranged so that the air is displaced into the burette. The 
permanganate should just reach the tap, which is then closed in 
this direction and opened towards the tap c. The air in the burette is 
driven out through c so that water again fills the burette as far as 
1 50 gram KMnO 4 + 25 gram KOH per 1000 c.c. 



DISTRIBUTION OF NITROGEN 93 

the tap <:, which is turned so as to shut off this part of the apparatus 
and to be in connection with D and the exit tube. 

7 c.c. of glacial acetic acid are put into A and run into D ; 30 c.c. of 
the sodium nitrite solution 1 are placed in A and also run into D. 
Sufficient should be used so that excess stands in A above the tap. 

The remaining air in the apparatus and that dissolved in the nitrous 
acid solution is removed by closing c by a quarter turn, leaving a open 
and shaking D. Rapid evolution of nitric oxide occurs which gathers 
in D and forces 10-15 c.c. of solution back into A. The cock c is now 
again opened and the nitric oxide, together with the air swept out of 
the solution, is forced out of D by liquid from A through c. This 
process is repeated twice to ensure removal of all the air. 

By closing c and shaking D a gas space of about 20 c.c. is now 
made to make room for the amino solution from B. 

Tap a is closed and tap c is opened to connect D and F . These 
manipulations require about two minutes. 

(2) Decomposition of the Amino Substance. 10 c.c. or less of the 
solution to be analysed is put into the graduated tube B ; any 
excess can be run off. The desired volume is run into D. The 
burette B need not be graduated and the desired volume can be in- 
troduced with a pipette into B, run into D, and B washed with a 
little water, which is also run into D. The evolution of nitrogen 
commences immediately, and D is thoroughly shaken for three to five 
minutes to complete the evolution of the gas. Only in a few cases is 
longer shaking necessary. 

The evolved gas passes into the burette F ; the residual gas in the 
bulb is driven into F by opening the tap a and letting liquid run from 
A into D as far as the tap c. 

(3) Absorption of Nitric Oxide and Measurement of the Nitrogen. 
The tap of the burette F is turned to connect it with the Hempel 
pipette and the gas is driven from F into the Hempel pipette by raising 
the levelling bulb. By shaking the gases with the permanganate the 
nitric oxide is absorbed. The residual nitrogen is run back into F, 
the permanganate filling the tubing as far as the tap/ The volume 
of gas in F is then measured by bringing the surface of the liquid in 
the bulb even with the meniscus. Generally, one shaking with the 
fresh permanganate suffices to remove all the nitric oxide, but it is ad- 
visable to test if the absorption is complete by returning the gas to the 
Hempel pipette and again measuring. The weight of nitrogen corre- 

1 3 grams per 100 c.c. 



94 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

spending to the volume of gas is calculated in the usual way ; the 
results are divided by 2 (see equation, p. 85). The weight may be 
taken from the table compiled by Van Slyke and reproduced on p. 1 36. 

Each milligram of amino substance gives off from 1*7-1 '9 c.c. of 
nitrogen. 

A correction should be made for the air 0*2 c.c. dissolved in 10 
c.c. amino solution, i.e., allowing for the oxygen which combines with 
the nitric oxide forming peroxide and is absorbed by the permangan- 
ate, O'i6 c.c. must be deducted from the volume of gas. This correc- 
tion is equivalent to 0-09 mgm. of amino nitrogen. No correction 
is necessary, if air-free water be used in preparing the amino solution. 

If proteins or other solutions which may froth are to be analysed, 
a few drops of capryl alcohol are first introduced into D through B, and 
capryl alcohol can be added subsequently from B. 1 

Except lysine, the natural amino acids all react quantitatively in 
five minutes : half an hour is required for this substance. Ammonia 
and methylamine require one and a half to two hours, purines and 
pyrimidines two to five hours, and urea eight hours. With these sub- 
stances the apparatus is allowed to stand for the required time and 
then shaken for two minutes. 

The completeness of the reaction may be tested by repeating the 
process and ascertaining if more gas is evolved. 

Glycine and cystine evolve a larger volume of gas than the theoret- 
ical ; the factor '926 may be used in estimating cystine ; 3 per cent, 
of the total volume of gas must be deducted for glycine. 

Micro Apparatus i. 

Van Slyke [1913, i] described a smaller form of apparatus for use 
in the analysis of amino acids in blood, tissues, etc., in which only 
minute quantities are present. It may be advantageously used in 
these estimations instead of the larger form of apparatus. Its dimen- 
sions are : 

(i) The gas burette : 10 c.c., the upper part of 2 mm. diameter 
measuring 2 c.c. and graduated in ^ c.c., the lower part wider and 
graduated in -^ c.c. 

A gas burette holding a total volume of 20 or 30 c.c. is better as 
frequently more than 10 c.c. of nitric oxide are evolved. 

This burette or its upper part should be enclosed in a water jacket, 

1 In later forms of apparatus another tube is sealed to D for introducing the capryl 
alcohol. 



DISTRIBUTION OF NITROGEN 



95 



since temperature variations of the atmosphere affect the small 
volumes of gas which have to be measured. 

(2) The deaminising bulb : 1 1-12 c.c. 

(3) The burette : 2 c.c. 

The quantities of reagents required are 10 c.c. of sodium nitrite 
solution and 2'5 c.c. of glacial acetic acid for which the correction for 
impurities amounts to 'O6--I2 c.c. It is not necessary to have a 
smaller Hempel pipette ; with the micro apparatus the permanganate 
lasts for a considerable time. 

The solution to be analysed may be introduced into the burette 
with a pipette and the burette is washed with six or seven drops of 
water. 

The error in the estimation need not exceed -005 mgm. when 
2 c.c. or less gas is measured ; with more gas -01 mgm. One- 
fifth of the amount of solution required for the macro apparatus is used 
in the micro apparatus. Not only is there an advantage economically 
with reagents, but also the apparatus is less fragile. 

O'5 mgm. of amino acid can be analysed with an accuracy of 
i per cent. The analysis is slightly more 
rapid : at 1 5-20 four minutes' shaking suffice, 
at 20-25 three minutes, above 25 two to 
two and a half minutes. 

It is essential that the burettes be ac- 
curate and the stopcocks be tight. The 
burettes are tested by weighing the water 
they deliver; the stopcocks by submitting 
them to a pressure of a column of water one 
metre high. 

The apparatus is cleaned with a mixture 
of potassium bichromate and sulphuric acid. 

Micro Apparatus ii. 
The size of apparatus can be reduced 
to one-half of the above, if the form of the 
gas burette be modified [Van Slyke, 1915, 
2]. This is shown in fig. 4. The zero- 
point, instead of being placed at the bottom 
of the stopcock, is located on a capillary 
which extends for a few millimetres below the tap. It permits 
marking off the upper boundary of the gas volume with an error of 




FIG. 4. 

After. J. Biol. Chem., 1915, 23, 
408. 



96 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

less than -oq|| c.c. The burette is of 3 c.c. capacity, and is graduated 
into O'l c.c. divisions about I mm. apart, so that by estimating tenths 
of a division gas volumes can be read to -ooi c.c. The burette must 
be carefully calibrated. I c.c. of amino solution is required for 
analysis. 

The removal of air from the apparatus is more easily effected as 
follows : 

The apparatus is shaken once until sufficient nitric oxide is formed 
to force the liquid in the chamber down to the mark at which the re- 
agent stands for introducing the amino solution. The tap a is closed 
and tap c is turned so that the gases can escape at c. The vessel is 
then shaken rapidly for two minutes with the motor. The air is more 
completely removed by this procedure than by the other procedure. 
The deaminising bulb is then connected through c with the gas 
burette. 

Soft heavy-walled rubber tubing must be used for the connections. 
"Stethoscope" tubing with a wall 3 or 4 mm. thick is very suitable. 



DISTRIBUTION OF NITROGEN 97 



II. Estimation of the Different Groups of Amino Acids. 

The estimation of the several groups of amino acids present in a 
protein is effected by the following series of operations : 

1. Hydrolysis. 3 grams of protein, or better 6 grams for duplicate 
analyses, are dissolved in 10 or 20 parts of 20 per cent, hydrochloric 
acid and boiled in a tared flask under a reflux condenser. 

(After six or eight hours the hydrolysis is stopped. Portions of 
i c.c. or 2 c.c. (enough to contain cri gram of protein) are withdrawn 
with a pipette and diluted to 10 c.c. In these portions the amount of 
amino nitrogen is determined, the reaction being allowed to proceed 
for five minutes standing and then for one minute with shaking. 
Under these conditions the same proportion of ammonia (15-20 per 
cent.) is decomposed in each determination. 

The hydrolysis flask is weighed and the hydrolysis continued for 
another period of six or eight hours, when amino nitrogen is again 
determined. 

Hydrolysis is continued until the amino nitrogen is constant. 

The object of weighing is to ascertain if the solution has become 
concentrated by loss of vapour and to allow of a correction for a de- 
crease of the volume. 

2. Estimation of Total Nitrogen. The products of hydrolysis are 
transferred to a measuring flask of 100 c.c. or 250 c.c. capacity. 
Total nitrogen is estimated by Kjeldahl's method in an aliquot portion 
containing 0-2 gram of protein. All the subsequent estimations are 
based upon this value. 

3. Amide Nitrogen. Since cystine is very easily decomposed by 
boiling with magnesia at 100 the determination of ammonia must be 
carried out in vacuo at 40, or at room temperature by the aeration 
method of Denis. 1 The method of distilling in vacuo is to be preferred 
as the same apparatus is repeatedly employed in the other estimations. 

The distillation in vacuo is performed in the apparatus shown in 
% 5 (P- 98)> or m some arrangement by which the ammonia vapour 
passes into the standard acid. 

The Claisen flask and receiver are of I litre capacity, the guard 
flask of 200 c.c. 

The hydrolysed solution is placed in the double-necked flask and 

*y. Biol. Chcm.,S, 427. 
FT. I. 7 



98 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



diluted to 200 c.c. ; 100 c.c. of alcohol are added to prevent frothing 
and then an excess of a 10 per cent, suspension of calcium hydrate, 
as shown by the turbidity and alkaline reaction of the solution. Air 
may be introduced through the stop-cock, if distillation starts too 
rapidly ; the stop-cock serves to release the vacuum when the distilla- 
tion is finished. The decinormal acid in both flasks is mixed and 
titrated with decinormal soda using alizarin sulphonate as indicator, 
which may be added previous to the distillation. 

4. Humin Nitrogen. The black colouring matter is absorbed by 
the lime, which is filtered off on a folded filter paper and washed with 
water until the washings are free from chlorides. The precipitate 




Jo-6oc.c."//o 



FIG. 5. 
From Biol. Chem., 1911, 10, 21. 

and paper are then submitted to Kjeldahl analysis, using 35 c.c. of 
sulphuric acid. 

^ 5. Precipitation and Washing of the Phosphotungstate Precipitate. 
The filtrate from the humin nitrogen is neutralised with hydrochloric 
acid, returned to the vacuum distillation apparatus, and concentrated 
^ about 100 c.c. 

It is then washed into a 250 c.c. conical flask, and 18 c.c. of con- 
centrated hydrochloric acid and 1 5 grams of phosphotungstic acid l in 
aqueous solution are added. 

The entire solution is diluted to 200 c.c. with water and heated in 
a water-bath, until the precipitate has nearly, or quite, redissolved. 
On cooling, the phosphotungstates separate in a crystalline form. 

1 This should be purified by Winterstein's method by shaking its acid solution with 
ether ; from the ethereal layer containing the substance the acid is recovered by recrystal- 
lisation, 



DISTRIBUTION OF NITROGEN 99 

After standing for at least forty-eight hours they are filtered off 
and washed in the following manner : 

A 3-inch Buchner funnel is covered with a hardened filter paper of 
such a size that it fits against the bottom and side walls ; the portion 
of the paper against the side walls is folded into about twenty plaits 
so that it fits snugly all round. 

The precipitate is poured into this pocket and the mother liquor 
removed by suction and by pressing down the precipitate with a 
flattened rod. 

The filtrate is returned to a beaker. 

Washing is effected with 10-12 c.c. of a solution containing 2*5 
grams of phosphotungstic acid and 3*5 grams of hydrochloric acid per 
100 c.c. ; this is first used to dislodge the particles remaining in the 
flask ; it is then poured upon the precipitate which is stirred up until 
all lumps are broken and until there is only a granular suspension. 
It is then sucked dry as before. The washing is repeated three to 
four times in this manner. Then the precipitate on the filter is 
washed five to ten times with the same solution from a wash bottle, 
commencing round the edges and sucking dry each time. 

It frequently happens that the later washings run through some- 
what turbid ; these are filtered through a folded paper. All washings 
are combined with the main filtrate. The washing must be continued 
till the liquid is free from calcium ; I c.c. of the filtrate when tested 
with oxalic acid in 3 per cent, sodium hydrate must give no turbidity 
even after standing for several minutes. 

The filtration and washing may be effected equally well on a 
2-inch Buchner funnel with a hardened filter paper pressed over the 
perforations in the usual manner, as was shown by Hartley [1914] and 
by Van Slyke [1915, i]. A steady and moderately strong suction is 
continued without interruption during the filtration and washing. 
The 10-15 c.c. of washing solution 1 are poured upon the precipitate, 
which is stirred up with each portion. The washing is completed as 
soon as a few drops of the liquid on being allowed to flow upon a 
10 per cent, solution of sodium hydrate containing sodium oxalate 
and on gently shaking to bring the liquids in contact show no cloudi- 
ness even after standing for several minutes. 

If there be any doubt as to the purity of the phosphotungstates 
they may be recrystallised by suspending in water and dissolving by 
adding sodium hydrate. The solution is acidified, heated, and the 

1 This should be cooled to o. 

7* 



ioo THE CHEMICAL CONSTITUTION OF THE PROTEINS 

bases thrown down by adding 15 c.c. of concentrated hydrochloric 
acid and 5 grams of phosphotungstic acid, the final volume being 
made up to 200 c.c. as in the original precipitation. Filtration and 
washing is carried out as above. 

6. Treatment of the Phosphotungstate Precipitate. The precipitate 
is removed with a spatula, as completely as possible, to a beaker of 
over I litre capacity. The filter paper is then spread out on the 
bottom of a basin and washed with water made just alkaline with a 
few drops of 20 per cent, sodium hydrate. The small folded paper 
is similarly treated. The particles of precipitate are dissolved by 
the soda, and any granules remaining in the original flask are dis- 
solved in the same way. The alkaline solutions and washings are 
poured into the beaker containing the main bulk of the precipitate. 
The whole is then carefully dissolved in soda by adding 50 per cent, 
alkali, drop by drop, with continual stirring. Phenolphthalein is 
added as indicator ; as soon as the solution becomes red, addition of 
the alkali must be stopped ; if the colour disappears, more alkali, but 
only to the excess of three or four drops, must be added. A red 
solution must finally result. 

The solution is diluted to 800 c.c. and the phosphotungstic acid is 
removed by slowly adding, in portions of a few c.c., a 20 per cent, 
solution of barium chloride, until a test portion gives an immediate 
granular precipitate with neutral sodium sulphate solution. If the red 
colour disappears in the process two or three more drops of alkali are 
added. A large excess of barium chloride should be avoided. 

The barium phosphotungstate is filtered off using the same funnel, 
paper and precautions as before, except that larger portions of wash 
water may be used. The final washing must give no reaction for 
chlorides. 

The filtrate and washings are concentrated in vacua, until they are 
reduced in volume to 50 c.c. The barium phosphotungstate, which 
separates out, is filtered off, and the filtrate and washings received in 
a 200 c.c. double-necked distilling flask ; the volume is then again 
reduced and made up to 50 c.c. in a measuring flask. 

Van Slyke [1915, i] found that the following procedure was 
preferable to the original as described above : 

The precipitate is removed from the filter by a spatula and by 
washing with water and transferred to a 500 c.c. separating funnel, 
using 200-300 c.c. of water. 5 or 10 c.c. of concentrated hydrochloric 
acid are added, and the mixture shaken with about 100 c.c. of a 



DISTRIBUTION OF NITROGEN 



101 




mixture of equal parts of amyl alcohol and ether. Complete solution 
of the precipitate occurs after shaking for one or two minutes. Should 
an oil appear at the bottom more of the amyl alcohol-ether mixture is 
added until the whole of it floats upon the surface. 

If the layers do not separate readily with a clear boundary between 
them, due to humin not having been completely absorbed by the 
calcium hydrate previously arid to its precipitation 
by phosphotungstic acid, the whole solution 
without separation of the layers is passed 
through a Buchner funnel with suction. The 
clear filtrate readily separates into two layers. 
The aqueous portion is extracted with three 
successive portions of amyl alcohol-ether using 
a volume of mixture equal to one quarter of the 
aqueous solution. The combined amyl alcohol- 
ether extracts are shaken once with water, and 
this portion of water, after being shaken once 
or twice with fresh amyl alcohol-ether, is added 
to the aqueous solution. This contains the bases 
and should be free from phosphotungstic acid 
as shown by testing a few drops with baryta 
solution ; a precipitate should not be formed. 
The solution is evaporated to dryness in vacuo 
to drive off the excess of hydrochloric acid and 
the residue dissolved and diluted to 50 c.c. 

This procedure is more rapid than the older 
and has the advantage that no barium is present, 
which may cause bumping in estimating the 
arginine. 

7. Estimation of Arginine. Arginine is 
stated by Van Slyke [1911, 4] to be quantita- 
tively decomposed by boiling with 50 per cent, 
alkali with the loss of half of its nitrogen. Its estimation is performed 
before that of the total nitrogen contained in the solution. 

25 c.c. of the solution are placed in the 200 c.c. flask of the 
apparatus shown in fig. 6. 

The Folin bulbs are connected to the flask either by a ground 
glass joint, or by a heavy piece of rubber tubing. These bulbs con- 
tain 15 c.c. of decinormal acid, coloured with alizarin sulphonate. 
1 2 '5 grams of solid potash and a piece of porous porcelain are added 




FIG. 6. 

From Journ. Biol. 
Chem., 1911, 10, 26. 



102 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

to the solution in the flask, and the solution is boiled gently for exactly 
six hours. Nearly all the evolved ammonia diffuses into the bulbs. 

The bulbs are disconnected and 100 c.c. of water are poured 
through the condenser into the flask. The flask is connected with the 
condenser of a Kjeldahl distilling apparatus, and the remainder of the 
ammonia driven off and collected in the acid from the bulbs, which has 
been transferred to a suitable receiver. Not more than 100 c.c. of the 
solution must be distilled over as the strong potash may destroy the 
other substances in the solution. 

The excess of acid in the receiver is titrated. Each c.c. of acid 
neutralised by the ammonia corresponds to 0*0028 gram arginine- 
nitrogen, or 0*0056 gram in the total solution. 

Plimmer [1916] showed that arginine was decomposed by boiling 
with 20 per cent, caustic soda for six hours. 1 The estimation is carried 
out preferably by adding an equal volume of 40 per cent, caustic soda 
to the arginine solution and boiling for six hours. A larger volume 
of liquid is thus present in the flask and the lower concentration of the 
alkali minimises breakage of the glass flasks through the action of the 
alkali. Instead of disconnecting the bulbs and distilling as recom- 
mended by Van Slyke so as to collect the remainder of the ammonia, 
it is only necessary to run the water out of the condenser and continue 
boiling for another fifteen to twenty minutes so as to drive the am- 
monia into the bulbs. 20 c.c. instead of 25 c.c. of the solution may 
be used. 

Cystine is usually not present in sufficient amount in proteins to 
cause any considerable error in this determinatiofi, but if a keratin be 
under investigation a correction must be made ; 1 8 per cent, of its 
nitrogen is evolved when it is boiled with alkali ; the nitrogen figure 
of cystine is obtained later in the process. 

8. Total Nitrogen of the Hexone Bases and Cystine. The solution 
remaining from the arginine determination is transferred to a Kjeldahl 
flask of 500 c.c. ; 35 c.c. of sulphuric acid are added and 0*25 gram 
of copper sulphate. The nitrogen is then estimated in the usual way. 
This estimation can be performed in duplicate, if the solution be 
diluted to 100 or 200 c.c. and divided into two halves, each half being 
oxidised with 20 c.c. of sulphuric acid. 

On account of persistent bumping from silica dissolved from the 
glass in the arginine estimation and consequent loss, Plimmer [1916] 

1 Histidine is decomposed to a slight extent by this treatment, but the error introduced 
is negligible. 



DISTRIBUTION OF NITROGEN 103 

prefers to estimate the total nitrogen in another portion, 5 c.c., of 
the solution. 

The sum of the number of c.c. of decinormal acid neutralised + 
the number of c.c. of decinormal acid previously neutralised in the 
arginine determination, multiplied by 0-0028, gives the total nitrogen 
content. 

9. Estimation of Cystine. Cystine is estimated by determining 
the total sulphur content by Denis' modification [1910, i] of Benedict's 
method [1909-10]. 10 c.c. of the solution of hexone bases + cystine 
from operation (6) are placed in a porcelain dish of 7-10 cm. diameter 
with 5 c.c. of a solution containing 25 grams copper nitrate, 25 grams 
sodium chloride, and 10 grams ammonium nitrate per 100 c.c. The 
mixture is evaporated to dryness on the water-bath and then gradually 
heated to redness, at which temperature it is maintained for ten 
minutes. The residue is dissolved in 10 c.c. of 10 per cent, hydro- 
chloric acid and diluted to 1 50 c.c. 10 c.c. of a 5 per cent, solution 
of barium chloride are slowly added to the boiling solution and the 
barium sulphate is filtered off and washed in the usual way. A 
correction must be made for the amount of sulphate in the reagents 
which should not exceed i -5 mgm. barium sulphate. 

i mgm. BaSO 4 corresponds to 0*06 mgm. cystine nitrogen in the 
10 c.c. analysed, i.e., to O'3 mgm. cystine nitrogen in the total solution 
of bases + cystine. 

10. Estimation of Amino Nitrogen. 10 c.c. of the solution are 
usually used for this estimation (p. 89) ; I c.c. or 2 c.c. if the micro 
apparatus be used. The reaction may be allowed to proceed for half 
an hour at 20 or longer at a lower temperature without shaking, 
or for five minutes with vigorous shaking ; similarly for the blank 
determination for the reagents. 

A correction must be made for cystine which gives 107 per cent, 
gas ; as before it applies only to those proteins keratins which con- 
tain a large amount of cystine. 

Calculation of Histidine Value. 

Total nitrogen of bases minus total amino nitrogen gives the total 
non-amino nitrogen, D. This is contained in the arginine and histi- 
dine. 

Since three-fourths of the arginine nitrogen and two-thirds of the 
histidine nitrogen are in this form, the total non-amino nitrogen minus 



104 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

three-fourths of the arginine nitrogen (found previously) represents two- 
thirds of the histidine nitrogen. Hence 

Histidine N = f (D - f Arg.) 

= i -5 D - 1-125 Arg. 1 

Calculation of Lysine Value. 

Knowing the other values, that of lysine is found by difference : 
Lysine N = Total N - (Arg. N + Cyst. N + Hist. N). 

1 1. Determination of the Total Nitrogen of the Mono-amino Acids. 
To the combined filtrate and washings from the phosphotungstate 
precipitate 50 per cent, caustic soda is carefully added, until the solu- 
tion becomes turbid by precipitation of lime and is just alkaline. 
Excess must be carefully avoided as the precipitate may then not 
completely dissolve in acetic acid, which is added to clear the solution 
when just alkaline. The acid solution is concentrated in vacuo until 
salt commences to crystallise out. The solution is then washed into 
a 150 c.c. measuring flask and diluted up to the mark. 

Total nitrogen is estimated in 25 c.c. portions by Kjeldahl's 
method, using 35 c.c. of sulphuric acid, 15 grams potassium sulphate 
and 0*25 grams of copper sulphate. The acid must be added care- 
fully on account of the evolution of hydrochloric acid. The digestion 
must be continued for three hours after the solution has become clear. 

12. Determination of the Amino Nitrogen of the Mono-amino Acids. 
10 c.c. portions of the solution are used for this purpose and the time 
for the reaction with nitrous acid is six to ten minutes. Smaller por- 
tions are used with the micro apparatus. 

The volume of nitrogen evolved by a given amount of amino 
nitrogen is 2*5 times the volume of decinormal acid neutralised, if the 
same amount is determined by the Kjeldahl method. Therefore, the 
portions of 25 c.c. and 10 c.c. used in these last estimations give 
results of similar accuracy. 

13. Determination of the Nitrogen in the Mono-aminodicarboxylic 
Acids. In estimating the nitrogen of the mono-aminodicarboxylic 
acids, ammonia, di-amino acids, glucosamine and organic acids must 
be absent from the solution ; further, calcium salts must not be 
present. 

Andersen and Roed-Muller [1915] therefore adopt a procedure 
differing from that of Van Slyke : 

VIn the original paper the formula 1-667 D - 1*125 Arg. was given [Van Slyke, 
, 3]- 



DISTRIBUTION OF NITROGEN 105 

5 grams of protein, containing approximately 800 mgm. of 
nitrogen, are heated with 20-25 parts of 3N hydrochloric acid on 
a water-bath until there is complete or nearly complete solution, 
and then in an autoclave at 150 for 1-5 hours which effects com- 
plete hydrolysis. 

The solution is concentrated on the water-bath as much as pos- 
sible, the residue dissolved in water, the solution filtered and made 
up to 250 c.c. The nitrogen retained in the filter paper, usually a 
negligible quantity, is estimated. Total nitrogen of the solution is 
determined in 5 c.c. and amide nitrogen in 20 c.c. by distilling in 
vacua with '5N baryta dissolved in methyl alcohol (saturated baryta 
in methyl alcohol, if much ammonia be present). 

200 c.c. are treated with saturated sodium carbonate solution, until 
the reaction of the solution is alkaline to turmeric paper, and evapor- 
ated to dryness in vacuo. The residue is dissolved in 100 c.c. of 
water and its reaction tested : if not alkaline, more sodium carbonate 
is added and the evaporation repeated ; this must be continued until 
the reaction is alkaline. The residue is dissolved in 50 c.c. of water 
and neutralised with hydrochloric acid ; 27 c.c. of concentrated hydro- 
chloric acid and 25 grams of phosphotungstic acid are added; the 
solution is diluted to 300 c.c. and heated on a water-bath until the 
phosphotungstates have dissolved. After standing for forty-eight 
hours, the crystalline phosphotungstates are filtered off by suction on 
a hardened filter paper in an ordinary funnel. This method of filtra- 
tion is preferred to that adopted by Van Slyke. The precipitate is 
washed ten times with 15-20 c.c. of 2*5 per cent, phosphotungstic 
acid and 3 -5 per cent, hydrochloric acid by stirring up with the wash 
solution and each time draining off completely. The precipitate is 
dissolved and the bases estimated as directed by Van Slyke. 

The precipitate of barium phosphotungstate is dried in the air, 
and the nitrogen contained in it estimated. This is humin-nitrogen I., 
which is thrown down at this stage. 

The filtrate is diluted to 1000 c.c., made distinctly red to phenol- 
phthalein with alkali and barium chloride added to remove phospho- 
tungstic acid ; the solution is kept distinctly alkaline and not too 
small an excess of barium chloride is used. The barium phospho- 
tungstate is filtered off and washed till free from chlorides. The 
nitrogen in the precipitate is estimated and forms humin nitrogen II. 

The filtrate is neutralised, treated with an amount of hydrochloric 
acid equivalent to the total nitrogen, and evaporated in vacuo until salt 



io6 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

crystallises out. 200 c.c. of alcohol are added, and after standing for 
one hour the salt is filtered off and washed with 80 per cent, alcohol. 
The salt is dissolved in water and the nitrogen estimated ; usually 
very little nitrogen is contained in it, but if it be large in amount, the 
solution is acidified, evaporated, and treated with alcohol as described. 

The alcoholic solution is evaporated in vacuo to dryness, dissolved 
in water and made up to 200 c.c. 

Total nitrogen is determined in 5 c.c., and 25, 30, or 40 c.c., 
according to the amount of nitrogen, are neutralised with sodium 
hydroxide, diluted to 50 c.c., and used for the determination of amino 
nitrogen. 

The mono-aminodicarboxylic acid nitrogen is determined by exactly 
neutralising 20 or 25 c.c. in a platinum basin to azolitmin paper 1 and 
evaporated to dryness, the concentrated solution being stirred with a 
platinum wire if salts crystallise out, and ashed over a spirit burner. 
The ash is dissolved in not too great an excess of o*2N hydrochloric 
acid, washed into a conical Jena-glass flask, boiled over a flame for 
five to ten minutes, cooled and titrated with O'2N alkali with phenol- 
phthalein as indicator. The difference between the amounts of acid 
and alkali is equivalent to the carboxyl groups of the mono-amino- 
dicarboxylic acids and is expressed in terms of nitrogen by multiplying 
the figure in c.c. by 2 -8. 

14. Corrections for the Solubilities of the Phosphotungstates. 
Although the conditions of precipitation are not always the same, the 
variation is not sufficient to cause significant change in the solubilities 
of the phosphotungstates. When precipitated from a volume of 200 
c.c. the following corrections should be added, and the sum of the 
figures for amino nitrogen and non-amino nitrogen subtracted from 
the figures for the mono-amino acids : 

1 Henriques and Sorensen (Zeitschr.physiol. Chem., 64, 133) describe the preparation of 
sensitive litmus paper as follows : 

0-5 gram of powdered azolitmin is dissolved in 200 c.c. water + 22*5 c.c. 'iN NaOH 
in a basin. 50 c.c. of alcohol are added after filtering. Strips of good, ash-less filter paper 
are drawn through the solution and dried by hanging on strings. The preparation takes 
about one hour. The paper must behave in the following way with Sorensen's phos- 
phate solutions : 

To 3 sec + 7 prim (P H = 6, 47) slightly acid reaction. 

To 5 sec + 5 prim (Pn = 6, 81) neutral reaction. 

To 7 sec + 3 prim (Pn* = 7-17) slightly alkaline reaction. 

The neutral point is chosen slightly more on the acid side (= 7-07) on account of 
ammonium salts and amino acids. 

If a litmus paper does not react in this way, the amount of iN NaOH used in its 
preparation must be altered accordingly. 



DISTRIBUTION OF NITROGEN 



107 





Total N. 
Gram. 


Amino N. 
Gram. 


Non-Amino N. 
Gram. 


Arginine N. 
Histidine N. 
Lysine N 
Cystine N. . . 

Sum .... 


0-0032 
0*0038 
0-0005 
0-0026 


0-0008 

0-0013 

0-0005 
0-0026 


0-0024 
0'0025 
O'OOOO 
O'OOOO 


0-0052 


0-0049 



This long method was rigidly tested by Van Slyke upon pure 
amino acids singly, and when mixed together, and upon typical 
proteins. The accuracy of the method was borne out by the figures 
under the column of total nitrogen (p. 132); usually, the nitrogen re- 
covered was within I per cent. In the duplicate analyses of proteins 
the maximum and average differences were : 





Amide 
N. 


Humin 

N. 


Cystine 


Arginine 


Histidine 

N. 


Lvsine 

"N. 


Mono-amino 

N. 


Non-Amino 
N. 


Maximum 
Difference 
between 
Duplicates. 


0'37 


0-39 


O*II 


I-2 7 


2'I 4 
0-93 


1-23 


I -60 
0-60 


I'2O 


Average 
Difference 


0'12 


O'2O 


0-05 


073 


079 


0-61 


0-63 


0-68 



The differences of 2*14 per cent, for the histidine (edestin) and i'6o 
per cent, for the mono-amino nitrogen (hair) were more than twice 
any other deviations from the figures in the series. The highest 
average difference was 079 per cent, for histidine and the lowest 0*05 
for cystine. 

The correspondence between the figures and the actual amounts 
of amino acid isolated from the protein is fairly good. The high pro- 
line content of gelatin was well known, but the non-amino nitrogen 
content of 1 5 per cent, of the total nitrogen of this protein is very 
striking. Further, the large amount of lysine in haemoglobin was un- 
expected ; haemoglobin has always been supposed to be composed 
chiefly of histidine. 

Van Slyke [1913, 2] published revised data for- caseinogen. They 
differ considerably from his earlier data and may be partly accounted for 
by the use of the new apparatus for determining amino nitrogen. The 
data for gliadin by Osborne, Van Slyke, Leavenworth and Vinograd 
[191 5] are in closer agreement with the older data of Van Slyke's ; the 



io8 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



histidine and lysine figures are higher. These data are found by dif- 
ference and depend upon the accuracy of the determination of arginine, 
of amino N and of total nitrogen. The figures for the di-amino acids 
are in general higher than the figures given by the Kossel and Patten 
method (p. 55). Thus in gram per 100 grams of protein : 





Arginine. 


Lysine. 


Histidine. 


Method. 


Gliadin 


270 
2 '97 


0*13 
1*21 


r 4 9 
2-19 


Kossel-Patten 
Van Slyke 


Lactalbumin 


3'oi 
3*47 


8'io 
9-89 


I'53 
2'6l 


Kossel-Patten 
Van Slyke 



Both methods give the same value for arginine, the value for lysine is 
higher and more accurate, while the value for histidine is more consistent 
by the Kossel-Patten method. These and other data are given on 
page 132. 

The method has been of service to Hartley [1914] in his study 
of the proteins of ox and horse serum. His results confirm the obser- 
vations of previous writers that the albumin and globulin of serum ex- 
hibit well-marked differences in composition. Albumin contains more 
di-amino nitrogen than globulin, which is in agreement with the data by 
Hausmann[i899], Gumbel [1904], and Gibson [1912]. Albumin also 
contains more nitrogen as cystine than globulin, a fact which agrees 
with the data of By waters and Tasker [1913-14] who found that albumin 
contained about 2 per cent, of sulphur and 0-25 per cent, of carbohy- 
drate, while globulin contained 1-2 per cent, of/sulphur and 3-2 per 
cent, of carbohydrate. Globulin was also found to contain phosphorus 
while this element is absent from albumin. The chief difference found 
by Hartley was the presence of more lysine in albumin than in globu- 
lin. The chemical differences between these two proteins are against 
the supposition of Moll [1904, 1906] that albumin can be converted 
into globulin. There is so little difference between the globulins that 
the chemical composition supports Chick's suggestion [1914] that 
under certain conditions pseudo-globulin may undergo a process of 
denaturation and that a substance is formed which is very similar to 
euglobulin. This substance may be a mechanical complex formed by 
the interaction and mutual precipitation of the two colloids, pseudo- 
globulin and lipoid. The euglobin of serum is a protein-lipoid com- 
plex. 

Crowther and Raistrick [1916] used the method for the analysis 
of the proteins of colostrum and milk and showed that the casein- 



DISTRIBUTION OF NITROGEN 109 

ogen, lactalbumin and lactoglobulin in these secretions were identical. 
They found that colostrum in the earliest stages of secretion was very 
rich in protein (18-20 per cent, of the total secretion), that it con- 
tained 4-5 per cent, of caseinogen, 07-1*5 per cent, of lactalbumin, 
and 6-12 per cent, of lactoglobulin. They separated the globulin into 
pseudo- and eu-globulin and found these to be identical. They con- 
sidered that euglobulin was a protein-lipoid complex as suggested by 
Chick (see above). Lactoglobulin gave analytical data so close to those 
given by serum-globulin [Hartley, 1914] that they were regarded as 
identical proteins. Lactalbumin differed greatly from serum albumin. 
The difference in chemical composition of the three proteins leaves no 
doubt that milk contains three distinct proteins. Their figures for 
caseinogen are very close to the figures determined by Van Slyke, only 
that for arginine being higher. The analysis of lactalbumin is similar 
to that by Osborne, Van Slyke, Leavenworth and Vinograd [1915]. 
The high lysine content of lactalbumin is the most noticeable differ- 
ence in the three proteins ; caseinogen has the least amount of mono- 
amino nitrogen and the greatest amount of non-amino nitrogen. 

Drummond [1916] analysed a large number of normal human and 
chicken tissues for comparison with pathological human tissues with 
special reference to cancer. In general, the pathological tissues showed 
a higher content of the di-amino acids and they are attributed to the 
fact that these tissues all contain more nuclear material rather than to 
a specific difference of cancer tissue as believed by Kocher [1915]. 
The hexone base content of the various organs of man and other 
animals in health and disease has been determined by Wakeman 
[1905, I, 2; 1908] by the Kossel-Patten method. No very marked 
differences were noticeable, but there was a slight lowering of the bases 
in the pathological conditions. In the human liver, the amount of 
arginine varied from 6*9 in the normal to 5*1 in the pathological, that 
of lysine from 6*6 to 4-9, and that of histidine from 2-0 to 1-7 ; these 
figures are in terms of the total nitrogen of the dry substance, which 
was 1 1 '9 percent, in the normal and varied from 10*8 to 12*9 in 
the diseased organ. For purposes of comparison of proteins this 
method of nitrogen distribution is thus of extreme value. 

Analyses of various cattle food-stuffs have been made by Grindley, 
Joseph and Slater [1915], Grindley and Slater [1915], and by Nollau 
[1915]. The figures given by these workers are not always in agree- 
ment, but are accounted for by a slight difference in manipulation. 
Grindley and his co-workers hydrolysed the feeding-stuff and carried 



i io THE CHEMICAL CONSTITUTION OF THE PROTEINS 

out the estimations in the resulting solution, whilst Nollau filtered the 
hydrolysed solution. In practically all cases the humin nitrogen value 
was found to be very high and is due to the effect of the presence of 
carbohydrate (see p. 65). In spite of the errors introduced in the 
analyses by the presence of carbohydrate, the authors consider that the 
data are of extreme value for judging the food-value of the food-stuffs, 
in respect of the constituent amino acids. The data are given on page 
1 34. Marked differences are noticeable in the various food-stuffs. 

The influence of the presence of carbohydrate upon the results of 
the analysis by the Van Slyke method has been investigated in the 
case of caseinogen by Hart and Sure [1916] and of fibrin by Gortner 
[1916]. Osborne, Van Slyke, Leavenworth and Vinograd [1915] 
hydrolysed lactalbumin under the same conditions. 

Hart and Sure found that variations of the different figures occur 
to a great extent and that they differ with the nature of the carbo- 
hydrate. The changes in the data are especially apparent in the 
amount of di-amino acids or hexone bases. Loss of nitrogen occurs in 
the mono-amino acids in the presence of starch and xylan. It is not 
possible to correct the data. They concluded that the Van Slyke 
method was not applicable to the determination of the distribution of 
nitrogen in food-stuffs, not even for comparative figures that could be 
of any value. Such great variations were not observed by Osborne, Van 
Slyke, Leavenworth and Vinograd, nor by Gortner. These workers 
used a limited amount of carbohydrate, which never amounted to the 
large quantity present in the natural food-stuff in relation to the amount 
of protein. In all cases the most noticeable change in the data was 
in the humin nitrogen. Gortner agreed with Hart and Sure in the 
general conclusions and pointed out that this method of analysis of 
proteins was accurate only with pure proteins, a statement emphasised 
originally by Van Slyke. Considerations of this nature must also be 
applied to Drummond's data with tissues which contain other natural 
substances as well as proteins. 

Conclusion. 

The analytical data show that our methods of determining the com- 
position of proteins are far from satisfactory. Some of the methods 
have been adapted too exclusively to the solution of one particular 
part of the whole protein ; co-ordination of methods should bring about 
a better knowledge. The introduction of any new method has always 
advanced the study of the chemistry of proteins, and the final solution 
will probably result from the employment of new methods. 



ANALYTICAL DATA 



in 



s 



rf 



c 
-2 



a 

o 
U 



j] 






TJ 


S 5 






"S p^i ^ ^ ' 




i +i i 1 1 i r+ 1 1 1 1 VG + i 


1 


?* rt 2? 


li! 

xi c 


11+ 1 1 1 1 1 + 1 1 1 IVoo 1 


1 


3^-3 1^ 

^ ro Jg T3 ro 


s g 






MN W ^H yj M 


' H 






rt N w 1 ^ 


S 3 




^ 




111 


1 1 1 1 1 1 + 1 1 1 1 1 1 1 g**o 1 

N M 


\t- 

PO 


11 









1-1 


_; 






c 


*""* 






"** *^ ^ 


ISs 


1 1 + 1 1 1 + 1 1 1 1 1 1 1 1 1 1 1 


1 


cfl rt T 









rH C , H . 


U 






U ^ 


111 


i i + 1 i r, i i i i i i i ?go i 
N 


ro 
m 


|l|| 










U 








W 

.S 3 






_ _ 8 ..s 




1 1 1 I.I l-f 1 1 1 M l+l'oo 1 


CO 

R 


iljillil 


>> >, 


CO 




r-l ' 'c ^ H ? ' ' 


O U 


+ 




M 


3 "3 


n 




sl^S^u^ 


II! 


r+ii'i i 1 1 1 + 1 1 1 i?" o i 


co 


Islflll 


V3 ^ 






rt M <^-> 


a w> 






^^1 g^^i^^.s . 


11! 

o re 


iv+ 1 1 r+i + 1 1 1 1 -TOO i 

CO 


N 

CO 


iiiji|||il 

' '<* rt rt 


lij 


1- <s* iN IIIIIIIIII^O O^ i 
+ l + ilillllillobeNl 

m M H 


ro 

CO 


Iljillil 


c 
o 








U E 


H 




00? 


VI 


H O> 


in 

ib 

M 

M 


n 


I 








v Q 


d d w ci 




s= . e ^ 


s 1^ 


l+CVl M?J2M 1 l-Tool 


jo 

b 


22 o ^ 


ctf *** ^ 


M CO 


M 


<^ eg CT> ^ A H 


W3 .S 




H 


rt ' M '^4 


04 






(M 












.... S l u ...llll . u2 


3 








H 





112 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



i! 



13 ,_-, 

k> O^ 



1030 



I I I 1 O N fx 

I I I I w Jo b 



jo in i op i N w i 1 
''' ' 



^l. l!J ^^^ 



J3 rtlS^fc 

1 1 i|| 

^ U.-J W5 ^ 



Oc 

.SSCQ 



- 7 ^ ^ i *? i 

4. ro H t> I 10 ' 

M 



EO 

uE 



I 

11 

I 1 " 



^ a . 



ANALYTICAL DATA 









oT 


Lactalbumin. 


r-lr-lrH <-!,_, ^ fHlH 71 <N Cl <N 

o c b I ci b I I V 1 w b I * Si w 


p 

u~> 


=^"-^ 

^ES 5 jj 

III! a 

"rt -> Pt3 

llfl! 

?2|ii 








d 


g 






c 


J 






rt t t 








g oj 


ECQ 

is 


o | I i i ig | I I I 1 5 l l l l I 


b 

H 


1* 


if 






*O rt 


1 


- 




<^ 


i 


P 




?1 


spi 






2^,0 g 1 ? 


^^s 


H t ~ N |Oi HWV O ir >O.Ht^ I i I I 





^ ^i!li2 ^ 


If: 

13 3 


ON CONONH oo t^ 


M 


S ^ 


I'M 






< UJ 3s 


u 






rH ^ ^ 








w eo 


c 






. 


S 

3 

ua 


I^ I oj i c*i o 1 ^ H | r^ 10 i w co o i 


<X 


uS ^-g 


? 


oo'io'wiwIbi-i'Mco'NNb' 


O 


^ M e TI 


8 






Sill sit 








^ V^ "G ^ ^ S 1 


4) ^ 


,3 N w> i* . H oo ^o i d M o>op y* fo 


CO 


c rt o'"'^ 1 -' 
rt"o3 < S" S 1 1" i & 


.5 B 




* 


cgQ' 'Set- 


=3^ 






22 eg -| ^S 


S 


-",.- V- o 


^ 






O 00 ~^" t^ 1 ^ H I O C*l 1 H 00 ~^~ 1 


N 


^ PS* 






CO 


<3 CO 




































4^ "^ 








^ ^ "5 *"* "o S >L'^ SL^ ^"^^ ^^^2 s 

O^^hJl^HOHE^C/iOOHO^OB" 1 *^^^*^ 


r 





PT. i. 



1 14 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



+ +oy>oo>N [vpt^i o <x> H txop i 

ON ' pvioobiopnb I 
O 



3 1 8.8 1 

H * 1 1 \~S M ? I CI I I I I I j, j as 

< 

H j_ o , N en' 7 i Tt- i *^<P . 1 



o 3 "S h 9 

111 

p i JO \OQp N vo i P ."* P | t I I "* "* 

J8J - - - S| 

s,iiiii?ninaiMii ^ 

D || Si 

f 

J w 
o 

a 

m N , t^ | cp m i o>o i y^ n _L i 
S H 

5^2 

5 ffi 

^ . ^i _ 

o 

S .S? 



5 H o 



ANALYTICAL DATA 



ll 






u d. N 




vocoinr^liool >|c^ o^^ o <o noo 


M 


Iffr 


il 

i* 


OOIHTO ' COCO CO 1 COOl"'i>HHH 

H H 


M 
VO 


1*1 


8$ 






H i 


J3.C 


vOO^cocol COM 1 OIO>| co-^+^OvO^O 


0> 


.0 -%& 


.S * 

*3 a 1 
jaO) 

go 


b H b tx COCO O01 CO01 rt-010l H 

H " Vt^ 1 *V 1 1^1 1*^ Mill 


in 

H 


l!llf 

S g> c /3 


to 13 
>> a, 

SI 

M 


b + b V ' N H ' ' H ' ' 


N 


S'-'S .a 3 

1 i ll 
? ? 3 

H 


Edestin from 
Sunflower Seed. 


m invo oioooiiooioio 1 ^ i i i i 

01 Vb N 1 Voi 1 N 1 COCO+ 1 1 1 1 

M 


in 


1 Abderhalden and 
Reinbold [1905]. 




6"d 
S 






1 

rt 

Ss w 


Edestin f 
Cotton S 


01 in rt >o|pr o : it 'iro|p" 1 f'^i i i i 

HV+inlJooibloiloiK+l 1 1 1 

M M 


01 

Si 


T3 W 

132^ 
j8i 

< 




i i i i | ! H | | | 'r 1 ~ i^i Vr-^ l 

1 CO ' ' Tf" ' 00 ' Tj- H 01 ' 

H H 


1 


r^i 

^^g" 

|!P 

^UJH C 


11 






o cf S 

?s^^ 


OT 

a 


oovo" 1 o>| ^Hcocoi^oin co" 1 rx o H 1 


r* 


^rt^^ 


B a. 


COCO"""O NWObwW'^tO'MHM 

01 H 


*0 


S^J.s 

il?^ 

3 8 9-a 




1 1 1 1 1 1 1 1 1 1 1 1 1 \Ysz i 

H 


1 


" w S 
3 s rt 

5 N 

< j^XJ ^3 

r^ * W 3 

d O J3 

-^ 

-J 




















. . .! . . .11... . 










1 





116 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



-H LJ1 ^ 

O O "^"00 I fO ro,-f CO < O O^ CN cO w CO 

I " I ... 

m W OHO !> 10 N O *o O Tt* 1*0 * r*^ ^ ro c*i r^j x . "^ . 

|cS -- 

- ffi 

o5 O 

pit^inipiCTii | co |p>l n |j- | ^7f' k p o 

MHlbcO'cOH' 'co'cO^'inNMN io 

b 



c 

cs >. r P !^ I P :* I > I P I P P H t^ ff 

eg OHHO^'fOMO'M'mrJ-'rt-Tj-NN -<* O ' 

ja s II 

PH 

13 

g l! s 

|S s^ssigs' i?i^i^&si 
H J3i 

o ^^ S^iJ 

o 

w 
Hj 

PQ 

< 2 

(, & iH iH iH iH 1-lrH rt rHpHrtiHrHr-li-l 

w 'I .* r 1 r*"? 3 I f 71 . r| "'^- | w |?'r o +; H pp^ ? S2* 

C5 3> OHHOO N(M Vh COCO H -^- N N OJ 

pq M MHO 

- -- 

i 

l-(-<iH 1 -'r-lrHiH l -101lHC<l-<C1 C^CO* 

^S rhHocovomNcooirxO'<t-H H St-icy 1 

I* b N ' co ' Jon b ro ' inK + Hln* N N o-o- 

S) MM 

^ 0^0^ 

i 

O in 01 rf I CO rl-/x. I M I p7f't O>> 7l"Np 

bbbb> CON Vj- inM"""cbinNN 

vu en "- 11 

O ili 

.s "^ 

tli!!!-i.iii!i'it!ill I 



ANALYTICAL DATA 



117 



51 

So 



p> co 

CO Vf 



*p I 

Th CO 



3 

1 



I 1 

ew 



ob 



00 

in 






a 
i 



rH r-( |H r-i rn . -! . - vj v i 

opinHOO I ^w+ 1^ I P^+P^f^| 
bMHvb CON N cob> vbwb 



2 



^ 



ndin from 
lmond. 



ii8 THE CHEMICAL CONSTITUTION OF THE PROTEINS 









oT 








s rt'i? 


O 4) 






c -5 ? 


o,2 


\ \ \ 1 1 I 1 |P| 1 1 1 I^Pl 


H 

CO 
H 


oT c 2 

<U faD 
> O 








J3 rt .S 




' 




IE- 


2'S 






a) g^ 





b V b o 1 9 V b O V i b PO + V H H V 


m 


|| 


II 


r-rH 'rt rt _ rH rt rt rt 




c 
rt 1-2, 
a, 00 
c ex ~ 


S "3 


Ol 1 vo 1 w PO 1 linlbN~'~r > rOPON 


in 


o JS o 


O 






"S ^ 2^ 








o '- 1 


. 






1 = 
ss^ 


Avenin f 
Oats 


p inoo p I e* 10 1 1 TJ- i p ^ 1 I 1 l i 

H N H in PO M ' in ij-00 
H H 


P 

PO 

in 


IB 


c 






III* 


1 






S^-a S 


O 


< PO I H 1 o 0> 1 |O|l^OiTj-NNio 


*>. 


2 S S g^ 


S 


bb'V'HH' 'Vh'bV V N M N 

N 


*. 


S So*"' 1 ^ 








Oj g 03 <U 









Igfll 


a * 






*%& 




Ov >O N fO i 00 PQr-i i N i ^ t^ CTiOO OO ^ 


N 


c " 


D^ 


b ^~ b H 1 ro oo" 1 ool mvb~'~/")p*c<H 


b 


o a. 


S S 






43 CX, 









?0 


G 






c ^ 


IP 


'vn'oM^ys tooio 1 ^ |O|H P o r ^,riOprifO 


PO 


c > 


|| 


b b b b> ' ^H ' ^' ^-coirifONH 


m 


o . M . 


$& 






3^ 








?K 




1 '.ll a 








llsllllslllllfilll 


1 






slfJ|iSfes|||fll 







ANALYTICAL DATA 



119 



1 






S 1 

rt l-l 
C CT> 
rt M 


c 


1 1 N 1 1 l-l 1 II 1 1+SfSS 


o 
K 


ss 

I! 

- 2 

M 


1> 

'3 

S 


^ + r* i P . w i i 1 i p op i p p p *? 

1 O w ' ^ O 1 1 HI ' H H 1 H O O N 

MM M 

w 

p 
2 


CO 

CO 

Tf 


S ~2 

o\ t^ o 

. >^ 

s fi 

J3 p,*rt 
r^O ^O-* 

S3 IK 





OOOOMO i^OOO i O I l^ N O <O O 00 D 


p 


C*c '"'S S 


.s 

3 


b bi M o> 1 i co H 1 bil-HvooHobfo 


ir> 

CO 


w rt m <u 

1*5 2 5 




i N COVO lCT>|^0|niTi-cO|N|<-| 




iltfJiii 




iNbcblW-lolvolHooiHib 1 

H n 




hJW N WOO 


x 

.2 






jig; 

8>J5 





o <* "i- o 1 moM 1 (* 1 co co 1 o* o n -f 





s 


a 

a 


o M M t^ m V o Sn H H 1 co o o V 

<a a c* 01 eq ci e> c* e* c* 
O-*-NrviOt-H| 1^1 i ^- , N O fO 


10 

t^ 

IT) 


il 1 ? 

g rt ^ 
j3 4) O 

^ H 


1 

i 


bbbvolioHi Iro' lb"'"NbH^ 


M 
t^ 


.SJ^ 

W - 

So 

rH M 


i 

c 






ii 

<U O 


1 


H fO 1 fn 1 J> N H 1 CO 1 COCO i N Th H 


CO 


c a\ 

U-i M 


Gliadin i 


OMbHOCfc'b*'i NOom 

CO 


CO 
VO 


II 

rt 




O O -^-*O 1 Tt-NNU-)N iVOt^ONN^ON 





^ M l -A r N 1 *S * 

(U O vD ^ (-, ^ g 


1 


b N ro^> 1 Nwbbjy 1 bcoHJnbbio 

COM W *P5 0)toOW 

Iwkolool 1 IMIMCO.ONNW 


CO 

CO 


3 CTi O M O aT tV, 

i e s&i If 

CO g-^S 03 g 
1-1 o 2* M rt e 





'bin'H 1 'K'bKlcoHN^ 

CO 

OS 




^2 " J5 U 5-T3 . 

.| "3 J >? 

C r*W T3 ^ T3 (j-^., CJ> 


Gliadin f 


t^t^Tj-p i vp 71- N 1 :* I p^lopp? 1 ^ 

ONO^O 1 NNO ' N ' Hib NOHTl- 
co 

ir> 

M 
CO 




*rt H "rt SSw.og^ 

J3 "^, C fi C " '^ > 
*-< ^^ fl) M C M CO CO r* 

^a^ .^^^s,? 

3^^060^ S 

rH J q, K-l 












S . .-a? 








'l "^^ S 

lisillisiiiiifisll 
Illliltlliljj 6}fl j 

O<>i-iMa*HwofiO'<OH<i5C'< 


rt 


H 





120 THE CHEMICAL CONSTITUTION OF THE PROTEINS 





g ^ 






*o 




& bfl 






rt 




C 


NO H CO H 


^ 


S ? 






1 I 1 (TM 1 M 1 I 1 1 1 1 1 M O H i 


o 


J= g 




II 


H 


H 




c 


s 








1 
i 


11 


1 1 1 2 1 1 2 1 1 1 1 1 1 1 ? 1 


CO 


||| 
















* 00 O\ O> I VO rh 1 INIMO, mOO O> CO 


in 









O b H C* 1 0* co ' 1 V ' N co "l" K V H H 


CO 


N 




bo 

M 






I .* 




M 


001-10 000 00 0000 








JM 
<U 
S 


in m moo t^oin. n. tx o i o N H N 

^Hilb^l^lb- 


N 

b 

N 


"c ^2* 




* 






*Td Q jQ g * 




C 


w M 




rt w 3 * >"o> 




1 


Sb'co'Hb' ' V ' O M * M Ji g ' 


V 

H 


jriii 






- ^--' - 


P 


infill 






^, + NH|MH| 1 1 b S 1 1 1 1 


T(- 


*>, H-4 1 ] O H-l 














c 


o N coop i CO t^ i i 0> i p | I i | i 

O H H 00 1 IN V ' 1 IN 1 H M 1 1 1 1 1 
M 


r^ 

CO 
CO 


^ 




jj 


CO 




^ O rt . 

S ^ g ^ 




s 

0) 


.Jn|>|p | |t | H+| | | | 
OH t^Nin rf n 


in 

CO 


&-S 84? 







P 




*O H 'O <U 


i 


O 






*^ ** *^ tin 


g 








^ ^ 










fH C 


1 




to to to to to to e w co 




J 7J 1 


I 




OHt>b>'co'b'r^"'"binl 1 1 1 1 




s^,s 


o 








M (U 2* 










in 4-> en ... 




u* 






o . 2^; 




S 


n N Th i n in m t^ co ri-io inoo o ino 


m 


Ji ^ rt *^ u 




U) 

I 


H 


% 


jSti&lil 

2 H H J < ^^ 
Iti'ig'gll 








CO 


llsllll 






OOHO con-o o coo H o H rj-inw H 


M 

m 


?i??ll? 
















g . . . -o^ 










!|ll|!l|l|ttlllil| 


3 

a 





ANALYTICAL DATA 



121 



g s 

H o 
O 









j5 O, O\ 


1 


*O O 1 *O i vO d O i O * lO O I 1 1 1 i 

<* V 1 H 1 b a\ N 1 H 1 N H 1 I 1 1 1 


m 


S^ 1 

'W *. 
Q C 








gc 
2 o 

*rt ^JDr i 


rt 5) 


tno , N i Vpop i p j p*V i i i i i 


t^ 


C C ?* 


11 


1 H 1 H H 1 H 1 + 1 1 1 1 1 





I1 S 


Chinese. 
Tailung. 


Vg 12 it+r+i 2+ 1 1 1 1 1 


i 


li 

^^ 

li C M 

d ""' 2^ 

11" 

r-4 








c 

^ 

llr^, 


s| 


inp , p inp , in , p p - i II 


t^ 


lit 


55 


N 00 MIHQOMlCtiNCIll II 


* 


S!> o> 

31^ 

<J rt 


1 






|| 


i 

3 


ttN|O>|OoON.O|MO| i i i 

inob lblHKH|H|NNl 

01 H 


Tf 


jC *^5 O 

5 s 


E 
.3 


- 




Is 


J'S, 

JO C 


p jo . . p op jo , , p p . i j , 

^00 |H|HI^H|H|NCO| 1 1 1 

M H 


? 


< i 


"S 1 






c 

la 


il 
51 


1 P | P | P J^ | Y* i ? <*> i | i | | 

* f W 1 M O> H 1 1 M M 1 1 | 1 1 


CO 


o3 CQ o 


a 






41 

llr* 


ss 

c c 

S3 


join.inoopinp.op. . . . . . 

rco|H|HOiH|H|ol 
co N 


N 


<u PQ c^ 


S 






Td <L) 


1 

S| 


t^op ,0 ?*> ? \ ?* i ?*** \ i i i | 
O>CO|H HC*H|M|(MH| 

M Cl 


M 


PI 


c 
2 


p e, \r> . to jnO I co - "* w - " 5 . 


<* 


Ifl^swl,-!- 


1 


OwOH 1 MOM | O l"ro 1 H-r+| 
CO N M 


K 


f2;s s aE s a 




C "^ ""* 

tlil|flillliltiiil 


I 





122 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



.2 






s . 


oS 






2 'M' 


01 O 


rt IH rv. r>.. o.. o.. 






s* 

rt 


+ + + + |++ | | + | + + o | | | | 


1 


^^ 


*j 






- uli 


S 






^ 


1 








n 
2 . 

re r ^ t 




+ + 1 + 1 1 + 1 1 + 1 ++ 1 1 1 1 1 


1 


n 








2 


03 






? 


a 






C f"" 1 


l| 






C O^ 


O p 

f 


+ + 1 1 1 1 + 1 1 1 1 1 1 1 1 1 1 


1 


11 

2| 
< 


VI 








8*jj 


Vco o " ^ """ 7- Vow 


oo 


s 


i^l 


H 


rf 


O 
C 


( 

*4-( *^ '3 






c 

re 


09 9 


CON.CO.^O. , rt .ovo.oo -*o oo 
ib &* i M 1 T t 1 l + |Hblcot>.Hb 


O 

CO 


P "o> 
j| 


<>* 






1^ 


<u 

u- " 






^ 


111 


His i if i r+ 1 s+ 1 s;-1r? 


8 


N 

3 
CO 








n W 


s / ^ s 






rt 


o bi 
'5 JJ 


/^ 




c ^ 


u CQ 

?1 


&S I" i" I I i&l?fi I I I I 

6) H 


V 

in 


*S 3 


|| 






2!] 


ll 


.H^r. rH ^ rt rtrt 


^ 


1* 




locolwi Idol Icol |H| |ir> M 

CO N H 


o 


g 

tt.^. 






















1 





ANALYTICAL DATA 



123 



rt 
10 o 10 p i to" 1 p - JO - p - - op vp co - 

~ > ~ HCT>bI-i|N + N|w|vblloo>H| ^ "01 

* S-g 

I 2 1 

rtrHrHrtr<r H M P M O 3 O 0> 

lONtxtot^vpiipi^l^ii,,, ^ fe'S O 

rorOHOOlWMllThlwiolllll ro .JM> ? 

1 :hT 

S3 Is . 

f-4 1-4 IH r-4 f-4 iH rH IH IH ClJl) I 1 

'o, c* t>< t^op i r *" prj iii ri i^pi r> iiiii r^ ^QO 

Jrfw POOMVictVllHtHtOlllM CO W^tO> 

^3 S M ^ *a s M 

^c3 ^ S" 

d ~ I m I TT" r+ 

pC.tx.pt^fsj^.pH..... 00 ^O 

VON|O|H<^O|O|HO| 

!o 

ad 

-o !f*P i ?P i f P ? i 9 i P P i i i i i 1 

2g roo|olwcy.H|olHolllll oo 

"""O CO M 

c 

3 

! J^l 2 I I 5+ Tb I 2? I I I I I 
3* 

d 

1000 . op ro p .^ . p , op cp , 

HO>l^loHo|fn|NM| 

Is 

rHilp-( iHr-frH rH r4iH 

?* ?*o. p i ^? r ? 3 i y^ i i p i 

'H 0\ 'iol ONvol N I NN | 
OO co 

SB 

i - i !'S 

V:. 1 1 r+s 1 1 1 ii iV+c i s 
^_ ?s 

'C! 

-.... y ^ p. . . . . 

a 



124 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



: o E 

21 J 



Keratin, fro 
Egg-membran 
of Testudo 
Graeca. 



"SSls- 



i 



Ovo-Kera 
Egg-membr 



,_ _ 

O>nHTt-| 1 _ 1 VO O 
fc S H K 1 1 1 tx V 



M 

g g-a 



N op I co 

H io I r? N 



*b 1 Vob ' in ro o 



r 

22 S"S 



p 

Invb M 
OJ 



p 

H 
VO 



co it c-i rn 



^-ONOOimOiCO 



Sg, 1 M 1 M SS 1 1 M II 



S 
si. 



si 



. 

^S 15 S"o^ &'g ^2 ^&-2 b^ 
^ > J5 ^ S K a o < o H < 



.9 

.s g 



ANALYTICAL DATA 



125 



I 






c 
1 




II 


f^ 1 f 1 1 P 1 1 5 s 1 S* f 1 1 1 1 1 

iO CO 1 u-> 1 1 M 1 1 <O 1 M Ok 1 ' ' 

M 


P 

d 


4 : i* 

*-} O 

^.S o* 




a 






< 










'co 




is 






PO 




|| 


* 




o* 






c* H 1 rj- I oo 10 1 oo i 1 I 1 -"! | | 1 1 


VO 


II 




80 


K. N 1 N 1 co b 1 PO 1 1 1 1 1 1 1 1 

H H 


^ 


3 




c/3 cq 






i 










PQ 




o 






r^i 





."3 






CO 


^ 


1 






O\ 


Cu, 


| 


COppPOirxOlJOJOi I O 1 I I I 


CC 


- 





8 

8 


HM^-OlNmlTj-col Icol 1 1 I 
H H M 


z 


1 


1 


1 






u 

P 
PQ 


y 



















f 


1 


. 






H 





1! 
I* 


JO H ."HO I CO N I tx I I INI I 1 I I 
00 10 N 00 ' N m 1 * ' 


H 




U 


CO 

C 


3 . 






H 
H 

CT> 

H 


C 
o> 
u 


S.2 

03 O 


* N JO 1 H 10 | 1 I -H 1 I I 1 I 


00 




r 




O^coinmiMcoliol ' 1 O ' ' 




>o 




lO 


*H 






C 


CTi 








rH 


* 









C H* 
H 






00 * txOO |OtxONvO|ioO^| I 1 1 1 


H 


2 g 






b v bb>fo'bio^'''t'c<'c*oo' ' ' 


U3 


*G - 




J3 




* 


il 










11 
















c ^^ 












H 







126 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



NHOob 



r> "VpTi-iio|Mroj I j I I 
+ PO b 1 on ' M N 1 



g> 
ll 



M i i i ? c is i .i i 1 1. 1 1 



7? 



s^ 
3S& 



c ^ 

II 



ir> lx i O c* 1 tx PI 



41 

S'M 



7*- PI 
o M 



10*0 



o v I 100 I ro 



~ " CT 



.u - 



ANALYTICAL DATA 



127 









a 




w 




s 




+ + + + 1 ++".". + | + f + | | | | 




c ii 








S M 








V 


09-2 


d 




s 


s| 


1 + 1 1 1 1 1 1 1 1 l-b-og 1 1 M 




CO 









g 












Vo + + | + IX. 1 + 1 1 + 1 1 1 1 




JUl 


Azotobacter 
Chroococeum. 


1 1 1 1 1 1 1 1 1 1 1 i 1 1 3SSS 

d- OvO O 


1 


1 Omeliansky 
and Sieber 
[1913]- 


Water 
Bacillus. 


1 1 1 1 1 r+ M + 1 1 r+igfi 


in 


i] 

rH 1 1 


2 . 






rtr-^l 


11 

J3 o 

S 


I i + + + i + i i + i i l+jlss ' 


M 


M 

t- 1 2 1 

i-t i i 


6 

~ s 
""i s 






3 w 


o.y o 


1 1^1 1^1 1 | H | 1 1 |(7)mcoH 


M 


g p^ 


ty 3 


'''io 1 'io 1 'wobb 


in 


Co M 


3s 






H ^ 


s ^ 














rt 


O gj 

*o 3 


rH rt r-l rH pH rl rH 






S3 


1 1 .* 1 1 ?* 1 1 1 J 1 1 1 1 r M^. H 


H 


g ^ 


11 
09 


'(^''tx 1 ' ' 'NOOO 


ro 
M 


rt H 


1 


v+i + n i 1 1 1 1 v+i 1 1 1 1 


1 


S^ 0^ 
T3 *O M 










c 

Id 






bfl 

C 

-* 


ss 

td 


SiT 1 b 1 ' b 1 1 v ' S 1 1 STb S 1 


jn 
b> 


S a* 


c 


M 


W 


C i_j 








M 

r-t 












' c * "S? ' 








iiiiiiliijiiltliil 


1 





128 THE CHEMICAL CONSTITUTION OF THE PROTEINS 









cr 








S g . 





i Ti'W IP^IIIIIIIpvHI 

1 ^^ w 1 H 1 1 1 1 1 1 1 WO ' 1 


IT) 

V 

co 


K 








ffi rt 


c 

* M 

ii 

S- 1 


+ + 1 + + II + I + + 1 VE g- 1 


CO 


|| 


| 






1^ 


fe 

|.sl 






I'" 3 * 


ill 


rt irjH ^-Hr^H|N|HMiT}-ioco| 
OOH N bbb'b'oH HWO' 


g 


ill 


- 






< ^ 


III 

S M W 

o S> 


,_,OOiO<O l 1 ^! 1 N 1 ^- O , tx N N 1 

OOH co 'H' ' b b "*" o it b 


H 
CO 


^bderhalden 
and Weil 
[1913. i]. 


X. 

M 






IsrA 


111 


rt oor>o 1 1 too" 1 N 11^4-^?" 1 


* 


I'^S 


'Cg >> 


O'OOH' 'OOOH^OOO' 


O 


<U ^ H 


Q 






^Si 


*H > 


rt rt rt -<N - 1 r-lrtrHrHrHrt 

rt voiOH| lirjOlH HN,OT>H| 

ob OH' 'bb'b OH^bbb 1 


N 
CO 


c 

IK 

sis 








<j rt 


1 






S^ 


> 

G 

c 


+ + 1 + 1 1 1 1 1 1 1 ++ 1 M 1 1 


I 


^3 (U w 

IH 


'S 

o 






2 gS 









^ 


1 








3 










* !*.*.* r, 

I+I+I++II+I+++ + 


I 


t| 


0, 


' ' s 




"" H 


a 






77 


1 


1 Ir- iTr I I | +*? I?''?- rr 

O * M O O M co 


CO 
CO 


2 w 


1 






" S 


IS 






1 





1 1 | ? |P + 1 |>|P 1 1 1 1 1 


W- 


11 


s 






d ^ 




4) 13 12 " * ' 

c *o o 

*S w ^ ^ S 








g sl3.| w |-S'l'l.s o'|| 


a 






'3 '5 S S c o c ^ ex, 13 5 cLS .S *z; 


o 






5^Jll^|^ol5Hl5ffil 







ANALYTICAL DATA 



129 



Pro- 
rom 



ris 
ria. 



tei 
A 



P .* 1 1 1 * 1 *> V \ 1 1 1 1 :* 1 1 

Old ' 1 1 N ' O ** 1 i I 1 1 H I 1 



9 I | . 1 P - - H . . oo . pvp H . . 
ol 1 N | V 1 1 <r> 1 1 <n 1 ro b 1 1 



VO ^ f>. Tj- I N J>s 

b ~ vb V I N H 



M co fomrom 



e 2 



<U ol iXD^ 

2 ^3 1 
? a 



I 



11 



1 + + + 1 + 1 ++ 1 + + + 



H t^OO t^ 1 N N ^ I 00 1 in JO H \f)\Q 00 

ft fn b H 1 n ei . i Js 1 V r> + t<* t^ H 



oi 

b' 






8$ 
IK, 



^ 



. (O . . 

s^ s1o^ w . |.g 






PT. I. 



130 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



Hemielastin 
(Elastose). 


1 1 1 1 1 i i 1 I 1 1 1 1 iVjVg 


p 


|| 


ill 


i 1 1 1 1 1 1 1 i 1 1 1 1 I^FS 


> 

H 


ll 


6 


M l 




T3 


S tt) 


o> >n M oo 




qj^j cS g 


ei 


^ PO N O 




ffll|I 


J.Q 


M^-mwOinm nm r^.m ^.QO oo t^ 


m 






OPOPOrOPOMPO '^^- Tj"Ql VO^HH 


UD 


r-i >. 'CJ c* 


fa 




m 


rt 


I, 






T3 
fl) M 


I! 

|i 


in mop op vp ^i-vp I t^ o 1 p vp 1 t^ Thop a\ 
HwbinHThV bn POO i^oo N b 


PO 


-ll- 








^^ 'TO' 


c 


rH rH rHr-t rH rH rH r-f rH rH 






*S 


NrH \O lOOl IVOlNO"" 1 M ^ ^- i 


in 


e ?75 " 


OT 


N r *" IT^ 1 H PO 1 1 N ^ N O "^" OJ H O 





g OT< S 


S 


M H 




j|a 


M C 






IJBA 


l| 


OOOO t>. |<OPOM jVOOt^M 1 " 1 >n t^OO 1 


9* 


C C^ oo" 


^ 


ON ^h lNPOHlVoMOO"T"HNbl 
H 


Vj- 


111 


. s" .a 

till 


1 1 1 1 1 1 1 1 1 1 1 1 1 iVioloi 


p> 


II 


"3 co* 






N 1 1 


i ^ C 








6 g _,-| 


G* (S IN IN 






Sill 

ri 


1 M 1 1 1 1 1 1 1 1 1 1 l?g 


p 

H 


ffl I M I 


c 






Js . 


c 
2 

c 


m o CTIOO 1 m w 1 1 PO 1 jnvp I H PO t^ p> 


PO 


lllll 


CO 






JD C ' ' ' ' 














1 

H 






5^>jlH^uo<OH<^| 







ANALYTICAL DATA 



II. Composition of Proteins by Nitrogen Distribution in Three Groups, 



Protein. 


Amide 
N. 


Humin 
N. 


Di-amino N. 


VIonamino 

N. 


Total 
N. 


Observer. 


Haemoglobin. 


1-07 





("072 (haematin) 
t4-07 


10-95 


16-81 


Hausmann 


Cryst. Egg Albumin 


i'34 


O-29 


3*3<> 


10-58 


15*51 


Osborne and Harris 




1-28 





3*20 


io'i7 


14*65 


Hausmann 


Conalbumin .... 


I-2I 


0-26 


4-16 


10-49 


l6'll 


Osborne and Harris 


Cryst. Serum Albumin . 


0'95 


0-15 


4-86 


8-81 


14*60 


Gumbel 


Albumin (Jack Bean) . 


1-16 


0-23 


373 


11-18 


16-30 


Jones and Johns 


L-eucosin (Wheat) . 


1-16 


0'43 


3'5o 


11-83 


16-93 


Osborne and Harris 


Legumelin (Pea) . 
Serum Globulin 


1-04 
1-41 


0-38 


3'7i 
3'95 


10-96 
10-81 


16-09 
16*17 


Hausmann 


Edestin (Hemp Seed) . 


1-88 


O'I2 


5'9i 


1078 


18-64 


Osborne and Harris 


(Cotton Seed) . 


1-92 





571 


II'OI 


18-64 


ii 


(Sunflower Seed) 


2'57 


0-2 4 


4-27 


11-52 


18-58 


ii 


Cryst. Globulin (Squash Seed) 


1-28 


0-22 


5*97 


1 1 '04 


18-51 




Globulin (Flax Seed) . 


2'00 


O*22 


477 


11-47 


18-48 


H 


Excelsin .... 


I- 4 8 


0-17 


576 


10-97 


18-30 


I 


Legumin (Pea) 


fi-66 
(172 


ro-27 

\O'2O 


(5-24 
\5'43 


r 10-74 

\io-55 


17-91 


l 


(Vetch) . . . 


i'75 


0-18 


5'i7 


10-90 


18-00 





,, (Horse Bean) . 


1-62 


O'll 


4*92 


"'34 


17-99 


>i 


(Lentil) . 


1-69 


O-II 


5'i6 


11-03 


17-99 


?J 


Phaseolin (Kidney Bean) ' . 


174 


O-29 


3*97 


10-18 


16-20 


n 


Canavalin (Jack Bean) . 


1-41 


0-28 


3'i7 


"55 


16*41 


Jones and Johns 


Glycinin (Soy Bean) 


2-ii 


0-12 


3'95 


11-27 


17*45 


Osborne and Harris 


Vignin (Cow- Pea) . 


1-91 


0-25 


4-28 


10-81 


17-25 


n 


Conglutin (Lupine) . . < 


(2-12 
(2-65 


0-18 

0-14 


5'2o 
5-I3 


10-38 
10-30 


17-90 
18-21 


ii 

j 


Vicilin (Pea) .... 


1-67 


0-26 


5-12 


10*00 


17-05 




Amandin (Almond) 


3*05 


0-17 


4'*5 


ii*55 


19-00 




Arachin (Pea nut) . 


2-03 


0-22 


4-96 


11-07 


18-28 


Johns and Jones 


Conarachin (Pea nut) . 


2*07 


0-22 


6'55 


9-40 


18*24 


ii 


Corylin (Hazelnut) 


2-20 


0*16 


575 


10-70 


19*00 


Osborne and Harris 


(Walnut) . 


178 


0-15 


S'4 1 


11-51 


18-84 


ii 


Globulin (Cocoanut) 


1-36 


0-14 


6-06 


10-92 


18-48 


ii 


Glutenin (Wheat) 


3-30 


0-19 


2-05 


ii'95 


17*49 




Gliadin (Wheat) . 


4*34 


0-07 


I*OO 


12-25 


1766 


,, 


Gliadin (Rye) 


4* J 5 


O'll 


0-87 


12-59 


17-72 


M 


Hordein (Barley) . 


4-01 


0-23 


077 


12-04 


17*21 


ii 


Zein (Maize) 


2-97 


0*16 


0-49 


12-51 


16*13 


ii 


Kafirin (Kafir) 


3H6 


0-17 


1-04 


11-97 


16-64 


Johns and Brewster 


Gliadin (Oat) 


3*55 


0*26 


1-04 


10-85 


15-70 


Osborne and Harris 


Caseinogen .... 


2'IO 





1*84 


11-93 


15-87 


Hausmann 


Caseinogen .... 


1*61 


0-21 


3'49 


10-31 


15-62 


Osborne and Harris 


Vitellin 


1-25 


0'22 


4'65 


10-16 


16-28 


fj 


Vitellin 


075 


0-25 


377 


10-56 


15*33 


Plimmer 


Livetin 


0-68 


0*26 


3-12 


10-90 


14*96 


" i 


Gelatin 


0-29 





6*45 


11-26 


18-00 


Hausmann ^ 


Keratin (Horn) 


1-17 


0- 4 2 


2'95 


11-81 





Gumbel 


Silk-fibroin .... 


0*56 












Wetzel 


Silk-gelatin .... 


8-24 





lO'OO 





__ 


99 


Conchiolin .... 


3 '47 





8-66 










Chicken Muscle . 


I'2O 


0'44 


4-82 


9-63 


16-09 


Osborne and Heyl 


Fish Muscle .... 


I'lO 


0'39 


4'95 


9-96 


16-40 


ii 


Scallop Muscle . . 


I -08 


O'40 


4*52 


11-05 


17*05 


Osborne and Jones 


'Ox Muscle .... 


0-89 


0'43 


4-42 


10-44 


16-18 


ii 


Protoalbumose A . 


0'25 




5*24 


11*24 


16-80 


Pick 


Protoalbumose 


1-26 





4'49 


12-32 


18-07 


Friedmann 


Heteroalbumose . 


1-16 





7*00 


10-32 


18-48 


Pick 


Heteroalbumose . 


0*36 





6-27 


10-03 


16-89 


Friedmann 


Heteroalbumose . 


4*4 


I 4 -8 


23*9 


76-1 


100-00 


Haslam 


Deuteroalbumose . 


5*3 


6'5 


31*4 


68-6 


100-00 


99 


Koilin 


1-26 


0*19 


3-36 


9'39 


13-88 


Hofmann and Pregl 


Egg Membrane, Hen 


0-89 


0-03 


277 


9-81 


13-60 


Buchtala 


,, ,, Scyllium 


070 


0*08 


2-17 


10-96 


1375 




Tortoise Carapace . 


o'43 


0-07 


0-44 


I3'4i 


14-14 


. 


Elephant Epidermis 


i '47 


O'2O 


0-32 


12*25 


14-26 




Armadillo Scales . 


1-25 


0-07 


0-56 


12-71 


14-21 




Snake Scales (Boa) 


0'59 


0*16 


o*33 


12-67 


13-80 




(Python) . 


0-17 


0-23 


0-56 


1374 


14-42 





132 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



s. 



o 



I 

1 

Q 



I 



I 



F 



L. 



z 



>u 



ss 





II 

oo 



s 

2 g, 

i i 



i i cc i , 

- r s s r s> 7 

>* oT rt " 

I I ^tl = 

> O v?<-> 



COOO O >n CT> O t^OO ro N VO 



H POVO Ot^O^O ro O O> O> PO t"N O O O O O O O O OCO ir> 10 '* 

iOMOpopppp<OMpMCpcofxHp> t^op p> jn t- N H vp p> 
b^ Kii) t^vb N <n w it M M H b H, fn Vw .Jowob o Jr> V M M 

H H 



ri- w N *O rh 

O OJO H t-x Jf 

in ir>> 



OO O O O 



VO O Oi N VO 



a 



<i-H CTN int^O O *<* rj-i5 CO O OOO 00 PO PO O O Ch Oi POOO N 
N N N OJO U-) invO N TfN f^P^f^J^t > -. << *' *OJD N J~vN POPON r>t^ 

V o o <b <b V V V " 



N H o in t^>. 

N PO N tx N 



OlH(Q U-JPOOVO POO M lOCTi 
1^00 IT) O PO t^OJD t^ t>. t^ ^^P 



o o o o 



O O t^ * O O I** T}-00 t^NONN CT>OO CTiOOiOOOON 
CTiN PON POPOO POHOO O^CTiOOO ^VO O OlOO O N CO CO ^ 



P 1 r* 

H b 






POOO N OlD N POU">NVOlOVO O NOO N f^t^toO'OOO O^O^ 
Tt^N PON N POO'I'OOJO H H M N H CTi^O Htpvpcp 1010^ 
Jr)HHHHNHHHNNNNNHt^bpOHPobbHb 



por>mu-)ini^.ixN roooo 



Tj-N NNNiTjb SbSH M ir>H N O>O N POO^N IT) 



ootx t^o 



' * rt * * ' cS ' g rt " 

T o If "S 5 

3 . 0> . g . 0) . .cn<U .-^ 

If s .li^B^if .1 



S 

1 





3 -. 

-Q -S- 



sS* 

S 0< 






' 



ANALYTICAL DATA 



133 



J 

s 



I 



I 



& 



- 



o 


8 



o o o o 

P oo vp op in p poo vp 

8 88 8SS ' 

H H M M 



v * in 



o 

G 
C/} 

.a 


-3 

5 



Q 

g 

I 

2 



en 

G 



VO O Q <O IO 

M p N ip OJO ^p*O M O> M 



oo -^ Tt- Th PO M 



-Amino 

N. 



O OOOu->ONt>. -"ifCO * N CO w O CTiOO 
>O N 00 t^ CTi O O> >OVO PO N O O O CTiOO fOVO N M 00 ^VO >O O fO N 00 



H r^ob OMO N H N H t^ inoo on CTI t^ 
o ioinu-)io^ovo^ovo ootoir>to-^- 



co m 10 o 



iO OOO >O MOOVOOOHrhPO^-ONkD "">vO PO 

o p p vp N M op o>oo ojo p> H op fO'p '^P P P^P^P '^p > i y^^p p p 



' 55 



IT) ip 00 OO 00 ^ N 

pr>vb in io "^ io io ir>sib t^ N oo to POOO t^ r^ o> t>- POVO >o ooo 



IO OOO IO OOiOO^OOOO^POO PO<O 00 N O O 



M b b b M M b> b>ob obdbbiHO^HbHNi-ibMMbbbMbb 



' 



., 
t^jno o>p TI-W M^O t^vp oop^pcjpPopMpiHaiopp MM I 

NPONMNMNPOMHMHbMbMMMbMbbM HM 



O 

vo 



M o 



P ? 







85 ' 



p*oo j^ppp N^P p^r^p 1 ?^ w w w p M i r*p 1 ?* r P* r 1 " r P* 

t>. Io tx t-s biob t^vb i> K t^do o oo vb o * 






1? 

.1^ 



3 K 
O WS 



If 



I I 



~li 




3 3 IS 3 = 

ffiffiouffi 



134 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



3 "g 3 s 3 



gl-al'g J'gJ -g! 

* o o rt i-'gsa^ 





i -1 

O'gOO 
O 



O 



3 *g 

Olrt 



K 

O "g 

O 



s 

w 

-g 

rt 

-a 



c vnc H mCTi H H roco 

O\ O\ O\ O O O QiO O O O O 



88 

H H 



6 . 
?S5 



N -* tx w O in O rfVO tx O CO CTiCO t^-VO IO M CO CTiCO O OCO t^COO N 00 <y> O\ 

*t- a\ N p in p^i p op p ro p^vp w M-vp fx in H rt-Tt-jno p> N w vp TJ-H TJ-CO tx 



mooo tx H N tx crioo 



t^roC^cot^o inHOOVO N CO>H CTi'n^^O N M O CTifOO NOO HI mCO N 

jn in co tx^p t^ CTICO *p H tx o> ^i-*p M p tx txcp p t^vp cpNcop^ropNNp 

5-J 



in m m m i ^- 



ro txoo O lx.00 



O^O^OCTiO O 
M t^ Tj-O O 



OOH TJ-NOO 
w O t^ 
vbrvVH co&o bvb V 



ro tx -^GO l> H ^ rl- O O N 

cbob ^-N M corocob*b "^ 



VO N in ^i* O> O covD tx in C4 d N VO O H tx. d N tx< 

M fxHcopN i .^? 1 ^ r*"" r*"? 1 ^^^ ! i r* 

CTl t>. Vf- d txCO NCTlHOOHNHHobtxNinCTlN invO H H VO O O CO Tt-VO rx 



I. 



??? 



ONCOOPlCONNHtxdinTl- ^"O ts iDOO O H t^CO tx 

NOroHcsi'^'fOw o^^O ino txr^p p*p pidcp N jncp 



ino>oco H Tj-inCTiHtxTj-o 
p x i<p Tj-op N p txtxdcp p^vp p>; 



ro n n 



H H co ^co tx t> O co in co 
in p p> H tx 

bi tx o rt- in H ^*- 



inCTiCO cOCTi^txiOVO CTi<O N woo CTirod t^io^O rorororoN O roH 

inovb b txtxCiinro^b corororooi N b N b -^-H Vj-b b^ininb b b>o>cb 



ANALYTICAL DATA 



135 




; z 



m t^vo M H oo o ^o 

t^vo in irj t^. PO w vp f 

CXDOOCJONNPOHO 
CTlCTiOOOOOO O 



-am 

N. 



H H ro m CTioo O ro O 00 
iOCTiMCOpNt^l^p JO 
N ro fx t>.oo b> PO PO N co 



N tOOO CTlOO O O 

mop moo CTt p r> 



r 



moo t^ CT oovp m jo jo 



H <O M If) O> t^ -<*-VO O 

b cb t^ t^ c-Nib vb io t^ 






bbbbbbboH M 



. 

2 



bbibbbobb^boo 

H H H H M 



m 

do 



^~* V V 

> en 



S Ocw^X 
' 13 _ w 



- 

,cn c H 



e B s 

Wibjo 

^ 



m CJ N 



TABLE OF MILLIGRAMS OF AMINO NITROGEN CORRESPONDING TO i c c OF 
NITROGEN GAS AT n-3o C. ; 728-772 mm. PRESSURE. 



1 


728 


730 


732 


734 


736 


738 


740 


742 


744 


746 


748 


750 


11 


0-5680 


05695 


05510 


0-5725 


0-5745 


0-5760 


0-5775 


0-5790 


0-5805 


0-5820 


0-5840 


0-5855 


12 


o 5655 


0-5670 


0-5685 


05700 


0-5720 


0-5735 


0-5750 


0-5765 


0-5780 


0-5795 


0-5815 


0-5830 


3' 


o 5630 


0-5645 


05660*05675 05695 


0-5710 


o-5?25 


0-5740 


0-5755 


0-5770 


0-5785 


0-5805 


14 


05605 


0-5620 


0-5635 


o 5650 0-5665 


0-5680 


0-5700 


05715 


0-5730 


05745 


0-5760 


0-5775 


5 


o 5580 


0-5595 


0-5610 


0-5625 


0-5640 


0-5655 


0-5670 


0*5685 


0-5705 


0-5720 


0-5735 


0-5750 


1 6 


Q'5555 


0-5570 


0-5585 


0-5600 


0-5615 


0-5630 


0-5645 


0-5660 


0-5675 


0-5690 


0-5710 


0-5725 


17 


0-5525 


0-5540 


05555 


0-5575 


0-5590 


0-5605 


0-5620 


0-5635 


0-5650 


0-5.665 


0-5680 


0-5695 


18 


0-5500 


0-5515 


0-5530 


0-5545 


0-5560 


0-5580 


0-5595 


0-5610 


0-5625 


0-5640 


0-5655 


0-5670 


'9 


0-5475 


0-5490 


0-5505 


0-5520 


0-5535 


0-5550 


05565 


0-5580 


0-5595 


0-5610 


0-5630 


0-5645 


20 


0-5445 


0-5460 


0-5475 


0-5495 


0-5510 


05525 


0-5540 


0-5555 


0-5570 


0-5585 


0-5600 


0-5615 


21 


0-5420 


0-5435 


0-5450 


0-5465 


0-5480 


o 5495 


05510 


05525 


0-5540 


Q'5555 


0-5575 


0-5590 


22 


0-5395 


0-5410 


0-5425 


0-5440 


0-5455 


0-5470 


0-5485 


0-5500 


0-5515 


0-5530 


0'5545 


0-5560 


23 


0-5365 


0-5380 


0-5395 


0-5410 


0-5425 


0-5440 


0-5455 


0-5470 


0-5485 


0-5500 


O'55i5 


0-5530 


24 


Q'5335 


0-5350 


0-5365 


0-5380 


0-5400 


0-5415 


0-5430 


Q-5445 


0-5460 


0-5475 


0-5490 


0-5505 


25 


0-531 


0-5325 


0-5340 


Q'5355 


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0'5385 


0-5400 


0-5415 


0-5430 


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0-5475 










! 
















26 


0-5260 


0-5295 


0-5310 


0-5325 


0-5340 


0-5355 


0-5370 


0-5365 


0-5400 


0-5415 


0-5430 


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27 


0-5250 


0-5265 


0-5280 


0-5295 


0-5310 


0-5325 


0-5340 


0-5355 


0-5370 


o-5385 


0-5400 


0-5415 


28 


0-5220 


0-5235 


0-5250 


0-5265 


0-5280 


0-5295 


0-5310 


0-5325 


0-5340 


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0-5385 


29 


05195 


0-5210 


0-5220 


0-5235 


0-5250 


05265 


0-5280 


0-5295 


0-5310 


0-5325 


0-5340 


0-5355 


30 


o 5160 


05175 


o 5190 


0-5205 


0-5220 


05235 


0-5250 


0-5265 


0-5280 


0-5295 


0-5310 


0-5325 


/ 


752 


754 


756 


758 


760 


762 


764 


766 


768 


770 


772 




11 


0-5870 


0-5885 


0-5900 


0-5915 


Q'5935 


0-5950 


0-5965 


0-1,980 


0-5995 


0-6010 


0-6030 




12 


0-5845 


0-5860 


0-5875 


0-5890 


0-5905 


0-5925 


0-5940 


0-5955 


0-5970 


0-5985 


0-6000 




3 


0-5820 


0-5835 


0-5850 


0-5865 


0-5880 


0-5895 


0-5910 


0-5930 


x -5945 


0-5960 


0-5975 




14 


05790 


0-5805 


0-5825 


0-5840 


0-5855 


0-5870 


0-5885 


0-5900 


0-5915 


0-5935 


o-595o 




'5 


0-5765 


0-5765 


0-5795 


0-5810 


0-5830 


0-5845 


0-5860 


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0-5905 


0-5920 




16 


0-5740 


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0-5770 


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0-5800 


0-5815 


0-5830 


0-5850 


0-5865 


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7 


0-5710 


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6-5745 


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18 


05685 


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'9 


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20 


o 5630 


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21 


0-5605 


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0-5635 


0-5650 


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22 


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23 


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24 


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25 


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26 


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Reprinted from Van Slyke's paper, " The Quantitative Determination of Amino Groups. 
II.," J. Biol. Chem., 1912, 12, 275. 



BIBLIOGRAPHY. 

REFERENCES TO INTRODUCTION. 

ABDERHALDEN, E. , UNO M. KEMPE [1907, i]. Beitrag zur Kenntnis des Tryptophans und 
einiger seiner Derivate. 

Zeitschr. physiol. Chem., 52, 207-218. 
ABDERHALDEN, E. f UND A. WEIL [1912, 2]. \ 

ABDERHALDEN, E., UND A. WEIL [1913, i]. I See under Results of Hydrolysis. B. Various 
ABDERHALDEN, E., UND A. WEIL [1913, 2]. j Proteins. 
ABDERHALDEN, E., UND A. WEIL [1913, 3]. J 
ADENSAMER, A., UND PH. HOERNES [1905]. Ueber die Hydrolyse des Eiereiweisses. 

Monatsh. f. Chem., 26, 1217-1230. 
FISCHER, E., UND E. ABDERHALDEN [1904], See under Results of Hydrolysis. B. Phos- 

phoproteins, Caseinogen. 

FOREMAN, F. W. [1913, 2]. Die Prolinfraktion bei der Hydrolyse des Caseins. Isolierung 
von Aminobuttersdure. 

Biochem. Zeitschr., 56, i-io. 
FUNK, C. [1911]. Synthesis of dl-$ : ^-Dihydroxyphenylalanine. 

J. Chem. Soc. Trans., 99, 554-557. 

GALIMARD, J., L. LACOMME ET A. MOREL [1906]. Sur la vraie nature des glucoproteines-a 
de M. Lepierre. 

Compt. rend., 143, 298-300. 

GORTNER, R. A. [191 1]. A New Decomposition Product of Keratin which gives Millon's 
Reaction. 

J. Biol. Chem., 9, 355-357. 
GUGGENHEIM, M. [1913]. Dioxyphenylalanin, eine neue Aminosdure aus Vicia Faba. 

Zeitschr. physiol. Chem., 88, 276-284. 
HECKEL, F. [1908]. Ueber Leucin aus Kasein. 

Monatsh. f. Chem., 29, 15-21. 

HUGOUNENQ, L., ET A. MOREL [1906, 1907]. Sur la nature veritable desleuceines et gluco- 
proteines obtenues par P. Schiitzenberger dans le dedoublement des matieres pro- 
teiques. 

Compt. rend., 142, 1426-1428 ; Bull. Soc. Chim. [4], i, 154-164. 

LEPIERRE, C. [1903]. Les Glucoproteines comme nouveaux milieux de culture chimiquement 
dejinis pour V etude des microbes. 

J. Physiol. et Path, general, 5, 323-330. 
SAMEC, M. [1908]. Ueber des Leucin aus Nackenband. 

Monatsh. f. Chem., 29, 55-58. 
SCHUTZENBERGER, P. [1875]. Recherches sur les matieres albuminoides. 

Compt. rend., 80, 232-236. 
SCHUTZENBERGER, P. [1879]. Memoire sur les Matieres Albuminoides. 

Ann. de Chim. et de Phys. [5], 16, 289-419. 
SKRAUP, ZD. H. [1904]. Ueber die Hydrolyse des Kaseins. 

Ber., 37, 1596-1597 ; Monatsh. f. Chem., 25, 633-656 ; Zeitschr. physiol. Chem., 

42, 274-296. 
SKRAUP, ZD. H. [1905, i]. Ueber die Hydrolyse der Eiweissstojfe. II. Die Gelatine. 

Monatsh. f. Chem., 26, 243-264. 
SKRAUP, ZD. H. [1905, 2]. Berichtigung uber die Diaminosauren aus Casein und Gelatin. 

Monatsh. f. Chem., 26, 683. 

137 



138 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

SKRAUP, ZD. H., UNO R. WITT [1907]. Ubcr die Einwirknng von Bronilauge auf Casein. 

Monatsh. f. Chem., 28, 605-624. 

STEPHEN, H., AND C. WEIZMANN [1914]. Synthesis of dl-Tyrosine and dl--$ : 4-dioxyphenyl- 
alanine. 

J. Chem. Soc. Trans., 105, 1152-1155. 

THUDICHUM, J. L. W. [1901]. Die Chemische Konstitution des Gehirus des Menschen nnd 
der Tiere. 

Franz Pietzcker, Tubingen. 

TORQUATI, T. [1913, i]. Ueber die Gegenwart einer stickstoffhalligen Substanz in den 
Keimlingen von Vicia Faba. 

Arch. Farmacol. sperim., 15, 213-223 ; Chem. Centr., 1913, II, 517-518. 
TORQUATI, T. [1913, 2]. Ueber die Gegenwart einer stickstoffhaltigen Substanz in der 
griinen Hulse von Vicia Faba. 

Arch. Farmacol. sperim., 15, 308-312; Chem. Centr., 1913, II, 518. 
WOHLGEMUTH, J. [1904, 1905]. See under Results of Hydrolysis. B. Various Proteins. 



BIBLIOGRAPHY 139 



REFERENCES TO HYDROLYSIS. 

ABDERHALDEN, E., UNO C. BRAHM [1909]. See under Results of Hydrolysis. B. Silk. 
ABDERHALDEN, E., UND C. FUNK [1907]. Beitrag zur Kenntniss der beim Kochen von Casein 
mit -2^-prozentiger Schwefelsdure und mit starker Salzsaure entstehenden Spal- 
tungsprodukte. 

Zeitschr. physiol. Chem., 53, 19-30. 

ABDERHALDEN, E., UND R. HANSLIAN [1912]. Ueber die Verwendbarkeit der Estermethode 
zum Nachweis von Monoaminosduren neben Polypeptiden. 

Zeitschr. physiol. Chem., 77, 285-288. 

ABDERHALDEN, E., F. MEDIGRECEANU UND L. PINCUSSOHN [1909]. VergleichendeHydrolyse 
von Seide durch kochende Salzsaure, -z^-prozentiger Schwefelsdure, 2.^-prozentiger 
Natronlauge und heiss gesdttigte Barytlosung. 

Zeitschr. physiol. Chem., 6l, 205-209. 
COHN, R. [1896-97]. Ueber die quantitative Eiweissspaltung durch Salzsaure. I. 

Zeitschr. physiol. Chem., 22, 153-175. 
COHN, R. [1898-99]. Ueber eine quantitative Eiweissspalttmg durch Salzsaure. II. 

Zeitschr. physiol. Chem., 26, 395-410. 

FISCHER, E., UND E. ABDERHALDEN [1903, i]. Ueber die Verdauung einiger Eiweisskdrper 
durch Pankreasfermente. 

Zeitschr. physiol. Chem., 39, 81-94. 

FISCHER, E., UND E. ABDERHALDEN [1903, 2]. Ueber die Verdauung einiger Eiweisskdrper 
durch Pepsinsalzsditre und Pankreasfermente. 

Zeitschr. physiol. Chem., 40, 215-219. 

HENRIQUES, V., UND J. K. GJALDBAK [igio]. Ueber quantitative Bestimmung der im Pro- 
teine oder in dessen Abbauprodukten vorhandenen Peptidbindungen. 

Zeitschr. physiol. Chem., 67, 8-27. 

HERZIG, J., UND K. LANDSTEINER [1914], Ueber die Einwirkung von alkoholischen Salz- 
saure auf Eiweissstoffe. 

Biochem. Zeitschr., 67, 334-337- 
HLASIWETZ, H., UND J. HABERMANN [1871]. Ueber die Proteinstoffe. I. 

Ann. d. Chem., 159, 304-333. 
HLASIWETZ, H., UND J. HABERMANN [1873]. Ueber die Proteinstoffe. II. 

Ann. d. Chem., 169, 150-166. 

HUGOUNENQ, L., ET A. MOREL [igo8]. Contribution a Vetude de la constitution des matures 
proteiques. Nouvelle methode d'hydrolyse a Vacide fluorhydrique. 

Compt. rend., 146, 1291-1293 ; Bull. Soc. Chim. [4], 3, 1146-1151 ; J. Pharm. 
Chim. [6], 28, 486-493- 

HUGOUNENQ, L., ET A. MOREL [1909, i]. Contribution d Vetude de la constitution des ma- 
tieres proteiques par Vaction de Vacide fluorhydrique. Obtention de peptides natu- 
relles definies. 

Compt. rend., 148, 236-238. 

HUGOUNENQ, L., ET A. MOREL [1909, 2]. Uhydrolysefluorhydrique des matieres proteiques : 
nouveaux resultats. 

Compt. rend., 149, 41-43. 
OSBORNE, T. B., AND H. H. GUEST [iQii]. See under Results of 'Hydrolysis. B. 

Caseinogen. 

OSBORNE, T. B., AND D. BREESE JONES [1910, 3]. See under Results of Hydrolysis. A. 
PFANNL, M. [1910]. Ueber den Verlauf der Hydrolyse von Proteinen mit wasseriger oder 
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Monatsh. f. Chem., 31, 81-85. 

PRIBRAM, B. O. [1911]. Ueber die Anwendbarkeit der Estermethode bei Stoffwechsel- 
versuchen. 

Zeitschr. physiol. Chem., 71, 472-478. 



140 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

SKRAUP, ZD. H., UND W. TURK [1909]. Notiz iiber die Hydrolyse von Casein mit Salzsdui'c 
und Schwefelsaure. 

Monatsh. f. Chem., 30, 287-288. 
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Biochem. Zeitschr., 7, 45-101. 

SORENSEN, S. P. L. f UND H. JESSEN-HANSEN [1907]. Ueber die Ausfuhrung der Formol- 
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Biochem. Zeitschr., 7, 407-420. 
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of the Nitrogen. 
VAN SLYKE, D. D. [1912, 2]. The Conditions for Complete Hydrolysis of Proteins. 

J. Biol. Chem., 12, 295-299. 

WEIZMANN, C., AND G. S. AGASHE [1913]. Hydrolysis of Proteins with an Alcoholic Solu- 
tion of Hydrogen Chloride. 
Biochem. j., 7, 437-440. 



BIBLIOGRAPHY 141 



REFERENCES TO ISOLATION AND ESTIMATION OF TYROSINE. 

ABDERHALDEN, E. [1912]. Notiz zur Darstellung und quantitativen Bestimmung von 
Ty rosin und Glutaminsaure. 

Zeitschr. physiol. Chem., 77, 75-76. 

ABDERHALDEN, E. [1913]. Nachtrag zu : Der Gehalt der Proteine an I- Ty rosin und die 
Genauigkeit der Bestimmung dieser Aminosaure. 

Zeitschr. physiol. Chem., 85, 91. 

ABDERHALDEN, E., UND D. FUCHS [1913]. Uber den Gehalt der Proteine an l-Tyrosin und 
die Genauigkeit der Bestimmung dieser Aminosaure. 

Zeitschr. physiol. Chem., 83, 468-473. 

ABDERHALDEN, E., UND L. LANGSTEIN [1910]. See under References to Results of Hydro- 
lysis. B. Phosphoproteins. Caseinogen. 

ABDERHALDEN, E., UND Y. TERUUCHI [1906]. Notiz zur Darstellung von Tyrosin aus 
Seide. 

Zeitschr. physiol. Chem., 48, 528-529. 

BROWN, A. J., AND E. T. MILLAR [1906]. The Liberation of Tyrosine during Tryptic Pro- 
teolysis. 

J. Chem. Soc. Trans., 89, 145-155. 

FOLIN, O., AND W. DENIS [1912, i]. On Phosphotungstic-phosphomolybdic Compounds as 
Color Reagents. 

J. Biol. Chem., 12, 239-243. 

FOLIN, O., AND W. DENIS [1912, 2]. Tyrosine in Proteins as Determined by a New Colori- 
metric Method. 

J. Biol. Chem., 12, 245-251. 

FOLIN, O., AND W. DENIS [1913]. On the Tyrosine Content of Proteins. A Reply to Ab- 
derhalden and Fuchs. 

J. Biol. Chem., 14, 457-458. 

HABERMANM, J., UND R. EHRENFELD [1902]. Eine quantitative Methode zur Trennung des 
Leucins und Tyrosins. 

Zeitschr. physiol. Chem., 37, 18-28. 
LEVENE, P. A., AND D. D. VAN SLYKE [1908, 2]. Hydrolyse von Wittepeptone. 

Biochem. Zeitschr., 13, 440-457. 
LEVENE, P. A., AND D. D. VAN SLYKE [1910]. Note on Insoluble Lead Salts ofAmino Acids. 

J. Biol. Chem., 8, 285-286. 
MARSHALL, E. K. jun. [1913]. On the Preparation of Tyrosine. 

J. Biol. Chem., 15, 85-86. 

MILLAR, J. H. [1903]. A New Method for the Direct Estimation of Tyrosine in Mixtures 
of Amides and Amino Acids. 

Trans. Guinness Research Laboratory, I, Part I, 40-44. 

OSBORNE, T. B., AND S. H. CLAP? [1907, 8]. See under Results of Hydrolysis. B. Gliadins. 
OSBORNE, T. B., AND H. H. GUEST [1911]. Hydrolysis of Casein. 

J. Biol. Chem., 9, 333-353- 

PLIMMER, R. H. A., AND E. C. EAVES [1913]. The Estimation of Tyrosine in Proteins by 
Bromination. 

Biochem. J., 7, 297-310. 

TOTANI, G. [1916]. Feeding Experiments with a Dietary in which Tyrosine is Reduced to 
a Minimum. 

Biochem. J., 10, 382-398. 



142 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



REFERENCES TO ISOLATION AND ESTIMATION OF CYSTINE. 

ABDERHALDEN, E. [1903]. See under References to Results of Hydrolysis. B. Histones. 
ABDERHALDEN, E., UND F. PREGL [1905, 2]. See under References to Results of Hydrolysis. 

B. Albumins. 

EMBDEN, G. [igoo]. Ueber den Nachweis von Cystin und Cy stein unter den Spaltungspro- 
dukten der Eiweisskorper. 

Zeitschr. physiol. Chem., 32, 94-103. 
FOLIN, O. [igio]. On the Preparation of Cystine. 

J. Biol. Chem., 8, 9-10. 

FRIEDMANN, E. [1902]. Beitrdge zur Kenntniss der physiologischen Beziehnngen der Schwe- 
felhaltigen Eiweissabkommlinge. I. Ueber die Konstitution des Cystins. 
Beitr. chem. Physiol. Path., 3, 1-46. 

HOPKINS, F. G., AND H. SAVORY [1911]. See under Results of Hydrolysis. B. Globulins. 
MORNER, K. A. H. [1901-2]. Zur Kenntniss der Bindung des Schwefels in den Protcin- 
stoffen. 

Zeitschr. physiol. Chem., 34, 207-338. 
OSBORNE, T. B., AND S. H. CLAP? [1906]. See under Results of Hydrolysis. B. The 

Vegetable Proteins. Albumins. 
OSBORNE, T. B, AND S. H. CLAPP [1907, 6]. See under Results of Hydrolysis. B. The 

Vegetable Proteins. Globulins (crystallised). 
PLIMMER, R. H. A. [1913]. The Separation of Cystine and Tyrosine. 

Biochem. J., 7, 311-317- 

WINTERSTEIN, E. [igoi]. Ueber eine Methods zur Abscheidung der organischen Basen aus 
den Phosphorwolframsaureniederschlagen und uber das Verhalten des Cystins gegen 
Phosphor wolframsdure. 

Zeitschr. physiol. Chem., 34, 153-156. 



BIBLIOGRAPHY 143 



REFERENCES TO ISOLATION AND ESTIMATION OF TRYPTOPHAN. 

ABDERHALDEN, E., UND M. KEMPE [1907, i]. Beitrag zur Kenntnis des Tryptophans und 
einiger seiner Derivate. 

Zeitschr. physiol. Chem., 52, 207-218. 

FASAL, H. [1912]. Ueber eine colorimetrische Methode zur quantitativen Tryptophanbes- 
timmung und ilber den Tryptophangehalt der Horngebilde und anderer Eiweisskorper. 

Biochem. Zeitschr., 44, 392-401. 

FASAL, H. [1913]. Ueber den Tryptophangehalt normaler und pathologischer Hautgebilde 
und maligner Tumor en. 

Biochem. Zeitschr., 55, 88-95. 

HERZFELD, E. [1913, i]. Ueber Indolbildung bei der alkalischen Hydrolyse der Eiweiss- 
korper. 

Biochem. Zeitschr., 56, 82-94. 

HERZFELD, E. [1913, 2]. Ueber eine quantitative Tryptophanbestimmungsmethode. 

Biochem. Zeitschr., 56, 258-266. 

HOMER, A. [1915]. A Method for the Estimation of the Tryptophane Content of Proteins, 
Involving the Use of Baryta as a Hydrolyzing Agent. 

J. Biol. Chem., 22, 369-389. 

HOPKINS, F. G., AND S. W. COLE [1901, 2]. A Contribution to the Chemistry of Proteids. 
I. A Preliminary Study of a Hitherto Undescribed Product of Tryptic Digestion. 

]. Physiol., 27, 418-428. 
KURCHIN, E. [1914]. Tryptophanbestimmungen in normalen und pathologischen Nieren. 

Biochem. Zeitschr., 65, 451-459. 

LEVENE, P. A., AND C. A. ROUILLER [1906-7], On the Quantitative Estimation of Trypto- 
phan in Protein Cleavage Products. 

J. Biol Chem., 2, 481-484. 

LEVENE, P. A., UND C. A. ROUILLER [1907]. Ueber die Tryptophangruppe im Protein- 
molekul. 

Biochem Zeitschr., 4, 322-327. 

NEUBERG, C., UND N. POPOWSKY [1907]. Ueber Indolaminopropionsdure und ihre Halogen- 
verbindungen (Tiyptophanreaktion). 
Biochem. Zeitschr., 2, 357-382. 

SANDERS, J. A., AND C. E. MAY [1912-13]. A Method for the Determination of Try ptophan 
Derived from Protein. 

Biochem. Bull., 2, 373-378. 



144 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



REFERENCES TO ISOLATION AND ESTIMATION OF THE OTHER MONO- 

AMINO ACIDS. 

ABDERHALDEN, E. [igio]. Ailgemeine Technik und Isolierung der Monoaminosduren 
Handbuch der biochemischen Arbeitsmethoden. Band II, 470-497. 

ABDERHALDEN, E. [1912]. Notiz zur Darstellung und quantitativen Bestimmung von 
Ty rosin und Glutaminsdure. 

Zeitschr. physiol. Chem., 77, 75-76. 

ABDERHALDEN, E., UND K. KAUTZSCH [1912]. Nachweis des l-Prolins als primdres Spalt- 
produkt der Proteine. 

Zeitschr. physiol. Chem., 78, 96-114. 

ABDERHALDEN, E., UND A. WEIL [1912, 3]. Ueber die bei der Isolierung der Monoamino- 
sduren mit Hilfe der Estermethode entstehende Verluste III. Infreiheitsetzung der 
Ester mit Bleihydroxyd. 

Zeitschr. physiol. Chem., 8l, 226-227. 
EHRLICH, F. [1904], Ueber das naturliche Isomere des Leucins. I. 

Ber. Deutsch. Chem. Ges., 37, 1809-1840. 
FISCHER, E. [1901]. Ueber die Hydrolyse des Caseins durch Salzsdure. 

Zeitschr. physiol. Chem., 33, 151-176. 
FISCHER, E. [1902]. Notizen. II. Quantitative Bestimmung des Glycocolls. 

Zeitschr. physiol. Chem., 35, 229-230. 
FISCHER, E., UND C. HARRIES [1902]. Ueber Vacuumdestillation. 

Ber. Deutsch. Chem. Ges., 35, 2158-2163. 
FOREMAN, F. W. [1913, i, and unpublished]. Studies in Protein Hydrolysis. 

]. Agric. Sci., 4, 431-434- 

FOREMAN, F. W. [1914, i]. Quantitative Estimation of A spar tic and Glutaminic Acids in 
the Products of Protein Hydrolysis. 

Biochem. J., 8, 463-480. 
HABERMANN, J. [1875]. Zur Kenntniss der Glutaminsdure. 

Ann. der Chem., 179, 248-256. 
HLASIWETZ, H., UND J. HABERMANN [1873]. Ueber die Proteinstoffe. II. 

Ann. der Chem., 169, 150-166. 
KUTSCHER, FR. [1903]. Beitrdge zur Kenntniss der Eiweisskorper. II. 

Zeitschr. physiol. Chem., 38, 111-134. 
LEVENE, P. A. [1905]. See under References to Results of Hydrolysis. B. Derivatives of 

Proteins. 
LEVENE, P. A. [1906], Glycocoll Picrate. 

J. Biol. Chem., i, 413-414. 

LEVENE, P. A., AND D. D. VAN SLYKE [1908, i]. Zur Methodik der Destination der Amino- 
sdurenestern mittels der Geryk-Pumpe. 

Biochem. Zeitschr., 10, 214. 

LEVENE, P. A., AND D. D. VAN SLYKE [1908, 2]. See under References to Results of Hy- 
drolysis. B. Derivatives of Proteins. 
LEVENE, P. A., AND D. D. VAN SLYKE [1909, i]. The Leucin Fraction of Proteins. 

J. Biol. Chem., 6, 391-418. 

LEVENE, P. A., AND D. D. VAN SLYKE [1909, 2]. The Leucin Fraction in Casein and 
Edestin. 

J. Biol. Chem., 6, 419-430. 

LEVENE, P. A., AND D. D. VAN SLYKE [1910]. Note on Insoluble Lead Salts of Amino 
Acids. 

J. Biol. Chem., 8, 285-286. 

LEVENE, P. A., AND D. D. VAN SLYKE [1912]. The Composition and Properties of Glycocoll 
Picrate and the Separation of Glycocoll from Alanine. 
J. Biol. Chem., 12, 285-294. 



BIBLIOGRAPHY 145 

LEVENE, P. A., AND D. D. VAN SLYKE [1913]. The Separation of d-Alanine and d-Valine. 

J. Biol. Chem., 16, 103-120. 

OSBORNE, T. B., AND R. D. GILBERT [1906]. The Proportion of Ghitaminic Acid Yielded 
by Various Vegetable Proteins, when Decomposed by Boiling with Hydrochloric Acid. 

Amer. J. Physiol., 15, 333-356. 

OSBORNE, T. B., AND D. BREESE JONES [1910, 2]. Some Modifications of the Method in 
Use for Determining the Quantity of Mono-amino Acids Yielded by Proteins when 
Hydrolyzed with Acids. 

Amer. J. Physiol., 26, 212-228. 

OSBORNE, T. B., AND L. M. LIDDLE [igio, 2]. The Separation and Estimation ofAspartic 
and Ghitaminic Acids. 

Amer. J. Physiol., 26, 420-425. 
PRIBRAM, B. O. [1910]. Ueber eine Modification bei der Fischer'chen Estermethode. 

Monatsh. f. Chem., 31, 51-54. 
SIEGFRIED, M. [1891]. Zur Kenntniss der Spaltnngsprodnkte der Eiweisskorper. 

Ber. Deutsch. chem. Ges., 24, 418-432. 

VAN SLYKE, D. D. [1911, 3]. Quantitative Determination of Prolin Obtained by the Ester 
Method in Protein Hydrolysis. Prolin Content of Casein. 

J. Biol. Chem., 9, 205-207. 

ZELINSKY, N., A. ANNENKOFF UND J. KULIKOFF [1911]. Ueber ein einfaches und beqitemes 
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Zeitschr. physiol. Chem., 7, 459-470. 



PT. I. 10 



146 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



REFERENCES TO ISOLATION AND ESTIMATION OF THE DI-AMINO ACIDS 

KOSSEL, A., UND FR. KUTSCHER [1900-1]. Beitrage zur Kenntniss der Eiweisskorper. 

Zeitschr. physiol. Chem., 31, 165-214. 
KOSSEL, A., UND A. J. PATTEN [1903]. Zur Analyse der Hexonbasen. 

Zeitschr. physiol. Chem., 38, 39-45. 

KOSSEL, A , UND H. PRINGLE [1906]. Ueber Protamine und Histone. Methode der quantita- 
tiven Spaltung des Histopeptons. 

Zeitschr. physiol. Chem., 49, 318-321. 

OSBORNE, T. B., C. S. LEAVENWORTH AND C. A. BRAUTLECHT [1908]. The Different 
Forms of Nitrogen in Proteins. 

Amer. J. Physiol., 23, 180-200. 
STEUDEL, H. [1903]. Das Verhalten der Hexonbasen zur Pikrolonsdure. 

Zeitschr. physiol. Chem., 37, 219-220. 
STEUDEL, H. [1910]. Isolierung von Histidin, Lysin und Arginin. 

Handbuch der bio-chemischen Arbeitsmethoden von E. Abderhalden, Band II, 

498-509. 

SCHULZE, E., UND E. WiNTERSTEiN [1902]. Ueber die Trennung des Phenylalanins von 
anderen Aminosduren. 

Zeitschr. physiol. Chem., 35, 210-220. 
WEISS, F. [1907]. Untersuchungen iiber die Bildung des Lachsprotamins. 

Zeitschr. physiol. Chem., 52, 107-120. 

WEISS, M., UND N. SSOBOLEW [1913]. Ueber ein colorimetristhes Verfahren zur quantita- 
tiven Bestimmung des Histidins. 

Biochem. Zeitschr., 58, 119-129. 



BIBLIOGRAPHY 147 



REFERENCES TO RESULTS OF HYDROLYSIS. A. 

ABDERHALDEN, E. [1910]. Beitrag zur Kenntnis der bei den totalen Hydrolyse von Pro- 
teinen auftretenden Aminosauren. 

Zeitschr. physiol. Chem. , 68, 477-486. 

ABDERHALDEN, E., UNO K. KAUTZSCH [1912]. Nachweis des l-Prolins als primilres Spalt- 
produkt der Proteine. 

Zeitschr. physiol. Chem., 78, 96-114. 

ABDERHALDEN, E., UND A. WEIL [ign, 2]. Ueber die bei der Isolierung der Monoamino- 
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Zeitschr. physiol. Chem., 74, 445-471. 

ABDERHALDEN, E., UND A. WEIL [1912, i]. Ueber die bei der Isolierung der Monoamino- 
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Zeitschr. physiol. Chem., 77, 59-74. 

ANDERSEN, A. C., UND R. ROED-MULLER [1915]. Zur Kenntniss der Eiweisskorper. II. 
Die Verbindung von Ammoniak in die Eiweisskorper. 

Biochem. Zeitschr., 70, 442-463. 

ENGELAND, R. [1909]. Ueber Hydrolyse von Casein und den Nachweis der dabei ent- 
standenen Monoaminosduren. 

Ber. Deutsch. chem. Ges., 42, 2962-2969. 
ENGELAND, R. [1910]. Ueber erschopfende Methylierung einiger Aminosduren. 

Ber. Deutsch. chem. Ges., 43, 2662-2664. 
ENGELAND, R. [1914]. Ueber den Nachweis von Monoaminosduren. 

Zeitschr. f. Biol., 63, 470-476. 

GORTNER, R. A. [1916]. The Origin of the Humin Formed by the Acid Hydrolysis of 
Proteins. II. Hydrolysis in the Presence of Carbohydrates and of Aldehydes. 

J. Biol. Chem., 26, 177-204. 

GORTNER, R. A., AND M. J. BLISH [1915]. On the Origin of the Humin Formed by the Acid 
Hydrolysis of Proteins. 

J. Amer. Chem. Soc., 37, 1630-1636. 
GRINDLEY, H. S., AND M. E. SLATER [1915]. See under References to Distribution of 

Nitrogen in Proteins. 

HENRIQUES, V., UND J. K. GJALDBAK [1910], Ueber quantitative Bestimmung derim Pro- 
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Zeitschr. physiol. Chem., 67, 8-27. 

MAILLARD, L. C. [1912]. Action des acides amines sur les sucres ; formation des melanoi- 
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Compt. rend., 154, 66-68. 
MAILLARD, L. C. [1913]. Otigin des bases cycliques du goudron de houille. 

Compt. rend., 151, 850-852. 
MAILLARD, L. C. [1916]. Sur la formation des bases pyridiques a partir des albuminoides. 

Compt. rend., 162, 757-758. 
OSBORNE, T. B., AND D. BREESE JONES [1910, i]. Some Points in the Analysis of Proteins. 

J. Biol. Chem., 7, Proc., viii-ix. 

OSBORNE, T. B., AND D. BREESE JONES [igio, 3]. A Consideration of the Sources of Loss 
in Analysing the Products of Protein Hydrolysis. 

Amer. J. Physiol., 26, 305-328. 

OSBORNE, T. B., C. S. LEAVENWORTH AND C. A. BRAUTLECHT [1908]. The Different 
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Amer. J. Physiol., 23, 180-200. 

PICTET, A., ET T. Q. CHOU [1916]. Sur la formation de bases pyridiques etisoquinoleiques 
a partir de la caseine. 

Compt. rend., 162, 127-129. 

10* 



148 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

PITTOM, W. W. P. [1914]. Studies in Protein Hydrolysis. 

Biochem. J., 8, 157-169. 

ROXAS, M. L. [1916]. The Reaction between Amino Acids and Carbohydrates as a Probable 
Cause of Humin Formation. 

J. Biol. Chem., 27, 71-93. 
SAMUELY, F. [1902]. Uber die aus Eiweiss hervorgehenden Melanine. 

Beitr. chem. Physiol. Path., 2, 355-388. 

SKRAUP, ZD. H., UNO E. v. HARDT-STREMAYR [1908]. Ueber den sogenannten Amidstick- 
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Monatsh. f. Chem., 29, 255-262. 

VAN SLYKE, D. D. [1912, 2]. The Conditions for Complete Hydrolysis of Proteins. 
J. Biol. Chem., 12, 295-299. 



BIBLIOGRAPHY 149 

REFERENCES TO RESULTS OF HYDROLYSIS. B. 
PROTAMINES. 

ABDERHALDEN, E. [1904]. Die Monoaminosauren des Salmins. 

Zeitschr. physiol. Chem., 41, 55-58. 

DEZANI, S. [1908]. Protein Bases of the Sperm and Ovaries of the Tunny Fish and their 
Products of Hydrolysis. 

Giorn. R. Accad. Med. Torino, 14 ; J. Chem. Soc. Abs., 1909, ii. 163-164. 
KOSSEL, A. [1896-97]. Ueber die basischen Stoffe des Zellkerns. 

Zeitschr. physiol. Chem., 22, 176-187 
KOSSEL, A. [1898]. Ueber die Constitution der einfachsten Eiweissstoffe. 

Zeitschr. physiol. Chem., 25, 165-189. 
KOSSEL, A. [1898-99]. Weitere Mittheilungen uber die Protamine. 

Zeitschr. physiol. Chem., 26, 588-592. 
KOSSEL, A. [1903]. Sur les Protamines et la Constitution des Matieres Albuminoides. 

Bull. Soc. Chim. [iii], 29, Conference faite le 30 Mai. 
KOSSEL, A. [1903-4]. Zur Kenntnis des Salmins. 

Zeitschr. physiol. Chem., 40, 311-315. 
KOSSEL, A. [1905], Einige Bemerkungen uber die Bildung der Protamine im Tierkorper. 

Zeitschr. physiol. Chem., 44, 347-352. 
KOSSEL, A. [1906]. Ueber das Scombrin. 

Zeitschr. physiol. Chem., 49, 310-311. 
KOSSEL, A. [1910]. Zur Chemie der Protamine. 

Zeitschr. physiol. Chem., 69, 138-142. 
KOSSEL, A. [1913]. W< itere Mitteilungen uber die Proteine der Fischspermien 

Zeitschr. physiol. Chem., 88, 163-185. 
KOSSEL, A., UNO H. D. DAKIN [1903-4]. Beitrdge zum System der einfachsten Eiweisskorper. 

Zeitschr. physiol. Chem., 40, 565-571. 
KOSSEL, A., UND H. D. DAKIN [1904]. Ueber Salmi n und Clupein. 

Zeitschr. physiol. Chem., 41, 407-415. 

KOSSEL, A., UND H. D. DAKIN [1905]. Weitere Beitrdge zum System der einfachsten 
Eiweisskorper. 

Zeitschr. physiol. Chem., 44, 342-346. 

KOSSEL, A., UND F. EDLBACHER [1913]. Ueber einige Spaltungsprodukte des Thynnins und 
Percins. 

Zeitschr. physiol. Chem., 88, 186-189. 
KOSSEL, A., UND FR. KUTSCHER [1900-1]. Beitrdge zur Kenntniss der Eiweisskorper. 

Zeitschr. physiol. Chem., 31, 165-214. 
KURAJEFF, D. [1898-99]. Ueber das Protamin aus dem Spermatozoen der Makrele. 

Zeitschr. physiol. Chem., 26, 524-534. 
MALENUCK, W. D. [1908]. Zur Chemie der Protamine. 

Zeitschr. physiol. Chem., 57, 99-112. 

MIESCHER, FR. [1874]. Das Protamin, eineneue organische Basis aus dem Samenfdden des 
Rheinlachses. 

Ber. Deutsch. Chem. Ges., 7, 376-379. 
MORKOWIN, N. [1899]. Bin Beitrag zur Kenntniss der Protamine. 

Zeitschr. physiol. Chem., 28, 313-317. 

PICCARD, J. [1874]. Ueber Protamin, Guanin und Sarkin, als Bestandtheile des Lacks- 
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Ber. Deutsch. Chem. Ges., 7, 1714-1719. 
TAYLOR, A. E. [1908-9]. On the Composition and Derivation of Protamin. 

J. Biol. Chem., 5, 389-398. 
ULPIANI, C. [1902]. Sulla base proteica dello sperma di tonno. 

Gaz. chim. ital., 32, ii, 215-234. 
WEISS, F. [1907]. Untersuchungen uber die Bildung des Lachsprotamins. 

Zeitschr. physiol. Chem., 52, 107-120. 



150 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



HlSTONES. 

ABDERHALDEN, E. [1903]. Hydrolyse des Krystallisirten Oxy haemoglobins aus Pferdeblut. 

Zeitschr. physiol. Chem., 37, 484-494. 

ABDERHALDEN, E., UNO L. BAUMANN [1907]. Die Monoaminosauren des Krystallisierten 
Oxy haemoglobins aus Hundeblut. 

Zeitschr. physiol. Chem., 51, 397-403. 

ABDERHALDEN, E., UND F. MEDIGRECEANU [1909]. Beitrag zur Kenntnis des Oxy haemo- 
globins verschiedener Tierarten. 

Zeitschr. physiol. Chem., 59, 165-169. 
ABDERHALDEN, E., UND P. RONA [1904]. Die Abbauprodukte des " Thymushistons ". 

Zeitschr. physiol. Chem., 41, 278-283. 
BANG, I. [1899]. Studien uber Histon. 

Zeitschr. physiol. Chem., 27, 463-486. 
EHRSTROM, R. [1901]. Ueber ein neues Histon aus Fischsperm i. 

Zeitschr. physiol. Chem., 32, 350 354. 

FISCHER, E., UND E. ABDERHALDEN [1902]. Hydrolyse des Oxy haemoglobins durch Salzsdure. 

Zeitschr. physiol. Chem., 36, 268-276. 
KOSSEL, A. [1883-84]. Ueber einen peptonartigen Bestandtheil des ZeUkerus. 

Zeitschr. physiol. Chem., 8, 511-515. 
KOSSEL, A. [1906]. Ueber Protamine und Histone. 

Zeitschr. physiol. Chem., 49, 307. 

KOSSEL, A., UND FR. KUTSCHER [1900-1]. See under Protamines. 
KUTSCHER, FR. [1903]. Beitrdge zur Kenntnis der Eiweisskorper. II. 

Zeitschr. physiol. Chem., 38, 111-134. 
LILIENFELD, L. [1894]. ZuY Chemie der Leucocyten. 

Zeitschr. physiol. Chem., 18, 473-486. 
MATTHEWS, A. [1897]. Zur Chemie der Spermatozoen. 

Zeitschr. physiol. Chem., 23, 399-411. 
MIESCHER, FR. [1874]. Die Spermatozoen einiger Wiebelthiere. 

, Die Histochemischen und Physiologischen Arbeiten. Band I. F. C. W. Vogel, 

Leipzig, 1897. 
PROSCHER, FR. [1899]. Ein Beitrag zur Erforschung der Constitution des Eiweissmolekuls. 

Zeitschr. physiol. Chem., 27, 114-122. 
SCHULZ, FR. N. [1898]. Der Eiweisskorper des Hamoglobins. 

Zeitschr. physiol. Chem., 24, 449-481. 






BIBLIOGRAPHY 151 



ALBUMINS. 

ABDERHALDEN, E. [1902-3]. Hydrolyse des Krystallisirten Serumalbumins aus Pferdeblut. 

Zeitschr. physiol. Chem., 37, 495-498. 
ABDERHALDEN, E. [1906]. Nachtrag. 

Zeitschr. physiol. Chem., 48, 518. 

ABDERHALDEN, E., UNDA. HUNTER [1906, i]. Vorlaufige Mitteilung uber den Gehalt der 
Eiweisskorper der Milch an Glykokoll. 

Zeitschr. physiol. Chem., 47, 404-406. 

ABDERHALDEN, E., UNO F. PREGL [1905, 2]. Die Monoaminosauren des Krystallisierten 
Bier albumins. 

Zeitschr. physiol. Chem., 46, 24-30. 

ABDERHALDEN, E., UNO H. PRIBRAM [1907]. Die Monoaminosauren des Albumins aus Kuh- 
milch. 

Zeitschr. physiol. Chem., 51, 409-414. 

ABDERHALDEN, E., UND A. SCHITTENHELM [1906]. See under Phosphoproteins. Caseinogen. 
ABDERHALDEN, E., UND SLAVU [1909]. Vergleichende Untersuchung iiber den Gehalt der 
Serumeiweisskorper verschiedener Blutarten an Tyrosin, Glutaminsaure und an 
Glykokoll. 

Zeitschr. physiol. Chem., 59, 247-248. 
CHAPMAN, H. G., AND J. M. PETRIE [1909]. The Hexone Bases from Egg-White. 

J. Physiol., 39, 341-345- 
HUGOUNENQ, L., ET J. GALiMARD [1906]. Sur les acides diamines derives de Vovalbumine- 

Compt. rend., 143, 242-243. 

HUGOUNENQ, L., ET A. MOREL [1906]. See under References to Introduction. 
MORNER, K. A. H. [1901-2]. Zur Kenntniss der Bindung des Schwefels in den Protein- 
stoffen. 

Zeitschr. physiol. Chem., 34, 207-338. 

OSBORNE, T. B M D. BREESE JONES AND C. S LEAVENWORTH [1909]. Hydrolysis of Cry- 
stallized Albumin from Hen's Egg. 
Amer. J. Physiol., 24, 252-262. 

OSBORNE, T. B., D. D. VAN SLYKE, C. S. LEAVENWORTH AND M. VINOGRAD [1915]. Some 
Products of Hydrolysis ofGliadin, Lactalbumin and the Protein of the Rice Kernel. 
J. Biol. Chem., 22, 259-280. 



GLOBULINS. 

ABDERHALDEN, E. [1905]. Abbau und Aufbau der Eiweisskorper im tierischen Organismus. 
Hydrolyse des Serumglobulins. 

Zeitschr. physiol. Chem., 44, 17-52. 

ABDERHALDEN, E., UND O. ROSTOSKI [1905, 2]. Beitrag zur Kenntnis des Bence-J 'ones 'chen 
Eiweisskorpers. 

Zeitschr. physiol. Chem., 46, 125-135. 

ABDERHALDEN, E., UND F. SAMUELY [1905, 2]. Beitrag zur Frage nach der Assimilation 
des Nahrungseiweiss im tierischen Organismus. 

Zeitschr. physiol. Chem., 46, 193-200. 

ABDERHALDEN, E., UND SLAVU [1909], See above, under Albumins. 

ABDERHALDEN, E., UND A. VOITINOVICI [1907, 3]. Wetter e Beitrdge zur Kenntnis der 
Zusammensetzung der Proteine. II. Hydrolyse des Blutfibrins. 

Zeitschr. physiol. Chem., 52, 371-374. 

HOPKINS, F. G., AND H. SAVORY [1911]. A Study ofBence-Jones Protein, and of the Meta- 
bolism in Three Cases of Bence-jfones Proteinuria. 

J. Physiol. ,42, 189-250. 
LEVENE, P. A., AND D. D. VAN SLYKE [1908, 2]. Hydrolyse von Wittepeptone. 

Biochem. Zeitschr., 13, 440-457. 

MORNER, K. A. H. [1901-2]. See above, under Albumins. 

NEUBERG, C., UND N. POPOWSKY [1907]. See under References to Isolation and Estimation 
of Tryptophan. 



152 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



THE VEGETABLE PROTEINS. 
ALBUMINS. 

OSBORNE, T. B., AND S. H. CLAP? [1906]. The Chemistry of the Protein Bodies of the 
Wheat Kernel. III. Hydrolysis of the Wheat Proteins. 

Amer. J. Physiol., 17, 231-265. 

OSBORNE, T. B., AND F. W. HEYL [1908, 3]. Hydrolysis of Legumelin from the Pea. 
J. Biol. Chem., 5, 197-205. 



GLOBULINS (crystallised). 

ABDERHALDEN, E. [1902]. Hydrolyse des Edestins. 

Zeitschr. physiol. Chem., 37, 499-505. 
ABDERHALDEN, E. [1903]. Nachtrag zur Hydrolyse des Edestins. 

Zeitschr. physiol. Chem., 40, 249-250. 

ABDERHALDEN, E., UNO O. BERGHAUSEN [1906]. Die Monoaminosauren -von aus Kurbis- 
samen dargestelltem Krystallinischen Eiweiss. 

Zeitschr. physiol. Chem., 49, 15-20. 

ABDERHALDEN, E., UNO B. REINBOLD [1905]. Die Monoaminosauren des " Edestins " ans 
Sonnenblumensamen und dessen Verhalten gegen Pankreasseft. 

Zeitschr. physiol. Chem., 44, 284-293. 

ABDERHALDEN, E., UND O. ROSTOSKI [1905, i]. Die Monoaminosauren des " Edestins " 
aus Baumwollsamen und dessen Verhalten gegen Magensaft. 

Zeitschr. physiol. Chem., 44, 265-275. 
KOSSEL, A., UND A. J. PATTEN [1903]. Zur Analyse der Hexonbasen. 

Zeitschr. physiol. Chem., 38, 39-45. 
OSBORNE, T. B., AND S. H. CLAPP [1907, 2]. Hydrolysis of Excelsin. 

Amer. J. Physiol., 19, 53-60. 

OSBORNE, T. B., AND S. H. CLAPP [1907, 6], Hydrolysis of the Crystalline Globulin of the 
Squash Seed. 

Amer. J. Physiol., 19, 475-481. 
OSBORNE, T. B., AND R. D. GILBERT [1906]. See under Isolation and Estimation of the 

other Mono-amino Acids. 
OSBORNE, T. B., AND L. M. LIDDLE [1910, i]. Notes on the Analysis of Edestin and Zein. 

Amer. J. Physiol,, 26, 295-304. 

SCHULZE, E., UND E. WiNTERSTEiN [1901, i]. Ueber die Ausbeute an Hexonbasen, die aus 
einigen pflanzlichen Eiweissstoffen zu erhalten ist. 
Zeitschr. physiol. Chem., 33, 547-573. 



BIBLIOGRAPHY 153 



GLOBULINS (amorphous). 

ABDERHALDEN, E. [1906]. Anmerkung. 

Zeitschr. physiol. Chem., 47, 358. 
ABDERHALDEN, E., UNO B. BABKIN [1906], Die Monoaminosauren des Legumins. 

Zeitschr. physiol. Chem., 47, 354-358. 

ABDERHALDEN, E., UND J. B. HERRICK [1905]. Beitrag zur Kenntnis der Zusammen- 
setzung des Conglutins aus Sam en von Lupinus. 

Zeitschr. physiol. Chem., 45, 479-485. 

ABDERHALDEN, E., UND Y. TERUUCHI [1905, i]. Die Zusammensetzung von aus Kiefern- 
samen dargestelltem Eiweiss. 

Zeitschr. physiol. Chem., 45, 473-478. 
FOREMAN, W. [ign]. Hydrolysis of the Protein of Linseed. 

J. Agric. Science, 3, 358-382. 
JOHNS, C. O., AND D. B. JONES [1917]. See under References to Analysis of Proteins by 

Distribution of Nitrogen. 
OSBORNE, T. B., AND S. H. CLAP? [1907, i]. Hydrolysis of Phaseolin. 

Amer. J. Physiol., 18, 295-308. 
OSBORNE, T. B., AND S. H. CLAPP [1907, 4]. Hydrolysis of Leguminfrom the Pea. 

J. Biol. Chem., 3, 219-225. 
OSBORNE, T. B., AND S. H. CLAPP [1907, 5], Hydrolysis of Glycinin from the Soy Bean. 

Amer. J. Physiol., 19, 468-474. 
OSBORNE, T. B., AND S. H. CLAPP [1907, 7]. Hydrolysis of Amandin from the Almond. 

Amer. J. Physiol., 20, 470-476. 

OSBORNE, T. B., AND F. W. HEYL [1908, i]. Hydrolysis of Vignin of the Cow-Pea (Vigna 
Sinensis). 

Amer. J. Physiol., 22, 362-372. 

OSBORNE, T. B., AND F. W. HEYL [1908, 2]. Hydrolysis of Vicilinfrom the Pea (Pisum 
Sativum). 

J. Biol. Chem., 5, 187-195. 
OSBORNE, T. B., AND F. W. HEYL [1908, 4]. Hydrolysis of Vetch Legwnin. 

Amer. J. Physiol., 22, 423-432. 

SCHULZE, E., UND E. WiNTERSTEiN [1901, i]. Ueber die Ansbeute an Hexonbasen, die 
aus einigen pflanzlichen Eiweissstoffen zu erhalten ist. 

Zeitschr. physiol. Chem., 33, 547-573. 
SJOLLEMA, B., UND I. J. RiNKES [1911-12]. Die Hydrolyse des Kartoffeleiweisses. 

Zeitschr. physiol. Chem., 76, 369-384. 
YOSHIMURA, K. [1910]. Ueber das Eiweiss aus Samen von Finns Koraienis Sieb. el Zucc. 

Zeitschr, Nahr. Genussm. , 19, 257-260. 



154 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



GLIADINS. 

ABDERHALDEN, E., UNO F. SAMUELY [1905, i]. Die Zusammensetzung des " Gliadins " des 
\Veizenmehles. 

Zeitschr. physiol. Chem., 44, 276-283. 

ABDERHALDEN, E., UNO F. SAMUELY [1905, 2]. Beitrag zur Frage nach der Assimilation 
des Nahrungseiweiss im tierischen Organismus. 

Zeitschr. physiol. Chem., 46, 193-200. 
JOHNS, C. O., AND J. F. BREWSTER [1916]. See under References to Distribution of Nitrogen 

in Proteins. 
KLEINSCHMITT, A. [1907]. Hydrolyse des Hordeins. 

Zeitschr. physiol. Chem., 54, 110-118. 
KOSSEL, A., UND FR. KUTSCHER [1901-2]. Beitrage zur Kenntniss der Eiweisskorper. I. 

Zeitschr. physiol. Chem., 31, 165-214. 
KUTSCHER, F. [1903]. Beitrage zur Kenntniss der Eiweisskorper. II. 

Zeitschr. physiol. Chem., 38, 111-134. 
LANGSTEIN, L. [1903]. Hydrolyse des Zeins durch Salzsdure. 

Zeitschr. physiol. Chem., 37, 508-512. 

OSBORNE, T. B., AND S. H. CLAP? [1906]. See under Vegetable Albumins. 
OSBORNE, T. B., AND S. H. CLAPP [1907, 3]. Hydrolysis of Hordein. 

Amer. J. Physiol., 19, 117-124. 

OSBORNE, T. B., AND S. H. CLAPP [1907, 8]. Hydrolysis of the Proteins of Maize, Zea 
Mays. 

Amer. J. Physiol, 20, 477-493- 
OSBORNE, T. B., AND S. H. CLAPP [1907, 9]. The Hydrolysis of Gliadin front Rye. 

Amer. J. Physiol., 20, 494-499. 

OSBORNE, T. B., AND H. H. GUEST [1911, 2]. Analysis of the Products of Hydrolysis of 
Wheat Gliadin. 

J. Biol. Chem., 9, 425-438. 

OSBORNE, T. B., AND L. M. LIDDLE [1910, i]. See under Vegetable Globulins (crystallised). 
OSBORNE, T. B., AND C. S. LEAVENWORTH [1913]. Do Gliadin and Zein Yield Lysine ? 

J. Biol. Chem., 14, 481-488. 

OSBORNE, T. B., D. D. VAN SLYKE, C. S. LEAVENWORTH AND M. VINOGRAD [1915]. Some 
Products of Hydrolysis of Gliadin, Lactalbumin and the Protein of the Rice Kernel. 

J. Biol. Chem., 22, 259-280. 
SUZUKI, U., K. YOSHIMURA AND S. FUJI [1909]. Proteins of Rice Seeds. 

J. Coll. Agric., Tokyo, I, 77-88 ; J. Chem. Soc. Abs., 1909, ii, 927. 



GLUTELINS. 

ABDERHALDEN, E., UND Y. HAMALAINEN [1907]. Die Monoaminosduren des Avenins. 

Zeitschr. physiol. Chem., 52, 515-520. 
ABDERHALDEN, E., UND F. MALENGREAU [1906], Die Monoaminosduren des Glutens. 

Zeitschr. physiol. Chem., 48, 513-518. 

KOSSEL, A., AND F. KUTSCHER [1901-2]. See under Gliadins. 
OSBORNE, T. B., AND S. H. CLAPP [1906]. See under Vegetable Albumins. 
OSBORNE, T. B., AND S. H. CLAPP [1907, 8]. See under Gliadins. 
OSBORNE, T. B., D. D. VAN SLYKE, C. S. LEAVENWORTH AND M. VINOGRAD [1915]. See 

under Gliadins. 



BIBLIOGRAPHY 155 

PHOSPHOPROTEINS. 
Caseinogen. 

ABDERHALDEN, E. [1905]. Abbau und Aufbau der Eiweisskorper im tierischen Organismus. 

Zeitschr. physiol. Chem., 44, 17-52. 

ABDERHALDEN, E., UND A. SCHITTENHELM [1906]. Vergleichung der Zusammensetzung 
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Zeitschr. physiol. Chem., 47, 458-465. 

ABDERHALDEN, E., UND L. LANGSTEIN [igio]. Vergleichende Untersuckttng des Caseins 
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Zeitschr. physiol. Chem., 66, 8-12. 
FISCHER, E. [1901]. Ueber die Hydrolyse des Caseins durch Salzsdure. 

Zeitschr. physiol. Chem., 33, 151-176. 

FISCHER, E. [1903]. Nachtrag zur Hydrolyse des Caseins mid Seidenfibroimns durch 
Sduren. 

Zeitschr. physiol. Chem., 39, 155-158. 

FISCHER, E., UND E. ABDERH \LDEN [1904]. Notizen iiber Hydrolyse von Proteinstoffen. 
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Zeitschr. physiol. Chem., 42, 540-544. 

FOREMAN, F. W. [unpublished]. Studies in Protein Hydrolysis. 

HART, E. [igoi], Ueber die quantitative Bestimmung der Spaltungsprodukte von Eiweiss- 
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Zeitschr. physiol. Chem., 33, 347-362. 

MORNER, K. A. H. [1901-2]. Zur Kenntniss der Bindung des Schwefels in den Protein- 
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Zeitschr. physiol. Chem., 34, 207-338. 

HOPKINS, F. G., AND S. W. COLE [1901,; 2]. A Contribution to the Chemistry of the Pro- 
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T. Physiol., 27, 418-428. 
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J. Biol. Chem., 9, 333-353- 
SKRAUP, ZD. H. [1905]. Ueber den Gehalt des Caseins an Glykokoll und Alanin. 

Monatsh. f. Chem., 26, 1343-1349. 

WEITZENBOCK, R. [1906]. Ueber das Vorkommen von Isoleucin im Kasein. 
Monatsh. f. Chem., 27, 831-837. 

Vitellin, etc. 

ABDERHALDEN, E., UND A. HUNTER [1906, 2]. Hydrolyse des im Eigelb des Huhnereies 
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Zeitschr. physiol. Chem., 48, 505-512. 

ABDERHALDEN, E., UND M. KEMPE [1907, 2]. Vergleichende Untersuchung iiber den 
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GALIMARD, J. [1904]. Sur une albumine extraite des oeufs de grenouille. 

Compt. rend., 138, I354- T 355' 
HEDIN, S. G. [1895-96]. Ueber die Bildung von Arginin aus Proteinkorpern. 

Zeitschr. physiol. Chem., 21, 155-168. 

HUGOUNENQ, L. [1904]. Sur une albumine extraite des oeufs de poissons ; chimie comparee 
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Compt. rend., 138, 1062-1064. 
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J. Biol. Chem., 2, 127-133. 

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Egg. 

Amer. J. Physiol., 24, 153-160. 



156 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



SCLEROPROTEINS. 
Silk. 

ABDERHALDEN, E. [1908, i]. Die Monoaminosauren des " Byssus " von Pinna nobilis L. 

Zeitschr. physiol. Chem., 55, 236-240. 

ABDERHALDEN, E. [1908, 2]. Vergleichende Untersuchungen uber die Zusammensetzung und 
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Zeitschr. physiol. Chem., 58, 334-336. 

ABDERHALDEN, E. [1911]. Weiterer Beitrag zur Kenntnis der Zusammensetzung verschie- 
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Zeitschr. physiol. Chem., 74, 427-428. 

ABDERHALDEN, E. , UND L. BEHREND [1909]. Vergleichende Untersuchungen uber die Zusam- 
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aus Canton-Seide. 

Zeitschr. physiol. Chem., 59, 236-238. 

ABDERHALDEN, E., UND C. BRAHM [1909]. Vergleichende Untersuchungen uber die Zusam- 
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^eitschr. physiol. Chem., 6l, 256-258. 

ABDERHALDEN, E., UND G. A. BROSSA [1909]. Vergleichende Untersuchungen uber die 
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Zeitschr. physiol. Chem., 62, 129-130. 
ABDERHALDEN, E., UND H. R. DEAN [1909]. Studien uber die Bildung der Seide. 

Zeitschr. physiol. Chem., 59, 170-173- 

ABDERHALDEN, E., UND R. INOUYE [1912]. Weiterer Beitrag zur Kenntnis der Zusammen- 
setzung verschiedener Seidenarten. XIV. Ergebnisse der totalen und partiellen 
Hydrolyse des Kokons des Ailanthusspinners und von Tailung -Seide. 

Zeitschr. physiol. Chem., 80, 198-204. 

.ABDERHALDEN, E., UND B. LANDAU [1911, i]. Ueber die Zusammensetzung des Gespintes 
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Zeitschr. physiol. Chem., 71, 443-448. 

ABDERHALDEN, E., UND A. RILLIET [1908]. Vergleichende Untersuchungen uber die Zusam- 
mensetzung und den Aufbau verschiedener Seidenarten. I. Die Monoaminosauren 
der " New-Chwang- Seide" 

Zeitschr. physiol. Chem., 58, 337-340. 

ABDERHALDEN, E., UND J. SCHMID [1910]. Vergleichende Untersuchungen uber die Zusam- 
mensetzung und den Aufbau verschiedener Seidenarten. VIII. Die Monoamino- 
sauren aus " Tai-Tsao-Tsam " Seide (China). 

Zeitschr. physiol. Chem., 64, 460-461. 

ABDERHALDEN, E., UND J. SINGTON [1909]. Vergleichende Untersuchungen uber die Zusam- 
mensetzung und den Aufbau verschiedener Seidenarten. IV. Die Monoaminosauren 
aus Bengal-Seide. 

Zeitschr. physiol. Chem., 6l, 259-260. 

ABDERHALDEN, E., UND W. SPACK [1909]. Vergleichende Untersuchungen uber die Zusam- 
mensetzung und den Aufbau verschiedener Seidenarten. VI. Die Monoaminosauren 
aus Indischer-Tussah. 

Zeitschr. physiol. Chem., 62, 131-132. 

ABDERHALDEN, E., UND E. WALDE [1910]. Vergleichende Untersuchungen uber die Zusam- 
mensetzung und den Aufbau verschiedener Seidenarten. IX. Die Monoaminosauren 
aus Chefoo-Seide. 

Zeitschr. physiol. Chem., 64, 462-463. 

ABDERHALDEN, E., UND W. WEICHARDT [1909]. Die Monoaminosauren des Korpers des 
Seidenspinners. 

Zeitschr. physiol. Chem., 59, 174-176. 

ABDERHALDEN, E., UND WORMS [1909]. Vergleichende Untersuchungen uber die Zusam- 
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aus dem Leim der Canton-Seide. 

Zeitschr. physiol. Chem., 62, 142-144. 



BIBLIOGRAPHY 157 

FISCHER, E. [1903]. Nachtrag Zur Hydrolyse des Caseins und Seidenfibroins durch Sauren. 

Zeitschr. physiol. Chem., 39, 155-158. 
FISCHER, E. [1907]. Uber Spinnenseide. 

Zeitschr. physiol. Chem., 53, 126-139. 
FISCHER, E., UND A. SKITA [1901]. Ueber das Fibroin der Seide. 

Zeitschr. physiol. Chem., 33, 177 192. 
FISCHER, E., UND A. SKITA [1902]. Ueber des Fibroin und den Leim der Seide. 

Zeitschr. physiol. Chem., 35, 221-226. 
INOUYE, R. [1910]. Experiments with Bombyx Mori. 

J. Coll. Agric. Tokyo, 2, 223-235 ; Chem. Centr., 1910, II, 1146. 
INOUYE, R. [1912]. Composition of the Silkworm at Different Stages of its Metamorphosis.' 

J. Coll. Agric. Tokyo, 5, 67-79 ; Chem. Centr., 1913, i, 1296. 
KATAYMA, Y. [1916]. Nitrogen Compounds of Mulberry Leaves. 

J. Chem. Soc. Abs., 1916, i, 875. 
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Arch. Farmacol. Sperim., 20, 225-258. 

Chem. Centr., 1916, I, 168-169; J. Chem. Soc. Abs., 1916, i, 525-526. 
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Zeitschr. physiol. Chem., 68, 273-274. 

STRAUCH, F. W. [1911]. Vergleichende Untersuchungen uber die Zusammensetzung und 
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Zeitschr. physiol. Chem., 71, 365-366. 

SUWA, A. [1910]. Vergleichende Untersuchungen uber die Zusammensetzung und den Auf- 
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Zeitschr. physiol. Chem., 68, 275-276. 

SUZUKI, U., K. YOSHIMURA AND R. INOUYE [1909]. Hydrolysis of Wild Silk. 
J. Coll. Agric. Tokyo, I, 59-75 ; J. Chem. Soc. Abs., 1909, i, 859-860. 



158 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



Gelatin. 

FISCHER, E. [1902]. Ueber eine neue Aminosdure aus Leim. 

Ber. Deutsch. chem. Ges., 35, 2660-2665. 

FISCHER, E., UNO E. ABDERHALDEN [1904]. Notizen uber Hydrolyse von Proteinstoffen. 
II. Hydrolyse von Gelatine (Bildung von Serin). 

Zeitschr. physiol. Chem., 42, 540-544. 

FISCHER, E., UND R. BOEHNER [1910]. Bildung von Prolin bei der Hydrolyse von Gelatin 
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Zeitschr. physiol. Chem., 65, 118-123. 
FISCHER, E., P. A. LEVENE UND R. H. ADERS [1902], Ueber die Hydrolyse des Leims. 

Zeitschr. physiol. Chem., 35, 70-79. 

HART, E. [1901]. Ueber die quantitative Bestimmung der Spaltungsprodukte von Eiweiss- 
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Zeitschr. physiol. Chem., 33, 347-362. 
KOSSEL, A., UND F. KUTSCHER [igoo-i]. Beitrdge zur Kenntnis der Eiweisskorper. 

Zeitschr. physiol. Chem., 31, 165-226. 
LEVENE, P. A., UND W. A. BEATTY [1906]. Analyse der Spaltungsprodukte der Gelatine. 

Zeitschr, physiol. Chem., 49, 252-261. 
SKRAUP, ZD. H., UND A. v. BIEHLER [1909]. Ueber die Zusammensetzung der Gelatine. 

Monatsh. f. Chem., 30, 467-480. 

SKRAUP, ZD. H., UND F. HECKEL [1905]. Ueber Gelatin. II. 
Monatsh. f. Chem., 26, 1351-1358. 



Elastin. 

ABDERHALDEN, E., UND A. SCHITTENHELM [1904]. Die Abbauprodukte des Elastins. 

Zeitschr. physiol. Chem., 41, 293-298. 

BERGH, E. [1898], Untersuchungen uber die basischen Spaltungsprodukte des Elastins beim 
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Zeitschr. physiol. Chem., 25, 337-343. 

HEDIN, S. G. [1898], Einige Bemerkungen uber die basischen Spaltungsprodukte des 
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Zeitschr. physiol. Chem., 25, 344-349. 

HORBACZEWSKI, J. [1885]. Ueber die durch Einwirkung von Salzsdure aus den Albumin- 
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Ber. der Kais. Akad. der Wiss. Math.-Nat. Classe, 92, II, 657-668. 
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Zeitschr. physiol. Chem., 25, 551-552. 

KOSSEL, A., UND F. KUTSCHER [igoo-i]. See under Spongin. 

SCHWARZ, H. [1894]. Untersuchungen uber die chemische Beschaffenheit der elastischen 
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Zeitschr. physiol. Chem., 18, 487-507. 



Spongin. 

ABDERHALDEN, E., UND E. STRAUSS [1906, i]. Die Spaltprodukte des Spongins mitSduren. 

Zeitschr. physiol. Chem., 48, 49-53. 
KOSSEL, A., UND F. KUTSCHER [1900-1]. Beitrage zur Kenntnis der Eiweisskorper. 

Zeitschr. physiol. Chem., 31, 165-226. 



BIBLIOGRAPHY 159 

Keratins. 

ABDERHALDEN, E., UND E. EBSTEIN [1906]. Die Monoaminosauren der Schalenhaut des 
Huhnereies. 

Zeitschr. physiol. Chem., 48, 530-534. 

ABDERHALDEN, E., UND D. FUCHS [1908]. Der Gehalt verschiedener Keratinarten an Glu- 
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Zeitschr. physiol. Chem., 57, 339-341. 

ABDERHALDEN, E., UND B. LANDAU [1911, 2]. Zur Kenntnis der Monoaminosauren der 
Barten des Nordwales. 

Zeitschr. physiol. Chem., 71, 455-465. 

ABDERHALDEN, E., UND E. R. LE COUNT [1905]. Die Monoaminosauren des Keratins aus 
Gdnsefedern. 

Zeitschr. physiol. Chem., 46, 40-46. 

ABDERHALDEN, E., UND E. STRAUSS [1906, 2]. Die Monoaminosauren des Keratins aus 
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Zeitschr. physiol. Chem., 48, 535-536. 

ABDERHALDEN, E., UND A. VOITINOVICI [1907, i]. Hydrolyse des Keratins aus Horn und 
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Zeitschr. physiol. Chem., 52, 348-367. 

ABDERHALDEN, E., UND A. VOITINOVICI [1907, 2]. Welter e Beitrdge zur Kenntnis der 
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Zeitschr. physiol. Chem., 52, 368-371. 

ABDERHALDEN, E., UND H. G. WELLS [1905]. Die Monoaminosauren des Keratins aus 
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ARGIRIS, A. [1907]. Zur Kenntnis des Neurokeratins. 

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BUCHTALA, H. [1907]. Ueber das Mengenverhdltnis des Cystins in verschiedenen Hornsub- 
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BUCHTALA, H. [1908]. Elementaranalyse der Eihdute von Scyllium stellare, Pristiurus 
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Zeitschr. physiol. Chem., 52, 472-473. 
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Zeitschr. physiol. Chem., 56, i-io. 



160 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



VARIOUS PROTEINS. 

ABDERHALDEN, E. [1905]. See under Phosphoproteins-Caseinogen. 

ABDERHALDEN, E., UNO C. BRAHM [1909, 2]. Ueber den Gehalt der Mnskelsnbstanz dgypti- 
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Zeitschr. physiol. Chem., 6l, 419-420. 

ABDERHALDEN, E., UNO F. MEDIGRECEANU [1910]. Beitrag zur Kenntnis der Bausteine der 
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ABDERHALDEN, E., UNO F. PREGL [1905, i]. Ueber einem im normalen menschlichen Harn 
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Zeitschr. physiol. Chem., 46, 19-23. 

ABDERHALDEN, E., UND P. RONA [1905]. Die Zusammensetzung des " Eiweiss " von 
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ABDERHALDEN, E., UND W. VOLTZ [1909]. Beitrag znr Kenntnis der Zusammensetznng 
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ABDERHALDEN, E., UND A. WEIL [1912, 2]. Vergleichende Untersuchungen iiber den 
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ABDERHALDEN, E., UND A. WEIL [1913, 2]. Ueber eine neue Aminosdure von der Zusammen- 
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Zeitschr. physiol. Chem., 84, 39-59. 

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Biochem. Zeitschr., 18, 372-374. 

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Chem. Centr. igo4, II, 1576-1577- 
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Zeitschr. physiol. Chem., 88, 445-45g. 



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Amer. J. Physiol., 23, 81-89. 

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Zeitschr. physiol. Chem., 89, 289-303. 

TAMURA, S. [1914, 2]. Zur Chemie der Bakterien. V. Ueber die chemische Zusammen- 
setzung eines Wasserbacillus. 

Zeitschr. physiol. Chem., 90, 286-290. 
WOHLGEMUTH, J. [1904]. Zur Hydrolyse des Leberproteids. , 

Ber. Deutch. chem. Ges., 37, 4362-4364. 
WOHLGEMUTH, J. [1905]. Ueber das Nucleoproteid der Leber. IV. 

Zeitschr. physiol. Chem., 44, 530-539. 



PT. I. II 






162 THE CHEMICAL CONSTITUTION OF THE PROTEINS 

DERIVATIVES OF PROTEINS. 

ABDERHALDEN, E., UND T. SASAKI [1907]. Die Monodminosduren des "Syntonins" aus 
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Zeitschr. physiol. Chem., 51, 404-408. 

HART, E. [igoi]. Ueber die quantitative Bestimmung der Spaltungsprodukte von Eiweiss- 
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Zeitschr. physiol. Chem., 33, 347-362. 

HASLAM, H. C. [1901]. Quantitative Bestimmung der Hexonbasenin Hetero-albumose und 
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Zeitschr. physiol. Chem., 32, 54-58. 
DENNSTEDT, M., UND F. HASSLER [1906]. Ueber den Abbau von Eiweiss. 

Zeitschr. physiol. Chem., 48, 489-504. 

GUPTA, N. [1909]. Ueber die Zusammensetzung der Produkte alkalischer Hydrolyse des 
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Monatsh. f. Chem., 30, 767-771. 
LEVENE, P. A. [1902-3]. Ueber die Spaltung der Gelatine. 

Zeitschr. physiol. Chem., 37, 81-85. 
LEVENE, P. A. [1904]. Ueber die Spaltung der Gelatine. II. 

Zeitschr. physiol. Chem., 41, 8-14. 
LEVENE, P. A. [1905]. The Cleavage Products of Proteoses. 

J. Biol. Chem., I, 45-58. 
LEVENE, P. A., UND D. D. VAN SLYKE [1908, 2]. Hydrolyse von Wittepeptone. 

Biochem. Zeitschr., 13, 440-457. 
LEVENE, P. A., UND D. D. VAN SLYKE [1908, 3]. Ueber Plastein. 

Biochem. Zeitschr., 13, 458-474. 

LEVENE, P. A., D. D. VAN SLYKE AND F. J. BIRCHARD [1910]. The Partial Hydrolysis of 
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J. Biol. Chem., 8, 269-284. 

LEVENE, P. A., D. D. VAN SLYKE AND F. J. BIRCHARD [1911]. The Partial Hydrolysis of 
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J. Biol. Chem., 10, 57-71. 

LAMPEL, H., UND ZD. H. SKRAUP [1909]. Uber Hydrolyse des Serumglobulins durch 
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Monatsh. f. Chem., 30, 363-375- 
SKRAUP, ZD. H., UND F. HUMMELBERGER [1908]. Ueber einige Gelatosen. 

Monatsh. f. Chem., 29, 451-469. 

SKRAUP, ZD. H., UND F. HUMMELBERGER [1909]. Ueber die Hydrolyse des Eiereiweisses 
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Monatsh. f. Chem., 30, 125-146. 

SKRAUP, ZD. H., UND E. KRAUSE [1910, i], Partielle Hydrolyse von Proteinen durch 
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Monatsh. f. Chem., 31, 142-148. 
SKRAUP, ZD. H., UND E. KRAUSE [1910, 2]. Ueber par tielle Hydrolyse von Casein. 

Monatsh. f. Chem., 31, 149-164. 
SKRAUP, ZD. H., UND R. WITT [1906]. Ueber Peptone aus Kasein. 

Monatsh. f. Chem., 27, 663-684. 
SKRAUP, ZD. H., UND A. W^BER [1909]. Ueber die partielle Hydrolyse von Edestin. 

Monatsh. f. Chem., 30, 289-310. 
SKRAUP, ZD. H., UND R. ZWERGER [1905]. Zur Kenntnis der Kyrine. 

Monatsh. f. Chem., 26, 1403-1414. 
WECHSLER, E. [igioj. Zur Kenntnis des Hemielastins. 

Zeitschr. physiol. Chem., 67, 486-488. 



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KOCHER, R. A. [1915]. The Hexone Bases of Malignant Tumours. 
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Amer. J. Physiol., 23, 180-200. 

OSBORNE, T. B., D. D. VAN SLYKE, C. S. LEAVENWORTH AND M. VINOGRAD [1915]. 
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J. Biol. Chem., 22, 259-280. 

PLIMMER, R. H. A. [1908]. The Proteins of Egg-Yolk. 
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INDEX. 



ACID albumin, 2. 

amide groups, 4. 
Adamkiewicz-Hopkins reaction, 28. 
Alanine, 5, 7, 38, 71, 77, 79, 82, 85, 89. 
amount in proteins, 111-130. 
characterisation, 51. 
constitution, 2. 
ester, 38, 42, 51. 
in humin formation, 65. 
isolation, 44, 45, 48-50. 
loss during analysis, 67, 68, 69. 
phosphotungstate, 48. 
separation from glycine, 51. 

valine, 48-50. 

Albumins, i, 73, 76. 
composition, 113. 
tryptophan, 31. 
vegetable, i, 74. 
, composition, 118. 
, N distribution, 131, 132, 133. \ 
Albumose, 2. 

Aldehydes in humin formation, 66. 
Alfafa hay, N distribution, 134. 
Alkali globulin, 2. 
Amandin, i, 75. 
, composition, 117. 
, N distribution, 131. 
, tyrosine, 20, 23. 

Amide nitrogen, 4, 64, 74, 75, 85, 86. See 
also Ammonia. 

, analytical data, 111-130, 131-135. 

, estimation, 15, 55-56, 87-88, 97-98, 

105. 

Amino-acetic acid, 2. 
Amino acid anhydrides, 54. 
Aminobutyric acid, 5. 
Aminocaproic acid, 5. 
Aminoglutaric acid, 3. 
Aminoguanidinevalerianic acid, 3. 
Aminoisobutyric acid, 5. 
Amino-isocaproic acid, 3. 
Amino-isovalerianic acid, 2. 
Amino-methyl-ethyl propionic acid, 3. 
Amino nitrogen, 44, 64, 85. 

, estimation, Sorensen's method, 

12-14. 

, Van Slyke's method, 12, 89-96, 

108. 

Aminopropionic acid, 2. 
Amino succinic acid, 3. 
Aminovalerianic acid, 71. 
Ammonia, 4, 64, 74, 75, 94. 
, amount in proteins, 111-130, 131-135. 
, estimation, 15, 55-56, 85, 88, 97-98. 



Amyloid, composition, 129. 
Analysis, by N distribution, 85-110. 

, loss during, 63-69. 

, results, 63-84. 
Analytical data, 111-135. 
Anhydrides of amino acids, 54. 
Antiaris toxicaria, protein, 82. 

, composition, 129. 

Antipeptone, 14. 
Arachin, composition, 117. 

, N distribution, 131, 132. 
Arginine, 70, 71, 72, 73, 74, 75, 79, 81, 82, 
83, 85, 109. 

, amount in proteins, 108, 111-130. 

, constitution, 3. 

, estimation, 55,59, 85, 101-102, 107, 108. 

, in humin formation, 65. 

, isolation, 55, 56-57, 59. 

, nitrogen, 70, 85, 103. 

, analytical data, 132-135. 

, estimation, 101-102, 103. 

, separation from histidine, 55. 
Armadillo scales, 80. 

, composition, 125. 

, N distribution, 131. 

Asparagine, 74, 89. 
Aspartic acid, 82, 85, 86, 89. 

, amount in proteins, 111-130. 

, characterisation, 52. 

, constitution, 3. 

, ester, 37, 42, 52, 53. 

, isolation, 34, 35, 52-53. 

, loss during analysis, 67, 68, 69. 

, separation from glutamic acid, 34. 

, leucine, 53. 

Aspergillus niger, 81. 

, composition, 127. 

Avenin, composition, 118. 
Azolitmin paper, 106. 
Azotobacter, 81. 

, composition, 127. 

BACILLI, 81. 

, composition, 127. 
Barley, N distribution, 134. 
Bau-steine, 4. 
Bence-Jones protein, 25, 73. 

, composition, 114. 

Benzaldehyde in humin formation, 66. 
Betaine, 69. 

Betaines of amino acids, 69. 
Blood corpuscles, histone, i, 72. 

, dried, N distribution, 134. 

meal, N distribution, 134. 



167 



i68 



INDEX 



Body resistant to enzymes, 14. 
Brain, protein, 5. 

, composition, 128. 

Bread, tryptophan, 31. 

Bromination of tryptophan, 22, 28, 32. 

tyrosine, 22-23. 

CANAVALIN, N distribution, 131. 
Caproic acid, 5. 
Carapace, tortoise, 77, 80. 

, composition, 125. 

Carbohydrate in proteins, 2, 6-7. 
Carbohydrates in humin formation, 65-67, 
no. 

in N distribution, no, 135. 
Carcinoma, 109. 

, tryptophan, 29, 30. 
Caseanic acid, 5. 
Caseinic acid, 5. 
Casein-kyrin, 83. 

Caseinogen, i, 5, 6, 12, 19, 21, 33, 35, 44, 63, 
64, 65, 69, 76, 81, 83, 107, 109, no. 

composition, 86, 120. 

derivatives, 83. 

N distribution, 131, 132, 135. 

tryptophan, 27, 29, 30, 31, 32. 

tyrosine, 19, 20, 23. 
Caseoglutin, tryptophan, 31. 
Caseoses, 2, 83. 

Cattle foodstuffs, 109. 

, N distribution, 134. 

Centrophorus-histone, composition, 112. 
Chicken muscle, 81. 

, composition, 129. 

, N distribution, 131, 133. 

Chromoproteins, i. 
Clupeine, i, 70, 71. 

, composition, in. 
Clupeovin, composition, 120. 
Collagen, i. 
Colorimetric estimation of histidine, 61-62. 

tryptophan, 28-31. 

tyrosine, 20-21. 

Colostrum proteins, 108-109. 

, N distribution, 132. 

Completion of hydrolysis, 12. 
Composition, elementary, 2. 
Composition of albumins, 113. 

, vegetable, 118. 

gliadins, 119. 

globulins, 114. 

, vegetable, 115-117. 

glutelins, 118. 

histones, 112. 

phosphoproteins, 120. 

protamines, in. 

protein derivatives, 130. 

scleroproteins, 121-126. 

various proteins, 127-130. 

Conalbumin, i. 

, N distribution, 131. 

, tyrosine, 20. 
Conarachin, composition, 117. 

, N distribution, 131, 132. 
Conchiolin, N distribution, 131. 
Conglutin, i. 



Conglutin, composition, 117. 
, N distribution, 131. 
, tryptophan, 31. 
, tyrosine, 23. 
Conjugated proteins, i. 
Corylin, N distribution, 131. 
, tyrosine, 20. 

Cotton-seed Meal, N distribution, 134. 
Cow- Pea Meal, N distribution, 134. 
Crenilabrine, composition, in. 
Cyclic bases, formation, 65, 66. 
Cyclopterine, i, 71. 
, composition, in. 
Cyprinine, i, 71. 
, composition, in. 
Cystine, 15, 17, 27, 28, 79, 80, 82, 94, 97, 

102, 108. 

amount in proteins, 111-130. 
constitution, 3. 
estimation, 24-26, 85, 103. 
in humin formation, 65. 
isolation, 24-26. 
loss during analysis, 68-69. 
nitrogen, 85. 

, analytical data, 132-135. 
, estimation, 102-107. 
separation from tyrosine, 25-26. 

DERIVATIVES of proteins, 2, 82-84. 

, composition, 130. 

, N distribution, 131. 

Deutero-albumose, composition, 130. 

, N distribution, 131. 

Diamino acids, 4, 7, 15, 18, 19, 39, 70, 71, 
72, 75, 7 8 > 79, 83, 109, no. 

esters, 38. 

estimation, 55-62, 85-86, 98-104, 108. 

isolation, 55-61. 

list, 3. 

loss during analysis, 63. 

nitrogen, ^5, 86, 108. 

, analytical data, 131. 

, estimation, 88, 102. 

origin in fish sperm, 71. 

precipitation, 16, 54. 

separation, 55-62. 

Diamino-adipic acid, 5. 
Diaminocaproic acid, 3. 
Diamino-glutaric acid, 5. 
Diaminotrioxydodecanic acid, 5, 6, 63. 
Diazobenzenesulphonic acid reaction in his- 
tidine estimation, 61-62. 
Dicysteine, 3. 

Differentiation of proteins by N distribution, 
85-110. 

, analytical data, 131-133. 

Digestion of proteins, 4. 
Dihydroxy-diaminosuberic acid, 5. 
Dihydroxyphenylalanine, 6. 
Di-iodotyrosine, 6. 
Dimethylaminobenzaldehyde in tryptophan 

estimation, 30-31. 
Dimethyl-glutamic acid, 69. 
Diphtheria bacillus, 81. 

, composition, 127. 

Distillation, in vacua, fractional, 40-42. 



INDEX 



169 



Distillation, in vacuo, loss during, 67. 
, residue, 54. 

Distillers' grains, N distribution, 134. 
Distribution of nitrogen, 85-110. 

, analytical- data, 131-135. 

Di-thio-aminopropionic acid, 3. 

EDESTIN, i, 74, 107. 

, composition, 83, 115. 

, N distribution, 131, 132. 

, tryptophan, 29, 31. 

, tyrosine, 20, 22, 23. 
Egg-albumin, i, 5, 7, 64, 73, 84. 

, composition, 83, 113. 

, N distribution, 131. 

, tryptophan, 31. 

, tyrosine, 20. 
Egg-membrane, i, 24, 80. 

, composition, 124. 

, N distribution, 131. 
Egg-white, 73. 
Elastin, i, 5, 79, 80. 

, composition, 124. 

, tryptophan, 31. 
Elastose, composition, 130. 
Elementary composition, 2, 84. 
Elephant epidermis, 80. 
, composition, 125. 
, N distribution, 131. 
Engeland's rnethylation method, 44, 69. 
Enzymes in hydrolysis, 9, 14, 15, 19, 22, 27, 

3. 3 1 - 
Epidermis, 80. 

, elephant, composition, 125. 

, N distribution, 131. 

, tryptophan, 29. 
Ester method, 7, 33-54. 
Esterification, 35-42. 

, loss during, 67. 
Esters, extraction, 37-40. 

, , loss during, 67. 

, fractional distillation, 40-42. 

, reconversion into amino acids, 42, 52. 
Estimation of units, 15-62, 97-107. 
Euglobulin, 108, 109. 

, N distribution, 132, 133. 
Excelsin, i, 74. 

, composition, 115. 

, N distribution, 131. 

, tyrosine, 23. 

FEATHERS, i, 80. 

, composition, 126. 
Fibrin, i, 72, no. 

, composition, 114. 

, N distribution, 132, 135. 

, tryptophan, 31. 
Fibrinogen, i. 
Fibrinpeptone, 2. 

Fir-tree seed, protein, composition, 117. 
Fish muscle, 81, 82. 

, composition, 129. 
, N distribution, 131. 
Foodstuffs, cattle, 109-110. 

, N distribution, 134. 

Formaldehyde in humin formation, 65, 66. 



Formalin titration method of Sorensen, 12-14. 
Fractional distillation in vacuo, 40-42. 
Furfural in humin formation, 66. 

GADUS-HISTONE, i. 

, composition, 112. 

Gelatin, i, 4, 5, 7, 21, 36, 39, 79, 80, 107. 

, composition, 124. 

, N distribution, 131, 132. 

, non-amino N, 107. 

, tyrosine, 20. 
Gelatin-kyrin, 83. 
Gelatin peptone, 82-83. 
Gelatoses, 82 
Gliadin, rye, 75. 

, wheat, i, 25, 63, 69, 75, 86, 107, 108. 

, , cystine isolation, 25. 

, , tyrosine, 20. 
Gliadins, i, 74, 75. 

, composition, 86, 108, ng. 

, N distribution, 131, 132. 
Globin, 2, 72. 

, compositipn, 112. 
Globulins, i, 20, 25, 73. 

, composition, 114. 

, N distribution, 131, 132, 133. 

, tryptophan, 31. 

, tyrosine, 20, 23. 

, vegetable, i, 74-75, 81. 

, , composition, 115-117. 

, , N distribution, 131, 132. 

, , tryptophan, 31. 

, , tyrosine, 20, 23. 
Globulose, 2. 
Glucoproteines, 7. 
Glucoproteins, 2, 7, 8l. 
Glucosamine, 6-7. 

Glutamic acid, 33, 73, 74, 75, 76, 77, 79, 80, 
82, 83, 85, 86, 89. 
amount in proteins, 111-130. 
characterisation, 53. 
constitution, 3. 
ester, 37, 42, 52. 
hydrochloride, 33, 35. 
in humin formation, 65. 
isolation, 33-35, 53. 
loss during analysis, 67, 68, 69. 
separation from aspartic acid, 34. 

- leuc : ne, 34, 53. 
Glutamine, 74. 
Glutelins, i, 75. 

, composition, 118. 

, tyrosine, 20. 
Gluten, 75. 

, composition, 118. 

, flour, N distribution, 134. 

, N distribution, 134. 

, tryptophan, 31. 
Glutencasein, tryptophan, 31. 
Glutenin, i, 75. 

, composition, 118. 

, N distribution, 131. 

, tyrosine, 20. 

Glycine, 5, 8, 33, 44, 50, 73, 75, 76, 77, 78, 
79, 80, 82, 83, 85, 89, 94. 

, amount in proteins, 111-130. 



INDEX 



Glycine, constitution, 2. 

ester, 38, 40, 42. 

, hydrochloride, 36, 38, 51. 

estimation, 36-37. 

in humin formation, 65. 

isolation, 36, 38, 44, 45. 

loss during analysis, 67, 68. 

phosphotungstate, 48. 

picrate, 51. 

separation from alanine, 51. 
Glycinin, composition, 116. 

N distribution, 131. 

tyrosine, 20, 23. 
Glycocoll, see glycine. 
Glycoleucine, 5. 
Glycyl-alanine, 2. 
Goose feathers, 80. 
Grey matter, brain, 128. 

H^EMOCYANIN, N distribution, 132. 
Haemoglobin, i, 2, 72, 107. 

, composition of globin, 112. 

, N distribution, 131, 132. 
Hair, i, 20, 24, 79, 80, 107. 

, composition, 126. 

, N distribution, 132. 

, tryptophan, 29. 

, tyrosine, 20. 
Hemi-elastin, composition, 130. 
Hemp seed, N distribution, 134. 
Hetero-albumose, 83. 

, composition, 130. 

, N distribution, 131. 
Heterocyclic compounds, 3-4, 85. 
Hexone bases, 15, 63, 70, 83, 85, 109, no. 

, total N, 88, 102. 

, separation, 55-62. 

Hickory Nut, N distribution, 134. 
Histidine, 4, 15, 65, 70, 71, 72, 73, 74, 75, 
79, 82, 83, 85, 102, 103, 104, 108. 

, amount in proteins, 108, 111-130. 

, constitution, 3. 

, diazobenzene sulphonic acid reaction, 
61-62. 

, estimation, 55, 56-58, 61-62, 85. 

, in humin formation, 65. 

, isolation, 55, 56-58. 

, nitrogen, 70, 85. 

, , analytical data, 132-135. 

, , estimation, 103-104, 107. 

, separation from arginine, 55. 
Histones, i, 2, 71, 72. 

, composition, 112. 
Hordein, i, 75. 

, composition, 119. 

, N distribution, 131. 

, tyrosine, 20. 
Horn, i, 20, 24, 80. 

, composition, 126. 

, N distribution, 131. 

, tryptophan f 29. 

, tyrosine, 20. 

Horse serum proteins, N distribution, 133. 
Humin nitrogen, 98, 105, no. 

, analytical data, 131-135. 

, estimation, 55-56, 88, 98, 105, 107. 



Humin nitrogen, formation, 9, 64, 65-67. 
Hydrolysis, 7, 9-14, 55, 63, 64, 97. 
, by alkalies, n. 
, by baryta, 9, 32. 
, by enzymes, 9, 14, 15, 19, 22, 27, 30, 

3i, 64. 
, by hydrochloric acid, 9-10, n, 17, 18, 

24. 33. 34. 64, 87, 97, 105. 
, by hydrofluoric acid, n. 
, by sulphuric acid, 9, u, 15, 17, 18, 33, 

34. 55- 

, completion of, 12, 64, 97. 

, loss during, 63-65. 

, oxidation during, 84. 
Hydroxyaminopropionic acid, 3. 
Hydroxyaminosuberic acid, 5. 
Hydroxyaminosuccinic acid, 5. 
Hydroxydiaminosebacic acid, 5. 
Hydroxyphenylaminopropionic acid, 3. 
Hydroxypyrrolidinecarboxylic acid, 3. 

ICHTHULIN, I, 76. 

, composition, 120. 
Ichthylepidin, composition, 125. 
Imidazole-aminopropionic acid, 3. 
Indole, 30. 

Indole-aminopropionic acid, 4. 
Infusoria, phosphorescent, composition, 127. 
lodoform from spongin, 65. 
Isolation of units, 15-61. 
Isoleucine, 5, 76, 85. 

, amount in proteins, 68, 111-130. 

, constitution, 3. 

, isolation, 44-46, 47-48. 

, loss during analysis, 68. 

, separation from leucine, 47-48. 

, valine, 45-46. 

KAFIRIN, 75. 

, composition, 119. 

, N distribution, 131. 
Keratins, i, 24, 79-80. 

, composition^ 26. 

, N distribution, 131, 132. 

, tryptophan, 29, 31. 
KoiKn, 80. 

, composition, 124. 

, N distribution, 131. 
Kossel's method, 55-61. 
Kyrins, 83. 

LACTALBUMIN, 73, 109, no. 

, composition, 108, 113. 

, N distribution, 132, 135. 

, tryptophan, 29, 30, 31. 

, tyrosine, 20. 
Lactoglobulin, 109. 

, N distribution, 132. 
Legumelin, 74, 75. 

, composition, 118. 

, N distribution, 131. 
Legumin, i, 74, 75. 

, composition, 116. 

, N distribution, 131. 

, tryptophan, 31. 

, tyrosine, 20, 23. 



INDEX 



171 



Leuceines, 7. 

Leucine, 5, 18, 38, 48, 75, 79, 82, 85, 89. 
, amount in proteins, 68, 111-130. 
, constitution, 3. 
, fraction, 5. 
, ester, 38, 42, 53. 
, in humin formation, 65. 
, isolation, 44-46, 47-48. 
, loss during analysis, 67, 68, 69. 
, separation from aspartic acid, 53. 
, - glutamic acid, 34. 
, isoleucine, 47-48. 

, tyrosine, 18. 

, valine, 45-46. 

Leucininide, 54. 
Leucosin, 74. 
, composition, 118. 
, N distribution, 131. 
Leucyl-glutamic acid, 2. 
Linseed protein, 75. 

, composition, 117. 

Liver nucleoprotein, composition, 128. 
Liver, protein, 5, 29. 
, N distribution, 133. 
, tryptophan, 29. 
Livetin, N distribution, 131. 
Loss during analysis, 63-69. 
Lota-histone, i. 
, composition, 112. 
Lysalbic acid, 83, 84. 
Lysine, 19, 70, 71, 73, 74, 75, 81, 82, 83, 85, 

94, 107, 109. 

amount in proteins, 108, 111-130. 
constitution, 3. 

estimation, 55, 60-61, 85, 104. 
in humin formation, 65. 
isolation, 55, 56, 60-61. 
nitrogen, 85. 

, analytical data, 132-135. 
, estimation, 104, 107. 
separation from arginine and histidine, 
55, G O. 

MAIZE-GLUTELIN, tyrosine, 20. 
Maize-glutenin, composition, 118. 

kernel, N distribution, 134. 
Melanin, 24, 36, 55, 65-67. See Humin. 
Membrane, fat particles of milk, 81. 
Metaproteins, 2. 
Methylamine, 94. 

Methylation method, Engeland, 44, 69. 
Methylene compound of amino acids, 12. 
Methylhygric acid, 69. 
Micro-organisms, 81. 

, composition, 127. 
Milk, membrane of fat particles, 81. 

, proteins of, 108-109. 

, , N distribution, 132. 

, tryptophan, 31. 
Mono-amino acids, 5, 7, 15, 16, 70, 71, 72, 78, 

isolation, 33-54. 

list, 2-3. 

loss during analysis, 63-69, no. 

nitrogen, 70, 85, 86, 104, 109. 

, analytical data, 131-135. 

, estimation, 87-88, 104, 107. 



Mono-amino dicarboxylic acids, estimation, 

85-86, 104-106. 
Mucin, 2, 8l. 
Mulberry leaves, 78. 

, composition, 123. 

Muscle, i, 8l, 82. 

, composition, 129. 

, N distribution, 131, 133. 
Mycobacterium, 81. 

, composition, 127. 
Myosin, i. 
Myosinogen, i. 

NAIL, 80. 

, tryptophan, 29. 
Nervous tissue, protein, 5, 82. 

, , composition, 128. 

Neurokeratin, composition, 124. 
Nitrogen, amide, 4, 64, 74, 75, 85, 86, 94. 

, analytical data, 111-130, 131-135. 

, estimation, 15, 55-56, 87-88, 97-, 8, 
105. 

amino, 44, 64, 85. 

, estimation, Sorensen's method, 12- 
14. 

, , Van Slyke's method, 89-96. 

arginine, 70, 85, 103. 

, analytical data, 132-135. 

, estimation, 101-102, 103. 

cystine, 85. 

, analytical data, 132-135. 

, estimation, 102-103, 107. 

diamino, 85, 86, 108. 

, analytical data, 131. 

, estimation, 88, 102. 

distribution, 85-110. 

, analytical data, 131-135. 

histidine, 70, 85. 

, analytical data, 132-135. 

, estimation, 103-104, 107. 

humin, 98, 105, no. 

, analytical data, 131-135. 

, estimation, 55-56, 88, 98, 105, 107. 

lysine, 85. 

, analytical data, 132-135. 

, estimation, 104, 107. 

mono-amino, 70, 85, 86, 104, 109. 

, analytical data, 131-135. 

, estimation, 87-88, 104, 107. 

non-amino, 85, 103, 109. 

, analytical data, 131-135. 

, estimation, 103, 104, 107. 

total, 4. 

, analytical data, 131. 
Nitrous acid, action of, 85. 
Non-amino nitrogen, 85, 103, 109. 

, analytical data, 132-135. 

, estimation, 103, 104, 107. 

Norleucine, 5, 82. 
Nucleic acid, i, 2, 71, 72. 
Nucleoprotein, livef , composition, 128. 
Nucleoproteins, i, 2. 

OAT-GLIADIN, N distribution, 131. 
Oats, N distribution, 134. 
Oryzenin, i, 75. 



172 



INDEX 



Oryzenin, composition, 118. 

, N distribution, 132. 
Ovalbumin, see Egg-albumin. 
Ovokeratin, 78. 

, composition, 124. 
Ovomucoid, 2, 20. 

, composition, 128. 

, tyrosine, 20. 
Oxidation during hydrolysis, 84. 
Ox muscle, 81. 

, composition, 129. 

, N distribution, 131. 
Ox serum, proteins, N distribution, 133. 
Oxyproline, 8, 15, 21, 74, 79, 85. 

, amount in proteins, 111-130. 

, characterisation, 54. 

, constitution, 3. 

, isolation, 54. 

, loss during analysis, 68. 
Oxytryptophan, 6, 21. 



PARAMUCIN, 81. 

, composition, 128. 

Pathological tissues, 109. 

, N distribution, 133. 

Peanut, proteins, 75. 

, N distribution, 134. 

Pecan, N distribution, 134. 
Pepsin, composition, 128. 
Peptone Roche, tyrosine, 23. 
Peptones, 2, 83. 

, composition, 83, 130. 
Percine, 70, 71. 

, composition, in. 
Peripheral nerves, composition, 128. 
Phaseolin, composition, 116. 

, N distribution, 131. 

, tyrosine, 20. 

Phenylalanine, 7, 14, 52, 73, 79, 80, 81, 82, 
83, 85, 86, 89. 

, amount in proteins, 111-130. 

, characterisation, 52. 

, constitution, 3. 

, ester, 42, 52, 53. 

, in humin formation, 65. 

, isolation, 52. 

, loss during analysis, 69. 
Phenylaminopropionic acid, 3. 
Phosphoproteins, i, 2, 76. 

, composition, 120. 

, N distribution, 131, 132. 
Phosphorescent infusoria, 8 1. 

, composition, 127. 

Phosphotungstic acid precipitation, 16, 25, 

32, 48, 54, 6r, 87, 88, 98-99. 
Phosphotungstic-phosphomolybdic reagent 

for tyrosine estimation, 20. 
Pinus Koraiensis protein, composition, 117. 
Placenta, composition, 129. 
Plastein, 84. 

, composition, 130. 
Polypeptides, 2, 8, 82. 
Potato, globulin, composition, 117. 

, , N distribution, 132. 
Prolamines, 75. 



Proline, 8, 14, 15, 43, 45, 69, 71, 75, 79, 82, 
83, 85, 107. 

amount in proteins, 111-130. 

characterisation, 43. 

constitution, 3. 

copper salt, 43. 

ester, 42. 

estimation, 43-44. 

in humin formation, 65. 

isolation, 43. 

loss during analysis, 68, 69. 
Protalbic acid, 83, 84. 
Protamine nucleus, 71. 
Protamines, i, 2, 63, 70-71, 72. 

, composition, in. 
, N distribution, 70. 
Proteins, differentiation by N distribution, 

^i-ias- 

, elementary composition, 2. 

, various, 81-82. 

t , composition, 111-130. 
Proteoses, 2, 82-84. 

, composition, 130. 

, N distribution, 131. 
Proto-albumose, 83. 

, composition, 130. 

, N distribution, 131. 
Pseudoglobulin, 108, 109. 

, N distribution, 132, 133. 
Pseudomucin, 8l. 

composition, 128. 
Purines, 2, 72, 94. 

Pyridine, origin in coal tar, 65, 66. 
Pyrimidines, 2, 94. 
Pyrrol idine car boxy lie acid, 3. 
Pyrrolidone carboxylic acid, 34, 35, 67. 

RANOVIN, composition, 120. 
Residue from ester distillation, 54. 
Resistant body to enzymes, 14. 
Results of analysis, 63-84. 
Rice, N distribution, 134. 

protein, 75. ; 

, composition, 118. 

, N distribution, 132. 

Rye-gliadin, 75. 

, composition, 119. 

, N distribution, 131. 

grain, N distribution, 134. 

SALMINE, i, 4, 70, 71. 

, composition, in. 
Sarcoma, tryptophan, 29. 
Scales, armadillo, fish, snake, composition, 
125. 

, , , , N distribution, 131. 
Scallop muscle, 81. 

, composition, 129. 

, N distribution, 131. 

icleroproteins, i, 69, 77-80. 

, composition, 121-126. 

, N distribution, 131-132. 

Icombrine, i. 

, composition, in. 

Separation of amino acids into groups, 15, 85. 
Serine, 7, 71, 77, 79. 



INDEX 



173 



Serine, amount in proteins, 111-130. 

, characterisation, 53-54. 

, constitution, 3. 

, ester, 42, 52. 

, isolation, 53-54. 

, loss during analysis, 68, 69. 
Serum-albumin, i, 7, 73, 108, 109. 

, composition, 113. 

, N distribution, 108, 131, 132. 
Serum-globulin, i, 73, 108, 109. 

, composition, 83, 114. 

, N distribution, 108, 131, 132. 

, proteins, 108. 

, , N distribution, 133. 
Silk, 77-79. 

, composition, 122, 123. 
Silk-fibroin, i, 63, 77. 

, composition, 121, 122. 

, N distribution, 131. 

, tyrosine, 23. 
Silk-gelatin, i, 77, 79. 

, composition, 123. 

, N distribution, 131. 
Snake scales, 80. 

, composition, 125. 

, N distribution, 131. 

Sorensen's titration method with formalin, 

12-14. 

Soy beans, N distribution, 134. 
Spiders' silk-fibroin, 63. 

, composition, 122. 
Spinal cord, composition, 128. 
Spongin, 69, 79. 

, composition, 124. 
Squash seed globulin, composition, 115. 
, cystine isolation, 25. 

1 N distribution, 131. 

, tryptophan, 31. 

, tyrosine, 20, 23. 

Sturine, i, 70, 71. 

, composition, in. 
Sulphur in hydrolysis, 65. 
Sunflower seed, N distribution, 134. 
Synthesis, 8. 
Syntonin, 82. 

, composition, 130. 

TANKAGE, N distribution, 134. 
Tetrapeptide, 2. 
Thymus-histone, i, 72. 

, composition, 112. 
Thynnine, composition, in. 
Tissues, 109. 

, N distribution, 133. 
Tortoise carapace, 77, 80. 

, composition, 125. 

, N distribution, 131. 

Total nitrogen, 4. 

, analytical data, 131. 

Trimethyl-alanine, 69. 
Trimethyl-leucine, 69. 
Trimethyl-phenylalanine, 69. 
Trimethyl-valine, 69. 

Tryptophan, 6, 15, 21, 22, 23, 7 1 , 75, 79, 8 5 
86. 

, amount in proteins, 111-130. 



Tryptophan, bromination, 22, 28, 32. 

constitution, 4. 

estimation, 27-32. 

in humin formation, 65. 

isolation, 27. 

loss during analysis, 68, 69. 

nitrogen, 85. 

separation from cystine, 27. 
tyrosine, 27. 

Tubercle bacilli, 81. 

, composition, 127. 

Tumours, 81, 109. 
, composition, 129. 
, N distribution, 133. 
Tyroleucine, 7. 

Tyrosine, 6, 8, 15, 24, 27, 28, 32, 35, 39, 61, 
7i 73, 75, 76, 77, 78, 79, 80, 82, 83, 
85, 86, 89. 
amount in proteins, 111-130. 

, by bromination method, 23. 

, colorimetric method, 20. 

constitution, 3. 

ester, 26, 38. 

estimation, bromination method, 22-23. 

, colorimetric method, 20-21. 

, gravimetric method, 17-18. 

in humin formation, 65. 

isolation, 17-19. 

loss during analysis, 68. 

separation from cystine, 25-26. 

leucine, 18. 

URAMIDO groups, 64. 

Urea, 94. 

Urine protein, composition, 128. 

VALERIANIC acid, 5. 
Valine, 18, 71, 75, 85. 

amount in proteins, 111-130. 

characterisation, 46. 

constitution, 2. 

ester, 42, 43. 

isolation, 44, 45-46, 48-50. 

loss during analysis, 68, 69. 

phosphotungstate, 48. 

separation from alanine, 48-50. 

leucine and isoleucine, 45-46. 

Van Slyke's amino nitrogen method, 12, 44, 
64,89-96. , 

, N distribution method, 85, 97-108. 
Various proteins, 81-82. 

, composition, 127-130. 

Vegetable albumins, i, 74. 

, composition, 118. 

, N distribution, 131. 

globulins, i, 74-75, 8i. 

, composition, 115-117. 

, N distribution, 131. 

, tyrosine, 20, 23. 

- proteins, 33, 74775, 81. 

composition, 115-119. 

Vicilin, 74. 

, composition, 116. 

, N distribution, 131. 
Vignin, 69, 74. 

, composition, 116. 



174 

Vignin, N distribution, 131. 

, tyrosine, 20, 23. 
Vitellin, i, 76. 

, composition, 120. 

, N distribution, 131. 

, tyrosine, 20. 

WALNUT, N distribution, 134. 
Water bacillus, 81. 

, composition, 127. 
Whalebone, 80. 

, composition, 125. 
Wheat, bran, N distribution, 134. 

, N distribution, 134. 
Wheat-gliadin, i, 63, 69, 75, 107. 

composition, 86, 108, 119. 

cystine isolation, 25. 

N distribution, 132. 

tyrosine, 20. 
Wheat-gluten, 75. 

composition, 118. 

N distribution, 131. 



INDEX 



Wheat- gluten, tryptophan, 31. 
Wheat-glutenin, i, 75. 

, composition, 118. 

, N distribution, 131. 

, tyrosine, 20. 

White matter, brain, composition, 128. 
Witte's peptone, 83, 84. 

, composition, 130. 

, tryptophan, 31. 

Wool, 6, 20, 80. 

, composition, 126. 

, cystine, 24. 

, tryptophan, 29. 

, tyrosine, 20, 24. 

YEAST protein, 8l. 
, composition, 127. 

ZEIN, i, 19, 21, 65, 69, 75. 

, composition, 86, 119. 
, N distribution, 131. 
, tyrosine, 20. 



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