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OPTICAL ACTIVITY AND 
LIVING MATTER 



by 



G. F. GAUSE 

Professor of Experimental Blologij 
UNIVERSITY OF MOSCOW 



No. 2 of a series of monographs on general physiology 

edited by B. J. Luyet 



Published by 

BIODYNAMICA, Normandy^ Missouri 

1941 



"^^Ai 



LI8RAR Y| ^ 



•f-^WCJ^lUK^^)' 



Judging by the bibliographical list of publications deal- 
ing with optical activity in biological material, one has 
the impression that this subject did not arouse among 
American scientists the same degree of interest as did 
many other problems of biophysics and biochemistry. It 
is thought that this review will contribute to focus the 
attention of more of the so active investigators of this 
country on the important role of asymmetry in the build- 
ing stones of protoplasm. 

In presenting ''(9p^«c«^ Activity and Living Matter" by 
G. F. Gause to scientists at large, the object of the editor 
of this series of monographs is to bring to the fore a 
subject w^hich seems to be of fundamental significance in 
the problem of the structure and the mechanism of action 
of living matter. 

The contents of this monograph with the exception of 
the General Bibliography, are reprinted from BIODY- 
NAMICA, Nos. 52 and 56, 1939; 62 and 63, 1940; 70 and 
71, 1941. 

The Editoe. 

Saint Louis, Missouri, May 1941. 



••(•- 



PREFACE 

Althongli the study of the asymmetry of protoplasm 
was begun by Louis Pasteur about a century ago, it did 
not receive from the biologists the attention which it de- 
serves. The observations on that subject are scattered 
and need to be brought together into a separate division 
of experimental biology. The author of the present mon- 
ograph, who has for several years been engaged in expe- 
rimental studies of the structure and of the activity of 
living systems as related to the asymmetric configuration 
of their constituents, intends to review here this scat- 
tered literature and to discuss the various problems which 
the subject involves. 

Pasteur would, no doubt, rejoice in the importance that 
a number of questions related to the asymmetry of proto- 
plasm have acquired in the development of medical 
sciences. The recent findings on anthrax, a subject to 
which Pasteur has contributed so much, illustrate this 
point. Bruckner and Ivanovics showed in 1937, in the lab- 
oratory of Professor Szent-Gyorgyi, that the unnatural 
optical isomer of glutamic acid, which was not found 
anywhere before in organic nature, enters into the compo- 
sition of the capsules which enclose the anthrax bacilli. 
The capsules are responsible for the virulence of the 
bacilli, and the investigators just mentioned suggest that 
the protective role of the capsules is due to the unnatural 
configuration of glutamic acid. 

The author wishes to express his thanks to Professor 
W. W. Alpatov (Moscow) and to Professor W. J. Ver- 
nadsky (Moscow) for their aid in the course of the prep- 
aration of this work, and to Professor B. J. Luyet (St. 
Louis) for the revision of the manuscript. 

G. F. Gause. 



TABLE OF CONTENTS 

Page 

PREFArE - 4 

TABLE OF CONTENTS 5 

PRELIMINARY CHAPTER 
PRINCIPLES AND DEFINITIONS 

1. Dissymmetry and Asymmetry 9 

2. Optical and Geometrical Asymmetry 11 

3. Dissymmetric Structure as a Basis of Optical Ac- 

tivity - - 12 

4. "Eelative Configuration" and "Biological Series" 

of Optical Isomers - 15 



CHAPTER I 
OPTICAL ACTIVITY OF BIOLOGICAL MATERIAL 

1. Dissymmetry in Organic and in Inorganic Nature.— 19 

2. Asymmetry as a Specific Property of Protoplasm.— 20 

3. Asymmetry of Primary Constituents of Protoplasm 21 

4. Asymmetry of Secondary Constituents of Proto- 

plasm 24 

5. Exclusiveness of Asymmetry-Sign in Primary Sub- 

stances 27 

6. Non-Exclusiveness of the Asymmetry-Sign in Sec- 

ondary Substances 28 

7. Relative Configuration of Biological Material 30 

8. Asymmetry as a Criterion of the Organic Origin 

of a Substance 31 

CHAPTER II 

THE ORIGIN AND MAINTENANCE OF OPTICAL 
ACTIVITY IN LIVING MATTER 

1. The Transmission of the Asymmetric State by 

Asymmetric Synthesis 35 

2. The Transmission of Asymmetry, from the Ther- 

modynamic and Kinetic Point of View 37 



6 

3. Maintenance of Optical Purity by the So-Called 

''Stereo-autonomic Substances" 43 

4. Procedures Used by Nature for Maintaining Opti- 

cal Puritv and Establishing a ''Fixed Internal 
Milieu" ■ 45 

5. Biological Advantages of Optical Purity 51 

6. The Origin of the Asymmetry of Protoplasm. 52 

7. General Survey of the Problem of the Origin and 

Maintenance of Optical Asymmetry 53 

CHAPTER III 

HEREDITY AND THE INFLUENCE OF ENVIRONMENTAL 

FACTORS ON THE OPTICAL ACTIVITY OF 

BIOLOGICAL MATERIAL 

1. The Impossibility of Inverting the Optical Prop- 

erties of the Primary Constituents of Proto- 
plasm 59 

2. The Impossibility of Modifying Protoplasm so as 

to Cause it to Invert the Optical Properties of 
the Products of its Metabolism 61 

3. Mechanisms Controlling the Production of a Given 

Optical Isomer 67 

a. Production of Dissyinmetrir Substances from 

Symmetric Phenyl-Glyoxal 67 

&. Production of Optical Isomers by Esterases 68 

c. Production of Optical Isomers bv Oi)tically Active 

Alkaloid Catalysts ' *.... 68 

d. Production of a Given Optical Isomer by a Chemi- 

cal Alteration of the Catalyst .'. 69 

e. Control of the Production of Optical Isomers by 

Intermediate "Pathways'' 70 

/. Control of the Production of Optical Isomers by 

an Inversion of the Waldeu Tyije 73 

CHAPTER IV 

ON THE RELATION BETWEEN THE INVERSION OF 

SPIRALLY TWISTED ORGANISMS AND THE 

MOLECULAR INVERSION OF THEIR 

PROTOPLASMIC CONSTITUENTS 

1. Morphological Dissymmetry and Morphological In- 
version - 79 



2. Mori)li()l()ij;ical J)issyiinnetry and Morphological In- 

version mBacillus Mycoides 81 

3. Morphological Dissymmetry and IMorphological In- 

version in the Snail, F ruticicola lantzi 83 

4. Some Physiological Properties of the Dextral and 

the Sinistral Strains of Bacillus mycoides 84 

5. Some Physiological Properties of the Dextral and 

of the Sinistral Strains of the Snail, Fruticicola 
lantzi 89 

6. On the Relation between Morphological Inversion 

and Molecular Inversion..... 91 

7. Morphological Inversions and the Theory of Spiral 

Growth _ i 93 

CHAPTER V 

ANALYSIS OF VARIOUS BIOLOGICAL PROCESSES BY 
THE STUDY OF THE DIFFERENTIAL 
ACTION OF OPTICAL ISOMERS 

Asymmetric Analysis 99 

Section- I. Analysis of the Mechanism of Toxic 
Action, 

1. Toxic Action of the Optical Isomers of Nicotine 100 

2. Toxic Action of the Optical Isomers of Organic 

Acids - 108 

Section II. Analysis of the Evolution of the 

Nervous S^^stem. I 

1. Stereo-coefficients of Action of the Optical Isomers 

of Nicotine in the Phylogenetic Series 116 

2. The Acetylcholine System and the Differential Ef- 

fect of the Optical Isomers of Nicotine 120 

Section III. Analysis of the Mechanism of Va- 
rious Physiological Functions in Protozoa. 

APPENDIX 

ASYMMETRY OF PROTOPLASM AND THE 

STRUCTURE OF THE CANCER CELL \ 

GENERAL BIBLIOGRAPHY 133 

SUBJECT INDEX 154 /^^^ 

AUTHOR INDEX 157 - * ■ 



1 2: 



PRELIMINARY CHAPTER 
PRINCIPLES AND DEFINITIONS 

1. Dissymmetry and Asymmetry. A survey of the litera- 
ture on optical activity of protoplasm reveals some confu- 
sion in the terminology. Terms such as dissymmetry and 
asymmetry, which are so often used, are not always clearly 
defined. Some preliminary definitions are, therefore, 
necessary. 

Dissymmetry is a property of the individual components 
of a system, that is, in the cases to be considered here, a 
property of molecules, while asymmetry refers to an aggre- 
gate of molecules. 

The term dissymmetry was used in this sense for the first 
time by Pasteur in the classical paper that he wrote in 1848 
on the relations between crystalline form, chemical compo- 
sition and optical rotation and that he summarized in the 
two well-known lectures delivered in 1860 before the Paris 
Chemical Society on the molecular dissymmetry of natural 
organic products. Pasteur says that when we study mate- 
rial objects of whatever nature, as regards their form and 
the repetition of their identical constituent parts, we soon 
recognize that they fall into two large classes which present 
the following characters. Those of the one class, placed 
before a mirror, give images which are superposable on 
the objects themselves, while the images of the others are 
not superposable on the objects. A cube, straight stairs, a 
branch with opposite leaves, the human body — these are of 
the former class ; an irregular tetrahedron, winding stairs, 
a hand — these belong to the second group. The latter are 



10 PRINCIPLES AND DEFINITIONS 

dissymmetric,^ and are defined as objects possessing non- 
superposable mirror images. 

Dissymmetric objects can exist in two forms, right and 
left. 

When the two forms of dissymmetric molecules are rep- 
resented in equal concentrations (racemic mixture),' the 
aggregate of molecules is symmetric. When they are rep- 
resented in unequal concentrations, the aggregate is non- 
symmetric. There may be a predominance of the right 
forms (dextrality) or of the left forms of molecules (sinis- 
trality). Pasteur did not propose any special term for such 
a deviation of the molecular aggregate from the racemic 
state. His views on this subject were somewhat uncer- 
tain.' Following Emil Fischer (1894) and Japp* (1898), 
we shall designate this condition by the term asymmetry. 

1 In the German (1891) and in the English (1897) translations of Pasteur's 
work (lectures of 1860), the word dissymmetry was arbitrarily replaced by 
the word asymmetry. 

2 Eecently Findlay (1937) pointed out that the term acide racemique, as 
applied to tartaric acid, was due to Gay-Lussac (1828), but its use in the 
sense accepted at present originated with Pasteur (1861). Pasteur wrote in 
1860 : ' ' We still need a word in chemical terminology to express the fact 
of a double molecular dissymmetry concealed by the neutralisation of two 
opposite dissymmetries, the physical and geometrical effects of which com- 
pensate each other exactly. ' ' 

3 Pasteur did not distinguish sharply the dissymmetry of individual mole- 
cules from the asymmetry of their aggregates in the sense given above. For 
him the molecules acquire dissymmetry by receiving a ' ' twist ' ' in living 
organisms or in contact with products of living organisms and they lose their 
dissymmetry by being untwisted. In 1860 he wrote that "the twisted organic 
group can be untwisted and so assume the ordinary character of artificial and 
mineral substances. ' ' The ' ' twisting ' ' was considered as characteristically 
' ' vital ' ' and destructible by energetic chemical reactions. According to mod- 
ern views, these reactions, instead of ' ' untwisting ' ' the molecules, produce a 
racemisation or an equalization in the concentrations of the right and left 
forms of a substance. While Pasteur is the discoverer of the fact that 
' ' Racemic tartaric acid of chemists, inactive as to the optical rotatory power, 
consists of two acids, the rotations of which mutually neutralize each other, 
as one of them rotates to the right and the other to the left, and both in the 
same degree," (Pasteur, 1848, p. 458) he thought that the molecules of the 
racemates were symmetric by their very nature, and that they became dis- 
symmetric in their separation from the racemate by crystallization of the anti- 
podes under the action of some dissymmetry forces which might be furnished 
by ' ' organic dissymmetric particles on the surface of the crystallization dish ' ' 



PRINCIPLES AND DEFINITIONS 11 

From what has been said, it follows that dissymmetry, 
or noii-siiperposability of mirror image on the original 
object, can exist without any asymmetry, as in racemic 
mixtures. Dissymmetric molecules have the possibility 
of forming symmetric or asymmetric aggregates ; asym- 
metry is the realization of one of these two potentialities. 
It is, therefore, obvious that dissymmetry represents a 
necessary pre-requisite condition for any asymmetric state. 

2. Optical and Geometrical Asymmetry. Asymmetry as 
defined here should be distinguished from geometrical 
asymmetry. A geometrically asymmetric figure is one 
which possesses no element of symmetry, that is, no center, 
no axis and no plane of symmetry, while dissymmetric 
figures (in the sense of Pasteur) might possess a complex 
system of axes of symmetry, although they cannot possess 

(Pasteur, 1884). According to our views, one half of a racemic aggregate 
consists of the right and the other of the left form of molecules, before, as 
well as after, crystallization. 

That Pasteur was mistaken in this particular point is evidenced by the fol- 
lowing investigations. Ostwald (1889) has shown by electric conductivity 
methods that, in dilute water solutions, racemic tartaric acid does not exist 
as such but is entirely dissociated into its dextrorotatory and laevorotatory 
components. Eaoult reached the same conclusion by cryoscopic methods. 
Wyrouboff (1884), Jungfleisch (1884), and Errera (1898) pointed out that 
the separate crystallization of antipodes from racemic tartrate depends on the 
relative solubilities of the individual components and of the mixture. These 
solubilities, in their turn, are controlled by the temperature. Thus, at ordi- 
nary room temperature the antipodes are less soluble than the racemic mix- 
ture, and they crystallize separately, while, at temperatures above 26° C, the 
order of solubility is reversed, and the racemate crystallizes. 

4 Emil Fischer (1894) introduced the concept of asymmetric synthesis, that 
is, of the production of molecules which exhibit a rotation of a given sign 
with full or partial exclusion of the antipode. But the term asymmetry for 
expressing the properties of aggregates of molecules was employed^ — for the 
first time, it seems — by Japp (1898) in his well known address, "Stereo- 
chemistry and Vitalism," which was followed by an interesting discussion in 
' ' Nature. ' ' Japp wrote that the simultaneous production of two opposite 
asymmetric halves is equivalent to the production of a symmetric whole, 
whether the two asymmetric halves be actually united in the same molecule, 
as in the case of meso-tartaric acid, or whether they exist as separate mole- 
cules in the loft and right constituents of racemic acid. This statement shows 
quite clearly that the author conceived asymmetry as the property of the 
aggregate of molecules and not as the configurational character of the indi- 
vidual molecules (the term enantiomorph was used in this latter sense). 



12 PRINCIPLES AND DEFINITIONS 

a plane, a center or an alternating axis of symmetry {cf. 
the definition of Lowry, 1935), these elements being incom- 
patible with the non-superposability of the image. So, 
dissymmetric molecules are not necessarily asymmetric in 
the geometrical sense. 

3. Dissymmetric Structure as a Basis of Optical Activ- 
ity. The fact that the rotation of the plane of polarized 
light is caused by a dissymmetric structure of molecules 
leaves no place for doubt, but the problem of the physical 
mechanism by which this is done did not yet receive a defi- 
nite solution. Two models proposed by Pasteur — irregu- 
lar tetrahedron and spiral line — have formed the basis for 
further theories. We shall consider separately the case in 
which optical activity is due to a dissymmetric spatial dis- 
tribution of atoms as found in entire crystals and the case 
in which it is due to a dissymmetric structure of isolated 
molecules. 

It is known that the optical activity of quartz depends 
on the structure of the crystal itself, since the rotation of 
the plane of polarized light disappears with the crystalline 
state. The optical effect also diminishes, and at last van- 
ishes when a plate cut out from a crystal of quartz passes 
from a position perpendicular to the direction of the ray 
to an inclined position. Consequently, the fundamental 
difference between the dissymmetry of quartz and the mo- 
lecular dissymmetry of organic substances lies in the fact 
that in the former case the crystal as a whole is anisotropic, 
i.e., possesses different properties in diiferent directions, 
while, in the latter, as it was ascertained by Pasteur, dis- 
symmetry represents a property of the separate molecules 
independent of their relative position in space. A sub- 
stance in which one of the two possible dissymmetric forms 
of molecules, right or left, predominates, will possess 
optical activity. 

It was Fresnel (1824) who suggested for the first time, 
that the structural dissymmetry of quartz may be ex- 
plained on the basis of the spiral distribution in space of 
the molecules of silicon. In one of the two optical anti- 



PRINCIPLES AND DEFINiriONS 13 

podes of quartz, these spirals would turn from right to 
left and, in the other, from left to right. This view was 
adopted by Pasteur (1860), and, about a hundred years 
after its formulation by Fresnel, it received full confirma- 
tion in the X-ray analysis of quartz made by Bragg (1913, 
1925). This investigator showed that crystals of quartz 
can be considered as giant molecules in which the constitu- 
ent units build up a three-dimensional network, w^liere 
every atom of silicon is linked to four atoms of oxygen, 
wiiilst every atom of oxygen unites two atoms of silicon. 
The complex aggregate thus formed has a spiral structure 
which is shown in Fig. 1. The lines uniting the centers of 




Fig. 1. Spiral structure in a crystal of quartz. The silicon atoms are 
represented by solid black circles, the oxygen atoms by lighter and larger 
circles. Three atoms of silicon form a spire. Each atom of silicon is in 
the center of a tetrahedron at the apices of wliicli are 4 oxygen atoms; only 
2 of the latter are represented in the figure. 

the atoms are spirals, and these spirals are twisted in 
opposite directions in dextrorotatory and in laevorotatory 
quartz. (For further details on the coordination of sepa- 
rate spirals in the so-called a and 3 form of quartz, cf. 
Bragg.) Let it be noticed, then, that it is the spiral type 
of structure which prevails in the dissymmetric spatial 
distribution of elements in crystals of quartz. 

What is the structure of dissymmetric organic molecules 
and its relation to optical rotation? Modern theories, a 
detailed account of which may be found in the excellent 
monograph by Lowry (1935), consider the irregular tetra- 



14 



PRINCIPLES AND DEFINITIONS 



lieclron with four different radicals situated in its corners 
as the basis for the explanation of the origin of optical 
activity. This structure accounts for both the existence 
as well as the approximate value of optical rotation in the 
simplest dissymmetric molecules. It should be noticed 
that a tetrahedric molecule presents a spiral type of dis- 
tribution of its atoms. In Fig, 2 (a) is represented an 





Fig. 2. Dissymmetric configuration of organic molecules; a) 1-isomer, 
b) d-isomer. 

irregular tetrahedron in the corners of which are placed 
four different groups. In the order of diminishing mag- 
nitude these groups can be arranged in the following- 
manner: Ri > R2 > R3 > R4. By joining the centers of 
these groups in the order just given a spiral is obtained. 
If the largest group (Ri) is placed nearest to a hypotheti- 
cal observer, the spiral represented in Fig. 2 (a) will 
appear to rotate counter-clockwise. According to Boys 
(1934), such a structure would correspond to the left abso- 
lute configuration of the molecule. If we interchange the 
groups R2 and Rs, we obtain a figure which is the mirror 
image of the preceding one ; the spiral twist will now as- 
sume a clockwise direction and the molecule will possess 
the right configuration. 

Recently an attempt has been made to adapt the concept 
of absolute configuration to the definition of the configura- 



PRINCIPLES AND DEFINITIONS 15 

tioii of natural a-amiiio-acids (see Eainey, 1937). We 
shall also mention as related to this problem the geometri- 
cal investigations of Study (1913) on the right and left 
structures in a system of points. Finally we wish to point 
out again that the spiral distribution of elements appears 
as basic in the mechanism of optical rotation in molecules 
as well as in crystals. 

4. ^'Relative Configuration" and ''Biological Series" 
of Optical Isomers. Emil Fischer (1894) drew attention 
to the necessity of distinguishing the relative configura- 
tion of a substance from the sign of its optical rotation, 
there being substances which possess the same relative 
configuration but rotate the plane of polarized light in 
opposite directions. The importance of this remark be- 
came more evident in the subsequent developments of 
stereochemistry. Changes in temperature, solvent, con- 
centration, etc., are often accompanied by a change in the 
sign of the optical rotation. As Lowry (1935) pointed out, 
these changes make it impossible to judge the configura- 
tion of a substance by the sign of its rotation. This may 
be demonstrated by the following example. Let us con- 
sider an optically active compound 

CH3 X 

\ / 

c 

/ \ 
/ \ 

containing a single asymmetric carbon atom, linked to 
methyl and ethyl and to two other radicals, X and Y. No 
matter what the influence of temperature and of solvent 
is, the sign of the rotation will be reversed but its magni- 
tude will be unaltered if the methyl and ethyl radicals are 
interchanged, i.e., if usual optical inversion takes place. 
The rotation will disappear completely if methyl is re- 
placed by a second ethyl radical, or conversely, since then 
the plane of symmetry will appear in the molecule. If 
methyl is replaced not by ethyl but by propyl, it is gen- 



16 PRINCIPLES AND DEFINITIONS 

erally admitted (Lowry, 1935) that the sign of the rotation 
will be reversed, i.e., that the molecules 

CH, X C,H, X 

\ / \ / 

\ / \ / 

C and C 

/ \ / \ 

/ \ / \ 

C,H, Y C,H, Y 

will have opposite rotations, although the position of the 
univalent radical C2H5CXY is identical, and although there 
has been only a substitution of one chemical group in the 
molecule by another. Such possibilities render illusory 
any conclusion as to the configuration of a substance on 
the basis of the direction of its rotation. 

To clarify this situation, Fischer (1894) proposed to 
take as a prototype of configuration that of a specific 
isomer of some definite substance and compare to this pro- 
totype the optical isomers of other substances. In this 
manner a series of optical isomers of different substances 
can be established, all the members of this series possess- 
ing the same relative configuration. Wohl and Freuden- 
berg (1923) suggested that the members of one such series 
be designated by the letter d and their antipodes by the 
letter /, while the sign of their optical rotation w^ould be 
indicated by (+) for a rotation to the right and by (-) for 
a rotation to the left. The decision as to which one of the 
two series should be marked by the letter d is, of course, 
arbitrary, the absolute configuration of the substance being- 
unknown. According to this system, a substance belong- 
ing, for example, to the left steric series, but rotating the 
plane of polarized light to the right will be marked by I (+). 
Fischer, furthermore, suggested to take as a standard of 
comparison dextrorotatory glucose, conventionally taking 
it as a r/-form. He proposed that, in writing the formulas, 
the aldehydic or ketonic group of sugars and the carbonyl 
group of monobasic acids be put on top and the chain of 
carbon atoms in a downward direction, the hydroxyl of the 
fifth carbon atom being to the right. If one figures out. 



PRINCIPLES AM) DEFINITIONS 17 

on llie basis of what is known on chemical structure, wliieh 
isomer of fructose presents the same position for the tifth 
carbon atom as rf-glucose, one finds that it is laevorotatory 
fructose. Thus d (+) glucose and d (-) fructose possess 
the same relative configuration in spite of their rotation 
in opposite directions. Both these isomers are found in 
living organisms and belong to the same *' biological 
series." 

Wohl and Freudenberg (1923) proposed to take glycer- 
ine aldehyde and not glucose, as a standard of comparison, 
conventionally considering the dextrorotatory form as a 
member of the <7-series and attributing to it such a struc- 
ture that the hydroxyl of the fifth carbon atom be again 
written to the right. 

SUMMARY 

1. Dissymmetry is the property of molecules of possess- 
ing non-superposable mirror-images. Dissymmetric mole- 
cules can exist in two forms, right and left. 2. Asymmetry 
is the property of molecular aggregates of presenting a 
predominance of the right or the left form of dissymmetric 
molecules. 3, Optical asymmetry is to be distinguished 
from geometrical asymmetry. 4. Optical activity is at- 
tributed to the spiral arrangement of atoms, either in 
entire crystals, as in quartz, or in single molecules, as in 
some organic compounds. 5. If, besides the sign of the 
optical rotation of a substance, one considers the config- 
uration of its molecules, one can classify the optical isomers 
into "biological series" as found in living organisms. 

BIBLIOGRAPHY 

BEAGG, W., Proc. Boi/. Soc. A., 89, 575, 1913; IM, 405, 1925. 
EERERA, G., Nature, 58, 616, 1898. 
FINDLAY, A., Nature, 140, 22, 1937. 

FISCHER, E., Ber. cliem. Ges., ^7, 3189, 1894; 3^, 3638, 1900. 
FRESNEL, A., Bull. Soc. Philomat., p. 147, 1824. 
GAY-LIJSSAC, L., Couvs de Chimie, Paris, 1828. 
JAPP, F. R., Nature, 58, 452, 1898. 

JUNGFLEISCH, M. E., Bull. Soc. Chim. Paris, 41, 222, 1884. 
LOWEY, T. M., Optical Eotatoiy Power, Longmans, Green & Co., Loudon, 
1935. 



18 PRINCIPLES AND DEFINITIONS 

OSTWALD, W., Z. physikal. Chem., 3, 369, 1889. 

PASTEUR, L., Ann. Chim. et Phys., 24, 442, 1848; 61, 484, 1861. 

, Recliei'clies sur la Dissymetrie Moleculaire des Produits Or- 

ganiques Naturels. Soc. Chim. Paris. English translation in Alembie 

Club Beprints, 14, 1860. 
, Bev. Scieniif. Hi, 4, 2, 1884. 



RAINEY, R. C, Nature, 140, 150, 1937. 

STUDY, E., Arcli. Math, unci Physilc, 31, 193, 1913. 

WOHL, A. AND FREUDENBEEG, K., Ber. chem. Ges., 56, 309, 1923. 

WYROUBOFF, G., Bull. Soc. Chim. Paris, 41, 212, 1884. 



CHAPTER I 

OPTICAL ACTIVITY OF BIOLOGICAL MATERIAL 

1. Dissymmetry in Organic and in Inorganic Nature. It 
has been repeatedly pointed out that all physiologically 
important substances possess a dissymmetric structure. 
This is precisely what Pasteur meant when he wrote: "On 
trouve la dissymetrie etablie notamment dans les principes 
immediats essentiels a la vie. ' ' But is there any essential 
relation between dissymmetry and life, in the sense that 
one is a necessary attribute of the other! Dissymmetry 
is certainlj^ much more general than life. We know that 
the dissymmetric structure exists in crystals of quartz. 
The same is true of several metallic compounds {cf. Lowry, 
1935). Recently Jaeger (1919), after having investigated 
a great number of inorganic compounds, came to the con- 
clusion that the dissymmetric structure might be much 
more general than we usually assume, but that, in inorganic 
nature, the existence of dissymmetry is often difficult to 
establish, there being no method for separating the anti- 
podes. Vernadsky (1934) made a similar remark. In 
such cases at least, dissymmetry^ has no obvious relation 
to life. But, if dissymmetry exists without life, life might 
not exist without dissymmetry. The possibility that life 
be the attribute of systems built of substances of such a 
level of complexity that dissymmetry is the very condition 
of their existence is not excluded. A suggestion which was 
recently made by Ackermann (1935), and which is practi- 
cally identical with that of Pasteur (1884), is that dissym- 
metry is characteristic of the basic components of proto- 
plasm, whilst such products of metabolism as urea, uric 
acid, creatinin and hippuric acid are devoid of dissym- 
metry and their molecules are structurally inactive. The 
simplest amino-acid of the protein molecule, glycocoll, is 



[on-metals 




C N 




Si P 


S 


As 


Se 




Te 



20 OPTICAL ACTIVITY OF BIOL. MATERIAL 

the only one devoid of dissymmetry, and, in metabolic 
processes, it is less important than the other amino-acids 
which are dissymmetric. 

It is of interest to mention here that the elements of 
which optically active compounds consist include twenty- 
one of them, as follows (Lowry, 1935) : 

Metals 

B C N Be 

Al 

Cr Fe Co Ni Cu Zn 
Ell Eh 

Ir Pt 

2. Asymmetry as a Specific Property of Protoplasm. It 
is generally established that all the substances which are 
produced in the laboratory or in nature, without the action 
of living organisms, have right and left forms represented 
in equal concentrations, the formation of both being equally 
probable. It has never been observed, for example, that 
in any quartz bed the right or the left crystals would pre- 
dominate to any extent (Tromsdorff, 1937; Lemmlein, 
1938). There is, of course, dissymmetry in individual 
components, but no asymmetry in their aggregation. 

On the other hand, all basic chemical substances of which 
living systems are made up or which are formed in connec- 
tion with the activity of living systems, deviate from the 
racemic state and are represented mainly by one antipode. 
In other words, asymmetry is a specific attribute of living- 
systems and an essential feature of their organization. 
This is one of the most significant principles of experi- 
mental biology ; it is based on a large number of observa- 
tions accumulated within the last hundred years, since the 
pioneer work of Pasteur. 

We shall study here, in some detail, which parts of living- 
systems consist of racemic compounds and which parts 
deviate from the racemic state and in what direction. It 
will appear that the asymmetric state of protoplasmic 
components is directly related to the role played by these 
components in metabolic activity. 



OPTICAL ACTIVITY OF BIOL. MATERIAL 21 

3. Asymmeiry of Primary Constituents of Protoplasm. 
From the view-point of their asymmetric molecular aggre- 
gation, the substances which enter into the composition of 
living systems may be divided into two groups. Physio- 
logists have for a long time been accustomed to call these 
two groups, respectively, the primary and the secondary 
constituents of protoplasm. To the group of primary sub- 
stances belong the proteins and the lipoids which form 
together the so-called lipoprotein complexes, and the carbo- 
hydrates which functionally are closely related to them. 
These primary substances, except for some stored carbo- 
hydrates and proteins, build up protoplasm itself and pre- 
side over the fundamental living processes. To the group 
of secondary constituents belong various products of trans- 
formation of the primary substances, which represent 
either storage material or excreta. 

We shall study, to begin with, the asymmetric structure 
of primary substances, and we shall consider, first, the 
degree in which they deviate from the racemic state, or, in 
other words, their optical purity. 

As far as proteins are concerned, the optical activity of 
which was already known to Pasteur, Emil Fischer was the 
first to express the idea that their constituent amino-acids 
are always found in protoplasm in the optically pure state, 
and that, when a total or partial racemisation occurs, it is 
due to the application of too coarse methods of isolation. 
For instance, serine from silk was known for a long time 
only in the form of a racemic compound, as it is rather 
easily racemised in the process of protein hydrolysis. 
However, Fischer (1907) succeeded in isolating from silk 
optically active serine, of which the specific rotation (in 
hydrochloride solution,^ at 18°) was + 11.6°, while the rota- 
tion of optically pure laevorotatory serine prepared syn- 
thetically {i.e., crystallized from racemate with alkaloids) 
was + 14.4° (at 20°). On the ground of these experimental 
data alone it is, of course, impossible to conclude that serine 
in silk is optically pure, and that it is partially racemised 

1 The hvdroc'liloride of laevorotatory serine is dextrorotatory. 



22 OPTICAL ACTIVITY OF BIOL. MATERIAL 

ill the process of isolation, but we shall see below that the 
principle itself is definitely established. 

Pringsheim (1910) had shown on asparagine that the 
optically pure form is gradually racemised by boiling with 
water, a step in the isolation procedure. 

With appropriate treatment, amino-acids both of vege- 
table and animal origin, always prove to be optically pure, 
that is, one isomer only of each amino-acid is present, its 
antipode being completely absent. In the case of leucine, 
this was established by the elaborate investigations of 
Ehrlich and Wendel (1908), the results of which are given 
in Table 1. 

TABLE 1 

Specific Eotation of Preparations of Leucine of Different Origin, 
IN Water at 20° (Ehrlich and Wendel, 1908) 

Synthetic, optically pure preparation - 10.3° 

From egg-wliite (chicken) - 10.4° 

From casein (cow's milk) -10.3° 

From yeast {Saccharomyces cerevisiae) - 10.8° 

The optical purity of tyrosine remained for a long time 
questionable as a result of a number of old contradictory 
observations (Lippman, 1884). But Schulze and Winter- 
stein (1905) have definitely shown that, after careful 
preparation from vegetable material, one obtains always 
optically pure substances and that racemisation and the 
consequent decrease of rotatory power are the result of 
the application of coarse methods of isolation (Table 2). 

TABLE 2 

Optical Eotation of Preparations of Tyrosine of Different 

Origin, in Hydrochloride Solution 

Synthetic, optically pure prep- 
" aration - 16.4° Fischer, 1900 

From Cow 's Milk ; hydrolysis | - 13.2° 

of casein by HCl \ - 11.6° ' ' 



From the bulbs of Dahlia 
variabilis; 80% boiling 
alcohol was used in the 
isolation procedure 



- 12.5° Schulze and Winterstein, 1905 

- 12.9° 



From embryos of Liipinus 

albvs; autolysis - 16.2^^ 



OPTICAL ArTTYTTY OF BIOL. MATERIAL 23 

The pveparation from cow's milk and that from the 
bulbs of Dahlia show a weak racemisatioii as a result of 
the treatment, while the preparation from the embryos of 
Lupinus is optically pure ; the autolysis procedure used in 
this last case prevents racemisation. 

One can, at present, consider as an established fact that 
all amino-acids entering into the composition of proto- 
plasmic proteins are optically pure ; not a single exception 
is known/ 

The fats or lecithins, which contain nitrogen and phos- 
phorus, and which are considered integral constituents of 
the fundamental units of protoplasm, are also optically 
pure, as it was, for instance, established by the investiga- 
tions of Mayer (1906). 

Among primary substances, the carbohydrates, as well, 
are for the most part optically pure. Brown and Morris 
(1893) have shown in an extensive investigation that glu- 
cose and other sugars are found in the optically pure form 
in the leaves of the plant Tropaeolum majus. 

An interesting exception to the general rule has been 
observed in sugars. Neuberg (1900) found in the human 
organism optically inactive, racemic sugar under patho- 
logical conditions. Salkowsky (1892), who had discovered 
that in this case a pentose (arabinose) is excreted in urine, 
instead of glucose as it happens in glucosuria, called the 
disease pentosuria. Neuberg established that the arabi- 
nose excreted in urine is optically inactive. These obser- 
vations were later confirmed by a number of other physi- 
ologists. In what relation the inactive arabinose stands 
to the active arabinose entering into the composition of the 
nucleo-proteids of our body is at present unknown. 

Racemic sugar, dl-galactose, was also found in plants. 
Oshima and Tollens (1901) isolated it from the Japanese 
marine alga, Porphyra laciniafa. 

The presence of racemic sugars in plants and animals is 

1 It seems preferable, for the present, to suspend judgment on the recent 
data of Kogl and Erxleben (1939) concerning partial racemisation of some 
amino-acids in proteins of malignant cells. 



24 OPTICAL ACTIVITY OF BIOL. MATERIAL 

very rare but it is particularly significant. Since sugars 
do not racemise when boiled in water, it seems that the 
racemic state does not result from the process of isolation 
but that the optically inactive forms actually enter into the 
composition of living systems. The origin of racemic 
sugars in living organisms is by no means clear. Neuberg 
(see Fiirber, Nord and Neuberg, 1920) remarks that it 
might not be a mere accidental fact that the two racemic 
sugars found are just arabinose and galactose. 

To conclude, among the primary substances, all the 
amino-acicls, the lecithins and the majority of important 
sugars such as glucose, fructose and many others are 
always present in protoplasm in the optically pure state. 

4. Asymmetry of Secondary Constituents of Proto- 
plasm. As one passes from primary to secondary sub- 
stances, the optical purity loses its obligatory character. 
This is particularly evident in organic acids which repre- 
sent intermediate products of metabolism. Their origin 
and their signification is still a source of controversy, 
especially in plants. Whether, in the latter, the forma- 
tion of organic acids is related to the metabolism of the 
amino-acids, or whether they represent a stage in the 
carbohydrate cycle cannot be decided. When the organic 
acids begin to appear, they are optically pure, as if bear- 
ing some birth marks from the primary substances, but as 
soon as they separate from the primary asymmetric sys- 
tem, beginning perhaps to play the role of storage material, 
they assume the character of racemic compounds. 

The experimental data on which these conclusions are 
based are principally those of Ruhland and of his school.^ 
Ruhland and Wetzel (1929), and later Schwarze (1932) 
observed that, in the leaves of different plants, malic acid 
is found especially in the two forms: laevorotatory and 
racemic (Table 3). 

1 The data of the Leipzig school and, particularly, the analytical part of 
the work were severely criticized by Bennet-Clark (1937). But, as far as 
optical activity is concerned, Euhland's data are reliable. Enzymatic race- 
misation of malic acid in plants, according to Bennet-Clark, was observed also 
by Naylor (unpublished Thesis, Manchester University, 1935). 



OPTICAL ACTIVITY OF BIOL. MATERIAL 25 

TABLE 3 

Content of Optically Active and of Racemic Malic Acid, in ml. of 

Molar Acid Solution per gr. of Dry Weight, in Leaves 

OF Different Plants (Schwarze, 1932) 



1-malie 


dl-malic 


acid 


acid 


0.422 


0.163 


0.133 


0.150 


0.869 


0.485 


0.067 


0.208 



Nicotiana tabacuvi .. 
Pelargonium zonule 
P. peltatiini 

Pub IIS iclaeiis 



According to Rulilancl and Wetzel, the newly formed 
malic acid is always optically active and only later does it 
pass into the racemic form. In Rheum liyhridum, laevo- 
rotatory acid was found to be racemised after the newly 
formed portions of it had penetrated into the roots. 

Bendrat (1929) observed that all malic acid, in the plant 
Sempervivum glaucum, is in the racemic form in the even- 
ing, that it increases during the night, and that, after this 
increase one can find some laevorotatory acid, in the morn- 
ing (Table 4). It seems, then, that the optically active 
form appears in metabolic processes and that it is race- 
mised later. ■ 

TABLE 4 

Content of Total and Laevorotatory Malic Acid, in ml. of Molar 

Acid Solution per gr. of Dry Weight, in the Middle 

Leaves of Sempervivum glaucum (Bendrat, 1929) 

Total malic l-malic 

acid acid 

Evening 0.140 

Morning 0.194 0.013 

Data on other organic acids, though incomplete, agree 
in general with the observations just mentioned. Thus it 
was known to Pasteur that d-tartaric acid as well as dl- 
tartaric acid are present in grape juice (see Thiele, 1911). 

Inactive lactic acid has been found in the leaves of the 
common ash, Fraximis excelsior (Gintl, 1869) and in a 
number of other plants (Stoklasa, 1907). 



26 OPTICAL ACTIVITY OF BIOL. MATERIAL 

Katagiri and Katahara (1937) have shown that, in bac- 
teria, optically pure lactic acid is formed first and that it 
racemises later under the influence of some environmental 
conditions. 

Inactive lactic acid was also recorded in comparatively 
rare post mortem observations in animals (Morishima, 
1900). 

As is well known, dextrorotatory lactic acid is found in 
vertebrates and in different organs of invertebrates and 
racemisation is rare. The tendency has been, for a long- 
time, to explain the presence of this racemic lactic acid 
(especially in the case of bacterial fermentation) by the 
inactivity of the intermediate product, methylglyoxal, 
which has no asymmetric carbon atom and from which 
racemic lactic acid could be formed without the participa- 
tion of an optically active enzyme. But, at present, 
methylglyoxal is no longer considered an intermediate 
product in the transformation of the carbohydrates, and, 
besides, the thorough investigations of Katagiri and Kata- 
hara (1937) have demonstrated an initial formation of 
active lactic acid, which racemises later. 

Racemisation of the secondary substances after they are 
detached from the primary asymmetric complex takes place 
also in the glucosides which, in plants, play the part of 
storage material. The nitrite of mandelic acid which is 
enzymatically synthesised in plants in the relatively pure 
dextrorotatory form is subsequently racemisecl, and in the 
leaves of Primus laurocerasus, a glucoside of racemic dl- 
nitrile is found (Kuhn, 1936; this subject will be examined 
in detail elsewhere). 

The terpenes which, in general, represent vegetable 
secretions but on whose origin and physiological function 
much remains to be investigated are also often found in 
plants in the racemic state. For instance, racemic limo- 
nene or dipentene has been observed in Pinus silvestris, 
Lauriis camphora, Valeriana ojjic'malis and many others 
(Bartelt, 1910, names 16 of them). But optically active 
limonene as well is found in the same or similar kinds of 



OPTICAL ACTIVITY OF BIOL. MATERIAL 27 

plants ; consequently, the secondary origin of the racemic 
form from initially active limonene appears to be probable. 
The same conld be said also of racemic borneol. 

The last group of secondary substances to be considered 
is that of the alkaloids. They seem to represent some 
moditied fragments of protein molecules which perhaps are 
some end products of metabolism. The question of the 
optical purity of the alkaloids in plants has been repeat- 
edly and extensively discussed. Apparently in a great 
number of cases racemisation results from the process of 
isolation. This seems to hold, in particular, for optically 
inactive atropine, which represents the product of race- 
misation of the laevorotatory hyoscyamine, the latter being- 
found in plants in the optically active state (McKenzie 
and Wood, 1919; Hess and Weltzien, 1920). It is known 
that hyoscyamine is very easily racemised by w^eak alkalis 
at room temperature. Some alkaloids, however, it was 
suggested, might be present in plants in the racemic state, 
for instance, coniine and scopoline. Since the racemisa- 
tion of these alkaloids proceeds very slowly even at high 
temperatures and pressures, an artificial racemisation in 
the process of isolation seems excluded (Hess and Welt- 
zien, 1920). The origin, in the plant, of racemic coniine 
and scopoline is therefore still a mystery. 

In spite of the presence of a number of racemic forms 
of alkaloids in plants, the majority of them are found in 
the optically pure state, for instance, nicotine, anabasine, 
etc. The alkaloids constitute, therefore, an exception 
among the secondary substances which have severed their 
connection with the primary complex. It is probable that, 
owing to peculiarities of chemical structure, the mobility 
of some groups in the molecule of several alkaloids is ex- 
ceptionally low; their optical purity would be due, then, 
to a too slow racemisation. In fact, it has not been pos- 
sible to attain racemisation of the alkaloid heliotridane by 
any of the means employed successfully in other cases 
(Menshikov, 1937). 

5. Exclusiveness of the Asymmetry-Sign in Primary 



28 OPTICAL ACTIVITY OF BIOL. MATERIAL 

Substances. As has been said, the primary organic sub- 
stances are obligatorily asymmetric and the secondary 
substances are optionally asymmetric. To this character- 
istic property one should add another which might be called 
the "replaceability" or "non-replaceability" of a given 
optic isomer by its antipode. Substances possessing ob- 
ligatory asymmetry are found in nature in the form of one 
only of the two optical isomers, whilst the secondary sub- 
stances are found as well in the dextrorotatory as in the 
laevorotatory form, often as inactive racemates. We shall 
describe this property as exclusiveness or non-exclusive- 
ness of the asymmetry-sign. 

Exclusiveness of the asymmetry-sign in primary sub- 
stances is a well established fact. In amino-acids, no 
exception has ever been recorded. Only dextrorotatory 
alanine, laevorotatory leucine, dextrorotatory valine, 
laevorotatory histidine, laevorotatory aspartic acid, etc., 
have been isolated from animal or plant tissues. All 
apparent exceptions to this rule could be traced to some 
experimental error as shown by Pringsheim (1910). 

The same holds true for the carbohydrates which pos- 
sess obligatory asymmetry. Only dextrorotatory glucose, 
laevorotatory fructose, etc., can be found in living material. 

The isomer which is present in the biological material 
is often called "natural," whilst its antipode which is pre- 
pared synthetically is considered unnatural, but it is evi- 
dent that the term "natural" as a synonym of "biological" 
is somewhat improper. 

6. N on-Exdusiveness of the Asymmetry-Sign in Sec- 
ondary Substances. Turning now to the substances in 
which optical purity is not obligatory, we find that one 
optical isomer is found in one species of plants and its 
antipode in another. 

Let us consider first the optionally asymmetric carbohy- 
drates. Arabinose, which is found in organic nature in the 
racemic state, can also be present in the form of the rela- 
tively pure dextrorotatory and of the relatively pure lae- 
vorotatory isomers. The left form is the most widely 



OPTICAL ACTIVITY OF BIOL. MATEBIAL 



29 



spread ; it was found, for example, in the leaves of Adonis 
verualis (Eken-stein and Blanksma, 1908), entering in the 
composition of their glucosides. The right arabinose was 
found in the gincoside from Barbados, the so-called barba- 
loine (Leger, 1910). 

Similar findings were recorded in alkaloids. The laevo- 
rotatory alkaloid sparteine, for instance, is widely spread 
in plants ; it was repeatedly isolated from Spartium sco- 
pariiim and Liipinus liiteus. Recently Orechoff, Eabino- 
witch and Konowalowa (1933) discovered the dextrorota- 
tory isomer of sparteine in SopJiora pachycarpa, a plant 
from Middle Asia. 

Blockmann and Roth (1935) reported to have isolated 
and obtained in a chemically pure state laevorotatory alca- 
nine, a red dye found in the roots of Alkanna tinctoria, a 
South-European species ; the dextrorotatory isomer of the 
same substance was obtained from the roots of the Japa- 
nese plant, Lithospermum erytlirorhizon. 

The terpenes were recorded often as dextrorotatory in 
one species and laevorotatory in another (Oudin, 1932; 
Branke and Parishev, 1937). We tabulated below (Tables 
5 and 6) some data on the distribution of the optical 
isomers of the two most important terpenes, borneol and 
limonene. 

It is clear, then, that in secondary substances, both opti- 
cal isomers participate in the composition of living sys- 

TABLE 5 

The Distribution of the Optical Isomers of Borneol in 
Different Plants (Bartelt, 1910) 



1-Borneol 


d-Borneol 


Finns maritima (Belloni, 1906) 


Amomum cordamomiim (Schimmel, 


Thuja occideiitalifi (Wallacli, 1901) 


1897) 


Andropogon nardus (Schimmel, 1899) 


Dryobalanops sp. (Schimmel, 1905) 


Asarum canadense (Power and Lees, 


Lavandula spica (Bouchardat, 1893) 


1902) 


Salvia officinalis (Schimmel, 1895) 


Blumea hdlsamifera (Haller, 1886) 




Fyrctlirum partheniiim (Schimmel, 




1894) 




Tanacetum vulgare (Schimmel, 1895) 





30 



OPTICAL ACTIVITY OF BIOL. MATERIAL 



TABLE 6 

The Distribution of the Optical Isomers of Limonene and of Its 
Eacemic Form in Different Plants (Bartelt, 1910) 



d-Limonene 


1-Limonene 


dl-Limonene 
(= dipentene) 


Pinus serofina 
Juniperus virginiana 
Andropogon sp. 

Laurus campliora 
Pittosporum tmdulaium 
Canarium sp. 
Citrus madurensis 
Barosma sp. 
Myrrlia electa 


Andropogon nardus 


Finns silvestris 

Andropogon citratus; 

A. nardus 
Laurus camphora 

Citrus madurensis 
Barosma sp. 


Carum carvi 
Foenicidum vulgare 
Anethum graveolens 
Apimn graveolens 
Mentha sp. 
Erigeron canadensis 
Lindera sericea 
Massoia aromatica 


Mentha sp. 


Foenicidum vulgare 
Mentha sp. 




Abies pectinata 
Monodora myristica 
Peumus holdus 
Croton eluteria 
E ucalyptus staige- 
riana 








Picea excelsa 
Piper nigrum 
Myristica officinalis 
Lindera sericea 
Xanthoxylum sp. 
Myrtus communis 
Thymus capitatus 
Valeriana officinalis 
Solidago canadensis 



terns, although one of them is usually found more often 
than the other. 

7. Relative Configuration of Biological Material. The 
results of numerous investigations undertaken to establish 
the relative configuration of organic substances may be 
summarized as follows. All biological isomers of amino- 
acids possess the same relative configuration, Fischer and 
Raske (1907) observed that from biological (-) serine can 
be obtained biological (+) alanine and biological (-) cys- 



OPTICAL ACTIVITY OF BIOL. MATERIAL 31 

tine. The suggestion that the reUitive configuration in all 
biological isomers of amino-acids is identical was made by 
Clougli (1918) ; it received confirmation from the work of 
a number of later investigators (Freudenberg and Rhino, 
1924; Langenbeck, 1925; Karrer and Ehrenstein, 1926; 
Levene and Mardaschew, 1937 ; Pfeitf er and Christeleit, 
1937). In general, it is established that the primary sub- 
stances, although they rotate the plane of polarized light 
in different directions, possess the same relative configura- 
tion and form a definite "biological series" of optical 
isomers. Their antipodes are excluded from participation 
in living processes. Not all the potentialities of dissym- 
metric configuration, therefore, are employed in the 
organization of living systems. 

8. Asymmetry as a Criterion of the Organic Origin of 
a Substance. From what has been said, it follow^s that 
every deviation from the racemic state, that is, every 
asymmetry of molecular aggregates represents actually a 
specific attribute of biological systems, and we do not know 
a single case when it would take place outside of living* 
organisms or of the products of their activity. Conse- 
quently, optical activity can be used as a criterion of the 
biological origin of such natural products as petroleum. 
Two theories of the origin of petroleum are generally held, 
one attributing it to an inorganic, the other to an organic 
source. The first suggestion concerning the inorganic, 
volcanic origin of this so-called mineral oil is due to Hum- 
boldt (1804). The theory of its organic origin is still 
older (Lemery, 1675; Lomonosoff, 1761; Spielmann, 1774). 
In its more modern form (see, e.g., Engler, 1906), this 
theory implies that the fat contained in the dead bodies of 
fishes, molluscs and other sea animals, and especially the 
stable palmitic, stearic and oleinic acids are the ancestors 
of petroleum. As a result of a breaking down of the chains 
of carbon compounds under high pressure, hydrocarbons 
with comparatively low boiling point could arise, a poly- 
merization of which, during geological periods, resulted in 
our present-day oil. It is also possible that in some cases 
the initial substance was of vegetable origin. 



32 OPTICAL ACTIVITY OF BIOL. MATERIAL 

The theory of the organic origin of oil entered a new 
phase when Tschngaeff and Walden (1900) pointed ont the 
significance of the forgotten observations of Biot (1835) 
on the optical activity of oil as a criterion of its origin (see 
for further confirmation of these views Vernadsky, 1934). 
Since the asymmetry of molecular aggregates and their 
optical activity represent an attribute of the material of 
living systems only, the theory of the organic origin of oil 
can be considered as based on solid ground. 

The genesis of the optical activity of oil is far from clear. 
Natural fats or glycerides, except lipoids of the lecithin 
type and fats with active acid radicals, are optically inac- 
tive and do not possess any structural dissymmetry. 
Neuberg (1907) outlined the following scheme for the 
transformations undergone by these structurally inactive 
fats in the process of oil formation. Inactive trioleine, 
which constitutes a considerable part of vegetable and 
animal fats, would be the original source. By oxidation 
or hydration, the structurally inactive free oleinic acid 
would be transformed into a dissymmetric racemic body, 
for instance, into dioxystearic acid. If now the racemic 
trioleine with oxidized or hydratated radicals is subjected 
to the asymmetry-producing action of the fat-splitting 
enzymes, optically-active fatty acids would arise. Neu- 
berg and Rosenberg (1907) performed all these transfor- 
mations experimentally; after having obtained optically 
active fatty acids out of structurally inactive material they 
transformed these active acids into optically active oil. 
According to another suggestion of Neuberg (1906), sup- 
ported by Trask (1937), the active constituents of oil may 
result from the transformation of proteins of dead bodies. 
In putrefaction and in autolysis, the transformation of 
amino-acids into corresponding fatty acids is possible ; 
dextrorotatory isoleucine, for instance, has been trans- 
formed into optically active capronic acid. The latter 
could, by further condensation, give the numerous optically 
active hydrocarbons of oil. 



OPTICAL ACTIVITY OF BIOL. MATERIAL 33 

SUMMARY 

1. Dissymmetric molecules are found in inorganic nature 
where they have evidently no relation to life, but it is ques- 
tionable whether life is possible without dissymmetric 
molecules. 

2. In inorganic nature, the two forms of dissymmetric 
molecules are always represented in equal concentrations 
and the aggregate of molecules thus formed is symmetric 
(racemic mixture). 3. Asymmetry of molecular aggre- 
gates is a specific property of protoplasm and of living 
systems. 

4. Primary constituents of protoplasm, such as the 
amino-acids, the lecithins and the majority of the impor- 
tant sugars are present in protoplasm in the form of only 
one of the optical isomers: they are obligatorily asym- 
metric. 5. In these substances, the sign of the optical 
activity is not replaceable by the opposite sign. 6. The 
primary constituents of protoplasm are structurally re- 
lated to each other and form ''biological series" of optical 
isomers. 

7. The secondary constituents of protoplasm which, 
functionally, represent storage material or excreta are not 
obligatorily asymmetric ; they are sometimes found in liv- 
ing organisms in the racemic state. 8. The sign of their 
optical activity is replaceable by the opposite sign, so that 
one optical isomer is sometimes found in one species and 
its antipode in another. 

9. The optical activity of mineral oil lends support to 
the theory of its organic origin. 

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OUDIN, A., C. B. Acad. Sc. 105, 185, 1932. 
PASTEUR, L., Bev. Scientif. Ill, 4, 2, 1884. 

PFEIFFER, P. AND CHRISTELEIT, W., Z. physiol. Chem., 245, 197, 1937. 
PRINGSHEIM, H., Z. physiol. Chem., 65, 89 and 96, 1910. 
RUHLAND, W., and WETZEL, K., Planta, 7, 503, 1929. 
SCHULZE, E. AND WINTERSTEIN, E., Z. physiol. Chem., 45, 79, 1905. 
SCHWARZE, P., Planta, 18, 168, 1932. 
STOKLASA, J., Z. physiol. Chem., 50, 303; 51, 156, 1907. 
THIELE, A., Abderh. Bioch. Handlex., 1, 1149, 1911. 
TRASK, P. D., Nature, 140, 857, 1937. 
TROMSDORFF, Neue Jahrb. Mineralogie, 1937. 
VERNADSKY, W., Problems of Biological Geochemistry, Leningrad (in 

Russian), 1934. 
WALDBN, P., Nat^irwiss. Bundschau, 15, 145, 1900. 



CHAPTER II 

THE ORIGIN AND MAINTENANCE OF OPTICAL AC- 
TIVITY IN LIVING MATTER 

There are, in living matter, some components which con- 
stantly prodnce optically active substances while other 
components always racemize. The mechanism which in- 
sures the constant production of asymmetric substances 
needs an explanation. It is somewhat surprising that, for 
a long time after optical asymmetry had been recognized 
as a characteristic of some constituents of living matter, 
the problem of the origin of this asymmetry and of the 
maintenance of the optical purity of protoplasm was so 
little investigated. 

1. The Transmission of the Asymmetric State hy Asym- 
metric Synthesis. Before studying how the asymmetric 
purity of the primary components of protoplasm is main- 
tained, let us consider how the asymmetric state is trans- 
mitted from one aggregate of molecules to another or, in 
other words, how the asymmetry of protoplasm is "multi- 
plied." To answer this question, Pasteur (1860) sug- 
gested that every asymmetry owes its existence to some 
asymmetric forces operating at the moment at wiiich the 
asymmetry appeared. In this manner, one asymmetric 
substance would bring into being another in the same way 
as life produces life. The principle "Omne viviim ex 
vivo" would be paralleled in the transmission of asym- 
metry, 

Pierre Curie (1894) expressed the same fundamental 
principle as follows: "If a phenomenon possesses a defi- 
nite asymmetry, the same asymmetry can also be detected 
in the causes which have given rise to the phenomenon." 
Curie admits that a given asymmetry gives birth to an- 



36 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

other asymmetry of the same order of magnitude, i.e., 
possessing the same degree of optical purity. We shall 
refer to this view as Curie's principle. 

Emil Fischer (1894), after having stated that "one 
active molecule gives birth to another," illustrates his 
statement by the following concrete example : ' ' The for- 
mation of sugar in plants, according to the observations 
of physiologists, takes place in chlorophyll grains, which 
themselves consist of optically active substances." (The 
optical activity of chlorophyll has been demonstrated 
recently by Stoll and Wiedemann, 1933.) "I assume," 
continues Fisher, "that, in the formation of sugar, there 
is a combination of carbon dioxide or of formaldehyde 
with these substances of the chlorophyll grains and that 
the subsequent production of sugar proceeds asymmetri- 
cally on account of the presence of the asymmetric mole- 
cules of chlorophyll." Thus Fischer considered chloro- 
phyll to be an asymmetric catalyst, the asymmetric state 
of which is transmitted to the molecules of the organic 
substance undergoing synthesis and these are, therefore, 
represented by only one optical isomer. The notion of 
asymmetric synthesis was thus introduced. 

Somewhat earlier, Fischer (1890) had proposed another 
explanation for the origin of the asymmetric state. He 
assumed that, in plants, as in the laboratory, racemic com- 
pounds would appear first and that these would subse- 
quently be split up by the plant itself into their optical 
antipodes. 

A few years after Fischer had expressed this opinion, 
Brown and Morris (1893), in a thorough study of sugar 
metabolism in plants, could find in them neither racemic 
nor laevorotatory glucose. This finding caused Fischer 
to abandon his previous idea. 

It is now well known that laevorotatory glucose does not 
occur in living organisms, that it practically does not fer- 
ment and that it is not used as food by plants or animals. 
Furthermore, a great deal of experimental data has been 
accumulated which shows that, in different enzymatic re- 



ORIGIN AND MAINTEN. OF OPT. ACTIVITY 37 

actions, no intermediate racemic glucose is formed, but the 
optically active product is obtained immediately (c/., 
Tomivasu, 1937). 

The catalytic transmission of the asymmetric state was 
later considered by Strong (1898) in a well known discus- 
sion on asymmetry and vitalism. 

Asymmetric syntheses were soon experimentally real- 
ized in the laboratory. Marckwald (1904) synthesized 
optically active valerianic acid from structurally inactive 
methylethylmalonic acid in the presence of active brucine. 
He defined asymmetric synthesis as a process "in which 
optically active substances are obtained from symmetric 
compounds through the intermediary of optically active 
substances." 

The same year McKenzie also realized some asym- 
metric syntheses. 

Since these pioneer investigations, the literature on this 
subject has expanded considerably and the synthesis of 
optically active compounds from structurally inactive 
material has been carried out by a number of other chem- 
ists. These researches have been well reviewed by Mc- 
Kenzie (1932, 1936) and by Ritchie (1933) to whom we 
refer the reader. 

But, on the question of the fundamental physical mecha- 
nism by which the asymmetric state of the catalyst is 
transmitted to the substance acted upon, there are only 
some still incompletely shaped theories, for example, the 
theory of the so-called asymmetric induction (see Ritchie, 
1933)*. 

To summarize, the authors whose views have been de- 
scribed in this section admitted, generally, that some opti- 
cally active, relatively simple compounds appeared once 
in nature and that, bv asvmmetric syntheses, the asvm- 
metric state has been transmitted to other compounds 
more and more complicated in structure. 

2. The Transmission of Asymmetry , from the Thermo- 
dynamic and Kinetic Point of Vieiv. Recent investiga- 
tions of the kinetics of asymmetric synthesis have con- 



38 



ORIGIN AND MAINTEN. OF OPT. ACTIVITY 



siclerably modified our ideas concerning the maintenance 
of the asymmetric state. 

Among the first observations on this subject, one should 
mention those of Bredig and Fajans (1908) and those of 
Fajans (1910) on the asymmetric splitting of racemic 
camphorocarbonic acid into camphor and carbonic acid in 
the presence of various catalysts. 

Almost simultaneously, Rosenthaler (1908) began his 
studies on the asymmetric synthesis of the nitrite of man- 
delic acid, which were later on repeated and extended by 
a number of other investigators and which constitute, at 
present, the basis for the general theory of asymmetric 
synthesis. He observed that, by combining the symmet- 
ric molecule of benzaldehyde with the symmetric molecule 
of hydrocyanic acid under the action of the asymmetric 
catalyst emulsine, one obtains an optically active nit rile 
of mandelic acid. A considerable excess of dextrorotatory 
over laevorotatory nitrite was recorded. Rosenthaler 
also pointed out that the optical activity of the product 
synthesized by emulsine reached a maximum value after 
a certain time and then decreased (r/. Table 7 ). 

TABLE 7 

Change in Optical Activity during the Enzymatic Synthesis of the 

NiTRiLE OF Mandelic Acid (Eosenthaler, 1908) 

(The numbers give the optical rotation of the synthetic product) 



Temperature 


Time from the beginning of the synthesis : 


1 hour 


3 hours 


24 hours 


25° C. 
30° C. 


1.8 
2.2 


2.8 
2.6 


2.0 
2.1 



Other observations on the change of optical activity dur- 
ing asymmetric synthesis were made by Nordefeldt 
(1922). The optical activity was found to tend asympto- 
tically to zero {cf. Fig. 3). 

Bayliss (1913), Krieble (1913) and Nordefeldt (1922) 
showed the important fact that the synthesis of racemic 
mandelo-nitrile takes place in the absence of enzymes and 



ORIGIN AXD MAINTEN. OF OFT. ACTIVITY 



39 



>- 

> 

H 
O 
< 

_J 
< 

o 

I- 

Q. 
O 




TIME — *■ 
Fig. 3. Change in optical activity, as a function of time, in the enzymatic 
asymmetric synthesis of the nitrile of mandelic acid (from Kulm, 1936). 

that the addition of the latter accelerates the reaction and 
gives to it an asymmetric character but does not influence 
its equilibrium constant. 

These observations made it possible for Werner Kuhn 
(1936) to undertake the theoretical analysis of the prin- 
ciples of the asymmetric synthetic action of enzymes. We 
shall summarize here his more important conclusions. 

It should be pointed out, first, that the separated active 
components of a given organic substance and the equi- 
molecular mixtures of these components (racemates) are 
not equivalent from the thermodynamic point of view. 
The mixing of the components into a racemate liberates 
energy, while their separation requires an expenditure of 
work. Consequently the optically active state is not a 
state of equilibrium as compared to the racemic state. 
The question arises, then, as to the manner in which such 
conditions of thermodynamic disequilibrium can be real- 
ized in catalytic reactions in living matter. One might 
first inquire whether such reactions are true catalytic 
reactions or not. 

Let us consider the characters of a true catalysis lead- 
ing to the formation of an asymmetric compound. Inas- 
much as the preparation of the left and that of the right 
antipode of a given substance in equal concentrations are 
equivalent from the standpoint of energy expenditure, the 



40 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

constant Ki of equilibrium between the /-antipode of the fi- 
nal substance and the initial product must be equal to the 
constant K^ of equilibrium between the c/-antipode of the 
final substance and the initial product. If c is the con- 
centration of the initial substance, c'l the concentration 
of the 7-antipode and c'a the concentration of the d-m\ti- 
pode of the final substance, one has 

c'l/c = K, = c'd/c = Kd (1) 

Condition (1) characterizes a true catalysis. If this con- 
dition is not fulfilled, the initial substance will be simul- 
taneously in equilibrium with different concentrations c'l 
and c\i of the two antipodes and the final product will be 
partially optically active. But this is thermodynamically 
impossible in the case of true catalysis. 

Another character of true catalysis is that the value of 
the equilibrium constant in equation (1) is the same ir- 
respective of whether a catalyst is used or not. The 
velocity constant A-,, in the formation of the f/-antipode 
from the initial material and the velocity constant k'^ in 
the reverse conversion are increased by the catalyst to 
the same degree. If the addition of an enzyme would in- 
fluence the two velocity constants differently and change 
the equilibrium constant, the reaction would not be a true 
catalysis and the final product would be optically active. 

Experimentally, as has been said above, it was found 
that, in the synthesis of the nitrile of mandelic acid, the 
use of the catalyst does not change the equilibrium 
constant. 

That both velocities I'a and k'a are accelerated to the 
same extent by the catalyst has been proved in optically 
non-specific enzymatic reactions {cf. Borsook, 1935). 

Furthermore, Nordefeldt (1922) has observed that if in 
the synthesis of mandelo-nitrile, one adds emulsine when 
the reaction has already proceeded for a while, the enzyme 
does not change anything in that which has already been 
transformed, it exerts its asymmetrical effect only on the 
material yet to be transformed. In a system which has 
reached the state of equilibrium without enzymes, the ad- 



ORIGIN AND MAINTEN. OF OPT. ACTIVITY 41 

ditioii of an enzyme does not change anything either. 
This does not leave any doubt that, in the case of these 
isolated enzymatic transformations, we are dealing with 
true catalysis. 

Finalh% in true catalysis, the optical activity of the sub- 
stance being formed represents only a temporary phe- 
nomenon which gradually disappears. This will be clearer 
when we have examined the dynamics of the two ways in 
which optical activity could be obtained in biochemical re- 
actions, namely, the splitting up of racemates and asym- 
metric synthesis. 

In the splitting of a racemate consisting of two anti- 
podes, Ai and A^, which change respectively into Bi and 
B,j, one can represent the process as follows: 



k. 
k' 



(2) 



If the left initial product Ai is transformed into Bi with 
a velocity constant A,, different from the constant A-^ with 
which Aa is transformed into B^, there results optical 
activity. If Jii = Jici the racemate will be split up sym- 
metrically. 

In asymmetric synthesis, a symmetric initial substance 
A is transformed with different velocities, A^^ and k^, into, 
respectively, Bi and B,„ according to the diagram 




(3) 



A- 
Kuhn integrated the systems of differential equations 



42 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

corresponding to these two cases and studied the dynamics 
of the change of optical activity in terms of time. 

In the case of the splitting up of a racemate (2), if 
ki/k,, >> J , and K >> 1, the substance Bj will be obtained 
almost exclusively at the beginning; its concentration 
might approach c„/2 (if c,, is the concentration of the initial 
substance) ; later, A^, will be transforming itself into B,i 
till, finally, the concentrations Bj and Ba are equalized. At 
the initial and final states the solutions will be optically 
inactive. 

In the case of asymmetric synthesis (3), assuming again 
that ki/ka >> 1 and K >> 1, there will be, at the begin- 
ning, an accumulation of the /-form, B,-, the whole initial 
material (c,,) will be practically transformed into this 
/-antipode, since the velocity constant A",, is supposed to be 
very low as compared to k,. The concentration of the initial 
substance A will approach cjK. A will also change very 
slowly into B,j and, as a result of this change, its concentra- 
tion will be reduced and the equilibrium between A and Bi 
will be disturbed: consequently, a certain quantity of B, 
will be transformed into A. This will cause a further 
transformation of the initial substance into B,i. The pro- 
cess will continue as long as the initial substance A is in 
equilibrium simultaneously with Bj and B,i or, in other 
words, until the racemic state is obtained. So the same 
catalyst which, at first, brought about the transformation 
of A into practically pure antipode B, later causes a com- 
plete racemization of the product. 

It is of interest to inquire what is the difference in the 
stability of the temporary state of optical activity in the 
case of the splitting up of a racemate and in that of asym- 
metric synthesis. Kuhn showed that the ratio H between 
the time T^ necessary for racemization and the time Ti 
necessary for the attainment of maximal activity is 
H = ki/ka ■ K/2 in the case of asymmetric synthesis and 
H = ki/kf, in the case of the splitting of the racemate. The 
factor K/2 is absent in the second equation. Since the 
constant K is large, it is evident that the stability of the 



ORIGIN AND MAINTEN. OF OPT. ACTIVITY 43 

optically active state will l)e considerably greater in 
asymmetric synthesis than in the splitting of racemates. 

The experimental data reported above, concerning the 
temporary character of optical activity {cf. also Bredig 
and Fajaiis, 1908; Bredig and Minaeft", 1932; Nordefeldt, 
1922) are in agreement with. Knhn's calculations. The 
conclusion to derive from this agreement is that 
true catalysis occurs in the isolated enzymatic systems 
considered. 

Furthermore, it should be noticed that asymmetric syn- 
thesis, which seems to take place in protoplasm rather 
than dissociation of racemates, is precisely the process 
which secures a longer duration of the state of optical 
activity. 

The maintenance of asymmetry in mineral oils is 
probably to be explained by the extreme slowness of the 
transformations which take place in them. 

3. Maintenance of Optical Purity by the So-C ailed 
" Stereo-aiitonomic Substances." If, in enzymatic sys- 
tems, one has to do with true catalysis, and in true 
catalysis there is a gradual decrease in optical activity, 
one might expect that a substance formed in an asym- 
metric synthesis be optically less pure than the compound 
from which it originates (Langenbeck and Triem, 1936). 
Curie's principle, postulating that any asymmetry origin- 
ates from another asymmetry of the same order would 
not hold then. So the presence of protoplasmic com- 
ponents in the form of pure optical isomers for an in- 
detinitely long time still lacks an explanation. The 
features which, in the organization of protoplasm, are 
responsible for the maintenance of optical purity are still 
to be found. 

Kuhn (1936) showed that, in some cases, the mainten- 
ance of optical purity in a system, despite the gradual 
decrease of optical activity in a single synthetic enzymatic 
process, can be explained by the behavior of some sub- 
stances that he called "stereo-autonomic." It is knowni 
that the right nitrile of mandelic acid, when synthesized 



44 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

by the plant, is stored not as such but combined with 
gentiobiose in the form of 3-giucoside (natural amyg'da- 
line). The latter easily crystallizes from water solutions, 
while the glucoside which consists of gentiobiose and of 
the left nitrite of mandelic acid possesses such a high 
solubility that it does not, in general, crystallize from 
water solutions (Walker and Krieble, 1909; Krieble, 
1912).^ So, the fact that pure natural amygdaline is de- 
posited in the plant does not necessarily postulate the 
existence of an optically specific enzyme, synthesizing only 
the right nitrite of mandelic acid. The right and the left 
nitrites may be produced; then the right component pre- 
cipitates in the form of gentiobioside ; the excess left 
nitrile can thereupon be racemized according to the re- 
quirements of true catalysis ; the right nitrile originating 
from this process is again bound to gentiobiose and the 
process continues until all the nitrile is converted into the 
less soluble gentiobioside of the right nitrile, i.e., into pure 
natural amygdaline. The same final state would evi- 
dently ensue no matter whether the enzyme possesses the 
capacity of preferential synthesis of the right nitrile or if 
it would synthesize racemic nitrile. In the latter case, 
however, the gradual catalytic transformation of the left 
component into the initial substance (benzaldehyde and 
hydrocyanic acid) and the resynthesis of the right com- 
ponent would demand a long time, which is evidently 
spared by the utilization of an optically specific enzyme. 
Natural optically active gentiobiose is a stereo-autonomic 
substance in the sense that it conditions the stable optical 
purity of the synthetic product. Kuhn sees a confirmation 
of his views in the fact that, in the fruits of Prunns 
laurocerasus, one finds a gentiobioside of the pure right 
nitrile of mandelic acid, while, in the leaves of the same 
plant, one finds a glucoside of the racemic nitrile. It is 
probable that the difference in solubility brought about 

1 Similar differences are found, in general, in diastereomers, that is, in 
substances consisting of one antipode of a substance A combined Avitli either 
of the tAvo antipodes of a substance B. For example, AiBi and AiBa are 
two diastereomers. 



ORIGIN AND MAINTEN. OF OPT. ACTIVITY 45 

by glucose as a component of the glucoside and by gen- 
tiobiose as a component of the gentiobioside is responsible 
for the fact that they are fomid in nature as indicated. 

4. Procedures Used by Nature for Mainfaiuing Optical 
Purity and Establishing a ''Fixed Internal Milieu." The 
evolution of living beings has consisted in a gradual in- 
crease of the number of fixed parameters of the internal 
milieu. For example, in the transition from poikilother- 
mic into homoiothermic animals, the body temperature has 
been fixed at a constant value. The non-dependence on 
the temperature of the external medium has given the 
homoiotherms important advantages over the cold-blooded 
animals in natural selection. 

Considering such cases of specific fixity acquired by the 
internal medium, Claude Bernard made his famous state- 
ment : * ' La fixite du milieu interieur est la condition de la 
vie libre " ( " The fixity of the internal medium is the con- 
dition of independent living"). 

The elaboration of mechanisms in living matter to 
maintain optical purity evidently contributes to the fixity 
of the internal milieu. The spatial parameters which de- 
termine the asymmetry of a substance are fixed in primary 
constituents of protoplasm in such a way that optical 
purity is maintained. 

Barcroft (1934) notes that two methods are used by 
nature to secure the constancy of internal medium, the 
method of evasion and the method of correction. 
a) Widely Different Velocities in the 
Formation of the Two Optical Iso- 
mers. As a mechanism of evasion nature uses a high 
ratio of the reaction velocities. A-,/ A',,. While, in the cases 
of catalysis more commonly encountered, this ratio is of 
the order of 1 to 2, in enzymatic reactions the ratio reaches 
100, 1000, or even greater values. This ratio evidently 
determines the degree of predominance of the right or of 
the left isomer in the substance synthesized. A great dif- 
ference between the velocity constants, Jii and k,i, makes that 



46 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

the non-iitilizable isomer appears only in insignificant con- 
centrations in the first stages of synthesis. A large value 
of the constant of equilibrium K, on the other hand, as- 
sures a more lasting stability of the active state. Both 
these factors contribute toward having the system 
"evade" for a time the effects of inevitable racemization. 
h ) Langenbeck and Triem's IVIechan- 
i s m . Recently, Langenbeck and Triem (1936) have 
shown that an increase of optical purity can be obtained 
in interrupted reactions between two optically impure 
substances. If an optically impure enzyme is acting upon 
an optically impure substance and if the reaction is not 
allowed to proceed to the end, the optical purity of the 
system may be increased. Let us suppose that two sub- 
stances, optically active but not optically pure, A and B, 
combine to form AB. Let us assume also that the 
laevorotatory isomers, Ai and Bi, predominate over their 
antipodes. A,, and B^. The following partial reactions 
will take place 

A. + B,->A,B. 

A, + B, ^ A,B, 

A, + Ba -^ A.Ba 

A, + B,^A,Bi 

Since [A.] > [AJ and [B,] > [BJ, we shall have, if 
we interrupt the reaction before it is completed, 
1A3J [AJ [A.B,] rB.] 

[A,B,] ^ [AJ "^'^^ [A,BJ ^ [BJ ' 

If, for instance, the concentrations of the initial sub- 
stances, Ai and A,,, are in the ratio 2 : 1, and if the concen- 
trations of Bi and B,, are identical respectively to those of 
Ai and A,,, i.e., are also in the ratio 2: 1, a time will come 
at which the ratio of the concentrations of the enantio- 
morphic products, AjB, and A^B,,, will be the product of 

2 X 2 
the ratios, tttTj that is, 4 : 1. If the reaction is inter- 
rupted at that time, the optical purity of the transformed 
material will be increased. (It is certain that, simultan- 



ORIGIN AND MAINTEN. OF OPT. ACTIYITY 47 

eoiislv, the remainder of the untranst'ormed substances 
will undergo a" corresponding decrease in optical purity.) 

Langenbeck and Triem (1936) proved experimentally 
that the optical activity can be increased in reactions of 
this type. They synthesized /-tyrosine anhydride from 
/-tyrosine methyl ether and observed a concentration of 
30.8% of /-tyrosine in the final product while the initial 
substance contained only 27.4%. 

It is possible that such processes have taken place in 
the enzymatic origin of ferments, that is, when one fer- 
ment has been synthesized wdth the aid of another opti- 
cally active ferment. Then the necessary decrease of 
optical purity of the initial material is of no importance 
since only the newly formed ferment, in the interrupted 
reaction, will transmit to some other substance its 
increased optical purity. 

It should be noted, in relation with the reactions 
described in this section, that the succession of synthetic 
processes which take place continuously in living systems 
might in itself be an important factor in the evasion of 
the effects of racemization. The incessant reconstruction 
of living matter should then, perhaps, be considered as 
an indispensible condition for the maintenance of the 
optical purity of stereo-autonomic substances. 

It is usually thought that, though nature might evade 
for a time the eifects of racemization, finally the latter will 
inevitably set in and that nature does not possess any 
method of correction by which it would remove the un- 
natural isomer and actively resist racemization. Kuhn 
(1936) not only accepted the idea of the absence of such 
active resistance, there being no enzyme know^n for per- 
forming this function, but he thought that the racemiza- 
tion which finally takes place might constitute, in part, the 
process of ageing. 

The fact that, when animals and plants are fed with 
racemic amino-acids, they principally consume the natural 
isomers of the left steric series and leave the other isomer 
intact, has been, in general, considered as proving that the 



48 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

organisms are devoid of enzymes suitable for catalyzing 
transformations of the unnatural isomers. 

Scliulze and Bosshard observed this selective action of 
one isomer in lower organisms already in 1886 and their 
data were later confirmed by a great number of authors 
and especially by Pringsheim (1910). 

Many similar observations were made on mammals. A 
dog which receives a racemic preparation of leucine 
(Abderhalden and Samuely, 1906) or of alanine (Abder- 
halden and Schittenhelm, 1907) consumes preferably the 
natural isomers and excretes in its urine a large portion 
of the unnatural amino-acids. The same was observed 
later by Abderhalden and Tetzner (1935) in rats, rabbits 
and dogs fed with racemic alanine. 

But another series of facts points out the possibility of 
the presence, in living protoplasm, of an active mechanism 
contributing, by a method of correction, toward maintain- 
ing optical purity and thus toward securing the fixity of 
the internal medium. 

(c) Krebs' Mechanism. Krebs (1933) who has 
undertaken extensive investigations on the oxidative de- 
amination of different amino-acids by tissue slices of liver 
and kidney from rat, pig, cat, dog and rabbit, discovered 
the very important fact that, while both optical isomers of 
amino-acids are deaminated, the unnatural forms of the 
right steric series are almost always deaminated much 
more rapidly than the natural ones {cf. Table 8). 

These observations were soon confirmed by Kisch (1935) 
and by Neber (1936). Some data of Kisch are given in 
Table's. 

Krebs (1935) assumes that there are two different 
enzymatic systems one of which catalyzes the deamination 
of the right and the other that of the left amino-acids. 
This assumption follows, in particular, from the fact that 
the deamiiiation of the left amino-acids is inhibited by 
octyl alcohol, Avhile that of the unnatural isomer of the 
right series is not affected by octyl alcohol of the same 
concentration. He further points out that the data con- 



ORIGIN AND MAINTEN. OF OPT. ArTIVITY 



49 



TABLE 8 

Deamination of Optically Active M/20 Amino-acids by Slices 
OF Rat Kidney (Krebs, 1933) 



Aiiiino-;u'id 


ml. of ammonia 


Ratio of the velocity 

of deamination of 

the unnatural to 


mg. of tissue x hours 


that of the 
natural 
isomer 


i(+)al;niiiie 
r7(-)al;inine 

Z(+) valine 
d(-) valine 

I (-) loiu'ine 
d (+) leucine 

?(-)phenyl-a]amne 
d (+) phenyl-alanine 

i(— )histidine 
d(+)histi(line 


3.36 ] 
37.80 ^ 

3.86 ] 
57.60 ^ 

6.68 ] 
34.90 1 

10.4 ] 
77.0 ( 

3.18] 
9.75 ( 


11.3 

14.9 

5.2 

7.4 

3.1 



TABLE 9 

DEAiriNATioN OF M/50 Amino-acids by Slices of Liver axd Kidney 
OF Different Animals (Kisch, 1935) 

(The velocity of deamination is expressed in mI/5000 of NH3 per 
gram of fresh weight of tissue in 2 hours) 



Animal 



Organ 



Number 

of 
experi- 
ments 



Amino-acid 



Velocity of deami- 
nation of the 



natural 
isomer 



0) 



unnatural 
isomer 



(d) 



Ratio of the 
velocity of 
deamination 
of the un- 
natural to 
that of the 
natural 
isomer 



Rat 


Kidney 


3 


Alanine 


15.7 


56.8 


3.6 


1 1 


1 1 


3 


Leucine 


3.9 


115.6 


29.6 


i I 


1 1 


2 


Isoleucine 


16.2 


141.1 


8.7 


1 1 


Liver 


2 


Alanine 


1.9 


11.2 


5.9 


Sheep 


Kidney 


2 


Alanine 


13.5 


81.6 


6.0 


< < 


< t 


2 


Leucine 


1.2 


24.0 


20.0 


( ( 


I i 


2 


Isoleucine 


13.2 


118.9 


9.0 


Pig 


Kidnev 


2 


Alanine 


16.9 


122.2 


7.2 


( ( 


i I 





Leucine 


2.8 


52.0 


18.6 


(< 


1 1 


4 


Isoleucine 


12.5 


136.5 


10.9 


( ( 


Liver 


5 


Alanine 


7.1 


23.3 


3.3 



50 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

cerning the oxidative deamiiiatioii do not contradict the 
fact of a more rapid consumption by the whole organism 
of natural isomers of amino-acids. Natural amino-acids 
can evidently be consumed without deamination ; consump- 
tion and deamination need not to coincide. 

From the standpoint of the problem of the maintenance 
of optical purity in protoplasm, Krebs' results are very 
significant. The study of catalysis has shown that in 
protoplasm composed of optically pure left isomers of 
amino-acids the appearance of small quantities of the 
right forms is inevitable. Krebs' data suggest that the 
organisms have developed a mechanism for removing the 
isomers of unsuitable spatial configuration. This mecha- 
nism would consist in a deamination of the inappropriate 
right forms as soon as they appear. The right isomers 
would be transformed into structurally inactive keto-acids 
identical with those which can be obtained from the left 
amino acids. In this manner, the organisms would by no 
means be so helpless in regulating the optical purity of 
their protoplasm as was assumed by Kuhn and they would 
possess an active method of correction for securing the 
fixity of their internal medium. 

It is to be noticed that Ritchie (1933), before any of the 
researches that we mentioned on oxidative deamination 
had been made, admitted a priori the possibility of the 
existence of such a method of correction. He wrote that, 
while one of the antipodes participates in cell metabolism, 
the other, which is formed simultaneously but at a much 
lower rate, almost certainly is removed as soon as it is 
formed. Ages of evolution would be responsible, accord- 
ing to him, for the development of such a physiological 
regulating system. 

One might, at first, be inclined to consider the existence 
of a special enzymatic system acting on unnatural isomers 
of amino-acids, as a chance happening without particular 
significance. But, then, what sense is there in talking of a 
"specificity" of any enzymatic reactions, and in explain- 
ing this specificity as a result of a long process of natural 
selection? {Cf., Eric Holmes, 1937.) 



ORIGIN AND MAI NT EN. OF OPT. ACTIVITY 51 

Krebs (11)36) proposed another interpretation of his 
data. Following Emil Fischer's somewhat archaic views 
on a possible synthesis in protoplasm of racemic amino- 
acids and of their subsequent splitting into optical iso- 
mers, he considered the deamination by a deaminase 
specific for right amino-acids as a process by which the 
organism decomposes the racemate and obtains the left 
amino-acids required. But it has been seen above that the 
experimental data available do not speak in favor of the 
hypothesis of a primary symmetric synthesis. 

5, Biological Advantages of Optical Purity. In the 
study of the methods used by nature to maintain optical 
purity, some authors have considered the advantages for 
living organisms of working with asymmetric material. 
Mills (1932) has attempted to show that living systems 
consisting of substances in the asymmetric state are more 
efficient than their hypothetical racemic competitors would 
be. On the basis of what is known on the stereo-specificity 
of the action of, for example invertase, in the hydrolysis 
of saccharose one can expect that the common invertase 
activate onlv the clextrorotatorv saccharose and that the 
optical isomer of this invertase act only on the left saccha- 
rose. Let us consider the initial stage of the reaction, 
when, with small concentrations of saccharose, the velocity 
of hydrolysis is approximately proportional to the con- 
centration of the enzyme and to the concentration of the 
substance acted upon. In an experiment with optically 
pure saccharose and corresponding invertase, every mole- 
cule of saccharose coming in contact with the enzyme will 
be subject to activation, while in an experiment with a 
racemic saccharose and a r//-invertase only one-half of the 
collisions will be effective. Consequently, the reaction in 
the racemate will take place at a considerably lower rate 
than that in the optically active system. 

It should be noticed that, in the case of two enantio- 
morphic systems of transformations working side by side 
(racemic material), the velocities of many processes might 
be decreased when the two dissvmmetric substances inter- 



52 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

act. Consequently, the synthesis of the components of 
new tissues and the growth of the latter will proceed more 
rapidly with asymmetric than with racemic material. 

If the fundamental physiological processes are more 
intense in asymmetric systems, the passage from racemic 
to optically active protoplasm was a significant physio- 
logical advance which contributed to the survival of asym- 
metric protoplasm in the process of natural selection. Be- 
sides, the development of asymmetry, by contributing to 
the fixity of the internal medium, increased the possibility 
of independent life for any given organism, in the sense 
of Claude Bernard. 

6. The Origin of the Asymmetry of Protoplasm. Assum- 
ing that the asymmetry of protoplasm is maintained by 
some mechanism devised by nature, a fundamental prob- 
lem still remains to be solved, that of the origin of the 
initial inequality of the right and the left components of 
protoplasm. 

In the study of the causes of the initial asymmetry, the 
authors have followed two directions. Some have at- 
tempted to correlate the origin of the asymmetry of 
matter with the asymmetric influence of terrestrial mag- 
netism; others have considered asymmetry as originating 
in a deviation from a statistical average. 

Since C^otton (1896) had shown that solutions of opti- 
cally active substances possess different coefficients of 
absorption for the right and the left circularly polarized 
light, it has been thought that the action of such light 
might furnish a promising method of obtaining active 
compounds from racemic ones. 

It is known that the circularly polarized light is found 
in nature, for instance, when the plane-polarized light 
from the sky is reflected on the surface of the sea. Byk 
(1904) suggested that, because of the rotation of the plane 
of polarization of light by terrestrial magnetism, there 
must be, in the total quantity of light circularly polarized 
at the surface of the earth, a predominance of one of the 
two forms of light. This predominant form acting for 



ORIGIN AXD MAINTEN. OF OPT. ACTIVITY 53 

long' periods of time on racemic compounds would initiate 
optical activity. 

More recently Kuhn and Braun (1929) and Kulni and 
Knopf (1930) have shown that, in laboratory experiments, 
when circularly polarized light is used in the photochemi- 
cal decomposition of racemates, it causes the appearance 
of optically active isomers. 

Ritchie (1933) and later Langenbeck and Triem (1936) 
supported the hypothesis just described. 

The second explanation of the origin of optical asym- 
metry (cf., Pearson, 1898; Fitzgerald, 1898; Bartrum, 
1898; Errera, 1898; Kipping and Pope, 1898; Byk, 1925; 
Mills, 1932) is based on the assumption that the equality 
of the right and left components represents a statistical 
mean value around which fluctuations occur. Kipping and 
Pope (1898) observed, for example, that, while the occur- 
rence of either right or left component, in crystallization 
experiments, furnished a mean value of 50.08% ± 0.11, 
the proportion varied from 24.14/^ to 77.36% in separate 
experiments (46 of them). An inequality of the right or 
the left form of a substance might have originated acci- 
dentally in this manner when some living systems were in 
formation and this inequality might have spread by asym- 
metric catalysis (Strong, 1898). 

Lately Spiers (1937) supported the chance deviation 
hypothesis of the origin of asymmetry. 

7. General Survey of the Problem of the Origin and 
Maintenance of Optical Asymmetry. The various stages 
in the development and maintenance of the asymmetric 
state are represented diagramatically in Fig. 4. 

Let us note, first, that there are two levels of stability 
for the state of symmetry or asymmetry: 1. the level of 
thermodynamic stability which characterizes the racemic 
state ; 2. the level of protoplasmic stability which is main- 
tained by living matter. In inorganic nature, the race- 
mates are stable because they possess the least amount of 
free energy. In living nature, optically pure forms are 
stable because they are the most advantageous in natural 



54 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 



t 

>- 

> 

I- 
o 
< 

< 

o 

I- 

Q. 
O 



< 

I- 

a 

D 



LEVEL OF PROTOPLASMIC STABILITY 



c\ \' \ A 



■f 

B 



LEVEL OF THERMODYNAMIC STABILITY 



TIME 



Fig. 4. Diagram representing the processes involved in the origin and 
maintenance of optical purity in living matter. The arrows A represent the 
Langenbeck and Triem mechanism; the upward arrows B, the Krebs mecha- 
nism; the downward arrows C, the Kuhn mechanism; the horizontal arrow 
3 illustrates Curie 's view according to which asymmetry is simply transmitted 
from one asymmetric molecule to another. 

selection. So the racemic state is stable in inorganic 
nature and unstable in living matter. 

For living systems to pass from the level of thermo- 
dynamic to that of protoplasmic stability and to stay at 
the latter level requires a series of mechanisms of which 
two have been described : the Langenbeck and Triem 
mechanism by which the optical purity is increased in a 
series of interrupted reactions (upward arrows in plain 
lines, A^ in the diagram) and the Krebs mechanism by 
which the unnatural optical isomers are removed (upward 
arrows in dotted lines, B). 

Since the racemic state represents a state of thermo- 
dynamic equilibrium, the initial asymmetry will have a 
tendency to disappear (Kuhn's mechanism, downward 
arrows, C, in the diagram). 

The eifect of circularly polarized light in inducing some 
asymmetry will probably not be sufficient to maintain the 
high degree of optical purity exhibited by protoplasm. 

It is possible also that the Langenbeck and Triem 
mechanism, which probably is not efficient enough to main- 
tain the almost absolute purity of protoplasm as we know 



ORIGIN AND MAINTEN. OF OPT. ACTIVITY 55 

it at the present time, was involved in the early stages of 
natural selection while Krebs' mechanism, which is more 
higlily efficient, was developed only later in evolution. 

The asymmetric state of protoplasm and its mainte- 
nance by regulative mechanisms appear, then, as a heri- 
tage of countless ages of transformations, and as a result 
of the elaboration by nature of systems which seem to tend 
to some sort of physiological perfection. 

SUMMARY 

1. According to the earlier authors, asymmetry, once 
originated, has been transmitted from one substance to 
another, as life is transmitted from one living being to 
another. 

2. Emil Fischer suggested that asymmetric catalysts 
synthesize asymmetric compounds from symmetric ones. 
Such asymmetric syntheses were soon realized in labora- 
tory experiments. 

3. It was then observed that, in an asymmetric synthe- 
sis, the optical activity reaches a maximum and then 
decreases, and that the enzyme does not influence the equi- 
librium constant of the reaction. These observations have 
been the basis of theoretical investigations by Kuhn on 
the thermodynamics of asymmetric synthesis. 

4. Kuhn pointed out that the separation of two optical 
isomers requires an expenditure of work, while their mix- 
ing into a racemate liberates energy, the optically active 
state being a state of disequilibrium. He further showed 
that the characters presented by enzymatic reactions are 
those thermodynamically expected in true catalysis. 

5. Optical purity might be conditioned in some cases by 
the behaviour of "stereo-autonomic substances," i.e., of 
substances whose properties, such as solubility, maintain 
one isomer in solution while the other separates out. 

6. To maintain the state of disequilibrium inherent in 
optical purity, nature, it seems, has developed regulating- 
mechanisms, such as: {a) The use of widely different 
velocities in the formation of the two optical isomers in 



56 ORIGIN AND MAINTEN. OF OPT. ACTIVITY 

asymmetric syntheses; (b) The succession of reactions 
which are interrupted before the optical activity has had 
time to disappear (Langenbeck and Triem's mechanism) ; 
(c) The more rapid deamination of the unnatural isomer 
which then separates out (Krebs' mechanism). 

7. Optical purity seems to impart to protoplasm some 
advantages in natural selection, in particular, it seems to 
increase the reaction rate and the growth activity. So, the 
establishment of a state of optical purity can be con- 
sidered as a method used by nature to stabilize the inter- 
nal milieu. 

8. The origin of asymmetry has been ascribed by 
several authors to the influence, on some reactions, of 
circularly polarized light, which would be predominantly 
right or left on account of its rotation by terrestrial mag- 
netism ; other authors think that the equality of right and 
left isomers is a statistical mean value and that the 
inequality resulted from fluctuations from the mean. 

BIBLIOGRAPHY 

ABDEEHALDEN, E. and SAMUELY, F., Z. physiol. Chem., 47, 346, 1906. 
ABDEEHALDEN, E. and SCHTTTENHELM, A., Z. physiol. Chem., 61, 

323, 1907. 
ABDEEHALDEN, E. and TETZNEE, E., Z. physiol. Chem., 232, 79, 1935. 
BAECEOFT, J., Features in the Architecture of Physiological Functions, 

Cambridge, 1934. 
BAETEUM, C. O., Nature, 58, 545, 1898. 
BAYLISS, W., Journ. Physiol., 46, 236, 1913. 
BOESOOK, H., Ergebn. Ensymforsch.. 4, 1, 1935. 
BEEDIG, G. and FAJANS, K., Ber. chem. Ges., 41, 752, 1908. 
BEEDIG, G. and MINAEFF, M., Biochem. Z., 249, 241, 1932. 
BEOWN, H. and MOEEIS, H., Journ. Chem. Soc., 63, 604, 1893. 
BYK, A., Z. physil-al. Chem., 49, 641, 1904. 

, NaUirwiss., 13, 17, 1925. 

COTTON, A., Ann. Chim. et Phys., 8, 347, 1896. 

CUEIE, P., J. de Physique, 3, 393, 1894. 

EEEEEA, G., Nature, 68, 616, 1898. 

FAJANS, K., Z. phy.nlcal. Chem., 73, 25, 1910. 

FISCHEE, E., Ber. chem. Ges., 23, 2138, 1890 ; -21, 3189, 1894. 

FITZGEEALD, G. F., Nature, 68, 545; 69, 76, 1898. 

HOLMES, E., The Metabolism of Living Tissues. Cambridge, 1937. 

JAPP, F. E., Nature, 68, 452, 1898. 

KIPPING, F. S. and POPE, W. J., Nature, 69, 53, 1898. 



ORIGIX AXD MAIXTEN. OF OPT. ACTIVITY 57 

KISCH, B., Biochcm. /., JSO. 41, l!tH5. 

KEEBR, H. A., Z. i)hysiol. Clicm., 217, 191, 1938. 

, Bioch'. Jom-n., 29, 1620, 1935. 

, Ann. Mev. Biochem., 5, 247, 1936. 

KEIEBLE, v., Journ. Am<r. Chcm. Soc, 34, 71(>, 1912; 35, 1643, 1913. 
KUHN, W., Ergehn. Enziimforscli., 5, 1, 1936. 

, Angetc. Chem., 49, 215, 1936. 

KUHN, W. and BRAUN, E., Xatiirwisn., 17, 227, 1929. 
KUHN, W. and KNOPF, E., Xaturwi.s.s., 18, 183, 1930. 
LANGENBECK, W. and TRIEM, G., Z. pluisil-al. Chem., 177, 401, 1936. 
MARCKWALD, ^\ ., Bcr. chcm. Gcs., 37, 349, 1904. 
McKENZIE, A., J. Chcm. Soc, S'5, 1249, 1904. 

— ■, Angew. Chem., 4.5, 59, 1932. 

• — , Ergcbn. Enzymforsch., 5, 49, 1936. 

MILLS, W. H., Journ. Soc. Chem. Industry, 51, 750, 1932. 

NEBER, M., Z. physio}. Chem., 240, 59, 1936. 

NORDEFELDT, E., Biochem. Z., 131, 390, 1922. 

PASTEUR, L., Reclierches sur la dissymetrie moleciilaire des produits 

organiques naturels. Soc. Chim. Paris, 1860. English translation in 

Alembie Club Reprints, 14, 1860. 
PEARSON, K., Nature, 58, 495 ; 59, 30, 124, 1898. 
PRINGSHEIM, H., Z. physiol. Chem., 65, 89 and 96, 1910. 
RITCHIE, P., Asymmetric Synthesis and Asymmetric Induction. Oxford, 

1933. 
ROSENTHALER, L., Biochem. Z., 14, 238, 1908. 
SCHULZE, E. and BOSSHARD, Z. physiol. Chem., 10, 134, 1886. 
SPENCER, H., Nature, 58, 592, 1898. 
SPIERS, C, Naturwiss., 25, 457, 1937. 

STOLL and WIEDEMANN, Uelv. Chim. Acta, 16, 307, 1933. 
STRONG, W. M., Nature, 59, 53, 1898. 
TOMIYASU, Y., Biochem. Z., 292, 234, 1937. 
WALKER, W\ and KRIEBLE, W., J. Chem. Soc, 95, 1437, 1909. 



CHAPTER III 

HEREDITY AND THE INFLUENCE OF ENVIRONMENTAL 

FACTORS ON THE OPTICAL ACTIVITY OF 

BIOLOGICAL MATERIAL 

Pasteur (1860) wondered how living beings would dif- 
fer from what they are if the basic chemical substances 
which compose them would change the sign of their opti- 
cal rotation. Emil Fischer (1890) attempted to show how 
this question can be answered experimentally. ''If it 
proves possible", he writes, '"to feed plants, moulds or 
yeasts with unnatural optical isomers of different sub- 
stances, should one not expect that such a change in the 
constructive material would result in a modification of 
the delicate molecular architecture and of the entire 
structure of organisms ? The biologists have not yet 
studied this question because the chemists have not given 
them the substances necessary for such experiments". At 
present, fifty j^ears after Emil Fischer made this state- 
ment, we know that such transformations of biological 
structures are impossible, as the following data will show. 

1. The Impossibility of Iiirertinf/ the Optical Proper- 
ties of the Primary Constituents of Protoplasm. It is now 
well established that, if one gives to microorganisms 
unnatural food material as, for instance, laevorotatory 
leucine, they are simply unable to make use of such food 
as they do not possess the suitable enzymatic outfit. This 
was soon ascertained by Fischer himself who found that 
laevorotatory glucose is practically not fermented by 
yeast cells (Fischer and Thierf elder, 1894). 

In the light of modern knowledge one would not expect 
that a simple change in nutrient conditions would deter- 

59 



60 orr.M rn .. h i:in:niTy a\d I':\] ihoxmext 

mine the sign of asymmetry of protoplasm, wliicli is, 
according- to all evidence, the result of a long evolution- 
ary development. 

Among more recent investigations on the inefficacy of 
culture media on the sign of optical activity of primary 
constituents of protoplasm, we shall mention those of 
Gause and Smaragdova (1938). These authors have 
studied the etfect on the yeast, Torula iitilis, of a pro- 
longed cultivation in the optical isomers of leucine. The 
yeast was cultivated at 27° C on pure left leucine in one 
series and on pure right leucine in another. During the 
iy2 months that the experiment lasted, 14 passages were 
made. At the end of this period the rate of growth in 
both series was measured. A similar procedure was fol- 
lowed for a study of the action of right and left valine. 
It was found that, though growth of Torula proceeds 
much more rapidly on the biological forms of these amino- 
acids (laevorotatory leucine and dextrorotatory valine), 
there is some growth on their optical antipodes. It should 
be noticed that the majority of other yeasts cannot grow 
at all on the unnatural isomers of amino-acids. The very 
possibility of a weak but unlimited growth of Torula 
utUis on the right leucine and on the left valine is corre- 
lated with the fact that these substances are first deamin- 
ated by yeast (leucine, according to Ehrlich, 1906, is 
transformed into iso-amyl alcohol), and the ammonia 
thus formed proves to be a sufficient source of nitrogen 
for the unlimited growth in Torula utilis. But the supply 
of nitrogen in the form of ammonia alone is not sufficient 
for most of the otljer species of yeast and a prolonged 
growth on the unnatural isomers of amino-acids cannot 
take place in them. 

As was to be expected, a prolonged cultivation of Torula 
on such nonbiological isomers had no essential influence 
on the asymmetric properties of the protoplasm of these 
yeasts, so that growth always remained more rapid on the 
natural amino acids. Structurally inappropriate isomers 



orr. ACT I v.. in:in:niTY \\i) r:\]Jh'o\]iE\T 61 

cannot be directly involved in nietabolisiii. I'lie slight 
growth observed on aniino-acids which can be deaminated 
and thus transformed into material deprived of dissym- 
metry, from which subsequently molecules of suitable 
properties are built, even confirms this view. This con- 
clusion is in accord with a number of recent observations 
(Conrad and Berg, 1937; Du Vigneaud et al., 1939; Rat- 
ner, Schoenheimer and Eittenberg, 1940). 

2. The ImpossihUity of Modifying Protoplasm so as 
to Cause it to Invert the Optical Properties of the Prod- 
ucts of its Metabolism. That some products of meta- 
bolism, that is, some secondary constituents of living mat- 
ter may be generated in either of the two optically 
isomeric forms, under the influence of external conditions, 
has been admitted for a long time by a number of inves- 
tigators. The case of lactic acid fermentation by some 
microbes is classical in this respect. Concerning this 
case we shall mention first the fundamental facts on the 
specificity of each bacterial strain in the production 
of one type of lactic acid and then we shall review the 
more important investigations on the effect of external 
conditions on such production. 

Nencki (1891) showed that the optical form of the lac- 
tic acid i^roduced in microbial fermentation is specific 
for the kind and strain of microbes. Some species pro- 
duce the pure dextrorotatory isomer, others give the 
pure laevorotatory one and still others produce a form 
of lactic acid which is either totally or partially racemic. 
The sign of the asymmetry of the secondary substances 
seems, therefore, to represent a stable hereditary char- 
acteristic of the physiological organization of the cell 
which has produced these substances and Nencki even 
proposed to employ it for the identification of bacteria. 
These observations were subsequently confirmed by Cur- 
rie (1911), Pederson, Peterson and Fred (1926) and by 
Katagiri and Kitahara (1937). 



62 OPT. A CTIY., HEREDITY AND ENVIRONMENT 

As to the problem of the possibility that different 
isomers of lactic acid be produced when conditions of 
cultivation are changed, the data of the literature have 
for a long time been somewhat contradictory. 

a. I n f 1 u e n c e of General Culture Con- 
ditions. It was Pere (1893) who claimed for the first 
time that one and the same line of Bacterium coli^ in dif- 
ferent culture conditions can produce the two inverse 
forms of lactic acid (cf. also Pottevin, 1898, and Pere, 
1898). But to what extent the strains of microbes used 
by them were bacteriologically pure is not clear. 

Pederson, Peterson and Fred (1926) showed that in 
mixed cultures of microbes consisting of producers of 
the right and left isomers of lactic acid, one can often 
observe a change in the form of the lactic acid produced 
when the temperature, for example, is changed. How- 
ever, in such cases, there is evidently no inverting mech- 
anism in protoplasm. The species and strains of mi- 
crobes which produce the right and the left isomers of 
lactic acid possess different temperature optima of 
growth and metabolism and consequently, in some con- 
ditions of cultivation, the strains which produce the right 
isomer and, in other conditions, those which give the left 
isomer of lactic acid predominate. With microbic mate- 
rial of a guaranteed purity having originated from one 
cell, the possibility of inverting the lactic acid produced 
has not been observed even in the most dift'erent culture 
conditions, though the only isomer produced may subse- 
quently undergo racemisation to various degrees. 

6. I n f 1 u e n c e of the Culture M e d i u m. 
In the literature the work of Kayser (1894) is often re- 
ferred to as confirming Pere 's data, whilst, as a matter of 

lit is to be pointed out that Bacterium coli does not cause a pure 
lactic acid fermentation. Approximately half of the sugar is fermented 
into lactic acid, the remainder is transformed into acetic acid, ethyl 
alcohol, carbonic acid and hydrogen (Neuberg and Gorr, 1925). After 
this was shown, the investigators began to work with true lactic-acid- 
producers such as Lactobacillus. 



Orr. ACT IV.. HFJihUUTY A^'D ENVIRONMEl^T 63 

fact, the results of these two authors do not agree. Kay- 
ser studied the formation of the optical isomers of lactic 
acid by different strains of microbes cultured on various 
sugars. He used 14 bacterial species or strains, with 
which he performed 61 experiments. His data, which are 
sunmiarized in Table 10, show that there are strains which 
always produce the laevorotatory lactic acid, others 
which always produce the dextrorotatory isomer and 
finally some which yield a racemic mixture. Kayser's 
results (1894), therefore, coincide not with Pere's (1893) 
but with the data of recent investigators, particularly 
with the thorough observations of Katagiri and Kitahara 
(1937). In only one of the 61 experiments of Kayser (the 
last one marked w^ith an asterisk in the table) was dextro- 
rotatory lactic acid produced on one sugar (glucose) and 
laevorotatory lactic acid on another (maltose). Since 
this single excej^tion might have resulted from an in- 
sufficient purity of the bacterial culture employed (cf. 
Pederson and his collaborators, 1926), one comes to the 



TABLE 10 

ixfluexce of various sugars i.\ the nutritive medium ox the optical 

Properties of the Lactic Acid Produced by Different Bacteria 

(Kayser, 1894) 
(The letters a, b, c, . . . refer to the species and strauis of bacteria, 
most of which were not completely identified; the letters 1, d. and dl 
indicate the optical rotation of the lactic acid produced; the figures 
(1) and (2) after Maltose and Lactose mean that two media of dif- 
ferent composition were used with each one of these sugars.) 



Sugar 


a 


b 


r 


c 


d 


e 


s 


g 


h 


1 


m 


n 





P 


Arabinose 
















1 






Xylose 
























1 






Mannite 
























1 






Glucose 


1 


1 


1 


1 


1 


d 


dl 


1 


d 


d* 


1 


dl+1 


1 


1 


Le\ailose 






1 








dl 










1 






Galactose 




1 












1 








1 




dl 


Maltose (1) 


1 




1 


1 


1 






dl 


d 


1 


dl 


dl 


dl 


dl 


Maltose (2) 
























1 






Lactose (1) 




1 




1 


1 


d 




1 


d 


dl 


1 






1 


Lactose (2) 














dl 








1 






1 


Sucrose 






1 






d 


dl 




d 




1 


1 


dl 


dl 


Melezitose 






















1 


1 






Trehalose 
























1 






Starch 






















1 


1 







64 OPT. A CTn ., HEREDITY A^D EX 1 IROXMEXT 

conclusion that Pere's results are by no means confirmed 
by Kayser's. 

This same table shows that racemization is greatly in- 
fluenced by the culture medium. A given strain of bac- 
teria, when cultured on a definite sugar, forms an almost 
optically pure lactic acid (Pederson and collaborators, 
1926, had shown that it is never entirely optically pure), 
while on another sugar, it forms a racemic mixture. This 
is the case, for instance, in the strains g, m, n, o, p. Ped- 
erson and his collaborators (1926) made further observa- 
tions on this point. 

There are also the recent researches of Tatum and his 
coworkers (1932) in which 4 strains of lactic acid bacte- 
ria producing laevorotatory acid, 3 strains producing the 
dextrorotatory acid and 13 different strains of Clostri- 
dium acetohutylicwn were used. These authors found 
that lactic acid bacteria produce the optically pure form 
of lactic acid when grown separateh/ and the racemic 
form when grown in association with the microbe caus- 
ing acetonebutylic fermentation {Clostridium acetohuty- 
licum ) . 

At first Tatum (1932) interpreted his results in the 
light of the hypothesis of Orla- Jensen (1919) according 
to which there are in the bacterial cell two independent 
enzymes, one of which produced the right and the other 
the left lactic acid. In consequence of the association 
of lactic acid bacteria with the butylic bacteria the meta- 
bolism of the former would change in such a manner that 
both optical isomers of lactic acid would start to be pro- 
duced. However, in his later work (1936), Tatum showed 
that, in the association of the two types of bacteria, lactic 
acid is always initially formed m the optically active 
state by the lactic acid bacteria, and that Clostridium is 
only responsible for the subsequent racemization. (It is 
interesting to note that racemization takes place in the 
presence of antiseptics, therefore it is of enzymatic na- 
ture.) The investigations were continued by Katagiri 



OPT. . 1 r '77 1 .. // i:ri:dit\ amj i:\ virosmext 65 



and Kitahai-a [VXu) who eslablislicd that the lactic-acid 
bacteria which produced racemic acid are also capable 
of racemiziiig a ready-made active acid. This is per- 
formed by a special enzyme, called by these authors race- 
miase. Those species and strains of bacteria which, in 
fermentation, form as a tinal product optically active lac- 
tic acid cannot racemize a ready-made active acid, they 
have no racemiase. The formation of racemiase is closely 
dependent on the culture conditions. 

The results of the experiments of Katagiri and Kita- 
hara (1937) are represented in Table 11. They show 
clearly that, with various strains of Leuconostoc mesen- 
teroides and Lactobacillus sake, a change of the condi- 
tions of cultivation never brings about a change in the 
type of the lactic acid formed ; a greater or lesser degree 
of racemization of the substance produced accounts for 
all the observed facts. 

c. I n f 1 u e n c e of T e m p e r a t u r e. Similar 
results were reported also in investigations on the in- 

TABLE 11 

Inflxjence of the Sugar Fermented ox the Optical Foi:m of the 

Lactic Acid Produced in the Fermentation 

(Katagiri and Kitahara, 1937) 

(The letters 1, d and dl indicate the optical rotation.) 



Microorganism Glucose ' Fructose 



Mannose Galactose Arabinose Xylose 



Leuconostoc 














No. 34 


1 


1 


1 


— 


1 


1 


No. 52 


1 


1 


— 


1 


1 


1 


No. 13 


1 


1 


— 


1 


— 


— 


No. 14 


1 


1 


— 


1 


— 


— 


Lactobacillus 














No. 41 


d 


— 


d 


d 


— 


— 




' No. 24 


d 


d 


— 


— 


d 


— 




' No. 53 


d 


d 


— 


— 


d 


— 




No. 37 


dl 


dl 


— 


— 


d 


— 




' No. 42 


dl 


dl 


— 


— 


d 


— 




No. 45 


dl+d 


— 


dl+d 


dl+d 


d 


— 




No. 57 


dl+d 


— 


— 


— 


d 


— 


" No. 58 


dl + d 


— 


— 


— 


d 


— 



66 OPT. ACTIV., HEREDITY AXD EXVTRONMENT 

fiuence of nitrogen nutrition and of temperature upon the 
optical form of the lactic acid obtained in bacterial fer- 
mentation. The effects of various incubation tempera- 
tures are presented in Table 12. One sees that the tem- 
perature does not influence the sign of the optical rotation, 
but that the degree of racemization of the acid regularly 
increases with the rise of temperature. 

Thus the old data of Kayser (1894) become clear. In 
ditferent conditions of culture, the lactic acid formed by 
a specific strain of bacteria possesses a different degree 
of optical purity depending on the quantity of racemiase 
contained in the bacterial cells. 

Recently Kopeloff (1937) has shown that racemiase is 
sometimes lost in the transition of the R-f orms of lactic 
acid bacteria into the S-forms. 

To summarize, the production of a specific optical 
isomer, in the case of such secondary substances of the 
protoplasm, or products of metabolism, as lactic acid, 
represents a fixed hereditary character which is not de- 
pendent on the conditions of cultivation. It is only such 
processes as the velocity of a catalytic racemization of 
secondary substances initially formed in the optically 
pure state and the formation of racemiase which are de- 
pendent on the culture conditions. The hereditary char- 



TABLE 12 

Influexce of the Temperature of Incubation upon the Optical Form 

OF the Lactic Acid Obtained in Fermentation 

(Katagiri and Kitahara, 1937) 

(The letters 1, d and dl indicate the optical rotation.) 



Microorganism 


Temperature 


30OC 


20°C 


6°C 


Leuconostoc No. 14 


1 


— 


1 


No. 6 


1 


1 


— 


Lactodacillus No. 41 


d 


— 


d 


No. 42 


dl 


— 


dl + d (17%) 


No. 24 


dl+d (39%) 


dl+d (59%) 


— 


No. 45 


dl-l-d (10%) 


dl+d (47%) 





orr. A(:ti\ .. // hredity asd environment 67 

acter of optical activity in secondary constituents of pro- 
toplasm and its independency on the external conditions 
indicates that some physiological mutations peculiar to 
some specific strains of bacteria must have occurred 
some time in the past in those of them which produce opti- 
cally unusual isomers. 

Let us now attempt to penetrate into the nature of the 
process by which a given isomer arises instead of its 
antipode. This problem is directly related with the study 
of some of the basic principles which underly the forma- 
tion of physiological mutations. 

3. Mechanisms Controlling the Production of a Given 

Optical Isomer. 

a. Production of Dissymmetric S u b- 
stances from Symmetric P h e n y 1 - G 1 y o x a 1. 
The observations of various authors concerning the trans- 
formations of phenyl-giyoxal, a substance deprived of 
dissjmimetry, into mandelic acid which possesses an asym- 
metric carbon atom, are important in the study of the 
question here discussed. These transformations are cat- 
alysed by enzymes known generally as ketonaldehydemu- 
tases. Starting from a symmetric initial product these 
enzymes synthesize directly, without any intermediate 
racemic stage, the optically active mandelic acid. Fur- 
thermore, ketonaldehydemutases of ditferent species of 
bacteria synthesize from the same initial product sub- 
stances which are optically inverse, as the results re- 
ported by different authors and represented in Table 13 
show. 

The action of the ketonaldehydenmtases is probably to 
be attributed to the asymmetric state of these enzymes. 

There are many observations more or less directly re- 
lated to those just given. Neuberg and Simon (1926), for 
example, found that an acetic-acid bacterium, B. ascendes, 
produced laevorotatory amyl alcohol from a racemic 
valeric aldehvde, while another bacterium of acetic acid 



68 OFT. ACTI v.. HEREDITY AND ENTIF0X3IEXT 

fermentation, B. pasteurianuni, formed dextrorotatory 
amyl alcohol (an excess of 5 to 18 per cent) from the same 
initial aldehyde. Analogous results were also obtained 
with Bacterium pasteurianum in acetone preparations. 

6, P r o d u c t i n of Optical Isomers by 
Esterases. The data of Willstatter and his collabor- 
ators on the stereochemical specificity of esterases, the en- 
zymes which catalyse the hydrolysis of the ethers of dif- 
ferent organic acids, are of special interest in the present 
problem. Some of these data are presented in Table 14, 
but for more complete information we refer the readers to 
the review by Rona and Amnion (1933). 

In the majority of cases the esterase from liver and 
the esterase from pancreas catalyze the hydrolysis of op- 
tically inverse forms in initial racemic substrates. 

c. P r o d u c t i o n of Optical Isomers by 
Optically Active Alkaloid Catalysts. 
Bredig and Fajans (1908) and Fajans (1910), in their 
classical investigations on the decomposition of racemic 



TABLE 13 

Optical Activity of Maxdelic Acid Produced by Various 
Microorganisms from Phenyl-Glyoxal 



Organism 


Initial 

Substance 


Mandelic Acid 
Obtained 


Author 


1. Bacterium ascendes 


Phenyl-glyoxal 


d{-) 


about 100% 


Mayer, 1926 


2. Lactobacillus 48 


(( 


1( + ) 


84% 


t( 


3. The same; Acetone 


(( 


1( + ) 




ft 


preparation 
4. B. (lelbruccki 


(< 


1( + ) 


82% 


Neuberg and 
Simon, 1927 


5. B. lactis aerogenes 


f ( 


d(-) 


10% 


ti 


6. B. proteus 

7. The same; Acetone 
preparation 


Phenyl-glyoxal 
hydrate 

it 


d(-) 
d(-) 


95% 

78% 


Hayashi, 1929 

t( 


8. B. fluorescens 


(( 


d(-) 


43% 


ti 


9. B. jyyocyaneum 


a 


d(-) 


37% 


it 


10. B. procligiosum 


it 


d(-) 


87% 


ti 


11. B. coli 


ti 


d(-) 


68 to 75% 


tt 


12. Parts of green 
plants 


Phenyl-glyoxal 


d(-) 


about 100% 


Binder- 
Kotrba, 1926 



OPT. ACTI \ ., HEREDITY AND ENVIRONMENT 69 

cam]ilioro-cai'boiiic acid into caini)lior aiul carbonic acid 
under the inHiience of catalysts (optically active alka- 
loids), have established that laevorotatory quinine cata- 
lyses a more rapid decomposition of the left camphoro- 
carbonic acid, while dextrorotatory quinidine causes a 
more rapid decomposition of the right camphoro-car- 
bonic acid. 

TABLE 14 

Sign of the Optical Rotation of the Component which is more Rap- 

inLY HYPitni.YSED IX A Race:\iic Ether rxDER the Action of 

Esterases of Different Origin 

(CF. Rona and Ammon, 1933) 



Initial i-acemic substrate 



Esterase from 
Pig's Pancreas 



Esterase from 
Pig's Liver 



Mandelic acid — methyl ether 
" " ethyl ether 

" " monoglyceride 

Phenylmethoxyacetic acid — methyl ether 
Phenylchloroacetic acid — methyl ether 
Phenylaminoacetic acid — propyl ether 
Tropic acid — methyl ether 



(-) 
(-) 
(-) 
(-) 
(-) 
( + ) 
( + ) 



( + ) 
( + ) 
( + ) 
( + ) 
(-) 
( + ) 
(-) 



A similar condition has been observed in the synthesis 
of optically active substances from structurally inactive 
material. Bredig- and his collaborators, in their study of 
the synthesis of nitrites from hydrocyanic acid and dif- 
ferent aldehydes with the aid of optically inverse cata- 
lysts of known chemical constitution, have obtained the 
results reported in Table 15. 

So the optically inverse catalysts, quinine and quini- 
dine, bring about the synthesis of optical antipodes of the 
nitrite of mandelic acid from initial structurally inactive 
material. Quinine behaves in this respect analogous to 
emulsin, while quinidine has the properties of the anti- 
pode of emulsin. If the optically active catalyst, there- 
fore, is of a given pign, it affects in a definite direction 
the products of the catalyzed reaction. 

''/. I^ r () d u c t i o n of a G i v e n p t i c a 1 
Isomer by a Chemical Alteration of 
the Catalyst. In the cases studied in the preced- 



70 O FT. A CTIY., HEREDITY AND ENVIRONMENT 

ing pages substances of different optical signs result from 
the action of optically active catalysts of different signs. 
One of the two inverse catalysts might have originated by 
an inverson from the other, but another possibility is that 
the original catalyst has been changed chemicall}^ so as 
to produce an optically inverse substance without being 
itself inverted. The following case illustrates this last pos- 
sibility. In the study of the enzymatic hydrolysis of 
racemic ethyl mandelate by the esterase of the human 
liver, it has been found that if one adds some strych- 
nine, one obtains a strongly laevorotatory mandelic acid 
instead of the usual dextrorotatory one (Bamann and 
Laeverenz, 1930). Strychnine does not influence the vel- 
ocity of hydrolysis of the right ether, but it strongly in- 
creases that of the left ether thus causing the formation 
of an excess of laevorotatory material (for further de- 
tails see Rona and Amnion, 1933). There was no optical 
inversion of the enzyme but the chemical properties of 
the latter have been changed by combination with strych- 
nine. 

e. C o n t r o 1 of the Production of Op- 
tical Isomers by Intermediate * ' P a t h- 
w ays ". Let us now consider in greater detail the case 

TABLE 15 

Optical Pkoperties of the Nitriles Synthesized from Hydrocyanic 
A( ID AND VAiiioiw Aldehydes undei: the Action of Different 

Organic Catalysts 



Initial aldehyde 


Catalysts 


Synthetic nitrile 


Author 


Benzaldehyde 


Emulsin 


d — nitrile 


Rosenthaler, 

1908 


(( 


Quinine 


d — nitrile 


Bredig and 
Fiske, 1912 


a 


Quinidine 


1 — nitrile 


** 


Cinnamic aldehyde 


Emulsin 


d — nitrile 


Rosenthaler, 
1909 


(( (( 


Quinine 


d — nitrile 


Bredig and 
Minaeff, 1932 


a « 


Quinidine 


1 — nitrile 


a 



OPT. ACTIY., HEREDITY AXD ENVIRONMENT 71 

so much investigated of the production of one optical 
form of hictic acid by one kind of organism and of the 
production of the other isomer of lactic acid by other 
organisms. 

An important contribution to the study of this phe- 
nomenon has been brought forward by Embden, Baldes 
and Schmitz (1912) who discovered that in the transfor- 
mation of glucose into lactic acid by different animal tis- 
sues dextrorotatory lactic acid is produced exclusively. 

Another important advance was the finding of Neuberg 
(1913) that extracts of animal tissues transform ethyl- 
glyoxal, a structurally inactive body, into laevorotatory 
lactic acid. A number of papers were then published on 
the methyl-glyoxal reactions. It was found that in all 
cases when methyl-glyoxal is converted into optically ac- 
tive lactic acid the latter is laevorotatory. This was ob- 
served, in particular by Neuberg and Kobel (1927) with 
the yeast, Sacfiharomyces cerevisiae, by Neuberg and 
Simon (1928) with Mucor javanicus and by Widmann 
(1929) with Bacterium fluorescens. 

From these observations Embden, Deuticke and Kraft 
(1933) drew the important conclusion that since in tis- 
sues of higher animals pure dextrorotatory lactic acid is 
always formed and since the same tissues transform 
methyl-glyoxal into laevorotatory lactic acid, methyl- 
glyoxal cannot be the precursor of the dextrorotatory 
lactic acid which appears in normal metabolism. Embden 
then developed his theory of glycolysis in muscle which 
received general acknowledgment. But, so far as we are 
concerned in the present review, the essential fact is that 
both optical isomers of a certain substance can appear in 
metabolism when different intermediate substances are 
involved. The left isomer of lactic acid is obtained 
from glucose if the intermediate is methyl-glyoxal, and 
the right isomer of lactic acid if the intermediate is, 
according to current views, pyruvic acid. Embden sug- 
gested that one or the other of these ^'pathways" could 



Z^-- 



'JU.( / , 



72 OPT. AVTl 1 ., HEREDITY AM) EX 1 Ih'OXMEXT 

be followed in the cells of different organisms and then 
the production of the right or the left isomer of lactic 
acid by various types of bacteria or tissues would be 
accounted for. A physiological mutation which brings 
about an optical inversion of the secondary protoplasmic 
constituents may consequently consist in a change of the 
intermediate pathways in the transformation of these 
substances. 

It appears then that the right and the left form of a 
substance should not be considered so fundamentally op- 
posed as far as their production is concerned, since one 
has only to change the path followed in the transforma- 
tions to obtain one or the other. 

What happens when a right or left isomer originates 
might also happen when the relative configuration of 
optically active organic substances is concerned. In the 
majority of cases optical isomers entering into the com- 
position of living systems possess the same relative con- 
figuration. Thus the configuration of natural alanine is 
the same as that of natural ephedrine (Freudenberg and 
Nikolai, 1934) ; the configuration of natural proline is the 
same as that of natural nicotine according to Karrer 
(see Pfeilfer and Christeleit, 1937). But there are also 
cases in which the substances which constitute living sys- 
tems belong to different series. Thus dextrorotatory lac^ 
tic acid, which is so generally found in the tissues of 
higher animals, possesses the same relative configuration 
as the unnatural laevorotatory tartaric acid, which is 
never found in organic material. (It also has the same 
configuration as natural dextrorotatory alanine; cf. 
Freudenberg and Rhino, 1924, and Freudenberg, Brauns 
and Siegel, 1923). The unity or the diversity of the rel- 
ative configuration of organic substances, as well as the 
character of being dextro- or laevorotatory, might depend 
onl}^ on the biochemical path followed in the formation 
of the substance. 



OI'T. A(TI\ .. H Eh'HniTY WD i:S \ I ROX M EXT 73 

/. V o 11 t r () 1 of the I * r o d u c t i o n o t' U p - 
t i c a 1 isomers by an Inversion of 
t li e W a 1 d e n Type. "Walden (1905) has shown 
that some optically active substances, when subjected to 
a series of substitution reactions, come out inverted. For 
example, dextrorotatory alanine treated w^ith bromides 
forms l-bromopropionic acid and, upon reaction with am- 
monia, alanine is again obtained, but laevorotatory alan- 
ine. Such a process is known as the ''Walden inver- 
sion" and, according to Eniil Fischer, it is "the most 
remarkable finding in the field of optical activity since the 
fundamental investigations of Pasteur". 

The mechanism of this inversion is far from clear. 
In reactions of a certain type the asymmetric carbon 
atom must be acted upon in such a manner that the con- 
figuration of the molecule is inverted. In the example 
given above one cannot even say if this is accomplished in 
the transformation of alanine into bromopropionic acid 
or in the transformation of bromopropionic acid into 
alanine. 

Mills (1932) had attempted to explain the Walden in- 
version on the basis of some peculiarities of substitution 
reactions. According to Levene, Rotheii and Kuna (1937), 
there does not seem to be any general agreement on this 
point. 

Some have assumed that in biochemical reactions the 
presence of a special enzyme, the waldenase, would be 
responsible for the Walden inversion of some amino- 
acids. This assumption, however, does not seem to stand 
a critical stud3\ 

Walden inversion might perhaps play a role in some 
biochemical processes, such as in the formation by some 
bacteria of laevorotatory lactic acid from dextrorota- 
tory glucose (see Freudenberg, Brauns and Siegel, 1923), 
while other bacteria form dextrorotatory lactic acid. This 
assumption, however, is not in the trend of current bio- 
chemical theories. It is generally .supposed that, in the 



74 OPT. A CTIV., HEREDITY AND ENVIRONMENT 

formation of the left lactic acid, the structurally inactive 
methyl-glyoxal represents an intermediate stage and, 
according to Embden's scheme, in the formation of dex- 
trorotatory lactic acid, the structurally inactive pyruvic 
acid is the intermediate stage. The dissymmetric con- 
figuration of the molecules is believed to disappear in the 
intermediate stages of transformation and then to re- 
appear again. In this interpretation one assumes that 
the asymmetry of molecular aggregates has disappeared 
in the intermediate stages because of the loss of dis- 
symmetry of the molecules. 

However, it is not necessarily so and Neuberg (1913), 
questioning such intermediate loss of dissymmetry, 
brought forward the theory of ''temporary dissymmetric 
substances." If, for instance, in the intermediate stages 
of transformation, methyl-glyoxal possesses some H and 
OH groups attached to it, the dissymmetric configuration 
of molecules will not disappear nor the asymmetric struc- 
ture of molecular aggregates; then it would be possible 
that in the production of one of the two optical isomers 
of lactic acid from an initial active glucose by one type of 
microbes a Walden inversion of the configuration of mole- 
cules takes place. 

The various questions studied in the last two sections 
suggest the two following generalizations which may be 
of significance in understanding the basic principles of 
vital activity : 1 . The impossibility of altering the optical 
properties of the primary substances of protoplasm fits in 
with the assumption that the activity of the fundamental 
physiological systems is based upon the principles of 
''fixed pathway", i. e., all the intermediate transforma- 
tions in these physiological systems would proceed along 
definitely fixed paths. On the contrary, the optical prop- 
erties of the secondary protoplasmic substances can be 
altered to a certain extent. Consequently, the formation 
and the transformations of the secondary substances are 
not bound by the principles of fixed pathway. 2. Fur- 



OPT. ACT IT., HEREDITY AND ENVIRONMENT 75 

thermore, in the fundamental protoplasmic systems, there 
are devices to avoid racemization, which were discussed 
previously; similarly it seems that there are devices to 
avoid inversion, such as those regulated by the ''prin- 
ciple of fixed pathway". Neither of these devices oper- 
ates in the transformations of secondary protoplasmic 
constituents. 



SUMMARY 

1. It is not possible to invert, by external influences, 
the asymmetric structure of the primary constituents of 
protoplasm which is the result of a long evolutionary 
process. 

2. As to secondary substances of protoplasm such as 
products of metabolism (for example, lactic acid in fer- 
mentative processes), the sign of their optical activity 
also represents a fixed hereditary character of the spe- 
cies or strain which elaborated them, but external in- 
fluences can affect the catalytic racemization of these 
products which were initially formed in the optically pure 
state. 

3. Concerning the mechanism by which the produc- 
tion of a given optical isomer is controlled in metabolic 
activities, one should note that: (a) From the same 
symmetric initial substrate, enzymes of ditferent organ- 
isms can synthesize optically dilferent substances; (b) It 
is often through optically inverse catalysts that optical 
antipods are synthesized; (c) Some catalysts can, after 
chemical alterations which do not constitute an inver- 
sion, synthesize substances in a form which is the optical 
inverse of the form that was synthesized before the al- 
teration of the catalyst; ( d) The sign of the optical rota- 
tion of the final product of a series of metabolic reactions 
may depend on the "pathway" followed in intermediate 
reactions; (e) Inversions of the Walden type (transfor- 
mation of one optical isomer into its antipod in a series 



76 OPT. A CTI I . , HEREDITY AND EN VI RON M EN T 

of chemical transformations) may play a role in biologi- 
cal phenomena. 

4. Physiological mutations which, in the evolutionary 
development of an organism, bring about optical inver- 
sions of the secondary protoplasmic constituents may con- 
sist in a change of the intermediate "pathways" in a 
series of reactions. 

5. Protoplasmic systems are provided wdth mechanisms 
by which "pathways" that would lead to an inversion of 
the asymmetric structure are avoided. Primary constit- 
uents of protoplasm are then regulated by a principle 
called here: ''Principle of fixed pathway". Such mech- 
anisms do not operate in the transformations of the sec- 
ondary substances of protoplasm. 

BIBLIOGRAPHY 

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BREDIG, G. and FAJANS, K., Ber. chem. Ges., .',1. 752, 1908. 

CONRAD. R. and BERG, C J. Biol. Chem., Ill, 351, 1937. 

CURRIE. J. N., Bioch. Bull., 1, 103, 191. 

DU VIGNEAUD, V., COHN M., BROWN, G., IRISH, O., SCHOEN- 
HEIMER. R. and RITTENBERG, D., J. Biol. Vhem., 131, 273, 1939. 

EHRLICH, F., Biochem. Z.. 1, 8, 1906. 

EMBDEN. G.. BALDES, K. and SCHMITZ, E., Biochem.. Z.. J,5, 108, 

1912. 
EMBDEN, G.. DEUTICKE, H. and KRAFT, G., Klin. Wochenschr., 12, 

213, 1933. 
FAJANS, K., Z. physikal. Chem., 73, 25, 1910. 
FISCHER, E., Ber. chem,. Ges., 23, 370, 1890. 

FISCHER. E. and THIERFELDER, H., Ber. chem. Ges., 21, 2031, 1894. 
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56, 193, 1923. 

FREUDENBERG, K. and NIKOLAI, F., Lieb. Ann. Chem., 510, 223, 
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FREUDENBERG, K. and RHINO, F., Ber. chem. Ges., 51, 1547, 1924. 
GAUSE, G. F. and SMARAGDOVA, N. P., Biol. J. (Russian), 1, 399, 
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KATAGIRI, H. and KITAHARA, K., Biochem. J., 31, 909, 1937. 

KAYSER, E., Ann. hist. Pasteur, 8, 737, 1894. 

KOPELOFF, L. and N., J. Bad., 33, 331, 1937. 

LEVENE, P., ROTHEN, A. and KUNA, M., J. Biol. Chem., 120, 111, 
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MILLS, W. H., J. Soc. Chem. Ind., 51, 750, 1932. 

NENCKI, M., Zhl. Bakt., 9, 304, 1891. 



OPT, ACTI 1 .. H EUEDl TY A\JJ ENVIRONMENT 77 

NEUBERG, C, Biochem. Z., 51, 484, 1913. 
NEUBERG, C. and GORR, G., Biochem. Z., KUl 482, 1925. 
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PASTEUR, L., Recherches sur la dissymetrie moleculaire des produits 
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PEDERSON, C, PETERSON, W. and FRED, E., J. Biol. Chem., 68, 

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CHAPTER IV 

ON THE RELATION BETWEEN THE INVERSION OF 

SPIRALLY TWISTED ORGANISMS AND THE 

MOLECULAR INVERSION OF THEIR 

PROTOPLASMIC CONSTITUENTS 



1. Morphological Dissymmetry and Morphological In- 
version. The attention of biologists has for a long time 
been attracted by the existence of dextral and sinistral 
spirally twisted forms in some animal or plant popula- 
tions. Ludwig (1932, 1936) published two extensive re- 
views in which he summarized a large number of scattered 
observations on this subject. These reviews show that 
practically all the studies of dextrality and sinistrality in 
plants and animals consist in descriptions of the morpho- 
logical aspects of the phenomenon and that the physio- 
logical mechanism which underlies the morphological 
processes has been left almost untouched. 

One of the basic attributes of spiral structures is their 
ability to undergo genotypic inversion. The work of 
Boycott, Diver, Hardy and Turner (1929) on the heredity 
of sinistrality in the mollusc Limnaea peregra has shown 
that the usual twist of the coil of this mollusc to the 
right (clockwise) is determined by a dominant gene, while 
the twist to the left is controlled by the recessive gene, 
and that the sinistral mutant individuals appear in the 
population from time to time. Consequently, in almost 
all the cases in which some experimental work was car- 
ried out with organisms possessing a spiral form, it 
was possible to detect among the usual, typical individ- 
uals a few hereditary inverse specimens. (We shall, 

79 



80 SPIRAL TMIST AXD OPT. AVTlYrVY 

hereafter, call these two kinds the typical and the in- 
verse individuals.) 

It may be supposed that the direction of the spiral is 
determined by some dissymmetric substance which is 
labile, in the sense that it can undergo an inversion of 
its molecular configuration with comparative ease, and 
that by means of such a mutation, the form of the or- 
ganism can change. Such an idea is due to Koltzoff 
(1934) and to Needham (1934), according to whom the 
origin of dextrality and sinistrality, as observed in the 
eggs of certain snails and later in their body, is connected 
with the stereo-chemical properties of some of their com- 
ponent protein molecules. But, at present, these rela- 
tions are very obscure. 

Koltzoff expresses himself as follows : ''Particularly in- 
teresting is the case when in a pond-snail, Limnaea ru- 
hella, in one and the same species genotypes are found 
which are characterized by either the left or the right 
spiral types of shell. The cleavage of the ovum in those 
genotypes proceeds, correspondingly, according to the 
right or left spiral types. Here, already at the first 
division of the ovum, the difference between both types 
is marked in a sufficiently distinct manner by the posi- 
tion of spindles. It is very probable that the right and 
the left types are distinct already in the unfertilised 
ovum, because they can be detected in the relative posi- 
tion of both directing bodies. Hybridological analysis 
shows that this character is determined by one pair of 
allelomorphs. The right twist of the spiral is determined 
by the dominant gene, the left twist by the recessive one. 
The mother homozygous as to the recessive gene can 
itself have the right spiral (because the ovocyte from 
which it evolved could be heterozygous), but all its eggs 
develop according to the left type, even if they were 
fertilized by the sperm of the homozygous dextral fa- 
ther. This shows that genotypical peculiarities of the 
male nucleus are not manifest on this stage. On the 



SPINA L TW Tf< T AND OPT. AdlVITY 8 1 

other hand, one can see here the proof of the fact that 
the basic features of the whole plan of structure of an 
organism can be the result of the action of a single gene. 

''What kind of influence this gene exercises on the 
structure of the ovum, we certainly do not know. As a 
hypothesis I can express the suggestion that this or 
other type of cleavage is here determined by the presence 
in the protoplasm of the ovum of the right or of the left 
optical isomer of some organic substance. This sub- 
stance goes out of the nucleus of the ovum during the 
ripening of the latter, forming itself preliminarily in 
connection with corresponding genes of the chromosome 
apparate of the ovocyte. Hence it may be inferred that 
both genes of a given couple of allelomorphs are optical 
isomers in respect to each other. ' ' 

Koltzotf 's hypothesis is, of course, not the only possible 
interpretation of the observed facts. The optical inver- 
sion of genes is certainly possible, but a change of their 
chemical properties, without the inversion of their con- 
figuration, may also be supposed. In the latter case the 
optical inversion of some organic substance determining 
the structure of the animal would take place only in a 
subsidiary reaction. The facts discussed in the preceding- 
chapter would confer about the same degree of proba- 
bility on the hypothesis of gene inversion and on the 
hypothesis of a chemical modification of the genes with- 
out inversion. 

The solution of some basic biological problems de- 
pends on the answer to this question. According to the 
first interpretation, the substance of the genes which 
determine the morphological structure of an animal would 
belong to the group of secondary protoplasmic constit- 
uents, those which play the role of storage substances or 
of products of metabolism. These products would then be 
quite important in the mechanism of evolution. 

2. Morphological Dissymmetry luuJ Morphological In- 
version in Bacillus Mycoides. The typical strain of Bacil- 



82 



SPIRAL TWL'^T A\D OPT. ACTIVITY 



lus mycoides, when grown on the surface of agar pep- 
tone medium, produces colonies spirally twisting to the 
left, i. e., counter-clockwise (according to the terminology 
adopted by Ludwig, 1932). After one has introduced a 
small quantity of inoculating material in the centre of a 
Petri dish of agar-peptone, one soon sees it grow ; the thin 
filaments of the growing culture begin to deviate to the 
left (cf. Fig. 5). 





L 



D 



Fig. 5. Dextral (D) and sinistral (L) spiral twisting of the growing 
filaments of colonies of Bacillus mycoides, as observed on peptone 
agar, in Petri dishes. 

The inverse form of this organism, growing in dextral 
coils, rarely occurs. It was first recorded by Gersbach 
(1922), who described this interesting case as an ''isom- 
erism in bacteria". He further established that the 
dextral and sinistral strains are entirely identical in all 
their properties. 

Later a single dextral strain among a great number of 
sinistral ones was observed by Oesterle (1929). 

Lewis (1932) isolated several dextral strains in Texas. 

In an extended series of investigations with Bacillus 
mycoides at the Microbiological Institute of the Academy 



SPIRAL TWIST AND OPT. ACTHITY 83 

of Sciences in Moscow, the dextral form was found only 
three times, though numerous isolations from different 
soils were made. 

The dextralitj" and sinistrality in Bacillus mycoidcs is 
a hereditar}^ feature. Dextral forms are always obtained 
from dextral forms, and sinistral from sinistral ones. 

It can be shown that the spiral form of the colonies of 
this organism is a secondary feature which is the result 
of the primary spiral structure of the growing cells which 
constitute the filaments. If one stains the filaments on 
the surface of the agar with neutral red or with toluidin 
blue (1:5000) and examines them under the microscope, 
one can occasionally observe the twisting of two filaments 
which have encountered each other. The motion of the 
growing filament consists of two components : an elonga- 
tion and a rotation around the axis of the filament, these 
will result in a spiral motion. Similar observations have 
been made also by Stapp and Zycha (1931) and by Eob- 
erts (1938). If during the free growth of a filament on 
the agar surface, the filament rotates around its longitu- 
dinal axis counter-clockwise, the interaction of the firm 
surface of the agar and of the growing filament will cause 
the latter to follow a spiral path in a counter-clockwise 
direction. Consequently, the secondary sinistral coil of 
the growing colony of bacteria will arise as a result of the 
primary sinistral spiral growth of the cells of the fila- 
ment. This is confirmed by the fact that a certain con- 
sistency of culture medium is necessary for the typical 
spiral growth of colonies (Pringsheim and Langer, 1924; 
Hastings and Sagen, 1933). The latter authors state that 
on agar of usual strength the growth of Bacillus my- 
coides spreads from the place of inoculation in the form 
of coarse filaments which twist counter-clockwise, form- 
ing a symmetrical pattern. On less consistent agar this 
pattern does not appear or is diffuse. 

3. Morphological Dissymmetry and Morphological In- 
version in the Snail, Fruticicola lantzi. We shall consider 



84 SPIRAL TWIST AXD OPT. ACTTTITY 

next the morpliological dissymmetry of an animal which, 
in the natural classification, stands far from the bacteria, 
namely, the land snail, Fruticicola lantzi. In this animal 
the typical individuals are dextrally spiralled as is the 
case in the majority of species of snails. Numerous ob- 
servations have led to the conclusion that, in snails, the 
sinistrally twisted individuals are ecologically weaker 
than the dextral forms. In joint occurrence of the two 
forms the sinistral ones often disappear in a rather 
short time. Zvetkov (1938) has recently shown, in a 
study of the distribution of Fruticicola lantzi in Middle 
Asia, that most of the populations consist of typical 
forms, dextrally spiralled. Populations consisting al- 
most exclusively of inverse, sinistrally spiralled forms 
were found only in some districts separated from the re- 
maining area by mountain barriers. Such isolated col- 
onies of inverse forms in both bacteria and hermaphro- 
ditic molluscs, it is thought, have originated from a sin- 
gle inverted ancestor. 

I-. Some Physiological Properties of the Dextral and 
the Sinistral Strains of BaciUus mycoides. a. A c t i o n 
of temperature. Recent experiments made by 
Gause (1939) have shown that there is a ditference in the 
action of temperature on the growth of the dextral and 
on that of the sinistral strains of Bacillus mycoides. 
Three series of experiments were performed as follows : 
A small quantity of inoculating material was placed in the 
center of the Petri dish, on agar, in the form of a circle 
0.5 mm. in diameter. Twenty hours after growth had 
started the diameter of the colony was about 6 mm. at 
20° C and about 25 mm. at 32° C. Taking then the diam- 
eter of colonies, either dextral or sinistral, growing at 
20° C, as a unit, curves of growth in terms of temperature 
were constructed. Such curves are represented in Figure 
6. If the rate of growth of the usual sinistral strain is 
normal, the inverted dextral strain presents the jdIic- 
nomenon of ''heat injury." 



SPIRAL TWIST A\D. OPT. ACTIVPTY 



85 



While the preceding experiment was made on the rough 
form (usually designated as the R-forni) of bacteria, we 
repeated it with the smooth form (S-form). By "disso- 




20° 24° 28' 



32"" 36°C 



Fig. 6. Growth-temperature relation in dextral (D) and sinistral 
(L) strains of Bacillus mycoides (R type) grown on solid medium. 
The three sets of curves represent three series of experiments. The 
size of the colonies grown at various temperatures is indicated in 
terms of the size of the colonies grown at 20° as a unit. (From Gause, 
1939.) 



86 



^^PIRAL 7'TT7>ST AND OPT. ACT I \ IT Y 



ciation" botli dextral, DR, and sinistral, LR, strains of 
Bacillus mycoides develop into smooth ones, which form, 
on growing on solid medium, flat spherical colonies, LS 
and DS (for literature see Arkwright, 1930). The study of 
the action of temperature was made according to the pre- 
vious plan. It was found that the phenomenon of heat 
injury characteristic of the dextral strains was about as 
marked in the S-f orms as in the R-f orms. 

It was decided then to try the experiment with the 
DS and LS strains in liquid medium. The rate of growth 
was now determined by a bacterial count in a Thoma 
chamber, under the microscope, forty hours after the 
inoculation. The relation of growth to temperature is 
represented in Figure 7. One sees that in the dextral 
strain the characteristic heat injury appears in the range 
of temperatures extending' from 24° to 28° C. 

Similar results were thus obtained with the R-forms, 
with the S-forms and on liquid as well as on solid culture 



200 



I 50 




100 - 



Fig. 7. Growth-temperature relation in dextral (DS) and sinistral 
(LS) strains of Bacillus ^nycoides, grown on liquid medium. Abscissae: 
Temperature in degrees C; Ordinates: Number of cells per 1/160 cc. 
(From Gause, 1939.) 



SPIRAL TWIST Ay D OPT. ACTIYTTV 87 

media. There is definitely in the dextral form a special 
sensitivity to heat injury at the temperatures indicated. 
It is interesting to compare this characteristic weakness 
of the inverted dextral form with the following property 
pointed out by Le^\ds (1933) and observed again by Gause 
(1939). 

b. Enzymatic properties. Lewis, working 
with dextral and sinistral strains of Bacillus mycoides, 
reported a specific physiological difference between them. 
It is known that this bacillus possesses the ability to 
decompose glucose and saccharose and to acidify the me- 
dium. On glucose the acid production is similar in sinis- 
tral and in dextral strains but not on saccharose. Accord- 
ing to Lewis the formation of acid on saccharose is rapid 
with the left spiralled strain and it ceases with the right- 
spiralled type. 

Gause (1939) repeated these experiments using the S- 
form of Bacillus mycoides. Tw^o per cent saccharose and 
a little quantity of a weak (0.02 per cent) solution of 
phenol red (according to Clark) were added to an agar- 
peptone medium of usual composition. At pH 7.7, which 
is the optimal hydrogen ion concentration for the gro^\i:h 
of Bacillus mycoides, phenol red has an orange tint. The 
culture w^as kept at 28° C. Nineteen hours after the be- 
ginning of the experiment the color of the sinistral 
strains differed very sharply from that of the dextral. 
The former showed a rapid production of acid, the pH 
of the colony being about 6.8. The dextral strains showed 
no production of acid. The reaction of the colony was 
alkaline, its pH being about 8.4. Lewis' results were 
therefore confirmed with the S-form of the bacillus. 

It might be worth mentioning the following detail. 
While the dextral strains are, without exception, unable 
to produce acid on sucrose, the sinistral strains were all 
able to form acid except in two cases, one mentioned by 
Lewis and another (doubtful) observed by Gause. 



88 



.SPIRAL TWIST AND OPT. ACTIVITY 



Lewis' reaction might be considered as a fermentative 
deficiency of the dextral strain. 

(7. G r o w t h on two optical isomers. It 
is of interest to know how an organism which presents a 
morphologically dissymmetric structure (right or left 
spiral) behaves when grown on a medium which is molec- 
ularly diss^anmetric. The dextral and sinistral strains 
of Bacillus mycoides were growm on the optical isomers 
of arginine (Gause, 1939). The results are summarized 
in Tables 16 and 17. 

A safe conclusion that one can draw from these data is 
that both the dextral and the sinistral strains of Bacillus 
mycoides, in both the rough and the smooth forms, grow 



TABLE 16 

Growth of Sinistral (LR) and of Dextkal (DR) Forms of Bacillus 
Mycoides on Optical Isomers of Arginine 



Amino 


Number of 


Ratio of 

growth on d-ar- 

ginlne to 

growth on 

dl-arginine 


Number of 


Ratio of 
growth on 
d-arginine 


acid 


cells in LR 


cells in DR 


to growth 
on dl-ar- 
ginine 


d-arginine 


31.3 1 
15.7 1 




19.7 i 




dl-arginine 


1.99 


12.4 f 


1.59 


d-arginine 


28.9 ) 




21.2 I 




dl-arginine 


14. 6 i 


1.98 


13.2 \ 


1.61 



TABLE 17 

Growth of Smooth Sinistral (LS) anu of Smooth Dextral (DS) 
Forms of Bacillus Mycoides on Optical Isomers of Arginine 



Amino 
acid 


Number of 
cells in LR 


Ratio of 
growth on d-ar- 
ginine to 
growth on 
dl-arginine 


Number of 
cells in DR 


Ratio of 
growth on 
d-arginine 
to growth 

on dl-ar- 
ginine 


d-arginine 
dl-arginine 
d-arginine 
dl-arginine 


40.1 ( 
31.9 1 
43.5 / 
23.3 ) 


1.26 

1.87 


66.8 [ 
31.0 t 
45.5 ) 
33.0 \ 


2.15 
1.38 



^^rih'AL T\\ IHT AND OPT. ACTIVITY 89 

better on iiahiral d-arginiiie than on the racemic dl-ar- 
giuine. Therefore, the dextrally and the sinistrally twist- 
ed organisms are alike in the optical properties of their 
basic protoplasmic constituents, since nutritive sub- 
stances of the natural configuration are more favorable 
for both of them. 

d. E e s p i r a t i o n. The oxidation of glucose by the 
dextral and sinistral strains of Bacillus mycoides was 
determined by the Warburg technique at three tempera- 
tures : 22°, 25° and 28°C. As is characteristic of biolog- 
ical processes generally, the velocity of respiration rose 
exponentially with the rise of temperature, and practi- 
cally at the same rate in the sinistral and in the dextral 
strains of the bacillus. Consequently the phenomenon of 
heat injury, which was characteristic of the growth of 
the dextral strain, was not observed in respiration on 
glucose. 

The general conclusions to draw from these investiga- 
tions is that the inverse, dextral strains of Bacillus my- 
coides are weaker than the typical, sinistral ones. This 
was observed in the rate of growth at 24° to 28° and in 
the deficiency of an enzymatic action. 

5. Some Physiological Properties of the Dextral and of 
the Sinistral Strains of the Snail, Fniticicola lantzi. 
Anabolic gain, I'esistance to starva- 
tion, mortality rate. The results of the in- 
vestigations on Bacillus mycoides are paralleled by the 
data obtained with Fruticicola lantzi. 

Gause and Smaragdova (1940) made a comparative 
study, under well controlled laboratory conditions, of the 
physiological behavior of the dextral and sinistral indi- 
viduals of this snail. They investigated (1) the velocity 
of anabolic assimilation as judged by the change in weight 
w^hen the snails were fed for a long time on carrots; (2) 
the velocity of the catabolic loss as determined by the 
decrease in weight when the animals were kept in a moist 
chamber without food; (3) the mortality rate in the sec- 
ond group of experiments. 



90 



SPIRAL TWLST AND OPT. ACTIVITY 



It was found that after having been fed for a prolonged 
time on carrots tlie typical, dextrally twisted individuals 
practically did not change their weight, while, under per- 
fectly identical conditions, the sinistrally twisted snails 
considerablv decreased in weight (cf. Figure 8). 



320 - 



300.- 



260 - 



240 - 



220 - 



7\ 




DEXTRAL 




^ -"' " "P^^ 




■ -u 


/ ,» -^ 


\. /^ 
















1 1 


t 1 




P'""^""^ 




SINISTRAL 




- 


■ 1 


T : 


9 



10 



20 



30 



40 



50 



60 



Fig. 8. Change in weight observed in the dextral and sinistral forms 
of the snail Fruticicola lantzi when they were fed for a long time on 
carrots. Abscissae: Time in days; Ordinates: Weight in milligrams. 
(From Gause and Smaragdova, 1939.) 



In the study of the behavior of Fruticicola in starvation 
it was observed that the sinistral individuals lost weight 
more rapidly and died off more quickly than the dextral 
forms (cf. Figure 9). 

The relative weakness of inverse left spiralled indi- 
viduals is evident in the three series of experiments. 
Similar results were obtained also in the study of the 
loss of dry weight. 



SPIRAL TWIST A\D OPT. ACTIVITY 



91 



uu7o 


V 










90% 


'v 


\^ 


X o 






80% 






5\ 


V^ 


"""^^ 


70% 


- 


I 




•^ 


^>--f 



e 



10 



Fig. 9. Average decrease in fresh weight in starving adult dextral 
(D) and sinistral (S) forms of the snails FrvAicicola lantzi. Abscissae: 
Time in days; Ordinates: Weight in % of the initial value. (From 
Gause and Smaragdova, 1939.) 

6. On the Relation hetiveen MorpJiological Inversion 
and Molecular Inversion. Having considered the physio- 
logical differences of the dextral and sinistral forms, we 
shall now turn to the problem of the possible relation 
between morphological and molecular inversions. Let us 
at first note that the heat injury in the dextral strains of 
Bacillus mycoides reminds one of the heat injury observed 
when different lower organisms, such as yeast, were cul- 
tured on unnatural isomers of amino acids (Gause and 
Smaragdova, 1938). When the yeast Torula utilis was 
grown on the natural isomer of leucine, which enters into 
the composition of all living organisms, the velocity of 
growth was that always observed in typical growth-tem- 
perature curves, but when it was cultured on the unnat- 
ural isomer of leucine, the increase in the velocity of 
growth became always less and less with the rise of tem- 
perature. 



92 



.Sl'Ux'AL T]\J,ST AM) OPT. ACTITITY 



These experiments were rei:)eated with the optical 
isomers of the following- amino acids : leucine, histidine, 
phenyl-alanine and valine (Gause, 1939). The results 
obtained are given in Figure 10. Typical heat injury at 
temperatures extending from 18° to 28° may be observed 
in the growth of Torula nfills on the unnatural isomers 
of leucine and of histidine but not on those of valine and 
phenyl-alanine. 



80- 



60 

40 
20 




25 



20 



140 iQO 22° 26° 



18° 22° 26°C 




10 
5 



Fig. 10. Growth of the yeast Torula iitilis on optical isomers of 
various amino acids, at different temperatures. Abscissae: Temperature 
in degrees C; Ordinates: Increase in the number of cells in 40 hours. 
(Prom Gause, 1939.) 



s/'iir\L T\\ 1ST A\n. oi'T. Acrn ITY 93 

Similar data were obtained also in the study of the 
growth of the- mould Aspergillus niger on the optical 
isomers of leucine and valine. 

The same relation between lenii)erature and rate of 
growth is thus observed in the unusual strain of Bacillus 
mycoides growni on natural substrates and in the yeast 
or fungi grown on unnatural substrates. In both cases, 
there nuist be an inhibitive factor of growth. It may be 
conjectured that in the case of yeast or fungi the un- 
natural isomer of the amino acid dissolved in the cul- 
ture medium surrounding the cells caused a retardation 
of growth because its spatial contiguration did not coin- 
cide with the spatial configuration of the basic constit- 
uents of protoplasm. In the case of Bacillus mycoides 
the retardation of growth in the unusual form would be 
caused by the presence inside of the cells of the unnatural 
optical isomer of some organic substance which partici- 
pated in the determination of the morphology of the 
cell. 

7. Morpliological Inversion and the Theory of Spiral 
Growth. The investigations on various physiological 
properties of dextral and of sinistral strains in Bacillus 
mycoides and in Fruticicola lantzi have brought out the 
two following points : ( 1) In the optical properties of their 
protoplasm these strains are alike. This follows from 
their behavior towards optically isomeric nutritive sub- 
stances. It has, furthermore, been confirmed by direct 
observations made by Kiesel, Efimochkina and Rail 
(1939), wdio isolated the same natural amino acids from 
both dextral and sinistral strains of the snail Fruticicola 
lantzi. (2) The inverted individuals of both bacteria and 
snails are physiologically weaker than the typical ones. 
These observations suggest that while in the typical in- 
dividuals the organic substances which participate in the 
determination of the twist might well have the same laevo- 
rotatory configuration as the other constituents of proto- 
plasm, in the inverted individuals some enzymatic dis- 
turbance might have occurred. 



94 SPIRAL TWIST A^W OFT. ACTIVITY 

Castle (1936) has recently undertaken the study of the 
mechanism of spiral growth in Phy corny ces. He claims 
that the spiral structure of the growing cell wall is not 
strictly predetermined but depends on the interaction of 
forces which exert their action in the growth region. The 
twist of the growing elastic elements of the wall may be 
the result of their resistance to turgor. Castle construct- 
ed a model to illustrate this process. If the elastic ele- 
ments of the wall are distributed symmetrically, dextral 
spirals ^\i\\ be obtained in 50 per cent of the cases and sin- 
istral spirals in the other 50 per cent. As the left direc- 
tion of the spirals is typical for Phycomyces, one must 
assume a dissymmetric distribution of the elastic ele- 
ments of the cell wall, which later, under the action of 
turgor, lead to the formation of sinistral spirals. 

As Castle himself points out, the mechanism of spiral 
growth can be different in different organisms, and one 
cannot directly transfer his explanation of the mechanism 
of twisting to the bacteria, the more so since the cell wall 
of the latter is thought to consist of some specific pro- 
tein material closely related by its nature and origin to 
cellular protoplasm (John-Brooks, 1930). 

But one can assume that in the spiral growth of the cell 
wall of a bacterium, as in that of Phycomyces, tw^o fac- 
tors are involved: (1) Some pre-existing asymmetric 
system (distribution of the elastic elements of the wall 
in Phycomyces; optically active secondary protoplasmic 
constituents in bacteria) ; (2) a system of forces directly 
inducing the spiral twist (turgor in Castle's experi- 
ments). The interaction of these two factors would bring 
about the dextrality or sinistrality of the spiral growth 
according to the following scheme : 

Secondary substance Asymmetric structure Sniral growth 

of metabolism ^ of the cell wall ^ ^ 

f 

Forces directly inducing 
* the spiral twist 



SPIRAL TWTST AND OPT. ACllYTTY 95 

One may conjecture that the inversion of the direction 
of the spiral growth in Bacillus mycoides is related to an 
optical inversion of some secondary substance in meta- 
bolism. The latter would bring about the inversion of 
some structures in the cell wall and, from the interaction 
of these with the forces inducing the spiral twist, there 
would result an inversion in the direction of the spiral 
grow^th of the cells. 

But to what extent can one assume that the secondary 
substances of metabolism which participate in the struc- 
ture of the wall of the bacterial cell can undergo an optical 
inversion? Some recent data on the chemical structure 
of bacterial capsules obtained by Bruckner and Ivanovics 
(1937) in the laboratory of Professor Szent-Gyorgyi, are 
of interest in this connection. As is kno^vn, the cell wall 
in bacteria consists of two layers: (1) A very thin in- 
ternal cuticle, and (2) An external gelatinous layer which 
is sometimes developed into an envelope called a capsule. 
(John-Brooks, 1930, remarks that the bacteriologists have 
come to look upon capsule formation as a general feature 
which is common to all bacteria, but which reaches the 
proportions that we know, only in certain species.) Bruck- 
ner and Ivanovics (1937) w^ho studied the chemical prop- 
erties of the capsule of Bacillus antJiracis and of some 
other species of bacteria, all of which are aerobic spore 
formers, standing near Bacillus mycoides in the classifi- 
cation, found that the capsule of these bacteria consists of 
a polypeptide substance, the hydrolysis of which yields 
d(-) glutamic acid. The laevorotatory isomer of this 
amino acid is unnatural, and it has not been found pre- 
viously anywhere in the organic nature. So, the presence 
of the unnatural glutamic acid in the structure of the 
envelope of the anthrax and of some other bacilli has 
already been recorded in the literature. Further investi- 
gations in that direction may reveal significant data on 
the present problem. 



96 S/'lh'AL TW'/sr A.\l> OI'T. ACTH IT) 

SUMMARY 

1. In organisms which possess a spiral structure, as in 
some bacteria, in snails, etc., one observes a larger num- 
ber of "typical" individuals, that is, of individuals twist- 
ed in one direction, while the "inverse" specimens are 
rarer. 

2. The properties of protoplasm related to optical ac- 
tivity are alike in dextral and sinistral forms. The same 
natural amino acids have been isolated from either the 
dextral or the sinistral snails {Fruticicola laiitzi). Both 
dextral and sinistral bacteria {B. my cold es) grow better 
on the natural than on the unnatural isomers of amino 
acids. 

3. The "inverse" forms are physiologically weaker 
than the "typical." When the culture temperatures are 
varied from 20" to 36% the "inverse" bacteria present a 
decreasing growth rate not observed in "typical" bacte- 
ria; furthermore, the "inverse" forms show some enzy- 
matic deficiencies. In the "inverse" snails the velocity 
of catabolic loss and the mortality rate, on starvation, 
exceed those of the "typical" individuals. 

4. It is suggested that some secondary substances which 
may determine the morphological inversion are optically 
inverted, or that some subsidiary process in metabolic 
activities is changed in the nmtant snails and bacteria, 
whereas the basic protoplasmic constituents are not. This 
would explain ihv disturbance in the enzymatic coordi- 
nation and the physiological weakness observed in the 
inverted specimens. 

BIBLIOGRAPHY 

BOYCOTT, A., DIVER. C, HARDY. S. and TURNER. F.. Proc.Roy.8oc: 
B.. !()'/. 152, 1929. 

BRUCKNER, V. and IVANOVICS, G., Z.physiol.Chem.. 2 ',7, 281, 1937. 

CASTLE, E.. Proc.Xat.AcSci.U.S.A.. 22. 336. 1936. 

CAUSE, G.F., Biol.BulL. Hi, 448, 1939. 

GAUSE, G.F. and SMARAGDOVA, N.P., Am. Naturalist. 7 ;. 1940. 

GERSBACH, A., Zbl.Bakt., Abt. I, .S,s, 97, 1922. 

HASTINGS, E. and SAGEN, H., J.Bact.. 2.), 39, 1933. 



Si/'lh'AL TWIST A\D O/'T. At'TH ITY 97 

JOHN-BROOKS, R., System of Bakteriol., 1, 104, 1930. 

KIESEL. A.. EFIRIOCHKINA, E. and RALL, J., C.r.Acad.Sci.U.S.S.R., 

25, 481, 1939. ' 
KOLTZOFF, N., Biol.J. (Russian) ,!, 420, 1934. 
LEWIS, J., J. Bad., 2',. 381, 1932; 25, 359, 1933. 
LUDWIG, W.. Das Rechts-Links Problem. Berlin. 1932. 

, Yer1i.Zool.Ges.. -iS, 21, 1936. 

NEEDHAM, J., Nature. W,, 277. 1934. 

OESTERLE, F., ZhL Bakt.. Abt. II, 7.';, 1, 1929. 

PRINGSHEIM, E. and LANGER, J.. ZM.Bakt., Abt. II, 61, 225, 1924. 

ROBERTS. J., Science. 87, 260, 1938. 

STAPP. L. and ZYCHA. H., Arcli.Mikrohiol.. 2, 493, 1931. 

ZVETKOFF, B., BuU.Soc.Natur. Moscow, 1938. 



chapt?:r V 

ANALYSIS OF VARIOUS BIOLOGICAL PROCESSES 

BY THE STUDY OF THE DIFFERENTIAL 

ACTION OF OPTICAL ISOMERS 

Asymmetric Analysis. When one analyses the action 
upon protoplasmic functions of dextrorotatory and of 
laevorotatory isomers of various organic substances, one 
often notices a difference in the effectiveness of the two 
isomers. The existence or the absence of such a dif- 
ference, as also its quantitative value, are evidently re- 
lated to the physical structure and the chemical compo- 
sition of protoplasm. One can, therefore, study the 
mechanism of various biological processes by examining 
how they are influenced by optical isomers of various 
substances. It is thought that this method of analysis, 
which we call ''Asymmetric Analysis," could contribute 
to the clarification of several important problems of 
comparative physiology. 

With the idea of elaborating some systematic methods 
of "asymmetric analysis", Gause and his associates have 
undertaken the following investigations : 1. An analysis 
was made of the mechanism of toxic action of optically 
isomeric nicotines upon lower and higher animals. 2. 
The mechanism of toxic action of optically isomeric or- 
ganic acids upon lower and higher animals was similarly 
studied. These two investigations will be reviewed and 
discussed in the first section of this chapter. The study 
of the effect of nicotine isomers in various animals led 
to important observations on the evolution of the ner- 
vous system. These observations will be discussed in the 
second section. 3. A study was made of the action of 
optically isomeric cinchonines upon various functions 
of the cell. This study will be summarized in the third 
section, 

99 



100 ASYMMETRIC ASALYSIS 

SECTION I 

ANALYSIS OF THE MECHANISM OF TOXIC 

ACTION 

A. TOXIC ACTION OF THE OPTICAL ISOMERS OF NICOTINE 

A Case of Identical Mechanism of Action in the Two 
Optical Isouters. Pictet and Eotscliv, in 1904, prepared 
pure dextrorotatory (unnatural) nicotine, tested its tox- 
icity on rabbits and guinea pigs, and ascertained that it 
was less toxic than the natural 1-nicotine. They ex- 
pressed the view, which subsequently was adopted by a 
number of authors, of a different mechanism of toxic 
action liy the dextro and by the laevo isomers, the symp- 
toms of poisoning having been found different. 

Maclit (1929) studied the pharmacological synergism 
of stereoisomeric nicotines. He found that the toxic 
action of a mixture of the 1- and dl- forms was stronger 
than the additive action of these isomers. The con- 
clusion that he reached then was that "an individual 
cell may possess receptor groups of a laevo and dextro 
type, and a mixture of two stereoisomers would thus have 
a double point of attack in place of a single one, in case 
only one of the optic isomers was used." The three fol- 
lowing points in the work of Macht are open to criti- 
cism. First, his important final conclusion is based on 
a very small number of experiments. Then, the author 
did not attempt to obtain a concentration-toxicity curve 
which would allow one to make some quantitive calcula- 
tions. Moreover, dl-nicotine is not the most suitable for 
such experiments, as it usually contains some hydronico- 
tine which influences the physiological effect (Gause, 
1936) ; only the purest dextro nicotine obtained by re- 
peated crystallizations with laevotartaric acid should be 
used. 

In the experiments of Gause and Smaragdova (1939) 
the dextro isomer, in accord with the data of Pictet and 
Eotschy, was found less poisonous than the laevo form. 



A^O'MMirr/nc AXALYSf,^ 101 

but tlu' relation ot* tlie increase in toxicity with the con- 
centration was the same in the two isomers and a com- 
plete identity of the temperature characteristics of toxic 
action of the dextro and laevo forms was also observed 
(in cold-blooded animals: fishes and tadpoles). The 
identity of the relation of toxicity to concentration and 
the identity of temperature characteristics are taken as 
an indication of the identity of the mechanism of toxic 
action of the two isomers. Both of them seem to act on 
the same link in the system of physiological processes, 
though with diiferent speed. 

To illustrate these conclusions, let us consider in some 
detail the results of recent experiments made with a brood 
of the fish, Leuciscus idus var. orfiis (Gause and Smar- 
agdova, 1939). The animals were placed in neutralized 
solutions of nicotine of different concentrations, pre- 
pared with redistilled water, and the killing time in sec- 
onds was recorded. Figure 11 (upper part) represents 
the relation of killing time to the concentration of nico- 
tine. 

It is to be pointed out that in the calculation of the 
relative toxicity of 1- and d- nicotine one cannot use indis- 
criminately results taken at various arbitrarily chosen 
concentrations. The relative efficacy of the isomers 
changes with change in absolute concentration. For com- 
parison of the physiological effect of the optical isomers 
one has to employ such characteristics of corresponding 
curves of toxicity which are determined not by any 
values of the absolute concentration of the poison but 
by some physiological action. The most convenient is to 
take the minimal lethal concentration of the poison (the 
constant n, cf. formula below). In 1-nicotine fh = 0.0022% ; 
in d-nicotine >/,i = 0.0064%. The coefficient of relative toxic 
action (a) = y?d/>^i, which indicates how nuicli the 1-isomer 
is more powerful than the d-isomer, is 2.91. (This co- 
efficient will be called, hereafter, the "stereo-coeffi- 
cient.") 



102 



ASYMMETRIC ANALYSIS 



C 500 




(U 

E 

'*- 3 

_c 

^ 2.5 

6) 
o 



2 



010% 



02 0% 



CONC. 




10 lb 20 25 

Log. of effective concentration i.v-ii) 
Fig. 11. Killing action of the optical isomers of nicotine on the fish 
Lencisciis idiis. The lower graph represents the toxicity curves in 
logarithmic coordinates. (From Cause and Smaragdova, 1939.) 

According to the principles of quantitative toxicology, 
concentration-toxicity curves can usually be expressed 
by the empirical equation of Ostwald : 

k 

^' ^ (x-n) ™ 
where u is the killing time, x the concentration of the 
poison, n its minimal lethal concentration, and k and m 
are constants. The constant m shows how rapidly the 
toxicity increases with the concentration; it thus charac- 
terizes the dynamics of the killing process. If one plots 
log y on ordinates and log {x-n) on abscissae, the relation 
between these variables will be represented by a straight 
line. The slope of this straight line is measured by the 
constant m. 



ASYMMETRIC ANALYSIS 103 

The lower part of Figure 11 represents the data on the 
toxicity of dextrorotatory and laevorotatory nicotines 
for Leuciscus, plotted in the manner just indicated. It is 
evident that the slopes of the straight lines, characteriz- 
ing the dynamics of the increase of toxicity with concen- 
tration, are identical. It is therefore reasonable to con- 
clude that in these experiments the mechanism of killing 
action in the two optical isomers of nicotine is identical 
in the sense defined. The unnatural dextro nicotine is 
weaker only in the sense that a higher dose is required to 
attain killing. Similar results were obtained also in ex- 
periments with birds {Acanfhis flammea), lizards {La- 
certa viridis) and tadpoles {Bana temporaria). Xo dif- 
ference in the symptoms of poisoning by the two optical 
isomers was observed. 

Investigations were then carried out to determine the 
temperature coefficients of toxicity of dextrorotatory and 
laevorotatory nicotine for various animals. It is known 
that if one plots the logarithms of the killing rate against 
reciprocals of absolute temperature, one usually obtains 
a linear relation. The slope of this straight line is gen- 
erally represented by |li, which is known as the tempera- 
ture characteristic (cf. Crozier, 1924). This characteris- 
tic shows how the killing process is speeded up by the 
rise of temperature. Physiological processes of differ- 
ent nature, i.e., in which ditferent mechanisms are at play, 
usually possess different temperature characteristics. 

The temperature characteristics of toxicity for tadpoles 
and for the fish Leuciscus were determined according to 
the following procedure. Two solutions of dextro and 
laevo nicotines were placed in a constant temperature 
bath. After the temperature equilibrium was attained, a 
number of fish or of tadpoles were immersed in the ves- 
sels and the killing time was recorded. Figure 12 shows 
the killing rate ( a value inverse to the killing time in 
seconds) in the fish Leuciscus, due to the action of optic 
isomers of nicotine at ditferent temperatures, Approxi- 



104 



AH \M METRIC ANALYSIS 



^ 0.020 - 



o 



c 



0.010 



0) 

o 



c 



o 



o 




2.0 - 



0.00340 



0.00350 



Reciprocal of absolute temperature 
Fig. 12. Effect of temperature on the killing rate of the fish Lciicis- 
ciis idus by the optical isomers of nicotine. The lower graph represents 
the killing rate plotted logarithmically. (From Cause and Smarag- 
dova, 1939.) 

mately isotoxic concentrations of the isomers, i.e., 0.007% 
for 1-nicotine and 0.014% for d-nicotine, were used. The 
temperature characteristics of the two isomers are prac- 
tically identical: in the d-form \i = 37,500, and in the 
1-form pi = 36,800. Such an identity of temperature char- 
acteristics was also observed in experiments with tad- 
poles; in the d-nicotine \x = 14,400, and in the 1-nicotine 
|ii ^ 14,600. Hence the relation of toxic action to temper- 
ature strongly supports the view that the mechanism of 
toxic action is identical in the two optically isomeric 
nicotines. 

The same relations, that is, (1) a high toxicity of the 
natural isomers, (2) the same relation between the in- 



.1 .s r .1/ 1/ r/rinc asal vms 



105 



crease in toxicity and the concentration, and ( .'5) tlie same 
temperature characteristics for the two isomers, have 
been ol)served by Gaiise and Smarag'dova (ll)o8, ]9o9) 
with nicotine on vertebrates (natural == laevorotatory), 
with tartaric acid on fishes (natural = dextrorotatory), 
and with cinchonine on ])aramecia (natural = laevoro- 
tatory. Figure 13 shows that the dynamics of toxic action 
are identical for dextrorotatory and for laevorotatory tar- 
taric acids on the brood of the fish Lehistes reticulatus. 
The temperature characteristics of toxic action for dex- 
trorotatory tartaric acid was found to be 10,200 (cf. 




02 04 06 08 10 12 

Log. of effective concentration i.v-ii) 

Fig. 13. Killing action of the optical isomers of tartaric acid on the 
fish Lehifites reticulatus. The lower graph represents the toxicity 
curves in logarithmic coordinates. (From Gause and Smaragdova, 
1938.) 



106 



ASYMMETRIC ANALYSIS 



Fig. 14) and for laevorotatory tartaric acid 9,700, in otlier 
words, they were of the same order of magnitude. Sim- 
ilar data were obtained also in experiments with the 
brood of another species of fish, Platypoecilus maculatus 
( Gause and Smaragdova, 1938). 



^ 0.00200 

10 



o 

I- 

en 

c 



000100 



o 

4- 

o 

i_ 

en 

_c 

M- 
O 

o 



3.3 
3.2 

3.1 

3.0 
2.9 
2.8 




L 
Ji = 9.700 



-L 



0.00330 



0.00340 



Reciprocal of absolute temperature 



Fig. 14. Effect of temperature on the killing rate of the fish Lebistes 
retiCKlatiis by the optical isomers of tartaric acid. The lower graph 
represents the killing rate plotted logarithmically. (From Gause and 
Smaragdova, 1938.) 



ASYMMETRIC ANALYSIS 



107 



Further, in experiments on the toxic action of opti- 
cally isomeric einchonines upon paramecia, it was found 
that the laevorotatory isomer inhibits the mechanism of 
ciliary movement more rapidly than does the dextro form 
(Gause, Smaragxlova and Alpatov, 1938). The dynamics 
of toxic action in dextro and laevo einchonines were found 
to be identical (cf. Fig. 15). A study of the temperature 
characteristics of toxicity has also shown that these are 
practically identical, 14,200 in the laevorotatory and 
14,000 in the dextrorotatory isomer (cf. Fig. 16). 




14 16 I 8 2 2 2 

Log. of effective concentration {.v-)i) 



Pig. 15. Killing action of the optical isomers of cinchonine on 
Paramecium caiulatum. The lower graph represents the toxicity curves 
in logarithmic coordinates. (From Gause, Smaragdova and Alpatov, 
1938). 



108 



ASYMMETRIC ANALYSIS 



B. TOXIC ACTION OF THE OPTICAL ISOMERS OF ORGANIC ACIDS 

1. A Case of Different Mechanism of Action of the Two 
Optical Isomers. In the cases reported so far the natural 
isomer was more powerful in its physiological action than 
the unnatural, the relation of increasing toxicity to con- 




0.00330 000340 

Reciprocal of absolute temperature 

Fig. 16. Effect of temperture on the killing rate of Paramecium 
caudatum by the optical isomers of cinchonine. The lower graph rep- 
resents the killing rate plotted logarithmically. (From Gause, Smar- 
agdova and Alpatov, 1938.) 



ASYMMF/rRIC . 1 A 1 /> ) N/N' 



109 



centration and the temperature characteristics of toxic 
action were identical for tlie two isomers. There are cases 
in which it seems that none of these relations hold. GaUvSe 
and Smarag-dova (1938) reported this situation in the 
action of malic acid on the brood of two species of vivi- 
parous fish, Lehistes rrticidafus and Platypoecilus macii- 
latiis. 

In these experiments they compared the natural laevo- 
rotatory with the racemic malic acid (the significance of 
the use of a racemate will be indicated below). It was 
found that the natural laevorotatory malic acid is less 
toxic than the racemic. The toxic action of weak 
(0.05%) solutions of the laevorotatory and racemic malic 
acids on Lehistes reUculatus at different temperatures 
(16% 18% 21°, 26°, and 31°) is recorded in Figure 17. It 
is quite apparent that the temperature characteristics of 



c 
o 
u 

0) 



(1) 
E 






0) 

o 



en 

c 



o 



en 
o 



1300 


• 


\ 






HOC 


• 


\ 
\ 


\ 




900 


■ 


X 


\ 




700 


■ 




X 




500 


■ 




^ 


N^ 


3.4 




I6» 


21* 26* 


3I»C 


3.2 


. 


*^ 






3.0 


■ 




>«=I2;300^ 


\M-9,3A.Q 


2.8 








^C^ 



0.00330 0.00340 O00350 

Reciprocal of absolute temperature 

Fig. 17. Effect of temperature on the killing time of the fish 
Lehistes reticulatus by the optical isomei-s of malic acid. The lower 
graph represents the killing rate plotted logarithmically. (From Gause 
and Smaragdova, 1939.) 



110 ASYMMETRIC ANALYSIS 

toxic action are different in the racemate and in the laevo- 
rotatory isomer. At temperatures from 16° to 26° C the 
racemate is more toxic than the laevorotatory isomer, 
whereas at the temperature of 31° C the latter is rela- 
tively more toxic than the racemate. The temperature 
characteristics of toxic action are also quite different. In 
the laevorotatory isomer n = 12,300, and in the racemic 
form |Li = 9,340. 

In similar experiments with the fry of Platypoecilus 
maculatus results of the same kind as those obtained with 
Lebistes were recorded (cf. Fig. 18). The temperature 
characteristic of toxic action of the left isomer of malic 
acid is 16,930 and that of the racemic form 12,880. 

The results obtained with fish were duplicated in a 
study of the action of optically isomeric malic acids on 
tadpoles of Rana temporaiia (Gause and Smaragdova, 
1939). 

Some experiments on the action of l(-) and of d( + ) 
leucine on the yeast Torula utilis (Gause and Smarag- 
dova, 1938) also bring confirmatory evidence that the 
unnatural form d(-|-) exerts a stronger action than the 
natural and that their effect is of different nature. 

Concerning the experiments in which racemic malic 
acid was used, it should be mentioned that, in dilute 
aqueous solutions, the racemic acid is completely disso- 
ciated into dextrorotatory and laevorotatory constituents 
(Ostwald, 1889). Therefore the greater biological activ- 
ity of the racemate must probably be attributed to a 
higher toxicity of the unnatural dextrorotatory compo- 
nent.^ 

If our last assumptions are correct there would be a 
series of cases in which, contrary to what has been de- 
scribed above, the unnatural optic isomers are physio- 
logically more effective and in which the mechanism of 

lit is to be remembered that the natural dextrorotatory tartaric acid 
and the natural laevorotatory malic acid belong to the same steric 
series. 



ASYMMETRIC ANALYSIS 



111 




0.00 300 0.00350 0.00400 

Reciprocal of obsolute temperature 

Fig. 18. Effect of temperature on the killing time of the fish 
Platypoecilus maculatus by the optical isomers of malic acid. The 
lower graph represents the killing rate plotted logarithmically. 



112 ASYMMETRIC ANALYfilS 

action of the two optic isomers is ditTerent. One could 
not, then, speak of a single receptive protoplasmic sub- 
stance which would simply react to a ditferent degree to 
the two isomers, as is probably the case when the mech- 
anism of action of the two isomers is the same. 

2. Dual Activity of Organic Acids. The mechanism of 
toxic action of the optical isomers of organic acids can be 
also investigated from another point of view. Heilbrunn 
(1928), among others, called attention to the dual nature 
of the action of organic acids upon living systems: (1) 
Organic acids produce an electro-chemical effect upon 
the surface of the cells, primarily due either to a destruc- 
tion of the negative charge of the cell surface by posi- 
tively charged hydrogen ions or to other physico-chem- 
ical surface 'pJienomena; (2) Owing to their relatively 
weak electrolytic dissociation, the solutions of organic 
acids contain a considerable proportion of non-dissociated 
molecules which penetrate into the interior of the cells 
where they produce transformations of a chemical nature. 
Koltzotf's experiments (1915) on the action of ditferent 
acids on the feeding activity of fresh-water vorticellids 
furnish an example of the first type of action. There the 
biological effect of the organic acid depends only on the 
pH and the mechanism of this action consists in electro- 
chemical changes upon surfaces directly accessible to hy- 
drogen ions. The sinniltaneous occurrence of the first 
and second type of effects is illustrated in the experi- 
ments of Stiles and Rees (1935) who showed that the 
killing action of monobasic organic acids of the aliphatic 
series first diminishes with the elongation of the chain 
of carbon atoms in the molecule, then reaches a minimum 
with valeric acid and finally again increases with the fur- 
ther elongation of the chain. This phenomenon was ex- 
plained on the idea that the degree of electrolytic disso- 
ciation diminishes with the increase in the weight of the 
molecule, while the killing action of the non-dissociated 
molecules increases with the increase of molecular weight. 



ASYMMETh'W ANALYSIS 113 

The observed lethal action, which is the resultant of the 
partial lethal action of hydroo^en ions and of that of non- 
dissociated molecules, would then decrease first and in- 
crease afterwards as we indicated. Other investigations 
on the mode of action of organic acids have been sum- 
marized by Lepeschkin (1937). 

Since optical isomers have all their physical and chem- 
ical properties identical, except those which are directly 
related to their structural configuration, one will observe 
that, if the common properties only are involved in the 
killing mechanism, the two isomers should produce the 
same effect, while, if the properties which are ditferent 
in the two isomers are involved in the killing action, the 
two isomers will produce a different effect. It is further- 
more assumed that the properties which are specific to 
each isomer will be involved in the interaction of these 
isomers with the protoplasm itself, within the cell, in op- 
tically active medium, while the properties common to 
the two isomers, such as the electric charge, the electric 
conductivity (observed by Ostwald, 1889, to be the same 
in the isomers of tartaric acid), the osmotic pressure, 
etc., will be involved in such processes as conduction to- 
ward the protoplasmic matter itself. Consequently, if 
our assumptions are correct, when solutions of dextroro- 
tatory and of laevorotatory acids are equally toxic for a 
given animal, one may infer that the killing results from 
physico-chemical injuries concerned with conduction or 
the like. If, on the other hand, the two optical isomers 
are not equally toxic, it is natural to think that the sur- 
face effects just described could not induce death, so that 
non-dissociated molecules have time to penetrate inside 
the cells and there carry out their stereo-specific destruc- 
tive actions. 

With these ideas in mind, Gause and Smaragdova 
(1938) determined the coefficient of relative toxicity of 
the optical isomers of tartaric acid on various fresh water 
animals. Some of their results are given in Table 18. 



114 ASYMMETRIC ANALYSIS 

TABLE 18 

Coefficients of Relative Toxicity of Dextbokotatory Tartaric Acid 

AND Significance of Differences in Relative Toxicity in 

Different Groups of Animals. 

(From Gause and Smaragdova, 1938.) 

(M is the coefficient of toxicity; P.E. is the probable error.) 



(1) Protozoa 



M±P.E. 0.981±0.019 



(2) Worms (3) Crustacea (4) Pisces 



1.048±0.010 



1.064±0.010 1.305±0.011 



The figures represent the mean vahie (M) of the data for 
all the animals of a given phylmn. The probable error 
(P.E.) from the mean is also given. 

The coefficient of relative toxicity in Protozoa is close 
to unity, which means that the dextrorotatory and laevo- 
rotatory tartaric acids are equally effective. On the con- 
trary, in fishes, the optical isomers of tartaric acid dif- 
fer strongly in their killing power, the stereo coef- 
ficient being 1.305. The other groups of invertebrates 
investigated occupy an intermediate position between the 
protozoa and the fishes in their differential sensitivity to 
the two isomers, the coefficient of relative toxicity reach- 
ing 1.0-1-8 in the worms and 1.064 in the Crustacea. 

Similar results were obtained also with the optical 
isomers of malic acid. 

According to the assumptions made, these data would 
show that, in the killing of lower animals by tartaric 
and malic acids, there predominates some electro-chem- 
ical surface injury, while in higher animals internal chem- 
ical injuries caused by non-dissociated molecules would 
occur. 

Cushny (1903, 1926) called into question the obser- 
vations on the differences in the killing power of the dex- 
trorotatory and the laevorotatory isomers of tartaric acid 
in vertebrates, because the weak specific action of tar- 
taric acids might, according to his opinion, be totally 
concealed by the more powerful effect of the hydrogen ion 



ASYMMETRIC ANALYSIS 115 

concentration which is known to be identical in tlie solu- 
tions of both optic isomers. But the objections of Cushny 
must be considered in the light of the following recent 
observations : 1. It is at present doubtful that the ef- 
fect of hydrogen ions is always dominant over the spe- 
cific action of non-dissociated molecules of organic acids 
(cf. Gause, 1936), 2. Furthermore, Sizer (1937), in a 
work on the stimulative effect of organic acids on various 
animals has shown that Balanus halanoides is more sus- 
ceptible to the action of hydrogen ions, while in Fimdulus 
heteroclitus the effect of these ions does not predom- 
inate over the specific action of nondissociated mole- 
cules. 

There is another essential point in the investigations 
of Gause and Smaragdova. If the animals studied are 
arranged in the order of increasing difference in the 
toxic jDOwer of the two optical isomers : Protozoa 
< Worms < Crustacea < Pisces, one obtains the phylo- 
genetic series of gradually increasing differentiation. 
This is not surprising if one considers the fact of the 
progressively diminishing relative vital importance of 
the physico-chemical injury of integuments when one 
ascends the animal series. The nature of susceptible 
integuments, the injury of which, according to our as- 
sumptions, brings about death in lower animals, is not 
known. It is possible that the respiratory surfaces are 
among the most susceptible. In Protozoa the whole sur- 
face of the cell is the respiratory surface. When one 
ascends the animal series, respiratory surfaces become 
more localized and more differentiated and the physico- 
chemical injury of these surfaces progressively dimin- 
ishes in magnitude as a cause of death. The results of fur- 
ther investigations along this line have recently been pub- 
lished by Gause and Smaragdova (1939). 



116 ASYMMETRIC ANALYi^hS 

SECTION II 

ANALYSIS OF THE EVOLUTION OF THE 
NERVOUS SYSTEM 

1. Stereo-coefficients of Action of the Optical Isomers 
of Nicotine in the Phijlogenetic Series. Since the two 
optical isomers of nicotine exert their killing action by 
the same mechanism but with a different strength one 
can, by measuring this difference of potency in various 
animals, study the properties of the specific receptive 
substance in different species. In higher animals, as has 
been already recorded above, there is some specific sen- 
sitive substance which is affected to dift'erent degrees by 
the toxic action of the dextro and the laevo isomers of 
nicotine. Protozoa do not possess, as some observations 
have shown, such a sensitive substance, and the dextro 
and laevo isomers of nicotine are for them equally toxic. 
The question arises of the nature of this specific sub- 
stance and of the stage of evolution at which it first 
appears. 

Greenwood, as early as 1890, carried out an extensive 
comparative investigation on the action of connnon laevo- 
rotatory nicotine on invertebrates, attempting to establish 
a parallelism between the toxic eifect of this alkaloid 
which affects, as is known, the nervous system of ani- 
mals, and the evolution of the nervous system. On the 
basis of purely qualitative observations he reached the 
conclusion that ''the toxic effect of nicotine on any or- 
ganism is determined mainly by the degree of develop- 
ment of the nervous system. Thus for Amoeba the sub- 
stance cannot be regarded as exciting or paralysing ; it is 
rather inimical to continued healthy life. As soon as any 
structural complexity is reached, the action of nicotine 
is discriminating in such a fashion that the nervous ac- 
tions which are the expression of automatism, that is, 
which imply coordination of impulses, are stopped first. 
This is seen dimly in Hydra, and it is more pronunced 
among the medusae. When structural development goes 



ASYMMKTIi'lC AX ALT SIS 117 

farther, the selective action of nicotine is traced readily, 
as for example in PaJaeniou". Greenwood writes fui'ther 
that: ''Animals which have enough in common to stand 
near each other in classification, may yet react differently 
to nicotine, each according to what I may perhaps call 
its own balance of organisation." 

Gause and Smaragdova (1939) made quantitative de- 
terminations of the toxic action of the two optically 
isomeric nicotines. The advantage of the use of the two 
isomers will appear in the discussion of the results. 

Experiments on vertebrates showed that the stereo- 
coefficients of toxic action of optical isomers of nicotine 
(a) are of the same order of magnitude in all ;ininials 
studied : 

Bird (AcanfJtis flaminra) a = 3.1 

Lizard {Lacerta viridis) a = 2.4 

Tadpoles {Rana temporaria) a = 3.0 
Fish (Leuclscus idus) a = 2.9 

Fish {Lebisfes reticulatus) a = 2.4 



Mean a = 2.8 
Inasmuch as the mechanism of toxic action is identical 
in optically isomeric nicotines, one can, by the difference 
of their effects, judge of the difference in spatial proper- 
ties of the specific receptive substance assumed. As the 
difference of effects remains constant, one can conclude 
that the chemical nature of the receptive substance in 
the vertebrates also remain essentially constant. 

Since the procedure for the introduction of nicotine 
was not the same for all the animals used — the poison 
was introduced in the muscle of Lacerta while Lehistes 
were immersed in the solutions of nicotine — the identity 
of the stereo-coeffcient is an experimental proof that the 
conditions of the penetration of nicotine do not affect 
significantly either the mechanism of toxic action or the 
reaction of the specific receptive substance. 

Furthermore, the absolute sensitiveness to nicotine 
in Acanihis is considerably higher than in Lacerta (0.8 



118 



ASYMMETRIC ANALYSIS 



mg. per 100 gr. of weight as compared to 5.6 mg. per 100 
gr.), but, practically, this difference in sensitiveness does 
not influence the stereo-coefficient. The constancy of the 
latter, despite a different sensitiveness, is also significant 
in the study of the properties of the specific receptive 
substance. 

The results of investigations on the toxic action of the 
two optical isomers of nicotine on fresh water and marine 
invertebrates are given in Table 19. One sees that all 
the invertebrates examined by Gause and Smaragdova 
can be divided into two groups. The first includes the 
animals for which the dextro and laevo isomers are equal- 
ly toxic, and for w^hich the toxicity curves of the two 
isomers fullv coincide. The second includes the organ- 



TABLE 19 
Comparative Killing Action of the Optical Isomers of Nicotine on 

Invertebrates 
(Tiie sign = means that d and 1 nicotines are equally toxic; the coeffi- 
cient (X indicates to what degree 1 is more toxic than d nicotine.) 



Animal 


Comp. 
Toxicity 




Animal 


Comp. 
Toxicity 


Protozoa 




Annelida 




1. Paramecium cauda- 




15. 


Saccocirrus papillo- 




tum 






cercus 


a — 2.3 


2. Euplotes patella 




16. 


Perinereis cultrifera 


a -1.9 


3. Stentor coeruleus 




17. 


Arenicola grubii 


a >1 


4. Spirostomtim ambi- 




18. 


Pristina longiseta 


a— 2.09 


guum 




19. 


Limnodrilus hoff- 




Coelenterata 






meisteri 


a— 3.45 


5. Hydra fusca 


— 


20. 


Helobdella stagnalis 


a— 4.0 


6. Cladonema radiatum 




21. 


Nais C07nm.unis 


a =2.41 


Platyhelminthes 




22. 


Chaetogaster langi 


a — 3.13 


Turbellaria 




23. 


Stylaria lacusti'is 


a >1 


7. PolyceUs nigra 


, , 


24. 


Aelosoma variegatum 


a -1.76 


8. Phaenocora sp. 




25. 


Aelosoma hemprichi 


a -1.84 


9. Dalyellia brevimana 






Chaetognatha 




10. Procerodes lohata 




26. 


Sagitta setosa 


a— 2.7 


11. Leptoplana tremel- ■ 






Arthropoda 




laris 




27. 


Daphnia magna 




Rotatoria 




28. 


Cyclops serrulatus 




12. Euchlanis triquetra 




29. 


Gammarus marinus 




13. Rotifer vulgaris 




30. 


Drosophila melan- 




Nemertinea 






ogaster 




14. Lineus lacteus 




(2-days old larvae were 
immersed in nicotine 
solutions.) 





ASYMMETRIC ANALYSIS 119 

isms in wliicli the laevo isomer of nicotine is more toxic 
than the dextro- isomer. Tlie two groups correspond to 
large divisions of the animal kingdom, and witliin each 
division, there are hardly any exceptions. 

All the representatives of Protozoa, Coelenterata, Tur- 
bellaria, Rotatoria and Nemertinea studied belong to the 
first group. They are devoid of spatially specific recep- 
tive substances in the process of poisoning by nicotine. 

It should be mentioned that the stereo-coefficients in in- 
vertebrates are not atfected by the differences in the ab- 
solute sensitiveness to nicotine, exhibited by various spe- 
cies, as it has been noticed in vertebrates. Thus, for ex- 
ample, Leptoplana is considerably more sensitive to nico- 
tine than Procerodvs, but both these turbellarians are 
characterized by an equal effect of the dextro and laevo 
isomers. There are many other examples of the inde- 
pendence of these characters. 

The lowest groups in the phylogenetic series, in which 
a stronger eifect of the laevo isomer of nicotine is ob- 
served, are the annelids, and particularly the Archian- 
nelids {Saccocirrus), the Polychaeta and the Oligochaeta 
and the primitive representatives of Deuterostomia {Sa- 
gitta setosa). In Arthropoda (Crustacea and Insecta) 
this etfect is absent, an equal toxicity of the dextro and 
the laevo isomers is again observed. 

Let us now compare the stereo-coefficients in verte- 
brates and in those invertebrates which show a higher 
sensitivity to the laevorotatory nicotine. The following 
values were recorded in invertebrates : 

Saccocirrus papillocerciis 

Perinereis cult rif era 

Pristina longiseta 

Limnodrilus Jiofmeisteri 

Helobdella stagnalis 

Nais communis 

Ch act og aster langi 

Aelosoma variegatum 

Aelosoma hemprichi 

Sagitta setosa 

Mean a 



a 




2.3 


a 




1.9 


a 


— 


2.1 


a 


= 


3.4 


a 




4.0 


a 


— 


2.4 


a 




3.1 


a 




1.8 


a 




1.8 


a 




2.7 


a 




2.6 



120 AswiMtyriiw A\ALysii<i 

The limits of error in measuring the coefficient a may 
extend over a rather wide range. Thus the following 
values were obtained in two independent measurements : 
in Nais 2.57 and 2.25; in Liwiwdrilus 2.9 and 4.0; in 
Aelosoma variegaium 1.79 and 1.74; in Aelosoma Jiem- 
prichi 1.50 and 2.18. Nevertheless the order of magnitude 
of the average is significant. We find nearly the same value 
in invertebrates (2.6) and in vertebrates (2.8). 

The data above mean that the Protozoa, Coelenterata, 
Turbellaria, Rotatoria and Nemertinea are deprived of 
the spatially specific receptive substance which responds 
differentially to the left isomer of nicotine. Annelides, 
Chaetognatha and Vertebrates possess this receptor, 
while in Arthropoda it is absent again. 

2. The Acetylcholine System and the Dlfferottial Ef- 
fect of the Optical Isomers of Nicotine. Considering that 
it is the nervous system in animals which is affected by 
nicotine and that there is an identity of stereo-coeffi- 
cient in invertebrates and in vertebrates, in spite of es- 
sential differences in the morphology of their nervous 
system, we come to the conclusion that there is some 
uniform receptive substance distributed in the various 
nervous systems of these animals. However, this chem- 
ical constituent is not an obligatory component of every 
nervous system; even some quite differentiated nervous 
systems of lower invertebrates (Turbellaria and Nemer- 
tinea) are deprived of it. 

A study of the present views on the mechanism of nic- 
otine toxic action will furnish more information on the 
nature of the receptive substance. Thomas and Franke 
(1924, 1928, 1933) have shown that it is the paralysis of 
the peripheral neuro-muscular junctions of the respira- 
tory muscles which is the cause of death of higher ani- 
mals in acute nicotine poisoning. This view was con- 
firmed by Gold and Brown (1935). We are thus led to 
the old classical observations of Langley (1904) that in 
the ''neuro-muscular junction" there is a certain sensi- 



A.SYMMI-JTinv A^ALYSI^S 121 

tive "receptive substance" wliicli is tlie tirst to be af- 
fected by nicotme. 

On tile other hand, since the cUissical works of Loewi, 
it is known that, in the transmission of impulses from 
nerves to effectors the various steps are as follows (1) 
nerve impulse — ^ (2) chemical mediator — ^ (3) receptive 
substance — v (-t) specific response (for literature see 
Cannon and Eosenblueth, 1937). There are some indi- 
cations that the chemical mediator in the voluntary mus- 
cles of higher animals is acetylcholine. Its action on the 
receptive substance in this case reminds one of that of 
nicotine: in small doses it excites, and in larger doses it 
paralyses, and according to the current views, nicotine, 
in case of an acute poisoning, atfects in some irreversible 
way the receptive substance, upon which acetylcholine 
mediation is no more effective. In other words, nicotine 
(at least in experiments of our type) acts upon neuro- 
effector synapses of voluntary muscles. In its action it 
reminds one of acetylcholine, the substance which trans- 
mits the excitation in these synapses. Consequently the 
receptive substance in nicotine poisoning has some close 
relation to the receptive substance for chemical media- 
tion. 

The experiments just described permit one to divide 
the animals into two groups according to the nature of 
the receptive substance atfected by nicotine. It might 
be that animals possessing a receptive substance differ- 
entially atfected and those possessing a receptive sub- 
stance identically affected by optically isomeric nicotines 
ditfer also in their receptivity to the normal chemical me- 
diator, and consequently in peculiarities of the transmis- 
sion of nerve impulses. 

An examination of the data on the distribution of 
acetylcholine in different groups of invertebrates will 
throw a new light on this problem. Despite the often 
questionable findings concerning the presence of this 
substance which is ascertained bv the action of extracts 



122 ASYMMETRIC ANALYSIS 

on different organs while not a single of the ordinarily 
used organs is strictly specific, as Cannon and Rosen- 
blueth (1937) pointed out, the results may be regarded 
as sufficiently reliable if they are repeatedly observed 
with several different procedures. The most extensive and 
elaborate investigations were carried out by Bacq (1935) 
at the Biological Station of Naples. He did not find 
acetylcholine nor the enzyme which destroys it, choline- 
esterase, in the tissues of different Coelenterates. The 
muscles of Annelids and of lower Deuterostomia (Holo- 
thuria) contained acetylcholine and choline-esterase. In 
the muscles of Crustacea he found so little acetylcholine 
that he concluded that the transmission of impulses 
from the motor nerve to the muscle in these animals is 
not accomplished by means of this mediator. He insisted 
on this point at the conference devoted to this problem 
held in Cambridge in 1937. On the other hand, there 
are some preliminary communications by Nachmanson 
(1937), according to which there is some choline-esterase 
in the ganglions of Crustacea. What is certain, however, 
is that neuro-effector synapses of the muscles of Crus- 
tacea are not typical acetylcholine systems, if only for 
the reason that they are extremely insensitive to the 
action of externally applied acetylcholine. 

In the accompanying table w^e compare the observa- 
tions of Bacq with those of Gause and Smaragdova. In 
six animal groups the two series of independently obtain- 
ed results coincide. If our suggestions are correct, the 
differential killing action of optical isomers of nicotine 
could be employed to detect the presence of the specific 
receptor characteristic for the acetylcholine system in 
the neuro-effector synapse of voluntary muscles (Gause 
and Smaragdova 1939). But further investigations are 
necessarv for a final conclusion on this problem. 



A S 1 -1/ .1/ /•; 77.' / (' - 1 XALYSIS 



123 



TABLE 20 

Comparative SmiY, of thic Presence in Various Animals of a Stereo- 
Differential Toxic Action of the Optical Isomers of Nicotine 

AND of A( KTYLCIIOLINE MEDIATION IN THE TRANSMISSION 

OF Nerve Impulse 





Animals 




Stereo-Differential 

Action of Nicotine 

(Gause and Smarag- 


Acetj'lcholine 

Mediation 
(Bacq. 1935) 








dova, 


1939) 




1. 


Coelenterata 




Absent 




Absent 


2. 


Annelida 




Present 




Present 


3. 


Lower Deuterostomia 










(Holothuria 


for ace- 










tylcholine and 


Chaetog- 










natha for nicotine) 


Present 




Present 


4. 


Crustacea 




Absent 




Absent 


5. 


Insecta 




Absent 




Absent 


6. 


Vertebrata 




Present 




Present 



SECTION III 

ANALYSIS OF THE MECHANISM OF A^AEIOUS 
PHYSIOLOGICAL FUNCTIONS IN PROTOZOA 

The following attempt at aii ''Asymmetric Analysis" 
of physiological functions in protozoa is based on the 
fundamental principle of dissociability of physiological 
processes. The action of the optical isomers of some 
organic substance will be studied on some particular 
function and the stereo-coefficient of action determined 
for that function. From the similarity or dissimilarity 
of the coefficient various conclusions can be drawn on the 
nature or mechanism of the function. 

It should be noticed that a similar method is followed 
in the temperature analysis of biological processes (cf. 
the recent discussion of this subject by Hoagland, 1935). 
"When two separate processes reveal different tempera- 
ture relations it is believed that they are not directly 
controlled by some common ''master reaction". (Con- 
cerning the caution with which the notion of "master 
reaction" should be used, cf. Burton, 1936 and Hoag- 
land, 1937.) 



124 Al^Y.}[}[ErRIC ANALYSLS 

The experiments of Gause, Sniaragdova and Alpatov 
(1938) to be reported here, consisted in the analysis of 
the action of the optical isomers of cinchonine on the 
rate of the following functions of the infusorian Para- 
mecium caudatiim: (1) The feeding rate, as measured by 
the number of food vacuoles formed in water suspensions 
of india ink; (2) The velocity of expulsion of gastric 
vacuoles; (3) The division rate; (4) The velocity of loco- 
motion in thin glass tubes, according to the method of 
Glaser (1924) ; (5) The death rate, death being diagnosed 
by the complete cessation of all motion. 

It was found that the stereo-coefficient of action of 
cinchonine (a) was of the same order of magnitude (about 
1.3) in the case of the inhibition of the following three 
functions : the formation of the gastric vacuoles, the ex- 
pulsion of these vacuoles, and the division rate. In other 
words cinchonine inhibits some susceptible system which 
controls these three functions. The gastric vacuoles in 
paramecia separate from the gullet, being, so to say, 
pulled away by the active protoplasm (cf. Metalnikoff, 
1910 and Bozler, 1924). Their formation and their ex- 
pulsion are evidently connected with the degree of activ- 
ity of the protoplasm. These two functions, as Avell as 
the rate of division, are, therefore, expected to be con- 
trolled by the rate of metabolism in the endoplasm of the 
Paramecium. In all probability optically isomeric cin- 
chonines, inhibiting one of the phases of metabolism in 
the endoplasm, depress all the three functions together. 

On the other hand, the cessation of the motion in para- 
mecia by cinchonine is evidently connected with the pois- 
oning of the svstem of locomotory cilia. This svstem is 
localized in the ectoplasm (cf. Kalmus, 1931). The stereo- 
coefficient of action of the optical isomers of cinchonine 
is significantly ditferent from that previously recorded, it 
reaches 1.98 (the left isomer being, as before, more pow- 
erful than the right). These data are presented in 
Table 21. 



yi^'vi/i//;77.'/r .i,v.i/.r.s7.s' 125 

TABLE 21 

Stereo-Coefficients of Action of the Optical Tso.mkus of Cinchoxine 

ox Vauioi's Functions in Paramecium caudatum. 

(Froji Gaise, Smaragdova and Alpatov, 1938.) 

Function Stereo-coefficient 



Inhibition of f 1. Formation of gastric vacuoles j ^ =: 1.36 



endoplasm ■{ 2. Release of gastric vacuoles 
[ 3. Division rate 



ot = 1.36 
a = 1.24 



Inhibition of \ 

ectoplasm ( 1. Mechanism of ciliary motion fi = ^-^^ 

The differences ol)served in the vakie of the stereo- 
coefficient suggest that, in the ectophism, cinchonine in- 
hibits a receptive substance diiferent from that of the 
endophism. A physiological differentiation of the cells 
into ectoplasm and endoplasm would then be brought into 
evidence in Paramecium caudatum by our method of 
''asymmetric analysis". 

However, in another species of paramecia {Para- 
mecium hursaria), the stereo-coefficients of the action of 
optically isomeric cinchonines on various functions did 
not disclose such a dilfereuce; the same stereo-coeffi- 
cient of inhibition was observed for the endoplasmic and 
ectopia smic function. 

For the study of the effects of the optical isomers of 
cinchonine on the rate of movement of paramecia the 
procedure Avas, in general, as follows. To 2 cc. of cin- 
chonine solution of a given concentration 5 drops of a 
culture of Paramecium caudatum were added. A little 
quantity of the cinchonine solution with infusoria was 
then transferred with a pipette into a thin glass tube 
and the latter was placed on a graduated glass plate on 
which the velocity of motion was measured with the aid 
of a stop-watch. The determinations were made every 
ten minutes for eighty minutes. 

While, for a time, no significant ditference in velocity 
could be observed in the control and in the right isomer 
solution, the paramecia in the left isomer of the same 



126 ASYMMETRIC ANALYSIS 

strength i^reseuted a considerable increase in the rate of 
movement. The left isomer of cinchonine definitely call- 
ed forth at first a strong stimulation of movement; sub- 
sequently the motion slowed down and finally the para- 
mecia died. With the right cinchonine the stimulation 
phase was entirely absent under all concentrations em- 
ployed, only the inhibition phase could be observed. 

So only the laevorotatory isomer has the specific power 
of stimulating the ciliary movement. One can suppose 
that the left isomer, because of peculiarities of its spatial 
configuration, interacts with the system of reactions 
which control the ciliary motion, while this system re- 
mains as if "closed" for the dextrorotatory isomer. In 
distinction from this stimulating effect, the less specific 
process of toxic destruction of the locomotory force of 
the cilia is carried out qualitatively in the same way by 
both optic isomers of cinchonine, the rate of the reaction 
only is different. This situation has its parallel in the 
following observation of Krebs (1936). He has recently 
pointed out that in the metabolism of amino acids some 
specific transformations such as the splitting of the imi- 
dazole ring in histidine (Edlbacher and Neber, 1934), or 
the oxidation of the ring in tyrosine (Bernheim, 1935), 
are open only to the natural amino acids of the left series 
and are closed for the right forms. On the contrary, in 
other less specific reactions, such as deamination, both 
optic isomers of amino acids can participate. 

Further data on the action of optical isomers of cin- 
chonine upon various protozoa are given in the orig- 
inal paper by Gause, Smaragdova and Alpatov (1938). 

SUMMARY 

1. The study of the mechanism of various biological 
processes by examining how they are influenced by opti- 
cal isomers of various substances is presented as a 
method of investigation called '^ Asymmetric analysis." 
This method is applied here in the study of (1) the 



ASYMMETRIC ANALYSIS 127 

meclianism of toxic action, (2) the evolution of the ner- 
vous system, (3) the mechanism of various physiological 
functions in protozoa, 

2. The two optical isomers of a toxic substance may 
exhibit different degrees of toxicity (the natural isomer 
being- more toxic) but possess the same mechanism of 
toxic action, as judged by the identity of the relation of 
increasing toxicity to concentration and by the identity 
of the temperature characteristics. Such conditions have 
been observed, in particular, in nicotine. There are cases 
in which none of the two relations just mentioned hold. 
The last series of cases cannot be accounted for by the 
assumption of a receptive substance diversely affected 
by the two isomers. 

3. The coefficient of relative toxicity of the two isomers 
of tartaric acid increases from 1 to 1.305 when one 
passes from the protozoa to the fishes through the worms 
and the Crustacea. The killing action, in the lower forms, 
seems, then, to be due to factors which are common to the 
two isomers, while, in the higher forms, it is due to factors 
which differ in the two isomers. It is suggested that the 
factors of the first type are those which act mostly on the 
surface of organisms, and the factors of the second type, 
those which act internally. The problem of the mode of 
action of toxic substances is then linked to that of the 
evolution of the integuments in fresh water animals. 

4. The study of the toxic action of nicotine in animals 
of variously developed nervous systems points to the 
absence of a spatially specific receptive substance in Pro- 
tozoa, Coelenterata, Turbellaria, Rotatoria and Nemer- 
tinea, and to the presence of such a substance in Annelids, 
Chaetognatha and Vertebrates. In Arthropoda it is ab- 
sent again. A comparison of its distribution with that of 
acetylcholine in different groups of animals leads to sig- 
nificant data on the evolution of the nervous system. The 
receptive substance in nicotine poisoning shows some 
close relation to the receptive substance for chemical 
mediation in the transmission of the nerve impulse. 



128 ASYM}fETRrC ANALYSTS! 

5. The results of the toxic action of the optical isomers 
of cinchonine on Paramecium caudatum bring into evi- 
dence a difference in the physiological functions con- 
trolled by the ectoplasm and those controlled by the en- 
doplasm. Of the two isomers of cinchonine only the 
laevorotatory showed the specific power of stimulating 
ciliary movement. 

BIBLIOGRAPHY 

BACQ. Z., Erg. Physiol.. :i7, 82, 1935. 

BERNHEIM, F., J. Biol. Chem., Ill, 217, 1935. 

BOZLER, E., Arch. Protistenk., J,9, 163, 1924. 

BURTON, A., J. Cell. Comp. Physiol.. 9, 1, 1936. 

CANNON, W. and A. ROSENBLUETH, Autonomic Neuro-Effector Sys- 
tems, New York, 1937. 

CROZIER. W., /. Gen. Physiol., 7. 1924. 

CUSHNY, A., J. Physiol.. SO, 176, 1903. 

, Biological Relations of Optically Isomeric Substances, Bal- 
timore, 1926. 

EDLBACHER, S. and W. NEBER, Z. physiol. Chem.. 22Jf, 261, 1934. 

GAUSE, G. F., Raumaufbau des Protoplasmas, Erg. Biol., l-l 54, 1936. 

, Nature. ISS. 976, 1936. 

, and N. P. SMARAGDOVA, Biol. Zhiirn.. 7, 1938; Phy- 
siol. Zooh. hi. 1939; Zool. Zhurn., 18, 1939; Bull. biol. et med. exp. 
Moscow, 7, 105, 108, 1939. 

, and W. W. ALPATOV, Biol. Zhourn., 7, 763, 



1938. 

GLASER, O., J. Gen. Physiol., 7, 177, 1924. 

GOLD, H. and F. BROWN, -/. Pharmacol.. J}, 463, 1935. 

GREENWOOD, M., ,/. Physiol.. 11, 573, 1890. 

HEILBRUNN, L.. The Colloid Chemistry of Protoplasm. Berlin, 1928. 

HOAGLAND, H., J. Cell. Comp. Physiol., 10, 29, 1937. 

KALMUS, H., Paramecium, Jena, 1931. 

KOLTZOFF, N., Trans. Shaniavsky Univ. Biol. Lab.. 1, 189, 1915. 

KREBS. H. A., Ann. Rev. Biochem.. .>, 247, 1936. 

LANGLEY, N., J. Physiol., 33, 374. 1905. 

LEPESCHKIN, W., Zell-Nekrobiose und Protoplasma-Tod. Berlin, 1937. 

MACHT, D., Proc. Nat. Acad. Sci., U.S.A., 15, 63, 1929. 

METALNIKOFF, S.,Trans. St. Petersburg Biol. Lab., 11, 3, 1910. 

NACHMANSOHN, D., Nature. 1937. 

OSTWALD, W., Z. physikal. Chem.. 3. 369, 1889. 

PICTET, A and A. ROTSCHY, Ber. chem. Ges.. 37, 1225, 1904. 

SIZER, S., Physiol. Zool., 10, 327, 1937. 

STILES. W. and W. REES, Protoplasma. 22, 518, 1935. 

THOMAS, J. and F. FRANKE, J. Pharmacol. 23, 150, 1924; 34. Ill, 
1928; .'/8, 199, 1933. 



APPENDIX 

ASYMMETRY OF PROTOPLASM AND THE STRUCTURE 

OF THE CANCER CELL 

When, in 11I2:I, ()tto Warburg reijortod thai the oxyda- 
tive metabolism of cancer cells is somewhat defective 
and that growth and multiplication in cancerous tissue 
is provided mainly by fermentative processes accompa- 
nied by the formation of large quantities of lactic acid, it 
was thought that the solution of the old mystery about 
the nature of cancer had been found. Numerous at- 
tempts of cancer therapy based on this principle, and 
consisting mostly in inhibiting anaerobic processes in 
malignant cells, were made. But all these attempts 
failed. It was then learned that anaerobic metabolism is 
not a characteristic feature of malignancy, it is observed 
in many embryonic tissues. 

In view of such failures, James Ewing, director of the 
Memorial Hospital, New York, remarked before the Na- 
tional Academy of Science (April, 1938), that the funda- 
mental nature of malignant growth is probably an in- 
solvable problem and that investigations on this prob- 
lem have consumed much time and money without pro- 
viding knowledge of practical value. There is, accord- 
ing to him, urgent need for greater support of clinical 
investigations which yield some practical results. 

In spite of that skeptical attitude toward the pros- 
pects of fundamental cancer research, the year 1939 
brought forth an important discovery on the structure of 
the cancer cell. Two distinguished Dutch chemists, Fritz 
Kogl and Hanni Erxleben isolated from proteins of ma- 
lignant cells the unusual optical isomer of glutamic 
acid (of the right steric series), which never occurs in 
proteins of healthy cells. This important finding was 
rapidly followed by significant practical applications. 
Waldschmidt-Leitz (1939) discovered in the serum of 
cancer patients proteolytic enzymes with unusual stereo- 

129 



130 ASYMMETRY OF PROTOPLASM AND CANCER 

chemical behaviour. These enzymes are absent from the 
sermn of healthy persons. 

Kogl and Erxlebeu also isolated several other amino 
acids from proteins of normal and malignant tissues and 
measured their optical rotatory power. Serine and pro- 
line, which undergo easily a partial racemization in hy- 
drolysis, were' obtained as partially racemic products in 
healthy tissues. Proline instead of specific rotation 
aa = ~ 84.9° gave a value of « = - 82.4°, and serine, in- 
stead of a = H- 14.45°, gave a = + 8.38°. But such amino 
acids as valine, leucine and glutamic acid were practically 
optically pure when isolated from healthy tissues, while 
partially racemic preparations were isolated from malig- 
nant tissues (Table 1). 

TABLE 1 

Rotatory Powek of Some Amino Acids Lsolated from Ovarial 

Carcinomes. 

• (Kogl and Erxleben, 1939.) 



Amino acid 


Expected specific 
rotation 


Observed specific 
rotation 


Leucine 
Lysine 
Valine 
Glutamic acid 


+15.40 
+14.60 
+28.80 
+31.70 


+13.20 
+13.50 
+26.90 
+ 4.60 



In the case of glutamic acid, racemization is most evi- 
dent, since as far as 42.7% consists of the unusual d(-) 
form. Such observations have led Kogl and Erxleben to 
conclude that the unusual optical isomers of some amino 
acids participate in the composition of cancer cells. The 
latter would, then, be characterized by some particular 
spatial molecular configuration, on account of which the 
growth-controlling enzymes would be disturbed. 

According to Kogl and Erxleben, partial racemization 
of glutamic acid in cancer tissues is most evident and it 
could be checked easily. This observation has been se- 
verely criticized by Chibnall (1939) and also by Graff 
(1939) who reported to have isolated only optically pure 



A8YMMETR Y OF PRO TO PLA^M AND CANCER 131 

1( + ) giutainic acid from malignant cells. Kogl and Erx- 
leben (1939) inmiodiately pointed out that their oppo- 
nents did not pay sufficient attention to the different sol- 
ubilities of the optical isomers. Racemic dl-glutamic 
acid, in the form of both chlorhydrate and barium salt, is 
two times more soluble than 1-glutamic acid. The pure nat- 
ural isomer consequently cristallizes first and, if the crys- 
tallization is not complete, the racemic isomer will be left 
in the mother liquid. 

Lipmann and his collaborators (1940) also opposed 
their findings to Kogl's and Erxleben's data. On ac- 
count of some difficulties in the ordinary isolation pro- 
cedures, Lipmann attempted to determine the total d- 
amino acid content of the human tumors and of normal 
tissues by means of d-amino acid oxidase with the aid of 
the Krebs enzyme. He found 1.85% of d-amino acids in 
hydrolvzates of normal tissues and 1.84% of d-isomers in 
those of cancer tissues, that is, practically the same value 
in the two cases. However, Lipmann himself admits that 
the accuracy of his method is not great. Moreover, since 
each hydrolysis inevitably leads to a partial racemization 
of such labile amino acids as serine and proline, the deter- 
mination of total d-amino acid content loses some of its 
significance. 

On the other hand Kogl's data have been confirmed by 
Arnow and Opsahl (1939). The glutamic acid which they 
isolated from normal tissues had an optical rotation of 
a — --h 31.0^, and that isolated from malignant tissue had 
an optical rotation of a = H- 5.5°. 

If one assumes that Kogl's data are correct, it is, how- 
ever, not clear whether the unusual optical isomer of glu- 
tamic acid pre-exists in the cancer cell, or whether the par- 
tial racemization observed is of a factitious nature. If 
glutamic acid, in the protein molecule of cancer cells, en- 
ters into some special labile compound different from that 
in which it exists in usual protein molecules, it is con- 
ceivable that hydrolysis could lead to a partial racemiza- 
tion in the cancer cell but not in the normal cell. This 



132 ASYMMETRY OF PROTOPLASM A^D CANCER 

possibility can be checked. Lipniaiiii reports that most of 
his hj^drolyses were carried out in HCl containing* heavy 
water. Subsequent determinations of the content of deu- 
terium attached to the alpha carbon atom of glutamic 
acid w^ill show, according to the suggestion of du Vig- 
neaud whether or not the partial racemization is due to 
the process of hydrolysis. 

If the recent data of Waldschmidt-Leitz and Mayer 
(1939) are confirmed, they will undoubtedly lead to new 
developments in the study of the problem of cancer from 
the viewpoint of protoplasmic asymmetry. According 
to Waldschmidt-Leitz, if unusual optical isomers of some 
amino acids really enter into the composition of malignant 
cells, there should be specific proteolytic enzymes to cat- 
alyze the spatially unusual metabolic processes. It is 
known that peptidases of healthy animal tissues do not 
split up such polypeptides which consist of unusual optic 
isomers of amino acids. Waldschmidt-Leitz showed this 
to be true also of the aminopoly peptidase and the dipep- 
tidase from the serum of healthy persons. But the prop- 
erties of peptidases from the serum of cancer patients are 
radically diiferent, they can split up polypeptides con- 
sisting of unusual optic isomers. This feature was used 
by Waldschmidt-Leitz in his diagnosis method. 

One can hope that more light be shed on this important 
problem in the near future. 

BIBLIOGRAPHY 

ARNOW, E. and J. OPSAHL, Science, 90, 257, 1939. 
BAYBRLE, H., Biochem. Z., 303, 251, 1939. 
CHIBNALL, A., Nature, 14',, 71, 1939. 
EWING, J., Science, 87, 399, 1938. 
GRAFF, S., J. Biol. Cheni.. 130, 13, 1939. 

KoGL, F. and H. ERXLEBEN, Z. physiol. Chem., 258, 57; 2G1, 154; 
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and A. AKKERMAN, Z. physiol. Chem., 262, 

141, 1939. 
LIPMANN, F. et at., Science, 01, 21, 1940. 

WALDSCHMIDT-LEITZ. E. and K. MAYER, Z. physiol. Chem., 262, IV 
1939. 

WARBURG, O., Uber den Stoffwechsel der Tumoren. Berlin. 1926. 



GENERAL BlI^LIOGRAPHY OF THE LITER- 
ATURE ON THE ASYMMETRY OF 
BIOLOGICAL MATERIAL 

(In Chronolofjicnl Onh r) 

This bibliograpliical list contains titles of publications 
which concern the problem of the Asymmetry of Biologi- 
cal Material, even if they were not mentioned in the text 
of this book. On the other hand, papers cited in the book 
but concerning- only indirectly our subject have been 
omitted. The list includes the literature published up to 
about the middle of the year 1940. 

1824 

Fresxel, A., Memoire sur la double refraction . . . Bull. 
Soc.Pliilomat., p. 147. 

1848 
Pasteur, L., Recherches sur les relations qui peuvent 
exister entre la forme cristalline, la composition chimique 
et le sens de la polarisation rotatoire. Ann.Chim.Pliys., 
2',, 442-459. 

1860 

Pasteur, L., Note relative au Peniclllium glaucum et a 
la dissymetrie moleculaire des produits organiques nat- 
urels. C.R.Acad.ScL, 51, 298-299. 

, Recherches sur la dissymetrie molecu- 
laire des produits organiques naturels. Soc.Chim.Paris. 
Legons de chiniie professees en 1860. English translation 
in Alembic Club Reprints, No. 14, 1897. 

1861 
Pasteur, L., Lettre aux redacteurs. Anii.Chim. et Phys., 
61, 484. 

1869 
GixTL, W.F., tjber einige Bestandtheile von Fraxinus 
excelsior. Journ.pract.Chem., JOG, 486. 

133 



134 GENERAL BIBLIOGRAPHY 

1874 

LeBel, J.A., Sur les relations qui existent entre les 
formiiles atomiques des corps organiques et le pouvoir 
rotatoire de leurs dissolutions. Bull.Soc.Chim,., 22, 337- 
347. 

van't Hoff, J.H., Sur les formules de structure dans 
I'espace. Arch.Neerland., 9, 445. English translation: 
The arrangement of atoms in space. New York, 1898, 

1884 

JuisTGFLEiscH, M.E., Sur le dedoublement des composes 
optiquement inactifs par compensation. Bull.Soc.cJiim. 
Paris, J,l, 222-233. 

LippMAN, E., tJber das Vorkommen von Leucin und 
Tyrosin in der Eiibenmelasse. Ber.chem.Ges., 11, 2835- 
2840. 

Pasteue, L., La dissymetrie moleculaire. Rev.Scient., 
J,, 2-6. 

Wyrouboff, G., Discussion. Bnll.8oc.Chim. Paris, Jjl, 
212. 

1886 

PiUTTi, A., Ein neues Asparagin. Ber.chem.Ges., 19, 
1691-1695. 

Schulze, E. and E. Bosshard, Untersuchungen iiber 
die Aminosauren, welche bei Zersetzung der Eiweisstoffe 
durch Salzsaure und durcli Barytwasser entstehen. 
Z.physiol.Ghem., 10, 134-145. 

1889 

OsTWALD, W., Tiber die Affinitatgrossen organischer 
Sauren und ihre Beziehungen zur Zusammensetzung und 
Konstitution derselben. Z .physical. Chcm., 3, 369-422. 

1890 

FiscHEE, E., Synthese der Mannose und Liivulose. 
Ber.chem.Ges., 2J,'370-394. 

, Synthesen in der Zuckergruppe. Ber. 

chem.Ges., 23, 2114-2141 ; also 27, 3189-3232, 1894. 



GENERAL BIBLIOCRAPHY 135 

1891 

Nencki, M., Die isomeren Milchsaurcn als Erkennimgs- 
mittel einzolnor Si)alt]nlzarten. ZhJ.Balt., 0, 304-306. 

1893 

Beowx, H. aistd H. Morris, A contribution to the chem- 
istry and physiology of foliage leaves. J.Cliem.Soc., 
63, 604-677. 

Chabrie, C, Sur la toxicite des acides tartriques ster- 
eoisomeres. C.R.Acad.Sci., 116, 1410-1413. 

Pere, a., Sur la formation des acides lactiques isome- 
riques. Ann.Inst. Pasteur, 7, 737-750. 

1894 

Curie, P., Sur la symetrie dans les phenomenes phy- 
siques. J.de Physique, 3, 393-415. 

Fischer, E. and H. Thierfelder, Verhalten der ver- 
schiedenen Zucker gegen reine Hefen. Berxliem.Ges., 27, 
2031-2037. 

Kayser, E., Etudes sur la fermentation lactique. Ann. 
Inst. Pasteur, 8, 737-784. 

1898 

Bartrum, CO., Chance or vitalism! Nature, 58, 545. 

Brion, a., tJber die Oxydation der stereoisomeren 
Weinsauren im tierischen Organismus. Z .pliysiol.Cliem., 
25, 283-295. 

Eerera, G., Asymmetry and vitalism. Nature, 58, 616. 

Fischer, E., Bedeutung der Stereochemie fiir die Phy- 
siologic. Z .pliysiol.Cliem., 26, 60-87. 

Fitzgerald, G.F., Chance or vitalism. Nature, 58, 545 ; 
59, 76. 

Japp, F.R., Stereochemistry and vitalism. Presidential 
address. Nature, 58, 452. 

Kipping, F.S. and W.J. Pope, Stereochemistry and 
vitalism. Nature, 59, 53. 

Pearson, K., Asymmetrv and vitalism. Nature, 58, 495 ; 
also 5.9, 30, 124. 



136 GENERAL BIBLIOGRAPHY 

Pere, a., Fermentation lactique des corps siicres par 
le Coli-bacille. Ami.Inst. Pasteur, 12, 63-72. 

PoTTEviN, A., Contribution a Petude de la fermenta- 
tion lactique. Ann.Inst.Pasteur, 12, 49-62. 

Spencee, H., Stereochemistry and vitalism. Nature, 

58, 592. 

Steoxg, W.M., Stereochemistry and vitalism. Nature, 

59, 53. 

1900 

Fischer, E., Spaltung- einig-er racemischer Amino- 
sauren in die ojjtisch-aktiven Komponenten. Ber.chrm. 
Ges., 32, 3638-36-t6. 

MoRiSHiMA, K., tjber das Vorkonnnen der Milchsaure 
im tierischen Org-anismus mit Beriicksichtigung der Ar- 
senvergiftung-. Aycli.exp.Path.Pharma'k., J/S, 217-244. 

Neuberg, C, Uber die Harnpentose, ein optisch-inak- 
tives, natiirlich vorkommendes Kohlenhydrat. Ber.chem. 
Ges., 33, 2243-2254. 

Walden, p., Fiinfundzwanzig Jahre stereochemischer 
Forschung. N at ur wis s. Rundschau, 15, 145-148. 

1901 
OsHiMA^ K. AND B. Tollens, Ubcr das Nori aus Japan. 
Ber.chem.Gcs., 3',, 1422-1424. 

1902 
Neuberg, C. and J. Wohlgemuth, Uber das Ver- 
halten stereoisomerer Substanzen im Tierkorper. I. 
Uber das Schicksal der drei Arabinosen im Kaninchen- 
leibe. Z .physiol.Cltem., 35, 41-69. 

1903 

CusHisTY, A.E., Atropine and the hyoscy amines — a study 
of the action of optical isomers. J.Physiol., 30, 176-194. 

McKenzie, a. and a. Harden, The biological method 
for resolving inactive acids into their optically active 
components. J.Chem.Soc, S3, 424-438. 



GEXERAL BIBIAOGRAPHY 137 

Neuberg, C. and p. Mayee, tJber das Verhalten ste- 
reoisomeror Substaiizoii im Tierkorper. II. Uber das 
Schicksal der drei Mannosen im Kaninchenleibe. Z.phy- 
siol.Chem., 37, 530-544. 

1904 

Byk, a., Zur Frage der Spaltbarkeit von Kacemver- 
biiidiiiigen durch zirkularpolarisiertes Licht, ein Beitrag 
zur priniareu Entstehung optisch-aktiver Substanz. 
Z.physikal.Che'm., ',9, 641-687. 

CoNDELLi, S., tJber die Spaltung der racemisclieii Wein- 
saure durcli Aspergillus niger. Chem.Zhl., 2, 824. 

Dakin, H.D., Tlie fractional hydrolysis of optically 
inactive esters by lipase. J.Pliysiol., 32, 199-206. 

Marckwald, W., Uber asymmetrische Synthese. Ber. 
cliem.Ges., 31, 349-354. 

JMcKenzie, a., Studies on asynnnetric synthesis. 
J.Chem.Soc, 83, 1249-1262. 

Pictet, a. and a. Eotschy^ Synthese des Nieotins. 
Ber.cliem.Ges., 31, 1225-1235. 

1905 

CusHNY, A.R. AND A. Peebles, Tlic action of optical 
isomers. II. Hyoscines. J.Physiol., 32, 501-510. 

ScHULZE, E. AND E. WiNTERSTEiN, Ubcr das spezi- 
fische Drehungsvermogen einiger aus Pflanzen darge- 
stellten Tyrosinpraparate. Z.physiol.Chem., Ji5, 79-83. 

Walden, p., Uber das Drehungsvermogen optisch- 
aktiver Korper. Ber.cliem.Ges., 38, 345-409. 

Wohlgemuth, J., Uber das Verhalten stereoisomerer 
Substanzen im tierischen Organismus. Die inaktiven 
Monoaminosauren. Ber.cliem.Ges., 38, 2064-2065. 

1906 

Abderhalden^ E. and F. Samuely, Der Abbau des Leu- 
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Z.physiol.Chem., )7, 346-353. 



138 GENERAL BIBLIOGRAPHY 

Ehrlich, F., tJber eine Methode zur Spaltung racemi- 
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Engler, C, Zur Frage der Entsteliuiig des Erdols. 
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Mayer, P., Uber die Spaltung der lipoiden Substan- 
zen durcli Lipase und liber die optischen Antipoden des 
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Meyerhoffer, W., Gleicligewiclite der Stereomeren. 

Neuberg, C, tJbor die Entstehung optisch-aktiver Fett- 
sauren in der Natur. Biochem.Z., /, 368-379. 

1907 

Abderhalden, E. and a. Schittexhelm, Studien iiber 
den Abbau racemischer Aminosauren im Organismus des 
Hundes unter verscliiedenen Bedingungen. Z.physiol. 
Chem., 51, 323-333. 

Fischer, E., Vorkommeu von 1-Serin in der Seide. 
Ber.chem.Ges., J/O, 1501-1505. 

Fischer, E. and K. Raske, Verwandlung des 1-Seriiis 
in d-Alanin. Ber.chem.Ges., JjO, 3717-3724. 

Neuberg, C, Die Entstehung des Erdols, Bildung von 
optisch-aktiver Naphtha aus drehenden Fettsauren. 
Biochem.Z., 7, 199-212. 

Neuberg, C. an^d E. Rosenberg, Verwandlung von op- 
tisch-inaktiven Triolein in ein optisch-aktives Glycerid 
und eine optisch-aktive Saure. Biochem.Z., 7, 191-198. 

Stoklasa, J., Uber die glykolytischen Enzyme im Pflan- 
zenorganismus. Z.physiol. Chem., 50, 303-360; also 51, 
156-157. 

1908 

Bredig, G. and K. Fajans, Zur Stereochemie der Kata- 
lyse. Ber.chem.Ges., J/l, 752-763. 

CusHNY, A.R., The action of optical isomers. III. 
Adrenalin. J.Physiol., 37, 130-138. 

Ehrlich, F., Uber die Spaltung racemischer Amino- 
sauren mittels Hefe. 11. Biochem.Z., 8, 438-466. 



GENERAL BIBLIOGRAPHY 139 

Ehrlich, F. axd a. Wende-l, Zur Kenntnis cler Leucin- 
fraktion des Eiweisses. Biocliem.Z., N, 399-437. 

RosENTHALEE, L., Duvcli Eiizyiiie bewirkte asymiiie- 
trisclie Syntlieson. Biochem.Z., 1'/, 238-253; also 17, 257- 
269, 1909. 

VAN Ekenstein, W.A, and S. Blanksma, Beitrag zur 
Kenntnis der Zucker. Chem.Zhl., 119. 

1909 

CusHNY, A.R., Eurtlier note on adrenalin isomers. 
J.PlujsioL, 38, 259-262. 

Neubeeg, C, Yerlialten von racemiselier Glutaminsaure 
bei der Faulnis. Biochem.Z., 18, 431-431. 

Neuberg, C. axd L. Karczag, Verhalten von dl-Valin 
bei der Faulnis. Biochew.Z., IS, 435-439. 

Walker, W. axd V. Krieble, The amygdalins. J.Chem. 
Soc, 0.'), 1437-1449. 

1910 

Bartelt, K., Terpene und Camplier. Ahderh.Bioch. 
Eandlex., 7, 266-538. 

Fajans, K., Uber die stereochemisclie Spezifizitat der 
Katalysatoren ( Optisclie Aktivierung durch asymme- 
trische Katalyse). Z.physikal.Chem., 73, 25-96. 

Karczag, L., Beitriige zur physiologischen Wirkung der 
Weinsauren. Z.Blol., o3, 218-231. 

Leger, E., Sur I'aloinose ou sucre d'aloine. C.R.Acad. 
ScL, loO, 983-986. 

, Sur I'aloinose cristallise; son identite avec 

I'arabinose -d. C.R.Acad.Sci., 150, 1695-1697. 

Pringsheim, H., Notiz fiber das Vorkommen von 
Rechts-Asparagin in der Natur. Z .physiol.Chem., Go, 89- 
95. 

, Studien iiber die Spaltung racemi- 
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109. 



140 GENERAL BIBLIOGRAPHY 

1911 

CuEEiE, J.N., The study of the optical forms of lactic 
acid produced by pure cultures of Bacillus hulgaricus. 
J.BioLChem., 10, 201-211. 

Neuberg, C. and S. Saneyoshi, tJber das Verhalten der 
stereoisomeren Weinsauren im Organismus des Hundes. 
B loch em. Z., M], 32-36. 

Thiele, a., Oxydicarbonsauren. Ahderli.Biocli. Hand- 
lex., 1, 1149-1182. 

1912 

Bredig, G. and p. Fiske, Durcli Katalysatoren be- 
wirkte asymnietrische Synthese. Biochem.Z., .'/(i, 7-23. 

Embden, G., Baldes, K. and E. Schmitz, Uber den 
Chemisnius der Milchsaurebildung' aus Traubonzucker im 
Tierkorper. Biochem.Z., 'i'), 108-133. 

Krieble, v., The amygdalins and their inter-relations 
with emulsin. J.Ani.Chew.Soc, 3'/, 71(1-735. 

1913 

Krieble, v., Asynnnetric synthesis through the action 
of oxynitrilases. J.Am.Chcm.Soc., 35, 1643-1647. 

Net'berg, C, Weitere Untersuchungen liber die bio- 
chemische Umwandlung von Methylglyoxal in IMilch- 
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484-508. 

Study, E., Die Begritfe Links, Kechts, Windungssinn 
und Drehungssinn. Arch.Mafh. mid Physih, 21, 193-226. 

1914 

Ehrlich, p., Uber asynmietrische und symmetrische 
Einwirkung von Hefe auf Racemverbindungen natiirlich 
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1916 

Peingsheim, H. and E., Die Chemotaxis von Bakterien 
gegen optisch-aktive Aminosauren. Z .physiol.Cliem., 97, 
176-190. 



(!i:\i:ir\i. iniUjiodRAPHY 141 

1918 

Clough, G.W., The relationship between the optical ro- 
tatory powers and the relative contiourations of optically 
active componnds. .1 .(liem.Soc, //.>, 526-554. 

1919 

CusHNY, A.E., On optical isomers. V. The tropeines. 
J.PlicumacoL, i.>, 71-93. 

Jaeger, F.M., Untersnchnngen iiber das Pasteursche 
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McKexzie, a. and J. Wood, The isomeric tropic acids. 
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Oela- Jensen, The lactic acid bacteria. Copenhagen. 

1920 

Faeber, E., F. Xord and C. Xeubp:rg, Die phytochem- 
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Hess, K. and W. Weltzien, Uber die Fahigkeit der 
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Pringsheim, H., Uber das Vorkonnnen optischer Anti- 
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1922 

Gersbach, a., Uber die Wendigkeit der Kolonieauslau- 
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KoTAKE, J. AND M. Okagawa, Ubcr den Einfluss des op- 
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NoRDEFELDT, E., Ubcr die asymmetrische Wirkung des 
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192 



Q 



Braun, J. AND W. Kaiser, Geruch und molekulare/^Gil C 
Asymmetric. Ber.chem.Ges., 56, 2268-2274. /'^'^ -'oi" 



^^,'^ 



LI5R 



142 GENERAL BIBLIOGRAPHY 

Freudenberg, K., F. Brauxs and K. Siegel, Die Konfig- 
uration der Manclelsiiure mid anderer d-Oxysauren. Ber. 
chem.Gcs., 56, 193-200. 

Jaeger, F.M., Sur le pouvoir rotatoire des composes 
cliimiques en rapport avec leur configuration moleculaire 
et la theorie des atoms dits asymetriques (Conference). 
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WoHL, A. AND K. Freudenberg, Die Bezeichnung ster- 
isclier Reilien. Ber.chem.Ges., 56, 309-313. 

1924 

Freudenberg, K. and F. Rhino, Die Konfiguration des 
Alanins. Ber.chem.Ges., 51', 1547-1557. 

1925 

Bragg, W. and R. Gibbs, The structure of a and |3 
quartz. Proc.Roy.Soc.A., 109, 405-427. 

Braun, J. and W. Teuffert, Geruch und molekulare 
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Byk, a., Zur Synthese der molekularen Asymmetric. 
Naturwiss., 13, 17-21. 

Langenbeck, W., Der Abbau des 1-Histidins zur 1-As- 
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Neuberg, C. and G. Gorr, tJber den Mechanismus der 
Milchsaurebildung bei Bakterien. Biochem.Z ., 166, 482- 
487. 

Okagawa, M., tJber den Einfluss des optischen Dre- 
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1926 

Binder-Kotrba, G., Die Umwandluug von Phenylgly- 
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CusHNY, A.R., Biological relations of optically isomeric 
substances. Baltimore. 

Karrer, p. and M. Ehrenstein, Zur Kenntnis einiger 
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GENERAL BIBLIOGRAPHY 143 

Mayek, p., Zur Biochemie des Asymmetrieproblems. 
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Neuberg, C and E, Simox, Beitrage zur Biochemie des 
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PEDERsoisr, C, W. Peterson and E. Fred, The forms of 
lactic acid produced by pure and mixed cultures of bac- 
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1927 

Neuberg, C. and M. Kobel, fiber die Bildung von reiner 
d(-) Milchsaure durch frische Hefen und Trockenhefe, 
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Neuberg, C. and E. Simon, tjber die quantitative Dismu- 
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1928 

Neuberg, C. and E. Simon, Weitere Untersuchungen 
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1929 

Bendrat, M., Zur Physiologie der organischen Sauren 
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Boycott, A., C. Diver, S. Hardy and F. Turner, The in- 
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Fitting, H., Uber die Auslosung von Plasma stromung 
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Hayashi, S., Experimentelle Untersuchungen iiber die 
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144 GENERAL BIBLIOGRAPHY 

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206, 223-'227. 

KuHN, W. AND E. Braun, Pliotocliemisclie Erzeuguiig 
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Macht, D., Pharmacological synergism of stereoisom- 
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Ruhland, W. and K. Wetzel, Zur Physiologic der or- 
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WiDMANN, E., Quantitative Umwandlung von Methyl- 
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1930 

Bamann, E. AND P. Laeverenz, Ubcr den Einfluss von 
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Erlenmeyer, H., Studieu zu einer dynamischen Stereo- 
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GEM:/f\L nffUJOCLW/'HY 145 

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SUBJECT INDEX 



Abies, 30 

Acanthis, 103, 117 
Adonis, 29 

Aelosoma, 118, 119, 120 
Alkanna, 29 
Amoeba, 116 
Amomum, 29 
Andropogon, 29 
Anethion, 30 
Apium, 30 
Arenicola, 118 
Asarium. 29 
Aspergillus, 93 
Asymmetric analysis, 99 

induction, 37 

state, transmission of, 35, 37 

synthesis, 35, 36, 37, 38, 39, 41, 
43, 45 
Asymmetry, and cancer, 129 

as a criterion of organic origin, 
31 

as a specific property of pro- 
toplasm, 20 

definition, 10 

of primary constituents, 21 

optical and geometrical, 11 

origin of, 52, 53 

Bacillus, 81, 82, 83, 84, 85, 86, 87, 

88, 89, 91, 93, 95, 96 
Bacterium, 62, 67, 68, 71 
Balanus, 115 
Barbados, 29 
Bar asm a. 30 
Biological series of isomers, 15, 17, 

31 
Blumea, 29 

Canarium, 30 

Caruvi, 30 

Chaetogaster, 118, 119 

Citrus, 30 

Cladonema, 118 

Clostridiuvi, 64 

Correction, in maintaining optical 

purity, 45, 50 
C rot on, 8 
Cyclops, 118 



Dahlia, 22, 23 

Dalyellia, 118 

Daphnia. 118 

Dextral and sinistral strains, physi- 
ology of, 84, 89 

Differential effect of optic isomers, 
99, 120 

Dissymmetric substances, produc- 
tion from symmetric, 67 

Dissymmetry, and configuration of 
organic molecules, 14 

and life, 19 

and optical activity, 12 

definition, 9 

in organic nature, 19 

in quartz, 13 

morphological, 79 

the loss of, 74 
Drosophila, 118 
Dryobalanops, 29 

Erigeron, 30 

Eucalyptus, 30 

Euchlanis, 118 

Euplotes, 118 

Evasion, in maintaining optical 

purity, 45 
Foeniculum, 30 
Fraxinns, 25 
Fruticicola, 83, 84, 89, 90, 91, 93, 

96 
Fundulus, 115 

Gammarus, 118 

Genes, optical inversion of, 81 

Helobdella, 118, 119 
Hydra, 116, 118 

Inversion, morphological, 79 
morphological and optical, rela- 
tion of. 91 
optical, 15, 59, 61 

Juniperus, 30 

Ketonaldehydemutases, 67 

Lacerta, 103, 117 
Lactobacillus, 62, 65, 66, 68 
Laurus, 26, 29, 30 



155 



156 



SUBJECT IXDEX 



Lavandula, 29 

LeMstes, 105, 106, 109, 110, 117 

Leptoplana, 118, 119 

Leuciscns, 101, 102, 103, 104. 117 

Leuconosioc, 65, 66 

Limnaea, 79, 80 

Limnodrilvfi, 118, 119, 120 

Lindera. 30 

Linens, 118 

Hthospermnm, 29 

Lupinus, 22, 23 

Massoia, 30 

Mechanism, of Langenbeck and 
Triem, 46, 54, 56 

of Krebs, 48, 54, 55, 56 

of Kuhn, 54 
Mentha, 30 
Monodora, 30 
Mucor, 71 
Myristica, 30 
Myrrha. 30 
Myrtus, 30 

2^ais, 118, 119, 120 

Natural isomers, 28 

Nervous system, evolution of, 116 

Nicotiana, 25 

Optical activity, and dissymmetry, 
12 

heredity and environment, 59 

stability of, 42 

Optical inversion, 15, 59, 61 

of genes, 81 

of the Walden type 73, 74 

Optical isomers, and intermediate 
pathways, 70 

differential action of, 99, 120 

mechanisms controlling produc- 
tion, 67 

mechanism of action, 100 

production by catalysts, 68, 69 

production by esterases, 68 

velocity of formation, 45 

Optical purity, advantages of, 51 
and 'fixed internal milieu," 45 
maintenance of, 43, 45 

Organic acids, dual action of, 112 



Palaemon, 117 

Paramecium, 107, 108, 118, 124, 125, 
128 

"Pathways", and optical isomers, 
71 

Pelargonium, 25 

Perinereis, 118, 119 

Petroleum, origin and asymmerty, 
31 

Peumus, 30 

Phaenocora, 118 

Phycomyces, 94 

Physiological mutations, 62, 72 

Picea, 30 

Pitivs, 26, 29, 30 

Piper, 30 

Pittosporum, 30 

Platyjioeciliis, 106, 109, 110, 111 

Polycelis, 118 

Porphyra, 23 

Primary constituents of proto- 
plasm, asymmetry of, 21 
exclusiveness of the asymmetry 
sign, 27 

impossibility of inverting, 59 

replaceability of, 28 
Principle, Curie's, 36, 43, 54 

of fixed pathway, 74, 75 
Pristina, 118, 119 
Procerodes, 118, 119 
Priimis, 26, 44 
Pyrethrum, 29 

Quartz, optical activity, 12, 13 

Racemates, splitting up of, 41 
Racemiase, 65, 66 
Racemic mixture, definition, 10 
Racemization, and ageing, 47 

and culture medium, 64 

and temperature, 66 
Rana, 103, 110, 117 
Relative configuration, 15, 30, 72 
Rheum, 25 
Rotifer, 118 
Rubus, 25 
Saccharomyces, 71 
Saccocirrus, 118, 119 
Sagitta, 118, 119 



SUBJECT INDEX 



157 



Secondary constituents of proto- 
plasm, asymmetry of, 24 
inverting of, 61 - 

non-exclusiveness of the asym- 
metry-sign, 28 
Sempervirum. 25 
Solidago, 30 
Sophora, 29 
SjXirtium. 29 

Spiral growth, theory of, 93 
SpirostoDiioti, 118 
mentor, 118 
Stereo-autouomic substances, 43, 

44. 55 
Stereo-coefficient, 101, 116 



Stylaria, 118 

Synergism, of stereoisomers, 100 

Tanacetum, 29 

Temporary dissymetric sub- 
stances, 74 

Thuja, 29 

Thy VI us, 30 

Torula, 60, 91, 92, 110 

Trapaeolum . 23 

Valeriana, 26, 30 

AValden inversion, 73, 74 

Xanthoxyluni, 30 



AUTHOR INDEX 



Abderhalden, 48, 137. 138, 146, 151 

Ackermann, 19, 146 

Akkerman, 151 

Alpatov, 107, 108, 124, 125, 126, 149 

Amnion, 68, 69, 70, 145, 146 

Arkwright, 86 

Arnow, 131, 149, 151 

Bacq, 122, 123 

Baldes, 71. 140 

Bamann, 70, 144 

Barcioft, 45 

Bartelt, 26, 29, 30, 139 

Bartium, 53, 135 

Bayerle, 150, 151 

Bayliss, 38 

Becker, 152 

Behrens, 152 

Belloni, 29 

Bendrat, 25, 143 

Bennet-Clark, 24, 147 

Berg, 61, 148 

Bergmann, 152 

Bernard, 45, 52 

Bernheim, 126, 146, 147 

Binder-Kotrba, 68, 142 

Biot, 32 

Blanksma, 29, 139 

Borsook, 40 

Bosshard, 48, 134 

Bouchardat, 29 

Boycott, 79, 143 

Boyland, 150, 151 

Boys, 14, 145 

Bozler, 124 

Bragg, 13, 142 

Branke, 29, 147 

Braun, E., 53, 144 

Braun, I., 141, 142 

Brauns, 72, 73, 142 

Bredig, 38, 43, 68, 69, 70, 138, 140, 
144 

Brion, 135 

Brockmann, 29, 146 

Brown. F., 120 

Brown, H., 23, 36, 135 



Brown, O., 150 
Bruckner, 95, 147 
Burk, 152 
Burton, 123 
Byk, 52, 53, 137, 142 

Caesar, 151 

Cannon, 121, 122 

Castle, 94 

Chabrie, 135 

Chibnall, 130, 150, 151 

Christeleit, 31, 72, 148 

Clough, 31, 141 

Cohn, 150 

Condelli, 137 

Conrad, 61, 148 

Cotton, 52 

Craft, 145 

Crozier, 103 

Curie, 35, 135 

Currie, 61, 140 

Cushny, 114, 115, 136, 137, 138, 139, 
141, 142 

Dakin, 137 

Deuticke, 71, 145 

Dittmar, 150 

Diver, 79, 143 

Dosser, 146 

Du Vigneaud, 61, 145, 148, 150 

Edlbacher, 126, 145, 152 

Efimochkina, 93, 150 

Ehrenstein, 31, 142 

Ehrlich, 22, 60, 138, 139, 140 

Ekenstein, 29, 139 

Embden, 71, 140, 145 

Engler, 31, 138 

Erlenmeyer, 144 

Errera, 11, 53, 135 

Erxleben, 23, 129, 130, 131, 150, 
151, 152 

Ewing, 129 

Faerber, 24, 141 
Fajans, 38, 43, 68, 138, 139 
Findlay, 10, 148 

Fischer, 10, 11, 16, 21, 22, 30, 36, 
51, 55, 59, 73, 134, 135, 136, 138 



159 



160 



AUTHOR INDEX 



Fiske, 70, 140 
Fitting, 143 
Fitzgerald, 53, 135 
Foster, 152 
Franlte, 120 

Fred, 61, 62, 143, 145, 147 
Fresnel, 12, 13, 15, 133 
Freudenberg. 16, 17, 72, 

144, 145 
Fruton. 152 



73, 142, 



Gause. 60. 84, 85, 86, 87, 88, 89, 90, 
91, 92. 99. 100, 101, 102, 104, 105, 
106, 107, 108, 109, 110, 113, 114, 
115, 117, 118, 122, 123, 124, 125, 
126. 147, 149, 150, 152 

Gay-Lussac, 10 

Gersbach, 82, 141 

Gerstner, 144 

Gibbs, 142 

Gintl, 25. 133 

Glaser. 124 

Gold, 120 

Gorr, 62. 142 

Goto. 148 

Graff, 130, 150, 152 

Greenwood, 116, 117 

Haller, 29 
Harden, 136 
Hardy, 79, 143 
Hastings, 83 
Hatschek, 153 
Hayashi, 68, 143 
Herken, 152 
Hess, 27, 141 
Hoagland, 123 
Holmes, 50 
Humboldt, 31 
Irish, 148. 150 
Irving, 152 
Ivanovics, 95, 147 

Jaeger, 19. 141, 142 
Japp. 10. 11, 135 
John-Brooks, 94, 95 
Johnson, 152 
Jungfleisch, 11, 134 

Kabit, 152 
Kaiser, 141 



Kalmus, 124 

Karczag, 139 

Karrer, 31. 72, 142 

Katagiri, 26, 61, 63, 64, 65, 66, 148 

Kayser, 62, 63, 64, 66, 135 

Kiesel, 93, 150 

Kipping, 53, 135 

Kisch. 48. 49, 146 

Kitahara, 26, 61, 63, 65, 66, 148 

Knopf, 53. 144 

Kobel. 71, 143 

Kogl. 23. 129, 130, 131, 150, 151, 152 

Koltzoff, 80, 112, 146 

Konikowa, 152 

Konowalowa, 29, 145 

Kopeloff. 66, 148 

Kotake, 141, 148 

Kraft, 71. 145 

Krebs. 48. 49, 50, 51, 126, 145, 146, 

147 
Krieble. 38. 44. 139. 140 
Kuhn. 26. 39, 41, 42, 43, 44, 47, 50, 

53, 55, 144, 147 
Kuna, 73, 148 



Laeverenz. 70, 144 
Langenbeck, 31, 43, 46, 47. 53, 

147 
Langer, 83 
Langley, 120 
LeBel, 134 
Lees, 29 
Leger, 29, 139 
Lehmann. 148 
Lemery. 31 
Lemmlein. 20, 151 
Lepeschkin, 113 
Levene, 31, 73, 148 
Lewis, 82, 87 
Lipmann, 131, 132, 152 
Lippman, 22, 134 
Loewi, 121 
Lomonosoff, 31 
Loring. 145 

Lowry, 12, 13, 15, 16, 19, 20, 146 
Ludwig, 79, 82, 144, 147 

Macht, 100, 144 
Marckwald, 37, 137 



142, 



Al THOR lyOEX 



161 



Mardaschew, 31, 14S 

Mathieu, 146 

Mayer. K., 132, 151, 153 

Mayer. P., 23, 68, 137, 138, 143 

McKenzie, 27, 37, 136, 137, 141. 144, 

147 
Mendel, 149 
Menschikov. 27, 148 
Metalnikoff, 124 
Meyerhol'fer, 138 
Mills, 51. 53, 73, 144 
Minaeff, 43, 70, 144 
Morishima, 26, 136 
Morris, 23, 36, 135 
Muller, 153 
Mundell, 149 

Nachmanson, 122 

Nay lor, 24 

Neber. 48, 126, 145, 147 

Needham, 80, 146 

Nencki, 61. 135 

Neuberg. 23, 24. 32, 62. 67, 68. 71, 

74, 136. 137, 138, 139, 140, 141, 

142, 143 

Nikolai, 72, 145 

Nord. 24. 141 

Nordefeldt, 38, 40, 43, 141 

Oesterle, 82 
Okagawa. 141, 142 
Opsahl. 131. 149, 151 
Orechoff, 29, 145 
Orla-Jensen, 64, 141 
Oshima, 23, 136 
Ostwald. 11, 102, 110, 113, 134 
Oudin. 29, 144 

Parishev, 29, 147 

Pasteur. 9. 10, 11, 12, 13, 19, 20, 21, 
25, 35, 59, 73, 133, 134 

Pearson, 53, 135 

Pederson. 61, 62, 64, 143 

Peebles, 137 

Pere. 62, 63. 64, 135, 136 

Peterson, 61, 62, 143, 145, 147 

Pfeiffer, 31, 72, 148 

Pictet, 100, 137 

Piutti. 134 

Podloucky, 151 



Pope, 53, 135 
Pottevin, 62, 136 
Power, 29 

Pringsheim, 22, 28, 48, 83, 139, 140, 
141 

Rabinowitch, 29, 145 

Rainey, 15, 148 

Rail, 93, 150 

Raoult, 11 

Raske, 30, 138 

Ratner, 61, 153 

Rees, G., 150, 151 

Rees, W., 112 

Rhino, 31, 72, 142 

Richter, 146 

Ritchie, 37, 50, 53, 145 

Rittenberg, 61, 150, 152, 153 

Roberts, 83 

Rona. 68, 69, 70, 145 

Rosenberg, 32, 138 

Rosenblueth. 121, 122 

Rosenthaler. 38, 70, 139 

Roth. 29, 146 

Rothen, 73, 148 

Rotschy, 100, 137 

Ruhland, 24, 25, 144 

Sagen, 83 
Salkowsky, 23 
Samuely, 48, 137 
Saneyoshi, 140 
Schimmel, 29 
Schittenhelm, 48. 138 
Schmitz, 71, 140 
Schoen, 147 

Schoenheimer, 61, 150, 153 
Schramm, 153 
Schulze, 22, 48, 134, 137 
Schwarze, 24, 25, 145 
Siegel, 72, 73, 142 
Simon. E., 67, 68, 71, 143 
Simon, M., 152 
Sizer, 115 

Smaragdova, 60, 89, 90, 91, 100, 101, 
102, 104, 105, 106, 107, 108, 109, 
110, 113, 114, 115, 117. 118, 122, 
123, 124, 125, 126, 149, 150, 152 

Spielmann, 31 

Spencer, 136 



162 



AUTHOR INDEX 



Spiers, 53, 149 
Stapp, 83 

Stiles, 112 

Stoklasa, 25, 138 

Stoll, 36, 145 

Strelitz, 149 

Strong, 37, 53, 136 

Study, 15, 140 

Szent-Gyorgyi, 95 

Tatum, 64, 145, 147 
Tetzner, 48, 146 
Teuffert, 142 
Thiele, 25, 140 
Thierfelder, 59, 135 
Thomas, 120 
Tollens, 23, 136 
Tomiyasu, 37, 149 
Town, 153 
Trask, 32 

Triem, 43, 46, 47, 53, 147 
Tristram, 150 
Tromsdorff, 20, 149 
Tschugaeff, 32 
Turner, 79, 143 



Van't Hoff, 134 
Vernadsky, 19, 32, 146 

Walden, 32, 73, 136, 137 
Waldschmidt-Leitz, 129, 132, 151, 
153 

Walker, 44, 139 
Wallach, 29 
Warburg, 129 
Webster, 147 
Weltzien, 27, 141 
Wendel, 22, 139 
Wetzel, 24, 25, 144 
White, 151 
Widmann, 71, 144 
Wiedemann, 36, 145 
Williams, 150, 151 
Willstatter, 68 
Winterstein, 22, 137 
Wohl, 16, 17, 142 
Wohlgemuth. 136, 137 
Wood, 27, 141 
Wyrouboff, 11, 134 

Zvetkov, 84, 149 
Zycha, 83 



Corrigenda 

P. 20, 18th line from the bottom, read 1939 instead of 1938. 
P. 29, line 13, read Brockmann instead of Blockmann. 
P. 59, 8th line from the bottom, read uilxtrorotatoky instead 
laevorotatory. 



of