FROM/THE
LIBRARY
OP
LEONARD WILLIAM
* BUCK*
UNIVERSITY QF CALIFORNIA
MEDICAL CENTER LIBRARY
SAN FRANCISCO
LEONARD W. BUCK, M.D.
rag '
GENERAL CHEMISTRY
OF THE
ENZYMES
BY
HANS EULER
Professor of Chemistry in tlie University of Stockholm
TRANSLATED FROM
THE REVISED AND ENLARGED GERMAN EDITION
BY
THOMAS H. POPE
FIRST EDITION
FIRST THOUSAND
NEW YORK
JOHN WILEY & SONS
LONDON: CHAPMAN & HALL, LIMITED
1912
152651
Copyright, 1912
BY
THOMAS H. POPE
SCIENTIFIC PRESS
ROBERT DRUMMOND AND COMPANY
BROOKLYN, N. Y.
PREFACE TO THE GERMAN EDITION
As the title of this book indicates, the author has attempted
to review the more important facts of enzymology from a general
standpoint and to fit them, so far as is possible, into their
proper places in the fabric of general and physical chemistry.
The aim has not been to give a complete synopsis of our know-
ledge of the enzymes, for already several such summa'ries are
available.
It may perhaps be asked: Is the time yet ripe for giving
a representation of the physical chemistry of the enzymes?
The author feels that this question must be answered in the
affirmative, although it is evident that extensive regions and
important problems in the subject are still entirely untouched.
The period during which the marshalling of facts was the most
essential task was followed by one in which it was sought to
harmonize the somewhat crude and imperfect experimental
data with the laws of theoretical chemistry. The deviations
from theory seemed to be wide and the peculiarities of enzymic
reactions numerous. Only in the most recent times has the
need for experimental revision of the quantitative data made
itself felt. Improvements have been effected in the practical
methods, while the factors participating in the reactions have
become more clearly understood and are hence more fully taken
into account. It is now being found that the results obtained
from these more exact and comprehensive investigations corre-
spond more closely with those required to satisfy physico-chemical
theories. At the stage which has thus been reached in the
development of enzymology a review such as that now pub-
lished does seem to be justified. The author has therefore
decided to allow the two reports on this subject which appeared
in the " Ergebnisse der Physiologic " in 1907 and 1910, to be
iii
iv PREFACE TO THE GERMAN EDITION
arranged and issued in book-form, despite the fact that many
problems still call for fuller treatment.
Although this monograph is intended more especially as an
aid to scientific research in enzymology, yet the author trusts
that it will be found useful by those concerned with the prac-
tical applications of enzymic actions. Thus an understanding
of the dynamics of enzyme reactions is indispensable for the
rational estimation of enzymic activities, such as that of pepsin
in the gastric juice or that of diastases in malt, and these examples
serve to show how theory may be of value to the physician and
to the technical worker.
An appendix to the book contains a short sketch of experi-
mental methods, more especially of those for which the original
literature is not readily accessible. Professor Bertrand has
kindly permitted the insertion of the tables prepared by him
for use with his admirable method of estimating reducing sugars.
A considerable part of the labour involved in preparing this
monograph has been undertaken by Miss Beth af Ugglas, Assistant
in the Biochemical section of the Chemical Laboratory here and
to her I wish to express my sincere thanks.
H. EULER.
STOCKHOLM, January, 1910.
PREFACE TO THE ENGLISH EDITION
ALTHOUGH only two years have elapsed since the first
publication of this work in the German language, the great
energy with which the study of enzyme chemistry is being prose-
cuted has rendered necessary numerous additions and alterations.
In view of the results of recent investigations, some of the
sections, e.g., those concerned with the glucosides and the
fermentation enzymes, have indeed been entirely rewritten.
To Mr. Pope's request to allow of the issue of an English
edition of the book the author acceded the more readily because
of the great success which has for a long time past attended
enzymological research in English-speaking countries. At the
present time, when various different paths have become clearly
marked in general enzymic chemistry, the opportunity is wel-
comed of laying the author's views before English workers in
this field.
To Mr. Pope the author is indebted, not only for a careful
translation of his book, but also for certain improvements and
additions in the part dealing with practical methods and for
the references to the literature.
In the initial treatment of so extensive a subject as enzyme
chemistry omissions are scarcely avoidable, and the author
would be grateful to any readers who may contribute, either
by sending him copies of their papers or by any other means,
to render a subsequent edition more complete.
H. EULER.
STOCKHOLM, March, 1912.
v
JOURNALS REFERRED TO BRIEFLY
Biochem. Z.: Biochemische Zeitschrift (Berlin).
Chem. Ber. : Berichte der deutschen chemischen Gesellschaft.
C. R. : Comptes rendus de rAcademie des Sciences (Paris).
H. : Hoppe-Seyler's Zeitschrift fur physiologische Chemie.
Hofm. Beitr.: Hofmeister's Beitrage zur chemischen Physiologic und
Pathologic.
Lieb. Ann.: Justus Liebig's Annalen der Chemie.
Pfliig. Arch. : Pfliiger's Archiv fiir die gesammte Physiologic.
Soc. Biol.: Comptes rendus de la Socie*te* Biologique (Paris).
vi
CONTENTS
PAOE
Introduction - 1
CHAPTER I
Special Chemistry of the Enzymes 5
Nomenclature 5
Classification 6
Sphere of Action of the Enzymes; Their Preparation and Puri-
fication 7
Esterases 9
Enzymes of the Higher Carbohydrates 13
'Enzymes of the Glucosides and Disaccharides 18
Other Enzymes which Hydrolyse Glucosides 27
Phytase 31
Hexosephosphatase 32
Pectinase 32
Qarbamases (Proteinases) '. 33
Pepsin 33
Trypsin 36
Erepsin 38
Proteolytic Enzymes of Plants 38
Nucleases 41
Urease 43
Amidases (Desamidases) 43
Coagulating Enzymes 45
Chymosin 45
Thrombin, Fibrin-ferment 49
Enzymes of Fermentation 50
Enzymes of Alcoholic Fermentation 51
Lactic Acid Bacteria-zymase 58
Oxydases 58
Alcoholase 60
Aldehydases 61
Laccase 62
Tyrosinase 64
vi i
viii CONTENTS
Peroxydases 65
Catalases 67
Reducing Enzymes (Reductases; Reducase) 68
Appendix 69
CHAPTER II
Physical Properties of the Enzymes 71
Adsorption 75
Solid, Neutral, Adsorption-media 81
CHAPTER III
Activators (Co-enzymes), Paralysors and Poisons 90
Kinases of Unknown Composition 91
Special Organic Activators 92
Acids, Bases, and Neutral Salts 94
Protective Agents 115
Inhibiting Agents (Paralysors) 115
Inorganic Salts 116
Organic Poisons and Inhibiting Agents 118
CHAPTER IV
Chemical Dynamics of Enzyme Reactions 124
Theoretical Principles of Enzymic Dynamics 125
Catalysis 127
Reversible Reactions 137
Experimental Data on the Course of Enzyme Reactions 146
Esterases and Lipases 146
Amylase 155
Invertase 158
Maltase 166
Lactase 168
Enzymes of Emulsin 171
Proteolytic Enzymes 175
Rennet (Chymosin) 200
Fibrin-ferment 205
Zymase 206
Catalases 215
Oxydases 219
Peroxydases 223
Tyrosinase 22S
Oxidation of Xanthine . . .230
CONTENTS ix
CHAPTER V
PAGE
Influence of Temperature and Radiation on Enzymic Reactions . 231
Influence of Radiation . 245
CHAPTER VI
Chemical Statics in Enzyme Reactions 251
Equilibria 252
End-states and Stationary States 255
CHAPTER VII
Enzymic Syntheses 261
CHAPTER VIII
Specificity of Enzyme Action 274
Conclusion 283
APPENDIX
Practical Methods 286
Tables for the Estimation of Sugars by Bertrand's Method 306
Index of Authors 313
Index of Subjects 321
GENERAL CHEMISTRY OF THE ENZYMES
INTRODUCTION
THE name enzymes or unorganised ferments is given to
animal or vegetable substances of unknown composition and
constitution which, in the organism itself or even independently
of the organ or cells in which they arise, are able to accelerate
chemical reactions. The term enzyme is thus included in the
much more general term, catalyst.
By catalyst we understand a substance which, without being
required by the accelerated reaction or appearing among the
final products, alters the velocity with which a chemical system
strives to attain its final condition. But enzymes are at least
with the degree of purity in which they have as yet been obtained
rarely ideal catalysts. Only with difficulty, however, can a
limit be set between these substances and ideal catalysts, this
being greatly dependent on the experimental conditions.
The literature of the last few years shows, indeed, that a
certain limitation in the meaning of the term enzyme is desirable ;
and, as a rule, those substances which are required in stoichio-
metric proportions by the reactions in which they participate
are not regarded as enzymes.
With non-enzymic catalyses the quantity of the accelerating
substance is mostly small compared with that of the substance
acted on indeed, an ideal catalyst should accelerate the trans-
formation of unlimited amounts of " substrate." Even in the
most minute quantities some enzymes certainly exert very con-
siderable amounts of action; but usually their activity becomes
limited with lapse of time and does not produce more than a
certain amount of change. As we shall see later, this limitation is
2 GENERAL CHEMISTRY OF THE ENZYMES
due, partly to the participation of the enzyme in the equilibrium
of the reaction and partly to the chemical instability of these
substances. One property which the enzymes exhibit and which
is generally regarded as characteristic of them, is that of becom-
ing inactive if their solutions are heated for a longer or shorter
time at a high temperature about 100. This is not an absolute
criterion, as inorganic catalysts and enzymes do not exhibit any
fundamental difference in this respect. As soon as any con-
stituent of an organ which accelerates a reaction is explained
chemically or identified with a known compound, no reason remains
for terming it an " enzyme"; whether the term enzyme is to be
retained for such substances, or whether the name scientifically
more accurate of catalyst is to be employed, is entirely a matter
for the future. But the choice of a definition is of subordinate
importance, as we are, in many cases, so far removed from any
chemical explanation of the enzymes that this term will certainly
persist for a long time.
The distinction between the enzymes and the toxines is also,
to some extent, arbitrary. Common to both classes of bodies
are their origin in the living organism, their capacity of forming
anti-bodies, and certain other properties, as also are their modes
of action. On the other hand, the toxines are characterised with
moderate sharpness by their poisonous action. We can, indeed,
omit a treatment of this extensive subject all the more readily,
as the physical chemistry of the toxines has undergone considerable
development during recent years.
Of far greater importance than deciding how the enzymes are
to be limited is the definition of the physical and chemical prop-
erties of typical representatives of these remarkable substances.
The aim in view is, of course, the exact description of the
enzyme by a chemical formula and by constants characteristic
of the pure substance. For the general chemistry of the enzymes,
clear views concerning the degree of purity and the composition
of the various members are of the greatest importance. In the
first place, the enzymes must be prepared and analysed, and here
physico-chemical investigations also afford valuable aid.
The first question to be decided is : Do the enzymes occur in a
state of true solution, or must they be classed with colloidal
substances? Or, speaking more strictly, which enzymes approach
the one and which the other limiting case, and what can be
INTKODUCTION 3
affirmed concerning their molecular magnitude and
degree of dispersion? As criteria on these points serve
diffusion, adsorption phenomena and also behaviour in the electric
field.
More recent measurements have shown that the influence of
temperature on enzyme action can be defined more exactly
than earlier data would have led one to suppose, and the " tem-
perature of destruction " and " optimum temperature," which
give little information, are now replaced by well-defined physico-
chemical magnitudes.
Undoubted and considerable success has followed the study,
during the past few years, of activators or co-enzymes; men-
tion need only be made here of the work of Harden and Young
and of Buchner and Meisenheimer, which has led to
the discovery of essential factors influencing alcoholic fermenta-
tion. Also, with reference to inactivators remarkable
regularities have been observed, those concerning the influence of
configuration calling for special mention. Further, the action of
poisons is now so far understood that, in enzyme investigations,
we can make use of substances which prevent bacterial infection
and yet have no harmful effect on the enzyme, thus avoiding the
errors which have been so often caused in work of this kind by
insufficient disinfection.
Although the majority of the anti-bodies, so important
physiologically, are classed among the toxines, yet such a large
number of observations have been made on the anti-fer-
ments that these must not remain unnoticed.
When we have, in the first part of this book, obtained infor-
mation concerning the chemical facts of enzymology, we
must turn to the second part of the question: In what manner
is a reaction induced or accelerated by an enzyme and how do
enzymic reactions proceed?
In the first place, we will consider the laws of chemical
dynamics which come into play in enzymic reactions and, in
particular, the results which have been obtained from a study of
catalysts. Comparison of non-enzymic reactions with enzymic
changes will show us that the same processes are being dealt with
in the two cases and that the deviations from the classical examples
of chemical dynamics, exhibited by many enzyme reactions, are
readily explained by the simple assumption that enzyme and
4 GENERAL CHEMISTRY OF THE ENZYMES
substrate unite to form more or less stable complexes, which are
to be regarded as the " active " molecules and hence bring about
the reaction. This assumption is adhered to all the more strongly,
because it corresponds with our general conception of the role
of catalysts.
In the fourteen years which have passed since the synthesis
of isomaltose by maltase was discovered (Croft Hill), the
number of enzymic syntheses has become quite considerable.
Not only do we now know enzyme reactions which proceed in
both directions, but in several cases the synthetic action of the
enzyme has been separated, and caused to take place apart, from
the decomposing action. A knowledge of these syntheses is
naturally of the utmost importance for the biochemistry of
animals and plants.
For chemistry in general these processes are the more import-
ant, since, as is well known, the enzymes are extremely sensitive
towards the steric configuration of the substrate and lead to the
formation of asymmetric products. The enzymes hence place
us in a position to effect asymmetric syntheses.
CHAPTER I
SPECIAL CHEMISTRY OF THE ENZYMES
NOMENCLATURE
THE already large and rapidly increasing number of enzyme
actions necessitates a rational system of nomenclature. According
to a proposal made by D u c 1 a u x (see Bourquelot, Les
ferments solubles, Paris, 1896), the name of the enzyme is
derived from that of the substance on which it acts: for example,
lactase is the enzyme which decomposes lactose. Unfortunately,
many departures have been made from this principle. In cases
where the name derived from that of the substrate is not suf-
ficiently definite, E. O. von Lippmann (Chem. Ber., 1903,
36, 331 ; see also B u c h n e r and Meisenheimer, Chem.
Ber., 1905, 38, 621) proposes that both the name of the substance
acted upon and that of the (principal) product formed from it
should be indicated in the name of the enzyme; for example,
amylo-maltase would be the enzyme which forms maltose from
starch. But as this enzyme is not a maltase, but belongs rather
to the class of amylases, it would be more convenient to term it
malto-amylase. Names in general use, such as pepsin, zymase
and erepsin, are retained. Although the employment of a rational
method of naming enzymes is to be desired, yet, on the one hand,
the right to give or alter a name must be left with the discoverer,
and, on the other, the region of action and the individuality of
enzymes, like pepsin are not yet so completely determined as to
allow of the adoption of a perfectly suitable name.
According to a suggestion by the author (H., 1911, 74, 13),
the names of synthesising enzymes should be made to indicate
the substances which they form and to terminate with the syllable
"ese"; thus phosphatese would be the enzyme which syn-
thesises organic esters of phosphoric acid, and nitrilese ( B-nitrilase
according to Rosenthaler) that which forms nitriles.
5
GENERAL CHEMISTRY OF THE ENZYMES
CLASSIFICATION
Since very little is known concerning the nature of the enzymes,
the classification of these bodies is based on the chemical reactions
which they induce. And it is to be expected that the classifica-
tion indicated by the chemical actions would also be brought out
in the physical and chemical properties of the enzymes.
An enumeration of all the enzymes described in the literature
of the subject does not fall within the scope of this work; indeed,
there are a very large number of such substances, the individuality
of which has not been sufficiently well established.
The following summary serves rather to indicate, in a general
way, typical reactions in which enzymes play a part.
Reac-
tion.
Substrate.
Products.
Enzyme.
r
Esters :
Fatty acids +alcohols
Esterases:
Fats
Higher fatty acids +glycerol
Lipases
Lower esters
Chlorophyll +alcohol
Lower fatty acids +alcohols
Crystalline chlorophyll +phytol
Butyrases
Chlorophyl-
luse
Higher carbohydrates :
Cellulose
Cellulase
Hemicellulose
Cytase
f Amylases and
Starch, glycogeu
Maltose (dextrins)
j amylo-pec-
[ tinases
Inulin
Fructose
Inulinase
Pectoses
Pectin
Pectase
Glucosides including
Polysaccharides :
Hexoses and glucoside-residues
a-Glucosides
Glucose
a-Glucosidase
1
/3-Glucosides
Glucose +sugar, alcohol or
phenol-residue
(Maltase)
0-Glucosidase
(Emulsin)
2
/3-Galactosides
Galactose
Lactase
T3
>>
Fructosides
Fructose +sugar residues
Invertase
n
Other glucosides
Other sugars +phenols, etc.
Rhamnase,
Myrosin, etc.
Phytin
Inositol +phosphoric acid
Phytase
Hexosephosphate
Hexose +phosphate
Hexosephos-
phatase
Digallic acid
Gallic acid
Tannase
(Tannin)
Carbamide derivatives:
R-CO-NH-R'
R-COOH+R'-NH 2
Carbamases,
proteinases
Proteins
Albumoses, peptones
Pepsin, pap-
ain
Proteins, albumoses,
peptones, peptides
> Peptides, amino-acids
/ Trypsin, erep-
^ sin
Arginine
Urea +ornithine
Arginase
I
Nucleic acids
Nuclein bases +phosphoric acid
Nuclease
SPECIAL CHEMISTRY OF THE ENZYMES
Reac-
tion.
Substrate.
Products.
Enzyme.
Acid amides:
1
Urea
Carbon dioxide +NH S
Urease
i .
Amines :
Desamidases
6
Amino-acids
Hydroxy-acids +NHs
Desamidase
W
Guanine
Xanthine +NH,
Guanase
Adenine
Hypoxanthine +NHj
Adenase
= . n
Hydrogen peroxide
Molecular oxygen +H*O
Catalases
|!l '
Hydroxvnitriles
Aldehyde +HCN
Nitrilases
, *
Benzaldehyde +HCN
Mandelic acid nitrite
d-Oxynitrilese
= ?. ,
Na2HPO4 +carbohy-
Ester of carbohydrate phos-
003
drate
phoric acid
Phosphatese
ill?
Peroxides
f Reduction products of per- \
\ oxides +O
Peroxydases
Sgo
>,
ll||
Casein
Paracasein ( +whey-albumen)
Chymosin
Fibrinogen
Insoluble fibrin
Fibrin ferment
?! S '
(thrombin)
5=^|S
Pectins
Pectinates
Pectinase
Glucose
Lactic acid
Zymase of lactic
a
acid bacteria
o
Glucose, fructose, man-
Alcohol +CO
Zymase (sum-
g'
nose, galactose
total of the
enzymes of al-
8
coholic f e r -
mentation)
Phenols
Quinones
Phenolases
Aldehydes
Acids
Aldehydases
f
Alcohol
Acetic acid
Alcoholoxy d ase
3
of acetic acid
bacteria
Certain enzymes which exert actions other than those given
above are mentioned hi the Appendix to Chapter I.
SPHERE OF ACTION OF THE ENZYMES. THEIR PREPARATION
AND PURIFICATION
In this section are given such data as appear necessary for
understanding the general behaviour of the enzymes.
The dynamics of the enzymes suffers in considerable measure
from the disadvantage that we know nothing of the composition
of these bodies and hence can form beforehand no idea of the
chemical processes taking place during their action. It is, there-
8 GENERAL CHEMISTRY OF THE ENZYMES
fore, all the more necessary to investigate experimentally all
the factors influencing enzymic reactions, in order to avoid the
danger of missing a secure foundation for the theoretical treatment
of the subject.
Especially would the author point out that it is no t possible
to pay too much attention to the preparation and purification
of enzymes for use in physico-chemical measurements.
Enzyme preparations are obtained either by subjecting the
organs to pressure or by extracting them with suitable solvents.
The consistency of the starting material, the admixtures which
are always present, the age and especially the previous history
of the preparation, influence not only the intensity, but
also the mode of action of the enzyme to a greater extent than
many investigators have supposed. A knowledge of the material
is hence indispensable to a critical examination of the exper-
imental results.
Extracts or preparations of organs often exert several enzymic
actions at the same time; thus, to choose an example from recent
literatune, a preparation from croton seeds has been found by
S c u r t i and Parrozzani (Gazzetta Chim. Ital., 1907, 37,
i, 476) to hydrolyse, not only fats and esters of monobasic acids,
but also cane sugar and proteins. From these results, it should
be concluded, not that an enzyme exists possessing a general
hydrolytic capacity, but that the preparation employed contains
several enzymes.
In order to study the separate components, the different
actions have to be separated, and when a preparation has been
obtained which exhibits only a single reaction, it is termed
biologically pure. There still remains, however, the
possibility that the various stages of the reaction are
ccelerated by different constituents of the enzyme. It has,
for example, been found to be probable that, in the hydrolysis
of amygdalin, three enzymes take part, one of them effecting the
resolution into glucose and the glucoside of mandelic acid nitrile,
a second hydrolysing the latter compound to mandelomtrile
and glucose, while the third decomposes the nitrile into benzal-
dehyde and hydrocyanic acid.
Even those enzymes which are biologically the purest are
still very far removed from the state of chemical purity and
we possess and on this stress must be laid no certain knowledge
SPECIAL CHEMISTRY OF THE ENZYMES 9
that even an approximate isolation of any hydrolytic enzyme
has yet been attained. This is explained by the instability of the
enzymes, which, when subjected to protracted and energetic
purifying processes, become inactive, so that their presence can
no longer be detected; and also by the extremely small con-
centrations in which the enzymes always seem to occur in nature,
and by the large amounts of impurities especially of colloidal
substances contained in the extracts.
Of the oxydases, which are to some extent stable to heat, we
have chemical knowledge of at least one member.
We shall see later that the behaviour of enzymes towards ex-
ternal influences, such as acids, alkalies, co-enzymes, etc., is often
determined, wholly or partially, by the impurities present.
On account of the importance which processes of purification
have for enzymology, the methods employed are treated some-
what in detail.
As may be again mentioned, consideration of the whole of
the literature on the different enzymes does not come within
the limits of this work. In the first place, investigations will,
of course, be omitted to which lasting value cannot be ascribed,
and no attention will be paid to those dealing with purely phys-
iological questions and with the distribution of the enzymes
in the animal and vegetable kingdoms, since these are not directly
connected with the general chemistry of the enzymes.
ESTERASES
The usual action of these enzymes consists in the hydrolysis
of esters. The enzymes of this group prove to be more or less
markedly specific, as will be shown more in detail in Chapter
VIII. It must, however, be mentioned that the lipase of the
stomach decomposes, not only true fats, but also the lipoids,
lecithin, jecorin and protagon (P. Mayer, Biochem.
Z., 1906, 1, 81; Schumoff-Simanowski and Sieber,
H., 1906, 49, 50). Also pancreatic juice, according to A b d e r-
h a 1 d e n and others, decomposes lecithin [but a negative result
was obtained by Kalaboukoff and Terroine (Soc. Biol.,
1909, 66, 176)]. A special class is formed by the
L i p a s e s , which resolve more especially the natural fats,
i.e., the glycerol esters of palmitic, stearic and oleic acids.
10 GENERAL CHEMISTRY OF THE ENZYMES
Animal lipases play an important part in the stomach
(gastric juice and mucous membrane l ), pancreas 2 and intestines 3
of the higher animals. Also serum contains lipases (Neuberg
and collaborators) and, according to Pagenstecher (Biochem.
Z., 1909, 18, 285), this is the case with all the organs, especially
the liver and spleen, of the ox. These animal enzymes decompose
both animal and vegetable fats and oils.
A lipase has also been found in the albumen of hens' eggs.
In general, it is relatively difficult to obtain active extracts from
animal organs containing lipase and Connstein is of the
opinion that it is best to employ the pancreatic juice of the crushed
glands themselves. Aristides Kanitz, however, seems
to have prepared active glycerol-extracts (H., 1905, 46, 482),
and Lewkowitsch and M a c 1 e o d have worked with
aqueous lipase solutions which attack neutral fat (Proc. Roy.
Soc., 1903, 72, 31). The extraction of lipase-preparations from
the pancreas has been investigated in detail by D i e t z and
Pottevin (Bull. Soc. Chim., 1906, [iii], 35, 693; see also E.
B a u r , Zeitschr. f. angew. Chem., 1909, 22, 97). . Rosen-
h e i m (Journ. of PhysioL, 1910, 40) has recently made the
interesting observation that the lypolytic enzyme can be
separated from its activator or co-enzyme by mere filtration
of the glycerol extract of pancreatic lipase. The substance
remaining on the filter is sensitive to heat, whilst that in the
nitrate is stable to heat. A mixture of these two exerts enzymic
action, but each separately is inactive.
There is a great amount of contradiction among the results
obtained with the lipases ; it does, however, seem established that
the lipases of the stomach and pancreas do not exhibit identical
properties. The two animal lipases appear to be related in the
same manner as the corresponding proteolytic enzymes, pepsin
and trypsin; but they both differ essentially from the lipases of
seeds.
*F. Volhard, Zeitschr. klin. Med., 1901, 42, 414 and 43, 397; W.
Stade, Hofm. Beitr., 1903, 3, 291; A. Zinsser, Hofm. Beitr., 1906,
7, 31; A. From me, Hofm. Beitr., 1905, 7, 51; A. Falloise, Arch.
Internat. de Physiol., 1906, 3, 396, and 1907, 4, 405.
2 H. Engel, Hofm. Beitr., 1905, 7, 77; U m b e r and Br u g s c h ,
Arch. f. exp. Path., 1906, 55, 164.
' W. B o 1 d y r e f f , H., 1907, 50, 394.
SPECIAL CHEMISTRY OF THE ENZYMES 11
Vegetable lipases are obtained from oily seeds, especially
of Ricinus. The first work on these lipases was carried out
by Reynolds Green (Proc. Roy. Soc., 1890, 48, 370)
and S i g m u n d. Further contributions to the knowledge
of them are due to Cojnnstein, Hoyer and Wartenberg
(Chem. Ber., 1902, 35, 2988), Nicloux (Soc. BioL, 1904, 56,
840 and Proc. Roy. Soc., B., 1906, 77, 454), H. E. Armstrong
(Proc. Roy. Soc., B., 1905, 76, 606) and others. A very good
monograph has been written by C o n n s t e i n for the " Ergeb-
nisse der Physiologic" (1904, 3). E. R o u g e (Centralbl. f.
Bakt., 1907, II, 18, 403) gives a resume of the literature deal-
ing more particularly with vegetable lipases.
Ricinus-lipase is only active in relatively strongly acid solu-
tion, and in the seeds, it is activated by the lactic acid present
(Hoyer, H., 1906, 50, 414). According to Braun and
Behrend (Chem. Ber., 1903, 36, 1142, 1900), the seeds of
Abrus precatorius, which are nearly related to Ricinus
seeds, decompose fats in neutral solution, but the effect is
comparatively slight. Characteristic of the Ricinus-enzyme, as
of most true lipases, is its insolubility in water; it is hence nec-
essary to bring the pressed mass, remaining after the removal
of the oil from the seeds, into intimate contact with the fat-
emulsion.
As to the other sources of plant lipases, mention may be made
of the fungi, both higher and also lower, like P e n i c i 1 1 i u m
(Gerard, C. R., 1897, 124, 370; Camus, Soc. BioL, 1897,
49, 192), Aspergillus niger (Camus, loc. cit.) and
especially yeast (Delbriick). Lipases have also been detected
in numerous bacteria; they cause the rancidity of butter and other
natural fats (for the literature see Fuhrmann, Bakterien-
enzyme, Jena, 1907).
Butyrases. Against the extended application of the
esters of the lower fatty acids and the monoglycerides to the study
of the lipases, objection has often been raised. In particular,
A r t h u s (Soc. BioL, 1902, 53, 381) and also D o y o n and
M o r e 1 (C. R., 1902, 134, 1001 and 1254) have pointed out that
H a n r i o t 's experiments with monobutyrin (Soc. BioL, 1896,
48, 925; C.R., 1896, 123, 753) which challenge criticism in many
directions, give no information as to the presence and action of
12 GENERAL CHEMISTRY OF THE ENZYMES
the true lipases. A distinction must therefore be drawn between
the lipases and esterases (buty rases). The latter enzymes,
which occur abundantly in many juices and organs (blood-serum,
liver, kidneys) decompose not only the monovalent alkyl and the
glyceryl esters of the lower fatty acids, but also amyl salicylate
(H. Chanoz and H. Doyon, Soc. BioL, 1900, 52, 116, 717)
and similar compounds. Schmiedeberg's histozyme
is perhaps identical with these enzymes.
D a k i n has effected asymmetric ester-decompositions by
means of li ver-esterases ; these will be considered in Chapter
VIII. Detailed studies on the same enzymes are due to K as tie,
Loevenhart and E 1 v o v e , whose quantitative measure-
ments will be referred to in the third chapter. They give the
following method for the
Preparation of li ver-esterases. The macerated liver
(10 grms.) is extracted with water (100 c.c.) and the extract
filtered through a linen cloth. Twenty c.c. of this extract are
diluted with 72 c.c. of water and 8 c.c. of a 0-OlN-solution of
hydrochloric acid added. When this mixture is heated to 40,
a heavy precipitate of protein separates and, on filtering, a clear
golden-yellow solution is obtained.
The action of pancreas-lipase is participated in by a co-
enzyme (R. Magnus, H., 1904, 42, 149), which is stable at a
boiling temperature and the essential constituents of which are
alkali salts of the bile acids (Magnus, H., 1906, 48, 376; see
also Chapter V).
Among the esterases must also be classed
Chlorophyllase. This very interesting enzyme, which
was discovered and described by Willstatter and S t o 1 1
(Lieb. Ann., 1911, 378, 18), accompanies chlorophyll and is
wide-spread in its occurrence. The reaction which it produces
is an alcoholysis.
Chlorophyll contains three carboxyl groups, one of which is
apparently free, and the others esterified with a methyl and a
phytyl (from phytol) group respectively. Only the latter reacts
with the alcohol, and then only under the influence of the enzyme,
the phytoxyl group being replaced by ethoxyl.
" In its action, chlorophyllase cannot be replaced by other
esterases. On the other hand, for the enzyme found in the
leaves, chlorophyll is a specific substrate. With phaeophytin
SPECIAL CHEMISTRY OF THE ENZYMES 13
this enzyme does not react so well and with an ordinary wax no
reaction takes place."
ENZYMES OF THE HIGHER CARBOHYDRATES
C y t a s e s . Whether true cellulose is decomposed enzym-
ically is still uncertain. More is known, especially from the
investigations of Mac Gil la wry, of H. T. Brown and
Morris (Journ. Chem. Soc., 1890, 57, 497), of Reynolds
Green (Annals of Bot., 1893, 7, 93) and, recently, of S c h e 1-
lenberg (Flora, 1908, 98, 257), of the action of cytase, the
substrates of which are the hemicelluloses and their reaction-
products, mannose and galactose; also pentose-polysaccharides
the pentosans are decomposed, but it is not yet known
whether the hydrolysis is complete. Such enzymes occur in the
intestines of herbivorous animals, in wood-destroying fungi
(C z a p e k , Lotos, 1898, 46, 235; Schorstein, Centrabl. f.
Bakt., 1902, II, 9, 446) and in bacteria. Also hydrocelluloses,
which, so far as is known, are nearly allied to the hemicelluloses,
are decomposed by cytases. To the same group of enzymes
belong caroubinase, which dissolves the caroubin in the
carob (C e r a t o n i a s i 1 i q u a) (E f f r o n t , C. R., 1897, 125,
116) and the enzyme described by Bourquelot and H e r i s-
s e y (C. R., 1899, 129, 228, 391, 614; 1900, 130, 42, 340, 741) as
s e m i n a s e , which occurs in lucerne, T r i g o n e 1 1 a and
other plants. As well as in plants, cellulases or cytases are found
in the animal body, especially in the intestines of graminivorous
animals (H. T. B r o w n , Journ, Chem. Soc., 1892, 61, 352), in
snails (Biedermann and M o r i t z , Pfltig. Arch., 1898, 73,
236) and in fishes (K n a u t h e) . Experiments by the author
(Zeitschr. f. angew. Chem., 1912, 25, 46) indicate the occurrence
in Merulius lacrimans (dry-rot fungus) of an enzyme
which decomposes cellulose-dextrin.
A m y 1 a s e s . These enzymes more accurately termed
malto-amylases include all those which break down starch and
glycogen forming maltose. Very little that is definite can
be asserted with regard to the individuality of the amylases.
After the view had been expressed by Brown and Mor-
r i s and by Reynolds Green that at least two enzymes
14 GENERAL CHEMISTRY OF THE ENZYMES
showing different biological relations are to be distinguished
(diastase of translocation and diastase of secretion), Maquenne
(C. R., 1906, 142, 124, 1059, 1387) carried out a series of notable
investigations which explained the saccharification from a
chemical point of view. Starch consists, indeed, of 80-85%
of amylose and 15-20% of amylopectin. Amylase attacks
dissolved amylose and also " soluble starch" very readily, but acts
on amylopectin (starch-paste) very slowly. Amylopectinase, on
the contrary, saccharifies amylopectin (starch-paste) with great
ease. The diastases of the very varying organs of plants and
animals must contain both of these diastatic enzymes. That the
saccharifying and liquefying actions of the " diastases " are often
parallel has been shown more especially by an investigation
made by Chrzascz (Zeitschr. f. Spiritusind., 1908, 31, 52).
Another recent noteworthy contribution on vegetable diastatic
enzymes is due to Butkewitsch (Biochem. Z., 1908, 10,
314). Further investigations in this direction are, however,
desirable, as well as a more detailed study of the diastatic
decomposition of glycogen.
Also the individuality of the true amylases, apart from amylo-
pectin, is doubtful, if we consider the far-reaching decomposition
necessary in order to pass from the highly-condensed starch
through the dextrins to maltose. After Miss T e b b (Journ. of
Physiol., 1894,15,421), Brown and Morris, Rohmann
(Chem. Ber., 1894, 27, 3251), Hamburger (Pflug. Arch.,
1895, 60, 543) and B e i j e r i n c k (Centralbl. f. Bakt., 1895,
II, 1, 221) had detected, in the mixture of enzymes which
saccharifies starch, an enzyme which effects the transformation
starch-to-maltose and another which further breaks down the
maltose to glucose, W i j s m a n (Rec. Trav. Chim. Pays-Bas,
1890, 9, 1), Pottevin and others assumed that the reactions
starch-to-dextrin and dextrin-to-maltose are also effected by
separate enzymes, 1 and recently Ascoli and Bonfanti
(H., 1904, 43, 156) speak of several amylases.
Occurrence. Enzymes which attack glycogen and starch are,
as was discovered by Claude Bernard, widespread in the animal
1 L i n t n e r ' 8 statement that isomaltose is formed during the
diastatic conversion of starch must, after subsequent work, more especially
by Ling and Baker (Journ. Chem. Soc., 1895, 67, 702) and by
Brown and Morris (ibid., 709), be regarded as disproved.
SPECIAL CHEMISTRY OF THE ENZYMES 15
kingdom. Their occurrence in blood-serum which was detected by this
investigator has been examined more closely by Bial, Pick, and
A s c o 1 i and B o n f a n t i (H., 1904, 43, 156). According to N a s s e
such an enzyme occurs in muscle-plasma, and this was confirmed by
Halliburton (Journ. of Physiol., 1887, 8, 182). Carlson and
Luckhardt (Amer. Journ. of Physiol., 1908, 23, 148) have found
amylase in numerous other body-liquids. The fact that saliva dissolves
starch has been known much longer, and this property was ascribed by
L e u c h s in 1831 to an enzyme, ptyalin. Foster and von
W i 1 1 i c h also found amylolytic enzymes in a great number of organs.
In addition to the liver and pancreas, the muscles are especially rich in
an enzyme which attacks glycogen; and Mendel and S a i k i (Amer.
Journ. of Physiol., 1908, 21, 64), by experiments on the pig, found that
this is the case in the embryo state, while other organs of different
animals usually become richer in enzyme as development proceeds
(P u g 1 i e s e and others). According to Roger (Soc. Biol., 1908,
64, 1137), an amylase occurs also in hens' eggs (white and yolk) and is
partially soluble in ether.
Of no less biological importance than animal amylases are
those of plants. The starch-decomposing action of germinated
barley was discovered by Kirchoff as long ago as 1814.
An enzyme-preparation was made in 1833 by P a y e n and
Persoz and was named " diastase"; this term is also much
used at the present time, but, in the interests of as rational as
possible a nomenclature, it should be replaced by the terms
amylase, amylopectinase, etc. 1 The whole of the saccharifying
enzyme-preparation together, that is, the mixture of amylase,
dextrinase, etc., may meanwhile be called diastase. 2
In accordance with the function of the diastases of effecting the
metabolism of the polysaccharides, these enzymes are widespread in
all parts of plants, and are especially abundant in shoots and leaves, par-
ticularly with the Leguminosse and grasses (Brown and Morris,
1 W i j s m a n ' s nomenclature is by no means an acceptable one. If
two enzymes really take part in the formation of maltose, they should be
distinguished as amylase and dextrinase.
2 It is most desirable that the use of the term "diastases" as a generic
name for enzymes should be abolished from the French literature. For
this use of the term there is, indeed, a historical explanation, but there is
no justification for its continuance, especially as it often gives rise to mis-
understanding.
16 GENERAL CHEMISTRY OF THE ENZYMES
Journ. Chem. Soc., 1893, 63, 604). Amylases have further been detected
in potatoes and the sugar-beet; also in germinating, starch-containing
pollen-grains (Reynolds Green), in the bark of many plants
(Butkewitsch, Biochem. Z., 1908, 10, 314), in the sap, in many
higher and lower fungi, especially in several species of yeast here the
diastase may be related to the glycogen-content and finally in bacteria.
Special mention must be made of the so-called t a k a - d i-
a s t a s e, the mixture of saccharifying enzymes from A s p e r -
gillus o r y z a e , a fungus contained in koji-yeast. It sac-
charifies starch and indeed, according to Stone and Wright,
and T a k a m i n e , more energetically than does malt-diastase.
Experiments with the view of preparing it in a pure state were
made byWroblewski (Chem. Ber., 1898, 31, 1130).
Preparation. Of the animal amylases, the ptyalin of
saliva is the best suited for preparation. According to J.
C o h n h e i m (Virch. Arch., 1865, 28, 241) the saliva is pre-
cipitated with freshly-prepared calcium phosphate. From the
precipitate the ptyalin is dissolved by means of water, and the
aqueous solution precipitated with alcohol. Another method
is given by K r a w k o w (J. Russ. Phys. Chem. Soc., 1887, 19,
387), who precipitates saliva-diastase by ammonium sulphate.
Direct precipitation of the saliva with alcohol also yields a sac-
charifying preparation.
C o h n h e i m has found saliva-diastase to be free from
protein, but he does not state on what absolute quantities of
the preparation the tests were made.
Von Wittich takes up pancreas-diastase in anhydrous
glycerol.
Larger and purer yields of amylase are obtained from vege-
table material. From malt Lintner prepared diastase as fol-
lows: one part of green malt (or air-dried malt) was extracted
for 24 hours with 2-4: parts of 20 J o alcohol, the extract being
precipitated with 2-5 times its volume of absolute alcohol and
the precipitate washed with absolute alcohol and ether.
Loew (Pflug. Arch., 1882, 27, 203; 1885, 36, 170) steeps ger-
minated barley in a little water and then extracts with 4%
alcohol. He precipitates the extract with lead acetate, suspends
the precipitate in water, removes the lead from the solution by
means of hydrogen sulphide, and finally precipitates the diastase
with a mixture of alcohol and ether.
SPECIAL CHEMISTRY OF THE ENZYMES 17
s b o r n e and Campbell (Journ. Amer. Chem. Soc.,
1896, 18, 536) and also Wroblewski (H., 1897, 24, 73) salt
out the diastase with ammonium sulphate.
E f f r o n t (Enzymes and their Applications, London and
New York, 1902, pp. 104 e t s e q.) proposes the extraction of malt
with water and, in order to diminish the quantity of the extractive
material possessing no diastatic action, he induces alcoholic
fermentation in the infusion by yeast previously rendered very
poor in nitrogen. E f f r o n t states that the fermentation
destroys a large quantity of carbohydrates, removes considera-
ble quantities of proteins and salts, and leaves the diastase
absolutely untouched.
Wroblewski (Chem. Ber., 1897, 30, 2289) gives the
following method:
Finely-ground malt is extracted, first with 70%, and then'
twice with 45% alcohol. Sufficient strong alcohol is added to
the last two extracts to bring the alcohol-content to 70%. The
precipitate formed is washed with absolute alcohol and ether and
dried in a vacuum.
Like O s b o r n e and Campbell (loc. ei t.), Wrob-
lewski (Chem. Ber., 1898, 31, 1130) effected further purifica-
tion by salting out with ammonium sulphate.
Wroblewski considered that, as a result of these exper-
iments, he had shown with certainty that diastase is a protein
substance nearly allied to the albumoses, whilst, according
to T. B. O s b o r n e (Chem. Ber., 1898, 31, 254), diastase is a
protein-like substance or "a compound of an albumin with a
proteose." For his most active preparation he gives the fol-
lowing composition (calculated for ash-free substance): C, 52-5;
H, 6-72; S, 1 -90; N, 16-10%. The solution gives the character-
istic reactions of the proteins. Wroblewski 's purest
preparation had a nitrogen-content of 16-5%.
If, however, the recent researches of S. Frankel and
Hamburg (Hofm. Beitr., 1906, 8, 389) should be confirmed,
diastase contains neither protein-groups nor reducing sugars.
The non-enzymic substances were precipitated with lead acetate,
the solution sterilised by filtration and further purified by fer-
mentation with yeast rendered poor in nitrogen and subsequent
filtration through a Pukall filter. After drying in a vacuum, the
syrupy liquid yields a powder free from fermentable and reducing
18 GENERAL CHEMISTRY OF THE ENZYMES
sugars and from protein. It represents a very active substance
which does not give the biuret reaction or reduce F e h 1 i n g ' s
solution but shows a faint M i 1 1 o n ' s reaction; it also gives
M o 1 i s c h ' s reaction and the pentose reaction slightly. When
dialysed into spring-water, the dissolved diastases are separated
into two principal groups: the saccharifying diastases diffuse
through the membrane, whilst the liquefying ones remain.
A distinct advance seems to have been made by H. C. S h e r-
m a n and M. D. Schlesinger (Journ. Amer. Chem. Soc.,
1911, 33, 1195). They found that pancreas-diastase keeps well
in 50% alcohol, and they purified such a solution by dialysis.
A very active preparation (saccharifying power, 5000 on Lintner's
scale at 40) contained 53-0% C, 6-6% H and 15-6% N.
Inulinase. In addition to hemicelluloses, starch and
glycogen, another carbohydrate, inulin, also occurs as a reserve
material. The enzyme, inulase or inulinase, accompanying this,
decomposes inulin into its simplest com-
ponent, fructose. Starch is not attacked by inulinase.
ReynoldsGreen (Annals of Bot., 1888, 1, 223) discovered this
enzyme, which occurs in many of the Compositse, in the tubers of H e 1 i -
anthus tuberosus (artichoke). Bourquelot (Bull. Soc.
Mycol., 1893, 9, 230; 1894, 10, 49) detected inulinase in Aspergillus
n i g e r and isolated it from the mycelium of this fungus. The enzyme
appears to be widespread in the Eumycetes; Dean (Bot. Gaz., 1903,
35, 24) found it also in Penicillium glaucum. The best
medium for its action is 0-001% hydrochloric acid.
The enzymes which decompose trisaccharides, such
as melicitase, etc., have not yet been sufficiently individu-
alised.
THE ENZYMES OF THE GLUCOSIDES AND DISACCHARIDES
E. Fischer has stated that the disaccharides may be
regarded as glucosides and can be classified with these according
as they contain the glucose in the a- or (3-form. From the results
of E. F. Armstrong (Journ. Chem. Soc., 1903, 83, 1305)
and C. S. H u d s o n (Journ. Amer. Chem. Soc., 1909, 31, 1242),
it appears that a-glucosides, which were originally characterised
SPECIAL CHEMISTRY OF THE ENZYMES 19
by the fact that they are hydrolysed by a constituent of yeast-
extract, generally yield a-glucose.
p-Glucosides are hydrolysed by a component of almond
extract, and from such a glucoside Hudson obtained ^-glucose.
a-G lucosidase; maltase. As a-glucosidases will be
designated those enzymes which hydrolyse a-glucosides specifically.
They are therefore limited, on the one hand, by ^-glucosidase,
which acts only on ^-glucosides, and, on the other, by lactase and
invertase, which accelerate the hydrolysis of the ^-galactosides
or fructosides.
Occurrence. In both the animal and vegetable kingdoms,
maltase almost always accompanies the diastases, from which it cannot
often be separated. Thus, this enzyme has been found in blood and in
serum (G 1 e y and B o u r q u e 1 o t , Soc. BioL, 1895, 47, 247; Ham-
burger, Pfliig. Arch., 1895, 60, 543; Tebb, Journ. of Physiol.,
1894, 15, 421 ; Fischer and N i e b e 1 , Sitzungsber. K. Akad. Berlin,
1896, 73) ; also in many tissues (Shore and Tebb, Journ. of Physiol.,
1892, 13, 19), especially in the liver, intestines and pancreas. As Miss
Tebb found, the maltase can be extracted from these organs in
both the fresh and dried states by means of chloroform water; the oppo-
site statement of Brown and Heron (Proc. Roy. Soc., 1880, 30,
393) is thus contradicted.
The maltases occur in great abundance in the vegetable kingdom.
Their occurrence in m a 1 1 and in y e a s t must -be especially mentioned.
These two maltases do not appear to be absolutely identical (Fischer,
H., 1894, 26, 74). The lactic acid yeasts and also kephir-grains always
contain lactases in place of maltases. Saccharomyces
Marxianus contains no maltase, but only invertase (E. C.
Hansen; E.Fischer and P. Lindner, Chem. Ber., 1895,
28, 984). Excepting in this case, it can be said that invertase always
accompanies maltases in yeast-extracts; according to Beijerinck
and to E. Fischer and P. Lindner (Chem. Ber., 1895, 28, 984),
invertase is lacking in Saccharomyces octosporus. In
Saccharomyces apiculatus neither maltase nor invertase
is found.
Preparation. As starting material for the preparation
of the yeast-enzymes, maltase and invertase, it is best to employ
pure cultures. The yeast is used as fresh as possible and is
well pressed and, according to E. Fischer (H., 1898, 26, 74),
ground and well shaken two or three times with the ten-fold
quantity of water. Removal of the mother-liquor is effected
20 GENERAL CHEMISTRY OF THE ENZYMES
most suitably by a P u k a 1 1 flask-filter. The yeast is pumped
as dry as possible and is then spread out in as thin a layer as
possible on poro.us tiles and dried in the air at the ordinary tem-
perature. Under these conditions it gradually shrivels up and
assumes a dark-grey colour. After 1-2 days, it is powdered as
finely as possible and left to dry in the air until it forms a loose
powder. The final drying may also be carried out at 30-35.
In this state the yeast can be kept for months without the maltase
being destroyed. When the enzyme is to be used, the yeast
is extracted with 10-15 times the quantity of water for 12-20
hours at 30-35, with occasional shaking, the liquid being then
filtered through paper. Toluene serves as a suitable antiseptic.
R 6 h m a n n (Chem. Ber., 1894, 27, 3251) heated the yeast
for an hour at 105^110 before extraction, but this procedure,
according to Croft Hill, is not to be recommended. The
latter investigator gives the following method (Journ. Chem.
Soc., 1898, 73, 636) : Good, pressed bottom-yeast is washed three
times with distilled water by decantation, collected on a covered
filter, spread out on a porous support and dried in a vacuum
over sulphuric acid. The yeast dries in about two days and is
then powdered and sieved through a cloth, a yellowish-white
powder being obtained. This powder is then spread out on a
double layer of fine tulle over the mouth of a glass vessel in an
oven previously heated to 40. In successive quarters of an
hour, the temperature is raised to 60, 70, 90 and 100, the
last being maintained for 15 minutes, after which the prepara-
tion is allowed to cool in a desiccator. The yeast is then weighed,
ground in a mortar with 10 times its weight of 0-1% sodium
hydroxide solution, filled into flasks with addition of toluene
and left at the room-temperature for 3 days. The extract is
now filtered, first through paper and afterwards through a Cham-
berland filter. If 1 c.c. of this fresh extract is added to 20 c.c.
of 2% maltose solution at 30, about 20% of the, sugar is
hydrolysed in 40 minutes.
Different yeasts appear to contain widely varying proportions
of maltase, so that not every species of yeast is suitable for the
preparation of maltase.
Trehalase. Trehalose, a disaccharide composed of two
molecules of glucose, is hydrolysed by an enzyme which was
SPECIAL CHEMISTRY OF THE ENZYMES 21
found by Bourquelot in Aspergillus and other fungi,
by Fischer (H., 1898, 26, 79) in the diastase of green malt
and in yeasts of the Frohberg type, and by Kalanthar (H.,
1898, 26, 97) in various other yeasts. Whether the enzyme,
which acts best with a very slight concentration of hydrogen-ions,
really differs from maltase is not yet established ; Bourquelot
(Soc. Biol., 1895, 47, 515) regards it as a separate enzyme, but
Fischer is not in agreement with this opinion.
The ^-glucosidases hydrolyse (3-methylglucoside and
also most of the natural glucosides, which are on this account
placed in the ^-series. 1
It has been shown recently by Hudson and Paine (Journ.
Amer. Chem. Soc., 1909, 31, 1242) that the hydrolysis of a typical
natural glucoside, salicin, under the influence of emulsin, yields
^-glucose. It is better here not to apply the ordinary principles
of nomenclature, but to name the glucosides according to the form
of glucose to which they give rise and the enzymes so that they
refer to the glucosides characterised in this way.
Of special glucosido-glucoses hydrolysed by emulsin, mention
may be made of:
Isomaltose (E. Fischer, Chem. Ber., 1895, 28, 3024;
compare also E. F. Armstrong, Proc. Roy. Soc., B, 1905,
76, 592).
Gentiobiose (Bourquelot and H e r i s s e y, C.R.,
1902, 135, 399).
Cellose or Cellobiose (E. Fischer and G.
Z e m p 1 e n, Lieb. Ann., 1909, 365, 1).
C e 1 1 a s e . From the results of fractional filtration of
extracts of Aspergillus niger by Holderer's method,
G. B e r t r a n d and M. H o 1 d e r e r (C. R., 1909, 149, 1385
and 1910, 150, 230) assume the existence of an enzyme which
differs from (3-glucosidase and acts specifically on cellose. An
enzyme extracted from apricot seeds hydrolyses only cellose and
not trehalose.
x The opportunity must not be neglected of pointing out that E.
Fischer, who introduced this method of reasoning, has issued a warning
that it must not be regarded as absolutely safe (Lieb. Ann., 1909, 365, 1):
"For it might be assumed that one and the same enzyme hydrolyses both
the alcohol-glucosides and the glucosido-glucoses. But as long as no pure
enzyme is obtained, complicated mixtures like emulsin or yeast-extract
having to be used, no proof of this exists."
22 GENERAL CHEMISTRY OF THE ENZYMES
Cellase occurs, together with other enzymes, in apricot kernels,
almonds, barley, and the mycelium of Aspergillus.
^-Methylgalacto sides are also hydrolysed by
the enzymes of the almond (E. Fischer, Chem. Ber., 1895,
28, 1429) and, since Fischer found that this enzyme likewise
effects the hydrolysis of lactose, the latter is to be regarded as a
p-galactoside. But we shall not go far wrong if we assume,
with B o u r q u e 1 o t and H e r i s s e y (C. R., 1903, 137, 56)
and with E. Fischer, that this latter action is not brought
about by the same enzyme as hydrolyses (S-glucosides, but
depends on the presence of an enzyme which decomposes (3-
galactosides and is hence either identical with, or nearly related
to, the lactase occurring in lactose-yeasts. The view that kephir-
lactase and emulsin- lactase are different, has been advanced by
H. E. and E. F. Armstrong and E. H o r t o n (Proc. Roy.
Soc., B, 1908, 80, 321). They assume that the one enzyme is a
galacto-lactase and the other a gluco-lactase, the first being taken
up by the galactose-residue and the latter by the glucose-residue
of milk-sugar.
A preparation which is biologically purer is obtained, accord-
ing to Pottevin (Ann. Inst. Pasteur, 1903, 17, 31), from
Aspergillus niger, Aspergillu s-emulsin hydrolys-
ing only ^-glucosides and not (3-galactosides or milk-sugar.
Amygdalin is resolved by the enzymes of the almond
into glucose, benzaldehyde and hydrocyanic acid, and mandelo-
nitrile glucoside, formed by the action of yeast-enzymes on
amygdalin, is also hydrolysed by emulsin into its simplest com-
ponents (E. Fischer, Chem. Ber., 1895, 28, 1508). The
glucosido-glucose contained in amygdalin is not identical with
maltose, since, on the one hand, maltose is not liberated by the
action of emulsin (C a 1 d w e 1 1 and Courtauld, Journ.
Chem. Soc., 1907, 91, 666; R o s e n t h a 1 e r, Arch, der Pharm.,
1908, 245, 684) and, on the other, maltose has no retarding action
on the hydrolysis by emulsin (A u 1 d , Journ. Chem. Soc., 1908,
93, 1276).
It is, therefore, best to indicate by "emulsin" the mix-
ture of glucoside-resolving enzymes and to characterise the prepara-
tion according to its origin, a distinction being drawn between
Aspergillus -emulsin, almond -emulsin, etc.
SPECIAL CHEMISTKY OF THE ENZYMES 23
For the enzyme-constituents, rational names are then chosen,
the results of H. E. and E. F. Armstrong and H o r t o n ,
C a 1 d w e 1 1 and Courtauld, and Rosenthaler
indicating at least four components of almond-emulsin, namely:
(1) Amygdalase, characterised by the reaction:
C 6 H 5 CH(CN) - O - CoHioO 4 O
Amygdalin
= C 6 H 5 CH(CN)
Mandelonitrile glucoside
(2) A (3-glucosidase, which acts on g-glucosides, among
them mandelonitrile glucoside:
C 6 H 5 CH(CN) O C 6 HiiO5+H 2 O
Mandeloaitrile glucoside
= C 6 H 5 CH(CN) - OH+C 6 Hi 2 6 .
Mandelonitrile Glucose
(3) A hydroxynitrilase:
C 6 H 5 -CH(CN)-OH = C 6 H 5 -CHO + HCN.
Mandelonitrile Benzaldehyde Hydrocyanic acid
In addition to these three substances which take part in the
decomposition of amygdalin, the existence in
emulsin must be assumed of:
(4) An enzyme which resolves milk-sugar, i.e., a lactase
(which Armstrong terms gluco-lactase).
According to N e u b e r g (Ergeb. der Physiol., 1904, 3, 446),
the conjugated glycuronic acids are also decomposed by emulsin.
The synthetic action of certain components of emulsin is
treated more in detail in Chapter VII.
Occurrence. 1. Phanerogams. As well as in almonds, emul-
sin is found in the leaves of Prunus laurocerasus (where
laurocerasin likewise occurs), in the seeds of many of the Rosacese, in
manihot (Guignard) and in extracts of numerous plants, such
asMonotropa, Polygala [Bourquelot, Journ. de Pharm.
et Chim., 1904, (5), 30, 433], Malus communis, Hedera
helix, etc. (H e r i s s e y , Thesis, " Recherches sur I'Emulsine,"
Paris, 1899).
2. Cryptogams. It was discovered simultaneously in P e n i c i 1 -
Hum glaucum by Gerard (Soc. Biol., 1893, 45, 651) and in
Aspergillus niger by Bourquelot, who also detected
enzymes capable of attacking glucosides in many other fungi, especially
24 GENERAL CHEMISTRY OF THE ENZYMES
in the Polyporus species found in wood. H 6 r i s s e y has found
emulsin in many lichens and mosses. Bourquelot has recently
observed hydrolysis of otherwise unknown glucosides by emulsin (Arch,
der Pharm., 1907, 245, 172). Fermi and Montesano (Centralbl.
f. Bakt., 1894, I, 15, 722), Gerard (Soc. Biol., 1896, 48, 44) and
Twort (Proc. Roy. Soc., B, 1907, 79, 329) have detected emulsin in
bacteria, 27 species out of 44 examined having the property of hydro-
lysing glucosides.
Worthy of note is the observation of Henry and A u 1 d (Proc.
Roy. Soc., B, 1905, 76, 568) that many yeasts also exhibit "emulsin"
action.
Animal enzymes closely related to emulsin were found by G e* r a r d
(Soc. Biol., 1896, 48, 44) in the kidneys of the horse and rabbit. In
molluscs B i e r r y and G i a j a (Soc. Biol., 1906, 58, 1038) found
enzymes capable of hydrolysing populin and phloridzin; extracts of
cross-spiders also resolve amygdalin (Robert and W. Fischer).
Decompositions of glucosides by animal extracts were also
noted byGonnermann (Pfliig. Arch., 1904, 103, 225; 1906,
113, 168) and, more recently by K o b e r t ; according to the
latter, extract of placenta hydrolyses amygdalin, arbutin, salicin
and helicin.
Preparation: Herissey (Thesis; compare Bour-
quelot, Arch, der Pharm., 1907, 245, 172).
One hundred grams of sweet almonds are steeped for about
a minute in boiling water and, after draining, are carefully peeled.
They are then ground as finely as possible in a mortar without
water, the product obtained being macerated at room-temperature
with 200 c.c. of a mixture of equal parts of distilled water and
water saturated with chloroform. After about 24 hours, the
mass is strained and pressed through a damp cloth. This pro-
cedure yields 150-160 c.c. of liquid, to which 10 drops of glacial
acetic acid are added to precipitate the casein. The clear nitrate
(120-130 c.c.) is added to 500 c.c. of 95% alcohol, the precipitate
thus formed being collected on a smooth filter and, after draining,
treated with a mixture of equal volumes of alcohol and ether.
After drying in a vacuum over sulphuric acid, horny, transparent
plates are obtained and, when ground, these give an almost
white powder.
Invertase ( = a-Fructosidase) . Invertin or sucrase owes
its name to its property of converting cane-sugar into invert-
SPECIAL CHEMISTRY OF THE ENZYMES 25
sugar ( = glucose + fructose). The sphere of action of invertase
extends to all synthetic a-methylfructosides; on the other hand,,
^-methylfructoside, a-glucosides and a-galactosides resist its
action. Apart from these synthetic glucosides, gentianose a
trisaccharide from Gentiana lute a is also attacked by
invertase, which resolves it into fructose and g e n t i o -
b i o s e. Further, melitriose (raffinose), a trisaccharide occurring
in the sugar-beet, is decomposed by invertase, yielding fruc-
tose and m e 1 i b i o s e .
Occurrence. The distribution of invertase in the yeasts is as
well known as important; in the majority of cases, it is accompanied by
maltase and, in the lactose-yeasts, by lactase. Invertase occurs alone
in only few yeasts, among them being Saccharomyces Marxi-
anus (Fischer, H., 1898, 26, 75). S. apiculatus contains no
invertase.
Of other lower organisms which contain invertase, mention may be
made of Fusarium, Streptococcus (Leuconostoc)
mesenterioides, Aspergillus oryzae and M o n i 1 i a
Candida. A long series of invertase-containing bacteria is also
known, especially owing to the investigations of Fermi and M o n -
t e s a n o (Centralbl. f. Bakt., 1895, 1, 482, 542).
With the higher plants, invertase is found especially in the green
leaves and young shoots (K a s 1 1 e and Clark, Amer. Chem. Journ.,
1903, 30, 422), in ripe bananas, in mulberries, in resting and, still more
abundantly, germinating pollen, and in wheat and barley embryos; in
the crown leaves of Robinia viscosa and pseudacacia,
Papaver rhoeas, Rosa species and Bougainvillea
bracts. Also in fruits, such as dates, which, when unripe, contain the
invertase as an insoluble endo-enzyme, this only becoming soluble when
the fruit ripens (V i n s o n , Journ. Amer. Chem. Soc., 1908, 30, 1005).
Invertase is found in human intestinal juice, even immediately after
birth (Kriiger), but not in that of cattle (F i s c h e r and Niebel).
Robertson (Edinburgh Med. J., 1894) found it in almost all organs.
Preparation. If yeast-cells are to be extracted with
water, it is first of all necessary to kill them, either by treatment
for a short time with ether or for a longer time with alcohol
(Osborne, H., 1899, 28, 399), etc., or by heating the dry yeast
at 105 (S a 1 k o w s k i) or by plasmolysis (I s s a e w).
After careful dehydration in a vacuum and subsequent heat-
ing, yeast may yield about 12% of its invertase on extraction
(Euler and Kullberg, H., 1911, 73, 94).
26 GENERAL CHEMISTRY OF THE ENZYMES
Living yeast also gives up invertase to the surrounding liquid
water or sugar solution but in relatively small quantities.
Presumably it is more especially the old cells from which the
invertase can be extracted directly.
' S u 1 1 i v a n and T o m p s o n left top-fermentation beer-yeast
for a month at 15 so that it became completely liquid; [it was then
pressed and the clear solution obtained precipitated with 47% alcohol.
The precipitate, after deposition, was dissolved in water and sufficient
alcohol added to bring its content in the liquid up to 28%; in this
solution the invertase remained dissolved, whilst the majority of the
protein substances separated. On raising the alcoholic content of the
filtered liquid to 47%, a precipitate was again formed and this was
washed with absolute alcohol and dried in a vacuum. The preparation
thus obtained was very active, but still not quite pure; it contained
about 5% of ash (magnesium and potassium phosphates), which the
English investigators regarded as an admixture. Their further puri-
fication experiments showed, as had already been indicated by the work
of Osborne (H., 1899, 28, 399) and of Salkowski, that invertase
is not a protein. Even the purest preparation contains, besides phos-
phoric acid, a carbohydrate. Wroblewski regards this as an
impurity, as also does shim a (H., 1902, 36, 42); the latter came
to the conclusion that yeast-gum consists of a substance which contains
d-mannose and a methyl-pentosan giving fucose on hydrolysis.
H a f n e r , who carried out a thorough examination of pure invertin
(H., 1904, 42, 1), regards it as by no means disproved that this peculiar
carbohydrate always adhering to invertin is an integral constituent of
the enzyme. A large part of the phosphorus of invertin preparations is
combined organically. The specific activity of the enzyme is not con-
nected with the presence of large nitrogenous groups like the albumoses
or peptones; the absence of peptones is also supported by the failure
of the biuret action. The nitrogen is probably present in the form of
smaller groups, which have, however, not been investigated.
Very active invertase solutions are also obtainable from
pure cultures ofAspergillus niger.
The best method for preparing invertase in as pure a form
as possible consists in removing protein by lead acetate and
kaolin, and in subsequently applying the following diffusion
process (E u 1 e r and Kullberg, H., 1911, 73, 335):
Bottom fermentation beer-yeast is subjected to autolysis for
3-10 days and then precipitated, as Hudson recommended,
with excess of lead acetate; the whole mass is then ground with
SPECIAL CHEMISTRY OF THE ENZYMES 27
kaolin and the liquid pumped off. The lead is precipitated by
means of hydrogen sulphide and the nitrate ground several
times with kaolin and a little charcoal and filtered. By means of
a collodion dialysor, the enzyme solution is freed from the impuri-
ties, which diffuse rapidly, and is finally precipitated with alcohol.
Two kilos of pressed yeast, treated in this way, give about
8 grms. of a pure white powder, which is freed from further
quantities of nitrogenous impurities by dialysis or diffusion.
The preparations are protein-free, and the content of
nitrogen varies between 0-3 and 2%. The molecular
weight exceeds 25,000.
The activity is d=0 = 10 minutes, i.e., 0-05 grm. of
the preparation, dissolved in 25 c.c. of an 8% cane-sugar solu-
tion, reduces the rotation of the cane-sugar to zero in 10 minutes
at a temperature of 20.
The sensitiveness of invertase to temperature (cf. Chapter
V) is such that the activity of an invertase solution is diminished
by one-half by heating for 30 minutes at 63 (H . E u 1 e r and
af Ugglas). Its optimum temperature is 53-56. The
influence of the acidity of the solution on the velocity of inver-
sion has been investigated in detail by Sorensen and by
Hudson (cf. Chapter IV).
A poisonous action towards invertase is shown by mercury
salts and potassium cyanide (and nearly all salts with an
alkaline reaction); hydrocyanic acid and chloroform are less
harmful, whilst thymol and toluene are without effect.
OTHER ENZYMES WHICH HYDROLYSE GLUCOSIDES
The number of different individuals in this group seems to
be very large, but the sphere of action and specificity of the
enzymes described are usually very indefinite. Closely related
to g- glucosidase is:
Gaultherase or betulase, the specific action of
which consists in hydrolysing the glucoside of methyl salicylate
(gaultherin) . Neither salicin nor amygdalin is attacked by this
enzyme.
Occurrence. Exclusively in plants. It was discovered by
Schneegans (Arch, der Pharm., 1894, 232, 437) in the bark of
28 GENERAL CHEMISTRY OF THE ENZYMES
Betula lenta. At the same time Bourquelot found it in
several Polygala and Azalea species, and in Spiraea
ulmaria, Monotropa hypopitys and Gaultheria
procumbens (C. R., 1896, 123, 315; J. de Phann. et Chim.,
1896, 3, 577).
Preparation, according to Bourquelot (loc. cit.) :
Monotropa plants are ground with sand and the glucoside
removed by digesting for half an hour with 95% alcohol. The
residue, which contains the enzyme, is quickly dried with alcohol
and ether, after which the enzyme can be extracted with water.
Cf. B e i j e r i n c k (Centrabl. f. Bakt., 1899, II, 5, 325).
Schiitzenberger mentioned an enzyme which hydro-
lyses populin and also phillyrin, a glucoside occurring in the
bark of Phillyrea latifolia, but he did not investi-
gate it further.
Sigmund (Monatsh. f. Chemie, 1909, 30, 77) found an
enzyme, which decomposes salicin but does not seem to be
identical with emulsin, in certain species of S a 1 i x and
P o p u 1 u s ; he also found one which hydrolyses arbutin in
Calluna vulgaris and Vaccinium myrtillus.
W . Sigmund (Monatsh. f. Chemie, 1910, 31, 657) dis-
covered an enzyme, capable of hydrolysing sesculin, in the seed-
coats of the horse-chestnut (Aesculus hippocastanum).
It does not appear to be either an amygdalase or a lipase, but is
not yet sufficiently defined. Sigmund proposes for it the names,
salicase, arbutase and aesculase.
According to T. Weevers (Rec. Trav. bot. J^Teerland.,
1910, 8) an enzyme which hydrolyses salicin specifically occurs
in Salix purpurea and Populus monilifera, and
one that hydrolyses arbutin in Vaccinium vitis idaea
and Pinus communis.
B i e r r y and G i a j a (Soc. BioL, 1907, 62, 1117) found,
in snails and Crustacea, an enzyme which is not identical with
" emulsin " but which hydrolyses populin and phloridzin. It
remains to be shown that these enzymes are not really general
g-glucosidases.
G e a s e is the name given by Bourquelot and H e r i s -
s e y (C. R., 1905, 140, 870) to a specific enzyme from G e u m
urbanum (Herb Bennett) and r i v a 1 e which
liberates eugenol from a glucoside contained in these plants.
SPECIAL CHEMISTRY OF THE ENZYMES 29
Elaterase, from Ecballium elaterium hydro-
lyses elaterin (Berg).
Rhamnase hydrolyses xanthorhamnin, yielding, according
to G . and C h . Tanret (Bull. Soc. Chim., 1899, [iii], 21,
1065), rhamninose and rhamnetin. Rhamninose is regarded as
a trisaccharide, which can be hydrolysed into 2 mols. of rhamnose
(methylpentose) and 1 mol. of galactose. The enzyme occurs in
Rhamnus infectoria.
Besides these glucoside-enzymes, another series is known
which hydrolyse glucosides of one or the other group in a specific
manner.
M y r o s i n decomposes sinigrin or potassium myronate into
glucose, potassium hydrogen sulphate and allyl mustard oil
(allyl isothiocyanate) according to the equation:
CioH 18 OioNS 2 K = C 3 H 5 -CNS + C 6 Hi 2 6 + KHS0 4
Sinigrin Allyl mustard oil Glucose
Also other sulphur-glucosides occurring in the Cruciferse are
hydrolysed by myrosin; but, according to E. Fischer
(Chem. Ber., 1894, 27, 3483), a- and ^-glucosides are not
attacked.
Occurrence. The distribution of myrosin has been shown by
the investigations of Spatzier (Pringsheim's Jahrb. f. wiss. Bot.,
1893, 25, 39) and, especially, of G u i g n a r d (C. R., 1890, 111, 249
and 920; also Journ. de Bot., 1894, 67 and 85). It is characteristic of
the Cruciferse and certain allied families and is found also inManihot-
species. It is localised in certain cells which are rich in proteins and
are dispersed through the tissues. G u i g n a r d has isolated mechan-
ically such cells and cell-layers, e.g., the pericycle of Cheiranthus.
Roots contain the enzyme mainly in the cork, whilst, in the stem, it is
met with especially in the pericycle. Leaves are often very rich in
myrosin, which occurs in the young mesophyll.
Poisons and antiseptics : The action of myrosin is
prevented by tannin in a concentration of 1% or by salicylic acid in
solutions stronger than 1-5%. Chloral in 1% concentration is less
harmful, and borax quite harmless. Cf. Reynolds Green, " Soluble
Ferments and Fermentation," 1899, p. 154.
Erythrozyme is the name given to an enzyme
(S c h u n c k , 1852) which" decomposes the ruberythrin or
ruberythric acid of madder into alizarin, dihydroxyanthra-
30 GENERAL CHEMISTRY OF THE ENZYMES
quinone and glucose. This hydrolysis is also effected, although
more slowly, by emulsin, with a constituent of which, ery-
throzyme is closely allied or identical.
Indigo-enzymes. Breandat founa in the leaves
of Isatis alpina, an enzyme which decomposes i n d i -
can, the glucoside of indoxyl, into indoxyl and a sugar (indi-
glucin). And according to Beijerinck (Malys Jahrb.,
1900) an enzyme exists capable of hydrolysing the allied isatan
(from Isatis tinctoria).
L o t a s e from Lotus arabicus decomposes the
glucoside lotusin into lotoflavin (1 : 3 : 3' : 5' tetrahydroxy-
flavone), glucose and hydrocyanic acid. According to D u n -
stan and Henry (Proc. Roy. Soc., 1900, 67, 224, and 1901,
68, 374), it is different from emulsin.
Phaseolunatase, investigated by Dunstan,
Henry and Auld (Proc. Roy. Soc., 5, 1907, 79, 315) seems
to be identical with the linamarase of Jorissen and
Hairs (Bull. Acad. Roy. Belgique, 1891, 21, 518) and possibly
with maltase.
As mentioned on p. 6, lactase decomposes milk-sugar
into d-galactose and d-giucose and, according to E. Fischer,
it hydrolyses p-galactosides generally. This action appears to
be strictly specific, since neither ex- nor (3-glucosides are attacked.
Maltase and lactase are found in various yeasts (E . Fischer,
H., 1898, 26, 81) and fungi (B o u r q u e 1 o t and H e r i s -
sey), and scarcely ever occur together. Eurotiopsis
G a y o n i forms an exception to this rule, as, according to
Laborde (Ann. Inst. Pasteur, 1897, 11, 1), it attacks both
maltose and lactose.
Occurrence. Its existence in lactose-yeasts was
assumed by Beijerinck (Centralbl. f. Bakt., 1889, 6, 44) but was
first definitely proved by E . F i s c h e r (Chem. Ber., 1894, 27, 3481).
In the animal organism it does not occur very largely. It is found in
both the freshly macerated placenta and in the dry powder. Human
intestinal mucus, as well as that of the calf and dog, contain lactase,
but only with the young organism. In the intestinal juice of adult
man no lactase is found (Hamburger and H e k m a, J. de Physiol.
et Pathol. gn., 1902, 4, 805). According to Plimmer (Journ. of
SPECIAL CHEMISTRY OF THE ENZYMES 31
PhysioL, 1906, 35, 20) lactase always occurs in carnivora and omnivora,
but with herbivora, with the exception of the rabbit, it is found only
in the young animal. W e i n 1 a n d (Zeitschr. f . Biol., 1899, 38, 606)
found this enzyme in the pancreas. Lactase must also occur in all those
bacteria which decompose milk-sugar with formation of lactic acid and
alcohol, the hexoses, which are formed as intermediate products, result-
ing from the action of this enzyme ; Fuhrmann (Vorlesungen liber
Bakterienenzyme, p. 96) mentions especially Bacillus acidi
laevolactici and Bacterium coli.
Preparation. (Fischer, Chem. Ber., 1894, 27,
2991 and 3481) . Air-dried yeast is carefully ground with powdered
glass, the mass being then digested with 20 times its quantity
of water for 20 hours at 30-35 and filtered through a P u k a 1 1
filter. The enzyme solution prepared in this way undoubtedly
possessed the property of converting milk-sugar into hexoses,
but its action was weaker than that of the aqueous extract of
kephir-grains.
M e 1 i b i a s e . Melibiose, a product of the hydrolysis of
ramnose, can be further hydrolysed to d-galactose and d-glucose,
so that it contains the same components as lactose. This
hydrolysis may be effected by an enzyme occurring in certain
bottom-yeasts, but not in top-fermentation yeasts. The
enzyme is closely allied to maltase and is perhaps to be regarded
as a maltase (Fischer, H., 1898, 26, 81).
PHYTASE
An enzyme which decomposes phytin or inositolhexaphos-
phoric acid, C6Ho[OPO(OH)2]6, into inositol and phosphoric
acid, was obtained by N. Suzuki, Yoshimura and
Takaishi (Bull Coll. Agric., Tokyo, 1907, 7, 503) from
rice- and wheat-bran. The preparation itself was free from
phosphorus and hydrolysed neither amylose nor proteins. Pre-
sumably the enzyme, like phytin, is widespread in the vegetable
kingdom. According to M c C o 1 1 u m and Hart (Journ.
of Biol. Chem., 1908, 4, 497), phytase is also contained in the
liver and blood, but not in muscle- or kidney-extract. Quite
recently, A. W. D o x and Ross Golden (Journ. of Biol.
Chem., 1911, 10, 183) have detected phytase in lower fungi.
32 GENERAL CHEMISTRY OF THE ENZYMES
HEXOSEPHOSPHATASE
According to Harden and Young (Proc. Roy. Soc.,
B, 1910, 82, 327), this enzyme, which occurs in pressed yeast
juice and also in yeast dried at room-temperature, separates the
phosphoric acid from hexosephosphate. See later under
" zymase."
PECTASE
It is undoubtedly most rational to employ the name p e c -
t a s e s for those enzymes which convert pectoses into pectin
and pectinic acids. The reaction, which yields also arabinose,
consists certainly of a hydrolysis, but the details of the chemical
changes occurring are unknown. The pectinic acids formed
exist in the plants as calcium salts. The work of M a n g i n
(C. R., 1888-1893) and of Devaux (Soc. phys. nat. de
Bordeaux, 3) can only be mentioned here. The enzyme here
termed pectase was obtained from malt-extract by B o u r -
quelot and Herissey (C. R., 1898, 127, 191; 1899,
128, 1241), who called it pectinase; according to the
general principle of naming the enzyme after the substrate,
this should be altered to pectase.
PECTINASE
By the term pectinase should be indicated the enzyme which
coagulates dissolved pectin-substances, e.g., in fruit-juices, in
presence of lime, to gelatinous calcium salts of the feebly acid
pectinic acids. Here also there is as yet no explanation of the
chemical reaction taking place. A high concentration of acid
retards or completely prevents coagulation, which, in the case
of acid fruit-juices, proceeds only after neutralization with lime.
The velocity of the reaction is conditioned by a certain equilib-
rium between the enzyme and the concentrations of the acid
and calcium salts. Without the action of the enzyme, the
lime is unable to coagulate pectins to calcium pectates;
soluble calcium salts can, indeed, induce pectins to coagulate,
but, in this case another product is formed, namely a pectinate
soluble in 0-2% hydrochloric acid. Calcium pectate, however,
yields insoluble pectinic acid with 0-2% hydrochloric acid. It
SPECIAL CHEMISTKY OF THE ENZYMES 33
should be noted that Bertrand and M a 1 1 e v r e , to
whom is due a thorough investigation of this enzyme (C. R.,
1894, 119, 1012; 1895, 120, 110, and 121, 726), named the latter
pectase.
Occurrence. It is found in nearly all plants, especially in
young, quickly-growing organs, shoots, leaves, roots, and fruit. In
great abundance and in an extremely active condition, the enzyme
appears in the extract of clover or lucerne, and also in the leaves of the
potato plant, rape, etc.
CARBAMASES (P r ot einases)
Three proteolytic enzymes or groups of enzymes are dis-
tinguished: the pepsin of the gastric secretion, the trypsin of
the pancreas and the erepsin of the intestinal mucus. It is
very probable that these three substances are not individuals,
but rather mixtures of enzymes, which yet act specifically on
certain protein complexes. These three enzyme-groups can,
however, be readily differentiated according to their origin.
More difficult is the division of the vegetable proteinases, where
classification is not possible according to either the localisation
of the enzymes or the media in which they act. Hence, a dis-
tinction between vegetable pepsins and vegetable trypsins can
hardly be drawn. The only classification at present apparent
depends, on the one hand, on a separation of the peptases from
the true proteinases, and, on the other, on a limitation of the
pepsinases, for which the absence of the lower hydrolytic products
is characteristic. It is, however, doubtful whether trypsin
itself carries the hydrolysis further than pepsin does, or whether
it owes this property to an accompanying peptase. In any
case, a strict division of the proteinases is at present difficult.
Pepsin: Pepsinases
The decomposition of proteins effected by pepsin extends,
so far as is known, to all proteins; the products formed con-
sist of albumoses and peptones, so that the hydrolysis
is incomplete, lower polypeptides and amino-
acids not, as a rule, appearing (Abder-
h a 1 d e n and R o s t o c k i , H., 1905, 44, 265).
34 GENERAL CHEMISTRY OF THE ENZYMES
Occurrence: In the gastric juice of all the vertebrates examined,
with the exception of certain fishes. The pepsin of Brunner's glands is
closely allied to gastric pepsin. In the gastric mucous membrane,
pepsin occurs not in an active form but as a pro-enzyme (pepsinogen).
It exists with new-born children and, with certain herbivorous animals,
even in the foetal state, in the mucous membrane, but is not present
at birth in those carnivora which have been examined, namely, the dog
and the cat. Proteolytic enzymes, similar to pepsin and active in
acid solution, have also been found in several invertebrates, but, with
some animals at least, these enzymes are not identical with ordinary
pepsin.
Preparation : Very active and stable pepsin-solutions
are obtained by extracting the gastric mucous membrane with
glycerol; the pepsin, together with protein, may be precip-
itated from the extract by means of alcohol. A considerable
amount of pepsin can also be extracted from this membrane
by acidified water. The best starting material is, however,
pure gastric juice, which can be prepared by P a w 1 o w ' s
well-known method from gastric fistulae.
Attempts at the purification of pepsin have, up to the present,
led to no final result, but Pawlow's gastric juice is the
best material to work on for this purpose. Such gastric juice,
on freezing, yields a solid product, which Mme. S c h u m o ff-
Simanowski has called " granular pepsin " (Korniges
Pepsin) .
The preparation of the enzyme in a pure state has been
worked at mainly by N e n c k i and S i e b e r (H., 1901,
32, 231; 33, 291) -and byPekelharing (H., 1902, 35, 8).
According to Pekelharing's method, P a w 1 o w ' s
gastric juice is dialysed for 20 hours at 0, the pepsin thus being
deposited in transparent granules- at the bottom of the dialyser.
The turbid liquid is centrifuged and the colourless residue pressed
and dried; in Pekelharing's opinion, this represents
pure pepsin.
The percentage composition of the enzyme is found to be :
C H N S Cl P Fe Ash
Nencki and
Sieber: 51-26 6-74 14-33 1-5 0-48 small variable 0-57
Pekelharing: 51-99 7-07 14-44 1-63 0-49 + 0-1
According to Nencki and Sieber, pepsin is com-
bined with lecithin. When boiled with acids, pepsin yields an
SPECIAL CHEMISTRY OF THE ENZYMES 35
albumose and a nucleo-proteid, which then yields purine-bases
(Pekelharing found xanthine) and pentoses ( F r i e -
denthal, Engelmann's Arch. f. PhysioL, 1900, 24, 181).
They regarded the pepsin molecule as performing (by three
different groups) the three functions of the gastric juice: protein
digestion, clotting and plastein formation, a view with which
Pekelharing agreed.
The precipitate formed when gastric juice is coagulated by
heat contains, according to Pekelharing, an acid pep-
sinic acid which has the percentage composition: C, 50-79;
H, 7-0; N, 14-44 and S,l-08 and gives M i 1 1 o n ' s and the
biuret reactions.
Formaldehyde does not act on " pepsin," and this fact
caused Bliss and N o v y to doubt the protein character
of pepsin. Further, as was shown by N e n c k i and S i e b e r
and also by Pekelharing, it is possible to prepare pepsin
solutions which vigorously digest proteins but do not show the
protein reactions; indeed, this was shown to be the case as
early as 1885 by Sundberg (H., 1885, 9, 319; cf . B r u c k e ,
Wiener Sitz.-Ber., 1861, 43, 601).
The preparation of a protein-free pepsin solution, which
has an energetic digestive action but does not produce clotting,
was described by Schrumpf (Hofm. Beitr., 1905, 6, 396).
The mucous membrane was separated from a fresh pig's stomach
and was then ground with kieselguhr and pressed under a high
pressure. The pressed juice was clarified by means of a Kitasato
filter-candle and was found to remain clear on addition of uranyl
acetate, ammonium sulphate, etc.
H e r 1 i t z k a (Atti Real. Accad. LinceL, 1904, [v], 13, ii,
51) has deduced a proof for the opposite view that pepsin
is a true protein from the fact that, in absence of hydrochloric
acid, pepsin gradually loses its activity, peptones appearing at
the same time. These may, however, result from admixtures,
so that the investigation proves nothing concerning the chemical
nature of pepsin.
As regards the individuality of pepsin, it has already been
indicated that the same molecule, by means of other side-chains,
causes rennet-action and plastein-formation, and this hypothesis
is not in discord with the facts at present known (cf . J a c o b y ,
Biochem. Z., 1906, 1, 53). On the other hand, justifiable
36 GENERAL CHEMISTRY OF THE ENZYMES
objections have been raised against P a w 1 o w ' s assumption
that the rennet-action is a reversal of the pepsin-action (Bang,
H., 1904, 43, 358; Schmidt-Nielsen, H., 1906, 48,
92; especially Hammarsten, H., 1908, 56, 18).
From its chemical characters, pepsin appears to be an acid,
and this view is sustained by a number of different observations
by various investigators. J a c o b y (Biochem. Z., 1907, 4,
471) found that pepsin, and also rennin, are soluble in alkali,
and adsorption experiments described by M i c h a e 1 i s indi-
cate marked adsorption by basic media. This view is also
supported by the observation that pepsin migrates to the
anode.
Pepsin is inactive in an alkaline medium, but on acidification
it recovers its activity more or less completely according to the
nature and duration of the previous alkalinity ( T i c h o m i -
row, H., 1908, 55, 107).
Pepsin exerts its optimal activity at 40 and its stability is
greater in acid than in neutral solutions and is further increased
by the presence of salts.
Trypsin: Tryptases
The enzyme of the pancreas, which is active in alkaline or
neutral solution, resolves the proteins into simple polypeptides,
these being to some extent further broken down. It does not
decompose all dipeptides, but presumably only those combina-
tions occurring in the organism (cf. Chapter VIII). On the
other hand, it can be stated that only those polypeptides are
attacked which contain naturally-occurring, optically active
amino-acids. Acid-amides, hippuric acid, etc., are not attacked
(Gulewitsch, H., 1899, 27, 540; Sc.hwarzschild,
Hofm. Beitr., 1903, 4, 155; Fischer and Bergell,
Chem. Ber., 1903, 36, 2592 and 1904, 37, 3103). With nucleo-
proteins the protein component is separated from the nucleic
acid and decomposed further. From certain nucleo-proteins,
the phosphoric acid is liberated ( B a y 1 i s s , Arch. Sci. Biol.
St. Petersburg, 1904, 11, Suppl., 281).
Not at all improbable is the assumption of Schaeffer
% and T e r r o i n e (J. de Physiol. et Pathol. gen., 1910) that
the trypsin of the pancreas is accompanied by an erepsin which
SPECIAL CHEMISTRY OF THE ENZYMES 37
attacks directly .(without kinase) all substances split off by
the gastric juice.
Occurrence. Trypsin occurs in the pancreatic juice and in
the tissues of the pancreatic glands as a pro-enzyme, which is trans-
formed by specific activators or kinases into the active condition.
Trypsins or tryptases have been found in all the vertebrata
tested for them. H e d i n (Journ. of Physiol., 1903, 30, 155 and 195)
discovered a tryptic enzyme in normal blood-serum ; it acts . in an
alkaline medium and decomposes casein, gelatine, and coagulated serum-
albumen, but globulins and coagulated egg-albumen are not attacked.
Besides in the pancreas, animal tryptases are found in the urine, and in
the spleen and other organs; also in hens' eggs.
Enzymes which must be termed tryptases are contained in the
leucocytes and, as Jochmann showed, not only in leucemic, but
also in normal, leucocytes. This occurrence is limited, so far as is known
to man, monkeys, and dogs (E r b e n , Munch. Med. Wochens., 1906
and 1907; Jochmann and Lockemann, Hofm. Beitr., 1908,
11, 449). The tryptase of the leucocytes is less sensitive to heat than
that of the pancreas; its optimal temperature is 55.
Tryptases have also been found in insects, protozoa, sponges, worms,
and molluscs.
Preparation. Active but impure solutions may be
obtained by extracting finely-chopped pancreatic glands with
chloroform- water. The best material is pancreatic juice obtained
by P a w 1 o w ' s method and activated by intestinal secretion.
The purification of trypsin has been recently attempted,
especially by Mays (H., 1903, 38, 428 and 1906, 49, 124
and 188). He obtained a relatively very pure and active prepa-
ration by salting-out the trypsin solution with sodium chloride
and magnesium sulphate; but his investigations indicate little
concerning the chemical nature of trypsin. It is not a nucleo-
protein and does not give the biuret reaction (Mays,
Schwarzschild).
In aqueous solution, it is very labile and sensitive to heat,
and, indeed, more so in alkaline than in neutral solution. Sub-
strate and decomposition products exert a considerable protecting
action (Bayliss, Vernon), so that the optimal tem-
perature is given as 40. Trypsinogen is less sensitive to heat
than trypsin. Acids denature it even in low concentrations.
In contrast to pepsin, which dissolves in alkali, trypsin is
38 GENERAL CHEMISTRY OF THE ENZYMES
soluble in acids. In the electric field, it behaves as an ampho-
teric electrolyte, that is, it migrates either to the anode or to
the cathode according to the reaction of the solution. It is
affected but slightly by chloroform or thymol.
Pollak (Hofm. Beitr., 1905, 6, 95) characterises glu-
tinase as a separate enzyme, owing to its resistivity towards
acids. It acts on glue, but not on certain other proteins.
On the other hand, A s c o 1 i and N e p p i (H., 1908,
56, 135) have established the fact that the specificity of glutinase
towards glue is only apparent and is due to the different proteins
being influenced differently by activators and paralysers
Er ep sin
See Cohnheim (H., 1901, 33, 651; 1902, 35, 134; 1902,
36, 13; 1906, 49, 64). This enzyme decomposes albumoses,
polypeptides, peptones and protamines completely into amino-
acids. With the exception of casein, proteins are not attacked,
but nucleic acids are decomposed (Nagayama, H., 1904,
41, 348).
Occurrence: in the intestinal juice and the intestinal mucous
membrane, the latter being the richer in the enzyme. It appears, especi-
ally after V e r n o n ' s investigations (Journ. of Physiol., 1904, 32, 33)
and those ofAbderhalden, to be the most widely distributed
proteolytic enzyme; it has been detected in nearly all the animals
examined for its presence.
Preparation. According to Cohnheim, it can
be prepared from the pressed juice of the intestinal mucous
membrane; also extraction of the latter with glycerol or water
yields very active solutions. From the aqueous solution, the
enzyme is salted out with ammonium sulphate. Pressed yeast
juice likewise contains much erepsin (Abderhalden).
The optimal temperature is about 38 (in alkaline solution).
PROTEOLYTIC ENZYMES OF PLANTS
Proteinases is the name given to those enzymes which
break down the true proteins. The decomposition appears to
proceed as far as the albumoses and peptones. It is seldom
that proteinases occur alone in the organs of plants where
SPECIAL CHEMISTRY OF THE ENZYMES 39
proteins are decomposed; they are almost always accompanied
by peptases, which correspond with the erepsins of the animal
body. Vines (Annals of Bot., 1904, 18, 289; 1905, 19, 149,
171; 1906, 20, 113; 1908, 22, 103; 1909, 23, 1) to whom we owe
a thorough investigation of the vegetable proteinases has, indeed,
recently found both enzymes in the seeds of Cannabis
s a t i v a .
Occurrence. Proteinases occur especially in germinating and
ungerminated seeds and more abundantly in oil-bearing than in starch-
containing seeds particularly in Cannabis sativa, Sinapis,
R i c i n u s and L i n u m ; further, in certain juicy fruits (F i c u s
c a r i c a ) and leaves (Agave). They are generally found in insect-
ivorous plants. Among those which have been thoroughly investigated
are
B r o m e 1 i n in acid banana-juice (Chittenden, Journ. of
Physiol., 1894, 15, 249); according to C aid well (Bot. Gaz., 1905,
39, 407), this consists of two components, a pepsinase and a tryptase; and
Papain or papayotin in the juice of the fruit, leaves and
stem of the papaw tree (Carica papaya). These two enzymes
are very closely allied if not absolutely identical. Animal proteins, such
as fibrin, are also hydrolysed by bromelin (Chittenden). From
the mixture of proteolytic enzymes in oily seeds, Vines isolated a
fibrin-digesting proteinase. A similar action is exhibited by the papain of
Carica which readily digests fibrin. After digestion of fibrin with
Carica- sap and with an enzyme similar to papain and obtained from
Bacillus fluorescens 1 i q u e f a c i e n s , Emmerling
(Chem. Ber., 1902, 35, 695) found in addition to albumoses and pep-
tones, also various amino-acids, such as leucine, tyrosine, etc. With
"papayotin Merck," Kutscher and Lohmann (H., 1905, 46,
383) obtained analogous results. According to Abderhalden and
Teruuchi, H., 1906, 49, 21), papain splits glycyl-1-tyrosine; this
also indicates the simultaneous occurrence of proteinases and peptases,
unless, indeed, papain is regarded as a tryptase with a wider sphere of
action.
Vegetable proteases accompany malt-diastase and taka-
diastase. These proteinases are extractable by alcohol in a
remarkable manner (Vines, Annals of Bot., 1910, 24, 213).
The statements made concerning the acidity or alkalinity
of the medium in which vegetable proteinases exhibit their
maximal activity differ considerably (cf . Reynolds Green,
Vines, Emmerling, Weis, and others) . This dis-
40 GENERAL CHEMISTRY OF THE ENZYMES
agreement depends partly on qualitative and quantitative
differences between the substrates employed and partly on
varying composition of the enzyme preparations. The peptase
present will always act best in neutral or faintly alkaline solu-
tions and the proteinases, on the other hand, in acid ones.
Proteinases occur abundantly in fungi and they can be extracted
from the mycelia by water or glycerol.
As extracellular enzymes, proteinases occur in insectivorous
plants. In these they are liberated from zymogens in certain
glands under the stimulative action of nitrogenous substances.
With Nepenthes, the enzyme is distributed in the anti-
septic, almost protein-free sap of the leaf -pitchers. This acid
sap decomposes proteins not merely into albumoses, but also
into amino-acids (Vines, Annals of Bot., 1897, 11, 563).
But Abderhalden found that glycyl-1-tyrosine is not
attacked. Hence uncertainty still prevails regarding the nature
of the enzyme or enzymes of Nepenthes. The leaf-glands
of D i o n a e a and D r o s e r a and the leaf -edge of P i n-
g u i c u 1 a yield acid, mucilaginous secretions which attack
proteins of cartilaginous and glutinous tissues. Extracellular
proteinases also appear to be produced by bacteria, especially
by those species which are capable of liquefying gelatine.
Another group of fungus-proteinases consists of typical endo-
enzymes; noteworthy among these is the
Endot-ryptase of yeast (Hahn and Geret).
So far as is known, its action extends to all proteins (gelatine,
fibrin, casein, egg-albumin), which it resolves into amino-acids.
It cannot be extracted from yeast by water, and is obtainable
only byBuchner's method.
Unlike papain, endotryptase is most active in acid solution.
Its temperature-optimum is 40-45.
^-Proteases. Papayotin, Hahn and G e r e t ' s
endotryptase and the enzymes which effect a partial decomposition
of the protamines and complicated proteins in faintly acid solu-
tion, are classed together by Takemura as ^-proteases.
The action of pepsin on protamines would thus be attributable
to the presence of a g-protease.
Peptases are those enzymes which decompose albumoses,
peptones and polypeptides into amino-acids. They generally
accompany the proteinases and, as has been shown by Vines
SPECIAL CHEMISTRY OF THE ENZYMES 41
(Annals of Bot., 1906, 20, 113), Abderhalden and S c h i t -
tenhelm (H., 1906, 49, 26) and Euler (H., 1907, 61,
244), they occur abundantly in the seeds of lupins, rape, peas
and maize. Abderhalden and his collaborators found it
also in pressed yeast juice.
According to certain investigators, among them W e i s
(H., 1900, 31, 79) and Vines (Annals of Bot., 1903, 17, 237
and 1904, 18, 289), the most rapid hydrolysis takes place in faintly
acid solution, whilst others, e.g., W i n d i s c h , assert that it
occurs in a slightly alkaline medium.
NUCLEASES
The decomposition of the nucleo-proteins begins with their
resolution into nucleic acids and protein-components, a reaction
which is brought about by pepsin or trypsin. The further
division of the nucleic acids is not, however, produced by the
true proteinases (Sachs; Abderhalden and Schitten-
helm, H., 1906, 47, 452), but by a special group of enzymes,
the nucleases. These, therefore, resolve the nucleic acids
into their constituents, purine or pyrimidine bases (adenine,
guanine, cytosine, thymin), pentoses, and phosphoric acid.
That the decomposition of nuclein, and the digestion of
Buchner's pressed yeast juice are enzymic in character
has been shown by Salkowski (H., 1889, 13, 506) and
H a h n and G e r e t (Chem. Ber., 1898, 31, 2335) respectively,
and more recent researches prove that a number of enzymes
act on the nucleic acids. These enzymes may be divided into
a number of groups (cf. B . B 1 o c h , Biochem. Centralbl.,
1907, 5, 561).
1. Nuclease, which liberates the phosphoric acid from
the nucleic acid molecule.
2. Hydrolytic enzymes, which split off ammonia from the
aminopurines and replace the amino-group by hydroxyl.
3. Oxidising enzymes which oxidise hydroxypurines to uric
acid.
4. Uric acid oxydases.
According to Sachs (H., 1905, 46, 337), pure trypsin has
no action at all on nucleic acids. Decomposition of nucleic
42 GENERAL CHEMISTRY OF THE ENZYMES
acids takes place only in fresh pancreas extracts, which exert
but slight proteolytic action.
Occurrence. Very frequent in animal organs, e.g., in the
spleen, liver (Jones), and in the pancreatic and thymus glands
(Kutscher, H., 1901, 34, 114). It performs an important function
in many organs of plants more particularly in germinating seeds in
synthesizing and decomposing nucleo-proteins (Z a 1 e s k i , Bot. Ber.,
1907, 25, 349). Worthy- of note also are the nucleases of higher (K i k-
k o j i , H., 1907, 51, 201) and lower fungi (I w a n o f f , H., 1903, 39, 31),
especially those of yeast.
P. A. Levene and Medigreceanu (Journ. of
Biol. Chem., 1911, 9, 389) have recently proposed the following
classification and nomenclature.
Nucleinases resolve the nucleic acid molecule into
nucleotides.
Nucleotidases decompose nucleotides into phos-
phoric acid and a carbohydrate-base complex (nucleoside) .
Nucleosidases hydrolyse nucleosides into ribose and
purine bases.
Arginase (Kossel and Dak in, H., 1904, 41, 321)
This enzyme hydrolyses arginine specifically into urea and
ornithine according to the following equation:
NH NH 2
+NH 2 CH 2 CH 2 CH 2 CH COOH.
I
NH 2
Whilst trypsin hydrolysis may be indicated by the scheme:
C CO-j-NH-C,
the action of arginase is represented by
Occurrence. In the liver, kidneys, thymus, and intestinal
mucous membrane of the calf and also in the muscles of the dog and
SPECIAL CHEMISTRY OF THE ENZYMES 43
the lymphatic glands of cattle. Shiga (H., 1904, 42, 502) found
arginase also in yeast.
It can be extracted from the organs by means of water.
Ur ease
This enzyme decomposes urea into carbon dioxide and
ammonia.
Occurrence. According to L e u b e (Virch. Arch., 1885, 100,
540), urease occurs inMicrococcus ureae, from which it passes
readily into the surrounding liquid. In cystitic urine the enzyme occurs
only when fungi capable of decomposing urea are present.
Lea (Journ. of Physiol., 1885, 6, 136) has attempted to
prepare urease in a pure state; by treatment of Micro-
coccus ureae with alcohol, extraction of the precipitate
with water and repeated reprecipitation with alcohol, a powder
is obtained which yields a clear aqueous solution but contains
protein. As later work has shown, urease is tenaciously retained
by living protoplasm. Moll (Hofm. Beitr., 1902, 2, 344)
also obtained the enzyme in a similar manner and from the
same material; it readily undergoes decomposition. Schit-
t e n h e 1 m obtained an enzyme-solution (free from purine-
bodies) from the kidneys and K i k k o j i found urease in a
pileate fungus.
AMIDASES (DESAMIDASES)
Decomposition of the proteins by trypsin and erepsin ceases
when the amino-acids are reached, but the desamidases attack
the latter, decomposing them into ammonia and hydroxy-acids.
Such simple desamination, as was discovered byH. Prings-
heim (Biochem. Z, 1908, 12, 15), takes place by virtue of
an enzyme present in acetone-yeast. Similar action is exhibited
by permanent preparations of Aspergillus niger (Shi-
bat a, Hofm. Beitr., 1904, 5, 384). It has been shown by
Pringsheim, Abderhalden and Schittenhelm
that the amidases of yeast do not pass into the press-juice. Of
biological importance is the detection of amidases in higher
plants (Butkewitsch, H., 1909, 63, 103; cf. Kiesel,
H., 1910, 65, 283).
44 GENERAL CHEMISTRY OF THE ENZYMES
The so-called " alcoholic fermentation of the ammo-acids "
discovered by F , E h r 1 i c h consists of a combination
of two reactions, namely the desamination of the amino-acids
to the corresponding hydroxy-acids and the subsequent loss of
carbon dioxide from the latter; the total reaction can hence
be formulated thus:
Reactions similar in principle to those brought about by
amidases are caused by the enzymes known as guanase
and a d e n a s e . These convert guanine and adenine into the
corresponding hydroxy-derivatives :
N = C NH 2 N = C OH
II II
CH C NH, + H 2 = CHC NH,
II II >CH || || >CH + NH 3 ;
N C - W N C - W
Adenine Hypoxanthine
and
NH CO
NH 2 -C C NH X +H 2 = OH-C
II II >CH || || CH+NH 3
N - C -- N^
Guanine Xanthine
Concerning the individuality and mode of action of these
enzymes of the purine-bases, various views have been expressed
by the different investigators who have studied them (Schitten-
helm, H., 1904, 42, 251; 1905, 43, 228; 45, 121, 152; 46,
354; Jones and his collaborators, H., 1904, 42, 35, 343; 1905,
44, 1; 45, 84; 1906, 48, 110; and also Burian, H., 1905,
43, 494) . Even if Schittenhelm's assumption that the
two enzymes are identical is not correct, they are certainly very
similar.
Occurrence. Guanase and adenase have been found in the
pancreas, spleen, lungs, and liver of the child, pig (not in the spleen),
cattle, and also in numerous other organs.
Preparation. (Schittenhelm, H., 1904, 42,
251). One or two spleens are extracted with about 2 litres of
water for 12 hours and the enzyme then precipitated with
SPECIAL CHEMISTRY OF THE ENZYMES 45
ammonium sulphate. The precipitate is dissolved in water
and freed from ammonium sulphate by dialysis.
Ni t r ilase
According to Rosenthaler (Biochem. Z., 1909, 19,
186; 1910, 28, 408), the decomposition of amygdalin is effected
by three enzymes an amygdalase, a ^-glucosidase and an
hydroxynitrilase. The last of these exerts a catalytic influence
on the reaction.
OH
C 6 H 5 -CH<f +H 2 = C 6 H 5 -CHO+HCN,
CN
and, in Rosenthaler's opinion, its action is solely
hydrolytic, i.e., it can be separated from the synthesising con-
stituent of emulsin; in Chapter VII of this book, hydroxynitrilase
(oxynitrilase) will be considered, together with the corresponding
synthesising enzyme, in greater detail.
COAGULATING ENZYMES
C h y m o s i n and parachymosin are those enzymes
which effect the clotting of milk, the casein of the latter being
changed in some way, as yet unknown. The two enzymes are
distinguished according to their place of origin. Chymosin
which has been long known and is the enzyme of the calf's
stomach, is, according to Bang (Deutsch. med. Wochens., 1899,
and Pfliig., Arch., 1900, 79, 425), to be distinguished from the
coagulating constituent of the human stomach, namely, para-
chymosin. The chemistry of rennet-action is still not
clear. With solutions containing pure casein and also rennet in as
pure a state as possible, the clear whey obtained after coagulation
contains a very small proportion of a protein whey-albumin
with only 13-2% of nitrogen. The bulk of the casein is precip-
itated as a substance, paracasein, very similar to casein
itself. Whether decomposition of the casein occurs is still
uncertain (cf . Hammarsten, Text-book of Physiological
Chemistry, 4th Edition, 1906, 442).
Occurrence. In the gastric juice, the gastric mucous membrane,
the pancreatic juice, the placenta, and certain other organs of many
46 GENERAL CHEMISTRY OF THE ENZYMES
different anLnals The enzyme often occurs in an inactive form as the
so-called prochymosin (Edmunds, Journ. of Physiol., 1895,
19, 466; Vernon, Journ. of Physiol., 1901, 27, 174), owing mainly
to the lack of an activator.
The activation of prochymosin can be effected instantly by
an acid and, after activation, the rennet is active in both
neutral and alkaline solutions.
Preparation. (Hammarsten, H., 1908, 56, 18).
This author has recently described the preparation of chymosin
from the stomach of the calf, horse, hen and pike. The first-
named material is treated as follows:
The innermost coat of the stomach is separated from the
intestines and from the three other stomachs, cut along the
small curvature and freed from contents by rinsing out with
water. The pylorus portion is then cut away together with at
least 3-5 c.m. of the large fold of the fundus portion. (The
pylorus part yields a more mucilaginous infusion and is, at the
same time, less rich in enzymes, than is the fundus.) The
remainder of the stomach is stretched out and thoroughly washed.
The glandular layer is then scraped from the two sides of a fold
with the edge of a watch-glass, weighed and introduced into
0-1-0-2% hydrochloric acid, 10-20 c.c. of the latter being taken
per grm. of the glandular matter. The acid is allowed to act,
with repeated shaking, for 12-24 hours at a low temperature
somewhat above the liquid being then filtered. The infusion
is neutralised and, if found to exert a vigorous coagulating action,
is precipitated with magnesium carbonate.
One grm. of magnesium carbonate is added to each 100
c.c. of the extract, which is shaken and quickly filtered; it
can then be tested for the presence of pepsin (see Appendix:
Practical Methods). If the filtrate still contains much of this
enzyme, it is again treated with magnesium carbonate, and
this procedure is repeated until a filtrate is obtained which
readily causes clotting of milk but has only a slight action on
fibrin.
Another process which Hammarsten gave for the
preparation of chymosin consists in freeing it from the greater
part of the pepsin by means of magnesium carbonate and pre-
cipitating the chymosin with lead acetate. The lead is removed
from the precipitate by sulphuric acid and the acid filtrate shaken
SPECIAL CHEMISTRY OF THE ENZYMES 47
with an alcoholic solution of cholesterol or stearic acid and a
little ether, so that the precipitated cholesterol or stearic acid
carries down the enzyme with it. If now the precipitate is
shaken with water and freed from the precipitant by treatment
with ether, a moderately pure chymosin solution is obtained.
The purest clotting enzyme obtained in this way did not give
the ordinary reactions for proteins.
Two different views are held as to the identity of chymosin
and pepsin. According to the one, which has been advanced
by Pawlow (H., 1904, 42, 415), Saw j alow (H., 1905,
46,307), Sawitsch (H., 1908, 55, 84) and Gewin (H.,
1907, 54, 32), both actions result from one and the same enzyme,
S a w j a 1 o w and Gewin regarding the clotting of milk as
the commencement of pepsin-digestion. On the other hand,
Nencki and Sieber (H., 1901, 32, 291), Pekelharing
(H., 1902, 35, 8), Schmidt-Nielsen (H., 1906, 48, 92),
Taylor (Journ. of Biol. Chem., 1909, 5, 399), and especially
Hammarsten (H., 1908, 56, 18) are of opinion that the
two enzymes are not identical but of different kinds.
In addition to the cholesterol method described above,
Hammarsten has recently given a second method allowing
of the separation of chymosin-action from that of pepsin. In
principle this method consists in heating the acid enzyme- solu-
tion to 40 or a rather higher temperature. In this way the
calf-chymosin is destroyed more rapidly than the pepsin, so
that after some time a solution is obtained which no longer
exerts a clotting action, but still digests proteins ( 1 o c . c i t . ,
p. 61).
Still more recently (H., 1911, 74, 142), Hammarsten has
succeeded in preparing pepsin-free chymosin solutions by mixing
an acid infusion of calf's stomach and a neutral alkali caseinate
solution in such proportions that the casein at first separating
just redissolves. To the acid casein solution thus obtained,
decinormal sodium hydroxide solution is added in sufficient
quantity to produce an abundant precipitation of casein and to
allow of ready filtration while the reaction still remains strongly
acid. Both the enzymes are carried down by the precipitated
casein, but the pepsin in much larger quantity than the chymosin.
The filtrate therefore contains a relatively high proportion of
chymosin.
48 GENERAL CHEMISTRY OF THE ENZYMES
Chymosin exhibits remarkable activity, one part of the
mucous membrane of the calf's stomach being sufficient to
clot 250,000 parts of milk. Purified chymosin coagulates
24,000,000 (Hammarsten) or 30,000,000 ( F u 1 d ) times
its weight of milk.
Chymosin causes clotting only in acid solutions; hydro-
chloric acid is the most favourable to its action and after this
come nitric, lactic, acetic, sulphuric and phosphoric acids. Its
optimum temperature is 37-39.
Chymosin is injured by chloroform (Benjamin), but
not by hydrocyanic acid ( F u 1 d and S p i r o ).
Milk-clotting Enzyme in Plants (Cynarase)
A large number of plants, such as Pinguicula v u 1 -
garis, Galium verum, artichokes, etc., possess the
property of rendering milk ropy.
Whether the chemical change underlying this coagulation
is or is not the same as that produced by the action of chymosin,
is unknown.
The vegetable chymases must, however, be quite different
from the animal enzymes. According to C h o d a t and
Rouge (Centralbl. f. Bakt., 1906, II, 16, 1), the syko-
c h y m a s e from Ficus carica investigated by them
acts in absence of calcium salts. Its optimal temperature is
75-80.
Further, as has been shown by Bruschi (Atti Real.
Accad. Lincei, 1907, [V], 16, ii, 360) and especially by G e r b e r
(C. R., 1907, 145, 689; 1908, 146, 1111; 147, 601, 1320; 1909,
148, 497, 992; 149, 137, 737; 1910, 150, 1202, 1357), the phyto-
chymases exhibit great differences among themselves. G e r b e r
distinguishes these enzymes according to the amounts of lime
they require for their action, and he has further shown that
some phyto-chymases coagulate raw milk, while others coagulate
boiled milk the more readily.
Occurrence. In addition to the plants mentioned above, the
following also exert a coagulating action on milk: Lolium perenne,
Anthriscus vulgaris, Plantago lanceolata, La-
mium amplexicaule and hybridum, Philadelphus
c oronarius , Geranium m o 11 e , C ap s e 11 a bursa pas-
SPECIAL CHEMISTRY OF THE ENZYMES 49
tor is, Ranunculus bulbosus,Medicago lupulina,
Centaurea scabiosa, etc. Reynolds Green (Proc. Roy.
Soc., 1890, 48, 370) found the enzyme in 'the germinating seeds of
Ricinus communisin the form of a zymogen, which is activated
by dilute acids. This enzyme is also contained in numerous other
seeds, for example, in those of Datura, Pisum, and L u p i n u s
hirsutus. Weis (H., 1900, 31, 79) found a clotting enzyme in
malt. The coagulating action of the fig, F i c u s c a r i c a , on milk
was known to the ancient Greeks. See also G e r b e r (C. R., 1909,
148, 992).
Lastly, coagulating enzymes have also been detected in many lower
fungi, among others Fuligo varians. C. Gerber has recently
examined 86 species and sub-species of Basidiomycetes, and
has found them to contain, in general, an "oxyphile " and a "calciphile"
coagulating enzyme.
Thrombin, Fibrin-ferment (Alexander Schmidt, 1872)
Thrombin causes blood to coagulate by converting dissolved
fibrinogen into insoluble fibrin.
The change taking place is probably as follows: When the
blood leaves the body, one of its constituents (possibly the
leucocytes) gives rise to a pro-enzyme, which is converted into
the active enzyme under the influence of the calcium salts.
This enzyme, without further action of calcium salts, then trans-
forms the fibrinogen into insoluble fibrin.
It is best prepared from blood-serum or defibrinated blood
by precipitation with 15-20 volumes of alcohol, which separates
the proteins at the same time. If the precipitate is then extracted
with water, part of the protein remains undissolved, whilst the
thrombin passes into solution. According to Hammar-
s t e n ' s method (Pflug. Arch., 1878, 18, 38) the globulins
are first precipitated by magnesium sulphate; the liquid is then
diluted with water and sodium hydroxide solution added so as to
precipitate magnesium hydroxide, which is accompanied by a con-
siderable amount of adsorbed fibrin-ferment. Pekelharing
dialyses the filtrate from the precipitate given by magnesium
sulphate. From the muscles of birds, F u 1 d obtained throm-
bin by extraction with 0-8% sodium chloride solution.
According to S h i g e j i and H i g u c h i , the placenta
contains a fibrin-enzyme, which can be extracted by means of
water or physiological salt solution.
50 GENERAL CHEMISTRY OF THE ENZYMES
Pekelharing regards thrombin as the lime compound of
pro-thrombin, the nucleoprotein which occurs in blood-plasma
and which he attempted to prepare in a pure condition (Verh.
d. k. Akad. v. Wetenschappen te Amsterdam, 1892, II, 1, No. 3;
and Zentralbl. f. PhysioL, 1895, 9, 102). The existence of this
nucleoprotein in blood-serum has been established, but its com-
position has not yet been investigated since the quantity of it
in blood is very small. In a concentrated solution of the enzyme,
which contained 0-417% of organic matter and 0-166% of inor-
ganic matter, Hammarsten found only 005% of nuclein.
The experimental results obtained by Huiskamp (H.,
1901, 32, 145), led this investigator to dispute Pekelharing 's
views. Huiskamp found that, in presence of calcium salts,
both the nucleohistone and the other nucleoprotein of the thymus
glands two essentially different substances acted on fibrin-
ogen in the same way as thrombin; for the calcium nucleo-
protein he gave the following percentage composition : C, 49 82 ;
H, 7-29; N, 15-81; P, 0-954; S, 1-188 and Ca, 1-337. The
question whether the protein itself is to be regarded as the
fibrin-enzyme or whether its action on the formation of fibrin
is due to an admixture with another substance is left undecided
by Hammarsten (Ergeb. der Physiol., 1902, 1, i, 339).
The optimal temperature for thrombin is 40.
For the chemistry of the coagulation of blood see Mora-
witz, Hofm. Beitr., 1903, 4, 381; 1904, 5, 133; L. Loeb,
Biochem. Zentralbl., 1907, 6, 829, 889; Pekelharing,
Biochem. Z., 1908, 11, 1.
ENZYMES OF FERMENTATION
Fermentation is not a chemically definite conception; by it
are understood those processes which are brought about by
lower organisms and the extent of which is great compared
with the mass of the organisms taking part.
From a chemical point of view, fermentation enzymes can
be contrasted with the hydrolytic enzymes in so far as fermen-
tation reactions consist of pure decompositions and take place
without any other substance, such as water or oxygen, being
taken up; the best-known example is the alcoholic fermentation
of the hexoses which is effected by zymase. On the other hand,
SPECIAL CHEMISTRY OF THE ENZYMES 51
the enzyme of the acetic acid fermentation is regarded as belonging
not to the same group but to the oxydases. The reactions under-
lying other fermentations have been investigated chemically
only in very recent times and attention must be drawn especially
to the work of Buchner and Meisenheimer; the
existence of the enzymes which presumably take part in these
fermentations has not yet been proved. The view that fermen-
tations in general are to be referred to enzyme actions is a con-
sequence of the discovery of E. Buchner, who, in 1897,
succeeded in producing alcoholic fermentation in a pressed yeast
juice free from cells and hence in showing that this fermentation
is not dependent on the action of the living yeast.
Enzymes of Alcoholic Fermentation
The term zymase, in its wider sense, is used to indicate the
sum-total of the enzymes which bring about the decomposition
of certain of the hexoses in the sense of the following equation:
C 6 H 12 6 = 2C 2 H 5 OH+2C0 2 .
d-Hexose. Ethyl alcohol.
As was assumed by Buchner and Meisenheimer
and by W o h 1 , this chemical change is to be regarded as taking
place in several separate stages. It was formerly thought that
glucose gives rise to lactic acid under the action of an enzyme
to which the name zymase was then applied in a more restricted
sense. Buchner and Meisenheimer have, indeed,
detected the formation of small quantities of lactic acid during
fermentation, but S 1 a t o r (Journ. Chem. Soc., 1906, 89, 128)
has shown that lactic acid, which is fermented with extreme
slowness, can be only a bye -product and not an inter-
mediate product of fermentation. It is now assumed that
compounds allied to lactic acid are formed at an intermediate
stage of the fermentation, which possibly passes through the
following stages:
52 GENERAL CHEMISTRY OF THE ENZYMES
Methylglyoxal
CHO CHO CHO CHO H-CO 2 H Formic CO 2
CH-OH C-OH CO CO CHO Acetaide > CH 2 -OH
CH-OH HCH -CH 2 CH 3 CH 3 CH 3
| ~~OH-^| 4-1 ->-
CH-OH CH-OH CH-OH CHO CHO CHO
CH-OH CH-OH CH-OH CH-OH~OH ""C-OH^CO
CH 2 -OH CH 2 -OH CH 2 -OH CH 2 -OH CH 2 CH 3
Glucose . Glyceraldehyde Methylglyoxal
There is also uncertainty concerning the occurrence of methyl-
glyoxal, which cannot be fermented by pressed yeast juice.
Boysen-Jensen (Bot. Ber., 1908, 26, 666; also Dis-
sertation, Copenhagen, 1910) supposes the intermediate product
to be dihydroxyacetone, an isomeride of glyceraldehyde, but it
cannot be said that this has been proved to be the case; this
assumption is, however, rendered probable by the fact that
dihydroxyacetone is fermented readily and glyceraldehyde only
slowly.
It is worthy of note that the transformation of glucose into
alcohol + carbon dioxide can be effected by purely chemical
means, the various reactions requiring, however, different catalysts:
Glucose ^lactic acid (alkali as catalyst).
Lactic acid >acetaldehyde+ formic acid (sulphuric acid as
catalyst) .
Acetaldehyde-f formic acid alcohol+ carbon dioxide (rhodium
as catalyst).
(Buchner, Meisenheimer and S c h a d e , Chem.
Ber., 1906, 39, 4217; Schade, Zeitschr. f. physikal. Chem.,
1906, 57, 1.) Further, the author's investigations (Arkiv
for Kemi, 1911, 4, No. 8) show that, in ultra-violet light, lactic
acid undergoes decomposition into alcohol and carbon dioxide.
According to a new and interesting investigation by Franzen
and Steppuhn (Chem. Ber., 1911, 44, 2915), formic acid is
fermented as well as formed by living yeast and must hence be
taken into account as an intermediate product.
As substrate for fermentation, mannose, galactose, or fructose
may be used instead of glucose. Glucose and fructose exhibit
no difference in their velocity of decomposition, and in the case
of mannose there is only a slight deviation. Galactose, on the
SPECIAL CHEMISTRY OF THE ENZYMES 53
other hand, is fermented only by certain species of yeast, including
bottom fermentation beer-yeasts, and by their pressed juices;
the fermentation takes place far more slowly than that of glucose
(E. Fischer and Thierfelder, Chem. Ber., 1894,
27,2031; E. F. Armstrong, Proc. Roy. Soc., B, 1905,
76, 600; SI at or, Journ. Chem. Soc., 1908, 93, 217). As,
however, has been shown by S 1 a t o r and by Harden
and Norris (Proc. Roy. Soc., 1910, 82, 645), the capacity
of yeasts for fermenting galactose can be increased by culti-
vating them in solutions containing this sugar. Mention must
also be made of S 1 a t o r ' s view that the different hexoses
are attacked by different enzymes: glucose and fructose by
gluco-zymase, mannose by manno-zymase and galactose by
galacto-zymase.
The mechanism of alcoholic fermentation is considerably less
simple than was formerly supposed, a number of enzymes and
subsidiary substances taking part in the formation of alcohol
and carbon dioxide.
The first separation of zymase into a " zymase in a restricted
sense " and a lactacidase must be given up, since the formation
of lactic acid as an intermediate product has been shown to be
improbable. And a special enzyme has now to be assumed
for each of the changes indicated in the above scheme of reactions.
Of great importance for the elucidation of the nature of
fermentation is Harden and Young's discovery of the
co-enzyme of zymase. By filtration through a film of
gelatine under a pressure of 50 atmospheres, pressed yeast juice
can be divided into a filtrate and a residue, which are separately
inactive towards sugar but produce fermentation when again
mixed (Harden and Young, Proceedings of the Physiol.
Soc., Nov. 12, 1904, see Journ. of Physiol., 1904, 32, i; Proc. Roy.
Soc., B, 1906, 77, 405).
The substance in the dialysate resists boiling (thermostable)
and undergoes hydrolytic decomposition and hence destruction
by enzymes (lipases) of the yeast juice.
On the other hand, the zymase itself which does not traverse
the gelatine filter is destroyed when heated and is presumably
a protein substance, being attacked by the proteinases or pro-
teases of the yeast juice. From this attack it is protected by a
thermostable substance a ntiprotease (Buchner and
54 GENERAL CHEMISTRY OF THE ENZYMES
Haehn, Biochem. Z., 1910, 26, 171). This antiprotease is,
like the co-enzyme, destroyed by lipase, but is more stable than
the co-enzyme towards hydrolytic agents and towards heat.
Both enzyme and co-enzyme are precipitated from the yeast
juice by acetone, but the latter less readily than the former,
so that a certain degree of separation can be attained by frac-
tional precipitation ( B u c h n e r and Duchacek, Bio-
chem. Z., 1909, 15, 221).
Alcoholic fermentation by means of pressed yeast juice is
facilitated by the addition of a phosphate. Harden and
Young (Proc. Roy. Soc., B, 1906, 77, 405) have made the
important discovery that, during the period of enhanced fer-
mentation, the amount of carbon dioxide produced exceeds that
which would have been formed in the absence of phosphate
by a quantity exactly equivalent to the phosphate added
C0 2 : R 2 / HP0 4 .
These two investigators consider that the phosphate reacts
with the hexose in the yeast juice in the following manner:
(1)
+2H 2 0+C6H 10 4 (P0 4 R 2 ) 2 .
The result is an ester of hexosediphosphoric acid, the salts
of which have been more closely investigated by Young
(Biochem. Z., 1911, 32, 177).
During the fermentation, the hexosediphosphate accumulates
in the solution, but as soon as fermentation ceases, this ester
undergoes hydrolysis in the yeast juice, thus.
(2) C6Hi 4 (P0 4 R 2 ) 2 +2H 2 = C 6 Hi 2 06+2R 2 HP04.
This hydrolysis is effected by a hexosephosphatase." These
reactions will be considered further in Chapter VII.
Occurrence. Zymases do not only occur in yeasts, but are
extraordinarily widespread throughout the whole of the animal and
vegetable kingdoms. There is now scarcely room for doubt that the
combustion of sugar in the animal organism and also in higher plants
begins with decompositions completely analogous to those brought about
by yeast. Deviation from these occurs only in the final phase of the
reaction, since in living animal organs and living plants alcohol is formed
only when oxygen is lacking. On the other hand, the intramolecular
respiration of sugar is absolutely identical with alcoholic fermentation.
SPECIAL CHEMISTRY OF THE ENZYMES 55
This v.ew, as far as the higher plants are concerned, is due especially to
E. Godlewski, Palladin and Kostytschew. Also the
change known as glycolysis, occurring in the animal body, must be
closely allied to fermentation.
Animal glycolytic enzymes or zymases have been prepared especially
from the blood, spleen, pancreatic tissues and muscle.
Mme. N. Sieber (H., 1903, 39, 484; 1905, 44, 500) has
obtained three glycolytic enzymes in the form of stable powders
from blood-fibrin and spleen:
(a) soluble in water,
(b) soluble in neutral salt solutions, and
(c) soluble in water or alcohol (peroxydase) .
All these enzymes contain nitrogen and give the reactions
of the proteins. Their ash contains iron, manganese and phos-
phoric acid, but not copper. The second of them gave the fol-
lowing mean composition: C, 52%; H, 7-5% and N, 15%.
Tincture of guaiacum is turned blue directly by enzymes
(a) and (b), but only in presence of hydrogen peroxide by (c).
11 6 h m a n n and S p i t z e r ' s reagent (a dilute alkaline
solution of a-naphthol and paraphenylenediamine) is coloured
by all three enzymes in absence of hydrogen peroxide. It is
doubtful if these oxydase- or peroxydase-reactions are related
in any way to the ability to bring about the combustion of
sugar. In any case the essential constituents of the enzyme-
complexes obtained by Mme. Sieber are fermentati^on-
enzymes and not oxidation enzymes.
Further the glycolytic enzyme found by Cohnheim
(H., 1903, 39, 336; 1904, 42, 401) in muscle, from which he
extracted it in an inactive state, is not oxydasic in character.
It is a decided endo-enzyme like yeast-zymase and is obtained
from the frozen, subdivided muscle by pressing or by extraction
with an ice-cold, isotonic solution of sodium oxalate; the oxalic
acid must then be precipitated with the calculated quantity
of calcium chloride (H., 1906, 47, 253). Corresponding with
this enzyme, there exists a co-enzyme or activator which, like
that of yeast-zymase, is thermostable. Cohnheim pre-
pared it from the pancreatic tissue of the cat.
The objections raised by Glaus and E m b d e n (Hofm,
Beitr., 1906, 6, 214 and 343) to the investigations of Cohn-
heim have been refuted by the latter author. Reference
56 GENERAL CHEMISTRY OF THE ENZYMES
must be made to Stoklasa's work (Chem. Ber., 1905,38,
664; Arch. f. Hygiene, 1904, 50, 165) which likewise indicates
the existence of a zymase in the muscles and also in the milk.
It is, however, remarkable that such a capable experimenter
as A. Harden, working in conjunction with H . Maclean
(Journ. of Physiol., 1911, 42, 64), was unable to detect alcoholic
fermentation by animal tissues (liver, kidneys, pancreas, flesh,
etc.) or by juices or powders prepared from them.
That the intramolecular respiration of plants is to be regarded
as a zymase fermentation has already been mentioned (Pal-
ladin and Kostytschew, H., 1906, 48, 214; Kost-
ytschew, Bot. Ber., 1908, 26, 167). Also by means of
seedlings of Hordeum distichum, Pisum sativum
and Lupinus luteus, Stoklasa, Ernest and C h o -
c en sky (H., 1907, 50, 303; 1907, 51, 156) have clearly shown
the action of the enzymes of intramolecular respiration. The
arguments opposed to Stoklasa's earlier experiments can
scarcely be advanced against the work just referred to.
Pal lad in (Bot. Ber., 1905, 23, 240) holds the view that
the carbon dioxide respired by plants arises in three ways: 1.
By the enzymes combined with the protoplasm; Palladin
calls this portion, nucleo-carbon dioxide and the corresponding
enzymes, " carbonases." 2. By the protoplasm itself (apparently
directly) under the influence of various irritants irritant-carbon
dioxide. 3. By the action of oxydases. In another place
(Zeitschr. f. physikal. Chem., 1909, 69, 187, Arrhenius-
Festschrift), the author has indicated that he is unable to agree
entirely with P a 1 1 a d i n ' s views, especially as regards car-
bonase and the role of oxydases and reductases in the respiration
process. The opportunity must, however, not be neglected to
direct attention to the many remarkable observations com-
municated by Palladin to the Berichte der deut. botan.
Gesellschaft.
Preparation of yeast-juice. One kilo of
washed and well-pressed bottom-yeast is mixed with 1 kilo
of fine sand and 300 grms. of kieselguhr and is ground, in 4-6
lots, in a large mortar until the mass becomes soft and doughy.
In this way a large proportion of the yeast-cells are broken.
The dough is then enveloped in a press-cloth and pressed in
a hydraulic press, the pressure being raised to about 90 kilos
SPECIAL CHEMISTRY OF THE ENZYMES 57
per sq.cm. and maintained at this value for an hour. About
400 c.c. of a clear, pale-brown, viscous juice, containing only a
very small number of living cells, are thus obtained ( E d u a r d
and Hans Buchner and M . H a h n , Die Zymase-
garung, Munich, 1903, p. 58).
From the yeast-juice the zymase can then be precipitated
with alcohol or acetone, which, however, throws down a large
amount of proteins, carbohydrates and salts at the same time.
In order to obtain very active preparations, the yeast-juice must
be poured into a large excess of acetone (10 volumes) ; . only in
this way can the precipitate formed be obtained so free from
water that the chemical changes occurring in it are reduced
to a minimum (Buchner and Duchacek, Biochem. Z.,
1908, 15, 221).
The following simple method for obtaining zymase-prepara-
tions has been recently discovered by von Lebedew (C.
R., 1911, 152, 49; Bull. Soc. Chim., 1911, [iv], 9, 744):
It consists in drying the yeast at a temperature of 25-30 and
macerating with water for 2 hours at 35; the filtered liquid
exhibits considerable fermentative activity. According to the
author's experience, Munich yeast (from Schroder's factory)
and many other yeasts are suitable for this purpose, but this is
not the case with all yeasts.
According to Rinckleben (Chem. Zeitung, 1911, 35,
1149), zymase can also be obtained by plasmolysing fresh yeast
with glycerol.
Preparation of Permanent Yeast. In addi-
tion to those described above, another method is known by which
the fermentative activity of yeast-cells can be separated from
the true life functions. When yeast is introduced into alcohol
or acetone (Albert, Chem. Ber., 1900, 33, 3775), the cells
are killed without their fermenting power being destroyed. Well
pressed yeast (500 grms.) is thoroughly disintegrated, placed
on a hair-sieve and the whole dipped into a basin containing
3 litres of acetone. By alternately raising and lowering the
sieve in the liquid, and by means of a small brush, the whole of
the yeast-cells are passed through the sieve in 3 or 4 minutes.
The yeast is then left in the acetone for 10 minutes, being fre-
quently stirred meanwhile. Most of the liquid is next poured
off and the yeast, after being pumped as dry as possible, is again
58 GENERAL CHEMISTRY OF THE ENZYMES
treated with acetone, filtered and drained. The residue is well
kneaded with 250 c.c. of ether, filtered, dried in the air, finely
ground and then dried for 24 hours at 45.
This preparation has been placed on the market under the name
of " zymin" by A. Schroder, of Munich.
The best antiseptics to use with yeast-juice are toluene and
thymol.
Lactic Acid B ac ter ia-zymase
This is the enzyme by means of which lactic acid bacteria
are enabled to decompose sugar into lactic acid. The enzymic
nature of this transformation was demonstrated by Buchner
and Meisenheimer (Chem. Ber., 1903, 36, 635; Lieb.
Ann., 1906, 349, 125); they succeeded in obtaining an active
permanent preparation of Bacillus Delbriickii which,
however, only gave rise to small quantities of ^-lactic acid.
Preparation. The organism was cultivated at 40-45
in wort prepared from malt and rye.
The bacteria were subsequently separated by means of a
centrifuge and dried on a porous tile. The mass was then intro-
duced into 15-20 times its weight of acetone, with which it
was ground for 10-15 minutes, the bacteria being pumped dry,
washed with ether and dried in a vacuum.
OXYDASES
The action of oxydases is assumed in changes of very dif-
ferent kinds: oxidations of purine bases, conversion of alcohols
and aldehydes into acids, transformation of simple and substituted
phenols, amines, amino-acids, and derivatives of these com-
pounds into quinone derivatives.
The following kinds are thus to be distinguished:
1. Purine-oxydases.
2. Alcoholases.
3. Aldehydases.
4. Phenolases.
5. Tyrosinase.
SPECIAL CHEMISTRY OF THE ENZYMES 59
To these true oxydases must be added the peroxydases,
which can hardly be separated sharply from the oxydases, and,
especially with the latter, there remains still a good deal that
is not clear, so that the division of this section can be considered
only as a provisional one.
For the recognition of oxydases use has been made of a
number of reactions, some of which are referred to below; they
are not, however, given by all oxydases, which are, to a
great extent, specific in their action.
Blue coloration of guaiacum tincture or of a-guaiaconic acid; violet
coloration of tetramethylparaphenylenediamine; brown coloration of
m- and p-phenylenediamine alone or in presence of hydrogen peroxide
(A so); reddening of aniline acetate (C. R., 1896, 123, 315) and blue
coloration of a-naphthol (B o u r q u e 1 o t , C. R., 1896, 123, 423; Soc.
BioL, 1896, 46, 896); reddening of alo'in (S c h a e r , Arch, der Pharm.,
1900, 38, 42; K a s 1 1 e); oxidation of phenolphthalin to phenolphthalein
(K a s 1 1 e and S h e d d , Amer. Chem. Journ., 1901, 26, 526) ; oxida-
tion of benzidine ; leucorosolic acid (K a s 1 1 e, Public Health and Marine
Hospital Service of the U. S. Hygienic Lab. Bull., 1906, No. 26, 7-22),
hydroquinone, pyrogallol, and guaiacol; oxidation of leuco-malachite
green to malachite green (quantitative, spectro-photometric method of
von Czyhlarz and von Fii r t h , Hofm. Beitr., 1907, 10, 358);
oxidation of aldehydes, e.g., salicylic aldehyde (S c h m i e d e b'e r g ,
Arch. f. exp. Path., 1881, 14, 288, 379), formaldehyde (Pohl; Arch. f.
exp. Path., 1896, 38, 65); oxidation of arsenious to arsenic acid; forma-
tion of diaminophenacin from o-phenylenediamine, and of indophenol
from p-phenylenediamine and a-naphthol (R 6 h m a n n and
S p i t z e r , Chem. Ber., 1895, 28, 567).
The separation of iodine from potassium iodide has also been regarded
as an oxydase reaction. But this action, as has been pointed out by
A s o and more recently by Wolff and d e S t o e c k 1 i n , C. R.,
1908, 146, 1415), must be attributed to the nitrous acid occurring in
plant juices.
As regards the oxidising enzymes of the purine
bases, the action of xanthine - oxydase is best under-
stood, owing especially to the work of B u r i a n . This enzyme
does not attack uric acid, which is so readily broken down by
ordinary oxidising agents. B u r i a n supposes it to be quite a
general oxidising enzyme for (cyclic?) amidines which is unable
to attack the double linking between the (4) and (5) carbon
atoms of the purine nucleus:
60 GENERAL CHEMISTRY OF THE ENZYMES
NH CO NH CO NH CO
CH C (4) NH\ CO C NHv CO C NH\
|| || >CH | || >CH | || >CO
N C (5) W NH C W NH C NEK
Hypoxanthine Xanthine Uric acid
An enzyme which oxidises uric acid to
allantoin was obtained by Wiechowski and Wiener
(Hofm. Beitr., 1907, 9, 232, 247, 295) from animal organs by
treating the powdered organ (the cells being completely ruptured)
with 05% soda solution.
The nitrates obtained after grinding with the soda solution
are inactive, but if the emulsions are subjected to dialysis into
0-05% soda solution for 5-6 days, the enzyme of dog's liver
passes completely, and that of ox-kidneys partially, into the
filtrate. Enzymic liquids were also obtained by centrifugating
the emulsions. Repeated precipitation and nitration yielded a
protein-free preparation, which exhibited the total enzymic
activity of the starting material.
Alcoholase : Oxydase of Acetic Bacteria.
This enzyme oxidises ethyl alcohol to acetic acid and thus
catalyses a reaction 'for which energetic oxidising agents are
otherwise necessary. The enzyme has not yet been separated
from the bacteria, but its existence can be proved by killing
the bacteria with acetone (Buchner and M e i s e n -
h e i m e r , Chem. Ber., 1903, 36, 637; B u c h n e r and Gaunt,
Lieb. Ann., 1906, 349, 140).
Preparation. Large quantities of acetic bacteria (best
Bacterium aceti, which can always be obtained by
leaving beer in a glass dish exposed to the air) are cultivated
on beer-wort to which 4% of alcohol and 1% of acetic acid
have been added. As culture-vessels, shallow glass basins, as
wide as possible, are most suitable; these are covered with a
layer of cotton wool in order to prevent the entry of germs
from the air. In the course of 4 or 5 days after inocu-
lation, the bacteria form a fairly thick coating on the surface of
the nutrient solution. The clear liquid underneath is syphoned
off and the residue centrifuged to remove most of the liquid;
the brittle bacterial membranes are then washed superficially
with water and dried on porous tile. On the following day the
SPECIAL CHEMISTRY OF THE ENZYMES 61
mass is introduced into 10-20 times its weight of acetone, rubbed
to a fine powder and left for 10 minutes, after which it is filtered,
washed with ether and dried in a vacuum over sulphuric acid.
This procedure yields a yellowish powder which, however, only
oxidises very small quantities of ethyl alcohol.
Aldehydases. Aldehydes, e.g., benzaldehyde and sal-
icylic aldehyde, are oxidised by the extracts of many animal
organs, but it is doubtful if this is an enzyme action. See the
remarks of B a c h (Chem. Ber., 1904, 37, 3791) arid the results
of Dony-Henault and van Duuren (Bull. Acad.
roy. Belgique, 1907, 577).
Of the vegetable oxydases mention must first be made of
the laccase from the lac-tree, which was discovered by Y o s h i d a
and studied in detail by Bertrand, who gives the follow-
ing method for its preparation (Ann. de Chim. et de Phys.,
1897, [vii], 12, 115).
The thick sap of Rhus succedanea is mixed with
5-6 times its volume of alcohol, by which means the laccase
is precipitated, whilst the phenolic derivatives which produce
the blackening of the lac pass into the alcoholic solution. The
solution is filtered through a cloth and the residue washed on
the cloth several times with alcohol. It is then taken up in
cold distilled water, only a small quantity of black substance
remaining undissolved. The filtered solution is again precipitated
with alcohol in large excess, the precipitate being collected on
the filter and dried in a vacuum.
Laccase is obtained as a white substance having a neutral
reaction and readily soluble in water. Bertrand regards it
as a protein, although his preparation contained only 0-44%
of nitrogen. He assumes its composition to be as follows :
Moisture (determined at 120) 7-40%
Gum (arabans and galactans) 84-95
Laccase 2-50
Ash 5-17
The most essential constituent of the ash is manganese,
which is present to the extent of 2-5%.
Laccase is extremely sensitive to acids (cf. Chapter III) and
is destroyed by short boiling of its solution.
62 GENERAL CHEMISTRY OF THE ENZYMES
From other plants, such as Medicago sativa and
Lolium perenne, Bertrand obtained preparations
which he termed laccases, since, in neutral solution and in
presence of manganese salts, they accelerate the oxidation of
polyphenols. As the author has pointed out (H., 1909,
61, 1), they are quite different from R h u s - 1-acease.
Preparation of Medieago-lacca.se. The
fresh plants, at the commencement of the flowering stage, are
chopped up and pressed. On standing, the juice obtained
deposits dark flocks, from which it is separated by filtration.
Alcohol is then added, the abundant precipitate thus formed
being, to a large extent, taken up in water and again precipi-
tated. This procedure is repeated thrice, the preparation ob-
tained, after drying in a desiccator, being a white, dusty, highly
hygroscopic powder, soluble in water with great readiness.
According to E u 1 e r and B o 1 i n , it consists mainly of the
calcium salts of aliphatic hydroxy-acids. The separation of the
mixture, effected by fractional crystallisation of the correspond-
ing barium salts, shows it to contain glycollic, citric, malic, and
mesoxalic acids. The oxidising action of these salts is described
in Chapter IV.
Numerous attempts have been made to prepare laccases artificially.
According to Bertrand, the oxidising agent of many plants is
composed of manganese and a protein with a specific action. T r i 1 1 a t
(C. R., 1904, 138, 94, 274) assumed that the laccase regarded as a
specific enzyme can be replaced by any protein or, at any rate, by
certain classes of proteins; he has, however, no good foundation for this
view.
Especially on the ground of his own experiments on R h u s - laccase,
the author also regards as unsuccessful Dony-He*nault's attempts
to attribute the action of laccase to the alkalinity of the preparations
and thus to show that lac case-action is only an oxidation by means of
manganese and hydroxyl-ions. The laccase preparations obtained by
Bertrand's method are not alkaline, but are extremely active.
Further efforts to prepare artificial oxydases and peroxydases have
been made by M a r t i n a n d (C. R., 1909, 148, 182), Wo 1 f f (C. R.,
1908, 147, 745), and d e S t o e c k 1 i n (C. R., 1908, 147, 1489).
With other oxydases, so little has been done as regards puri-
fication and isolation that is has not been decided to what class
of bodies these substances belong
SPECIAL CHEMISTRY OF THE ENZYMES 63
Slowtzoff (H., 1900, 31, 227) attempted to prepare the " lac-
case" of potatoes in a pure state, his purest preparation containing
12- 8% NandO- 53% S.
Sarthou (J. de Pharm. et Chim., 1900, [vi], 11, 482, 583; 1901,
[vi], 13, 464) obtained quite different numbers for his schinoxydase,
namely, 6-28% N, 0-2% S, and 1-34% ash, from which he concluded
the enzyme to be a nucleoprotein. A s o and L o e w regard the
oxydases as albumoses. But Rosenfeld's investigations (Disser-
tation, St. Petersburg, 1906) on the oxydase of the radish (Raphanus
s a t i v u s) appear to indicate that this enzyme does not belong to
the proteins. According to R o s e n f e 1 d the oxydase would be a
crystalline substance, containing C, N, S, P, and K but not Fe or Mg,
S pn t z e r came to the conclusion that the oxydase of the liver
is a nucleoprotein, but this enzyme, which effects the oxidation of
salicylic aldehyde to salicylic acid, was further purified by J a c o b y
(H., 1900, 30, 135). It is found to be soluble in water and non-diffusible,
and to become inactive on heating, while it does not give the reactions
characteristic of the proteins.
According to Tschirch and Stevens (Arch, der Pharm., 1905,
43, 504), the oxydase of Japanese lac shows the pyrrole reaction.
Bach and C h o d a t are of opinion that the oxydase consists of a
peroxydase and an oxygenase, the former alone giving the pyrrole
reaction (Bach, Chem. Ber., 1908, 41, 226).
Theoenoxydaseof apples (L i n d e t , C. R., 1895, 120, 370),
pears, plums, grapes, and the fungus Botrytis cinerea, parasitic
to grapes, must be a tannin-oxydase ; by its action the flesh of the
fruit is turned brown on exposure to the air.
Oxydases of doubtful individuality have been detected in
numerous plants, e.g., Arum maculatum, olives (" olease ")>
barley and malt (" spermase "), coffee beans and yeast.
Should B a 1 1 e 1 1 i ' s observation (C. R., 1904, 138, 651) on
the oxidation of formic acid to carbonic acid by oxydases in
presence of hydrogen peroxide be confirmed, an interesting
organic oxidation will present itself. In this connection, mention
may be made of Loevenhart's discovery (Chem. Ber.,
1906, 39, 130) that formic acid is oxidised to carbon dioxide
by hydrogen peroxide in presence of iron, copper, etc.
A very remarkable oxidation, which is not yet understood,
has been described by Z a 1 e s k i and R e i n h a r d (Bio-
chem. Z., 1911, 33, 449); it consists in the oxidation of oxalic
acid in 1% solution to carbonic acid.
64 GENERAL CHEMISTRY OF THE ENZYMES
For information concerning other oxydases, see Battelli
and Stern's resume in Ergeb. der Physiol., 1912, 12, 95.
Ty rosinase
(G.Bertrand, Bull. Soc. Chim., 1896, [iii], 15, 791.)
From tyrosine and its derivatives this enzyme forms, by oxidation,
melanins dark-coloured substances of unknown chemical com-
position.
Interesting data concerning the chemical aspect of the action
of tyrosinase have recently been obtained by Abderhalden
and Guggenheim (H., 1908, 54, 331). They were able
to show that ozone and tyrosine alone do not yield melanins,
but that these are synthesised by oxidation from tyrosine or
polypeptides containing it, on the one hand, and from phenols
or amino-acids on the other. Similar colorations are obtained
if the oxidation is effected with potassium dichromate instead
of tyrosinase, and it is hence probable that tyrosinase contains
an oxidising agent, the action of which is exerted along with
that of the amino-acids of the plant-juice.
Tyrosinase attacks not only Z-tyrosine itself, but also dl-
tyrosine, tyrosine anhydride (Bertrand and Rosen-
blatt; Chodat), and a large number of polypeptides
containing tyrosine. Suprarenin (adrenaline) is likewise oxidised,
and all the cresols, resorcinol, m-toluidine, o-, m- and p-xylenols,
thymol, carvacrol and naphthol; also, according to Ber-
trand, phenol.
The opinion expressed by Gonnermann that the
specific action of tyrosinase consists of a hydrolysis, which is
then followed by an oxidation (not specific), has been combated
by Chodat and Bach.
According to Chodat, Zahorski and F r e e d e -
rickz (Arch. Sci. phys. nat., 1909, 27, 306), the specificity of
tyrosinase is conditioned by the presence of an activator which
is stable to heat.
For the preparation of the enzyme, the disintegrated
fungus is extracted with water and the extract precipitated
with alcohol.
Occurrence. In numerous fungi of the species Boletus,
Russula, Lactarius, Coprinus, Paxillus, etc. Also
SPECIAL CHEMISTRY OF THE ENZYMES 65
in Merulius lacrimans, beet-juice, dahlia bulbs, potato
skins, and V i c i a f a b a . Tyrosinase is often accompanied by lac-
cases.
From sepia, C. Neuberg has extracted an enzyme
which resembles tyrosinase and acts on adrenaline.
PEROXYDASES
Under this name are included those substances which activate
peroxides. Their typical reaction is the transference of oxygen
from hydrogen peroxide to guaiaconic acid or to polyphenols.
Bach and Chodat (Chem. Ber., 1903, 36, 600) have
given a method for the preparation of peroxydase from pump-
kins and horse-radish roots. Bach and Tscherniak
have recently (Chem. Ber., 1908, 41, 2345) obtained a peroxy-
dase in the following manner:
Thirty kilos of turnips were pounded up and pressed and
the juice obtained (20 litres) mixed with 2 litres of 96% alcohol
in order to coagulate the gummy matters. After nitration,
the alcoholic juice was precipitated by 130 litres of strong
alcohol, the precipitate being filtered off, washed with alcohol
and freed from precipitant in a vacuum. The crude peroxydase
thus obtained (52 grms.) was kneaded with 600 c.c. of water,
only a small portion of the substance passing into solution.
The undissolved residue was filtered off and washed with a little
water, and to 600 c.c. of the filtrate, containing only about 7
grms. of dry matter, 40 grms. of powdered basic lead acetate
were added; the precipitate was pumped off and the clear filtrate
(600 c.c.) treated with powdered sodium carbonate (21 grms.)
until no further turbidity was produced. The alkaline filtrate
was dialysed through parchment into distilled water. After
13 days the dialysate (670 c.c.) was mixed with 4-5 litres of
99% alcohol, and the precipitate thus formed collected, after
24 hours, on a hardened filter, washed with absolute alcohol
and freed from the latter in a vacuum.
This preparation contained 7 87% of water, 81 66% of organic
matter and 1-47% of ash; the percentage of nitrogen, calculated
on ash-free material, was 3 44.
The peroxydase prepared by E. de Stoecklinin
C h o d a t ' s laboratory from Cochlearia armoracia
66 GENERAL CHEMISTRY OF THE ENZYMES
contained 1 1 41% of water, 65 88% of organic matter and 22 71%
of ash; the nitrogen-content was 3-43%. The activating power
of this peroxydase was only one-tenth part of that of the above-
mentioned preparation of Bach. Neither de Stoecklin
nor C hod at (Schweiz. Woch. Chem. u. Pharm., 1905, 43)
obtained protein reactions with their peroxydases.
The author's experience (H., 1909, 61, 1) indicates
that dialysis is the best known method of purifying peroxydase
preparations. From horse-radish E u 1 e r and B o 1 i n ob-
tained a preparation which increased continuously in activity
when subjected to dialysis. The preparation showing the greatest
activity per unit of weight contained 10-4% N and 2-5% of ash.
Dialysis is rendered far more effective if the enzymic juice is
previously treated with kaolin or other suitable adsorption
agent in order to remove the proteins. D e 1 e a n o ( Biochem.
Z., 1909, 19, 266) proposes the use of colloidal ferric hydroxide
for this purpose. Bach (Chem. Ber., 1910, 43, 362) suggests
the preliminary removal of the gummy matters by means of
magnesium sulphate.
During recent years it has been repeatedly pointed out that
the action of peroxydases can be obtained partly by purely
inorganic materials (see Wolff, C. R., 1908, 146, 781 and
M a r t i n a n d , C. R., 1909, 148, 182), and partly by synthetic
organic preparations (de Stoecklin, C. R., 1908, 147,
1489).
As Moitessier, Lesser, von Fiirth and von
C z y h 1 a r z , and Bertrand and Rogozinski (C.
R., 1911, 152, 148) have shown, the well-known guaiacum-blue
reaction of the blood depends not on an enzyme, but on the
haemoglobin; it appears with undiminished intensity after boiling.
Oxyhsemoglobin acts not only as a peroxydase but as an oxy-
dase as well (de Stoecklin). Further, the oxidation
phenomena in milk cannot depend on the presence of a peroxydase.
Thermolabile peroxydases do, however, exist and these must
for the present be classed as enzymes.
SPECIAL CHEMISTRY OF THE ENZYMES 67
CATALASES
( O . L o e w , Rep. U. S. Dept. of Agric., 1901, No. 68.)
After the ability to decompose hydrogen peroxide had been long
regarded as a general property of the enzymes, O . L o e w
demonstrated the existence of special catalases. The discoverer
distinguished two catalases occurring in plants, viz., an a-catalase
which is not extractable by water and was regarded as a nucleo-
protein, and a g-catalase soluble in water which was regarded as
an albumose.
The chemical action of the catalases is analogous to that of
the colloidal metals ( B r e d i g ), molecular (inert) oxygen being
formed, together with water. Ethyl hydroperoxide is not decom-
posed (Bach and Chodat, Chem. Ber., 1903, 36, 1757).
Occurrence : Extremely widespread in the animal and vege-
table kingdoms. In blood (S enter; Lesser, Zeitschr. f. Biol.,
1906, 48, 1) ; in numerous organs (K a s 1 1 e and Loevenhart ;
Liebermann; Battelli,C. R., 1904,138,923); in milk(Raud-
nitz, Zeitschr. f. Biol., 1901, 42, 91; Reiss, Zeitschr. klin. Med.,
1905, 56, 1; Faitelowitz, Dissertation, Heidelberg, 1904).
Catalase is also found in virtually all plant-juices. Especially rich in
this enzyme are many leaves, e.g., of clover, Rosa, Picea, which
mainly contain the insoluble form of the enzyme; and certain seeds
e.g., of the apple and peach, in which the enzyme occurs principally in
the soluble form. Highly active catalases are obtained from fungi,
e.g., Boletus scaber (E u 1 e r , Arkiv for Kemi, 1904, 1, 357),
and from the lower fungi, yeasts, and bacteria.
The preparation is usually carried out by precipitating
the aqueous extracts with alcohol. S e n t e r gives the follow-
ing method of obtaining the catalase of the blood: Defibrinated
ox-blood is mixed with 10 times its volume of carbonated water
and left over night. It is then centrifuged and filtered and the
liquid precipitated with an equal volume of alcohol, the alcoholic
solution of haemoglobin being poured off and the reddish-brown
precipitate repeatedly washed with 50% alcohol. The precipitate
is dried in a vacuum, ground to a fine powder, stirred with
water and allowed to stand in ice for 2 or 3 days in order
that the enzyme may be extracted completely. The solution
is filtered through hardened filter-paper until it becomes quite
68 GENERAL CHEMISTRY OF THE ENZYMES
clear; the faintly yellow liquid thus obtained vigorously decom-
poses hydrogen peroxide with evolution of oxygen.
For the preparation of highly purified catalase-products,
pig- or ox-fat is the most suitable starting material. The fat is
disintegrated by means of sand and extracted with water at
about 30, the enzyme being then precipitated with alcohol and
further purified by repeatedly dissolving in water and precip-
itating with alcohol (Euler, Arkiv for Kemi, 1904, 1, 357;
Bach, Chem. Ber., 1905, 38, 1878).
In his first experiments ( 1 o c . c i t . ) the author obtained
a preparation which still showed faint protein reactions and
contained 14-5% N and 1-2% S but no phosphorus. A prepa-
ration made from defibrinated blood and similarly purified gave
14-1% N. A continuation (not yet published) of this investi-
gation has yielded enzyme-preparations which are certainly of
a higher degree of purity. The nitrogen-content diminishes as
the purification proceeds, the final product, which exhibits con-
siderable activity, containing 6 2% N and exhibiting M i 1 1 o n 's
and M o 1 i s c h ' s reactions. Fat-catalase appears therefore
to be not a protein, but mention must be made of the
opposite results obtained by Bach (loc. cit.). The question
of the protein character of catalase hence requires further
investigation.
Reducin.g Enzymes (Reductases; Reducase)
A substance termed " philothion " was described by d e
Rey-Pailhade and was regarded by him and also by
Pozzi-Escot as a reducing enzyme. With this view,
however, the author is unable to agree, since the reactions described
by de Rey-Pailhade and Pozzi-Escot are not
enzymic in character. The existence of reducing enzymes has,
indeed, not yet been demonstrated with absolute certainty.
In most cases in which reduction has been observed, no attempt
has been made to show that it is really due to an enzyme-action,
i. e., a catalytic reaction, and is not merely a stoichiometric
reduction by a readily oxidisable substance.
A substance which, at 70, accelerates the reduction of meth-
ylene blue by formaldehyde, has been found to exist in milk
(Schardinger). That this is a catalytic action has been
SPECIAL CHEMISTRY OF THE ENZYMES 69
rendered probable by the investigations of S. Oppenheimer
(Arb. a. d. Inst. f. exp. Therapie in Frankfurt, 1908, 4) and
of Trommsdorff (Centralbl. f. Bakt., 1909, 49, 291).
Schardinger's reaction is also hastened by colloidal
platinum or iridium ( B r e d i g and S o m m e r , Zeitschr.
f. physikal. Chem., 1910, 70, 34).
Bach has recently (Biochem. Z., 1911, 31, 443) endeavoured
to ascertain if Schardinger's enzyme, for which he
proposes the name perhydridase, is related to the reduc-
ing enzymes of the liver and other tissues. He is of the opinion
that the " reducase " of the liver is a mixture of enzymes and
contains that of Schardinger. Further, the same reaction
underlies the action of the systems : palladium methylene blue
hypophosphate water; palladium methylene blue aldehyde
water; milk-enzyme methylene blue aldehyde water, this
reaction consisting in decomposition of the water by the oxidis-
able substance with the help of a catalyst which forms a labile,
strongly reducing compound with the hydrogen of the water.
In the researches of Abelous and his collaborators on
horse-kidneys, bacterial action was not excluded. Indeed, as
Abelous himself stated and the author has confirmed, no
reduction takes place if the extract is filtered through a Chamber-
land candle (cf . the work of Heffter, Arch. f. exp. Path.,
Schmiedeberg- Festschrift, 1908, 29).
In the roots of plants and in seedlings, strongly reducing
substances occur but these are not enzymic metabolic products
(O. Schreiner and M. Sullivan). The same holds
for reduction by micro-organisms. K a s 1 1 e and E 1 v o v e
(Amer. Chem. Journ., 1904, 31, 606) have also given an interesting
study on the reducing actions of plant-juices.
APPENDIX
In the above short account, mention has only been made
of those enzymes, of the individuality and mode of action of
which something definite is known. A large number of enzymes,
which have received special names, have not been considered,
as they do not differ essentially from the better-known repre-
sentatives of the same groups.
But certain other enzymes, which have found no place in the
70 GENERAL CHEMISTRY OF THE ENZYMES
main enzyme groups, may be briefly referred to here, since their
further study appears to be not without interest.
1. An isomerising enzyme, which converts man-
nose into glucose, is thought by Gat in (Soc. BioL, 1908,
65, 903), to exist in the seeds of Borassus flabelli-
f o r m i s .
2. According to J. Parnas (Biochem. Z., 1910, 28, 274)
the liver contains a soluble enzyme which is able to accelerate
Cannizzaro's aldehyde transformation, i.e., it converts aldehydes
anaerobically into the corresponding alcohols and acids. Par-
nas suggests the name aldehydemutase, whilst
Battelli and Stern (Biochem. Z., 1910, 29, 130) apply
the term aldehydase to a similar enzyme studied by
them.
3. Kotake (H., 1908, 57, 378) refers to an enzyme of
ox-liver which demethylates caffeine, giving xan-
thine, hypoxanthine, ^-methylxanthine, etc.
4. In the kidneys and liver, Gottlieb and Stangas-
singer (H., 1907, 52, 1; 1908, 55, 295, 322) found substances
which convert creatine into creatinine and are
apparently enzymic in character.
CHAPTER II
PHYSICAL PROPERTIES OF THE ENZYMES
ALTHOUGH the courses followed by most enzymic reactions
can be represented by formulae which hold for catalyses in homo-
geneous systems, and although also dynamics as yet affords little
means of taking the state of solution or the colloidal condition
of the enzymes into account, yet, in experiments with enzymic
liquids, adsorption phenomena always make themselves more
or less strongly felt and have, indeed, a determining influence
on the general chemical behaviour of the enzymes.
Wherever a liquid is bounded by a vaporous space, there
is formed at the surface a layer possessing properties different
from those of the body of the liquid this layer is termed the
surface-layer. The latter has a tendency to diminish, and it
is to this that the well-known capillary phenomena are due;
on the interior of the liquid a pressure is exerted, termed the
internal pressure.
The thickness of this surface-layer, which differs from the
remainder of the liquid, is very small.
The pressure with which the surface-layer presses on the
internal liquid is also very small, unless the relation between
the surface of a substance and its volume " the specific sur-
face " is very large, i.e., the distribution of the substance is
considerable. The latter is especially the case with the so-
called colloidal solutions.
1 We shall begin with a short theoretical consideration of
surface phenomena, employing the method of representation
given by Maxwell.
The wire EF (Fig. 1) is to be regarded as capable of moving
freely along the rectangular wire A BCD. Within the frame
EBCF is a layer of liquid which, in consequence of the surface-
tension, tends to diminish and so draw the side EF upwards.
71
72 GENERAL CHEMISTRY OF THE ENZYMES
The weight is so chosen that it exactly compensates the pull
of the liquid layer on the wire; increase of the load G would
then result in rupture of the layer and
decrease, in rise of EF.
The pull of the layer is caused by
its surface-tension, and, since both
surfaces of the layer are active, cor-
responds with double the surface-
tension.
If now the movable wire is displaced,
by means of the weight G, from its
F IG i highest position BC to the position EF,
an amount of work has been done
against the surface-tension which is proportional to G (made
equal to the surface-tension) and to the magnitude of the sur-
face produced. 1 This work evidently represents the surface-
energy and is given by :
Surface-energy = Surface-tension X surface.
The surface-tension may also be expressed by the energy
acting on unit-surface or by the force acting on
unit-length, and is usually given in dynes per cm.
Owing to the small absolute values possessed by the known
surface-tensions of liquids and aqueous solutions about 100
dvnes
the surface-energy of a liquid attains a considerable
cm.
magnitude only when the surface is large, and hence becomes
comparable with the other forms of energy of a substance only
when the ratio of surface to volume the " specific surface "
(Wo. Ostwald ) exceeds a certain value, namely, about
10,000.
The physiologically most important and most interesting
phenomena in this region are met with not in pure liquids, but
partly in solutions and partly in " heterogeneous systems," in
which a substance occurs in a very fine state of division and
which Wilh. Ostwald has named disperse systems.
Solutions. It can be stated generally that the con-
1 The surface-tension of liquids is independent of the expanse of the
surface.
PHYSICAL PROPERTIES OF THE ENZYMES 73
centration at the surface of a solution is different from that
prevailing inside the solution. This can be demonstrated experi-
mentally and also follows from thermodynamical considerations.
The derivation given below follows that given by M i 1 1 n e r
[Phil. Mag., 1907, (6), 13, 96] and was also deduced by H .
Freundlich in his" Kapillarchemie."
Suppose n molecules to be dissolved in a certain volume v. The
concentration has then not the uniform value --, but is greater (or
smaller) in the surface-layer (or interior). If the excess per unit of surface
w is indicated by a, the excess of concentration in the whole surface will
be aw and the concentration in the interior of the liquid, c = - .
v
Consideration of a thermodynamic cycle leads to the differential
equation:
dr dp
where T is the surface-tension and p the osmotic pressure.
This expresses the fact that the surface-tension changes with the
volume and, therefore, also with the concentration, if the osmotic
pressure changes with the magnitude of the surface, and this can only
happen if the concentration of the solution depends on the magnitude
of the surface and is therefore different from the concentration in the
latter.
If equation (I) is developed as a function of c, it gives rise to
dv dc dv v dc (II)
dt dr dc c
dv dc
and
-r~ = T'7~- 'T" (HI)
aw dc aw v dc
The excess of concentration per unit of surface is hence given by
C?T <j dp
dc c dc'
(IV)
If the change cf surface-tension with concentration is known, a can
be calculated from (IV). If the osmotic pressure obeys the simple
gas-laws, then -j- = RT, and hence
- or .
74 GENERAL CHEMISTRY OF THE ENZYMES
So far as the surface-tension of inorganic solutions has been measured,
it exhibits a linear increase with the concentration, i.e., is positive
and constant and therefore a is negative and constant. Hence we
arrive at the result:
If the surface-tension of a solution dimin-
ishes with increasing concentration, the
dissolved substance is more abundant in
the surface; but if the surface-tension of
a solution increases with augmented con-
centration, the concentration is less in
the surface than in the interior.
The law is also expressed as follows :
A dissolved substance is adsorbed if it lowers the surface-
tension; in the opposite case the adsorption will be " negative."
It is, however, not advisable to designate as adsorption, the
accumulation of dissolved substance at the surface of the solvent.
To the substances which lower the surface-tension of water
belong most compounds which are not strong electrolytes, such
as alcohols, glycerol, fatty acids, etc. When the surface-con-
centration attains a certain value owing to the tendency of these
substances to collect at the surface of their aqueous solutions,
the head of the osmotic pressure is held in equilibrium.
Unlike these substances, many salts increase the surface-
tension of water towards air and, presumably, also towards other
media, so that an increase of the concentration at the surface,
like that just described, cannot then take place. On the other
hand, it must be pointed out that D o n n a n and Barker
(Proc. Roy. Soc., A, 1911, 85, 557) have recently found, in cer-
tain cases, values for the surface-concentration (adsorption)
which agree in order of magnitude with those calculated from
G i b b s ' s equation.
What is understood by the surface-tension of a solution
must be more strictly defined. Since the surface-tension depends
on the concentration and since in every fresh surface a con-
centration is gradually attained which differs from that inside
the liquid, it is evident that a newly-formed surface, which
has not reached a condition of concentration-equilibrium with
the interior of the solution, possesses a different surface-tension
PHYSICAL PROPERTIES OF THE ENZYMES 75
from one already in stable equilibrium. The latter value is
suitably termed the static and the former the dynamic
surface-tension.
ADSORPTION
Of the possible cases of adsorption, those exhibited at the
limiting surface between a solution and a solid body are of the
greatest interest in this connection. Use is often made of such
adsorptions in the study of enzymes.
These phenomena have, to some extent, been known for
a long time, although the facts have only recently been satis-
factorily collated.
Attempts have been made, especially during the last few years,
to conceive of adsorption as a capillary phenomenon. But the.
above thermodynamical law showing the connection between
the change of surface-tension and the adsorption has up to the
present not proved very fertile. In the experimental proof it
was necessary, except in one special case, to assume that the
surface-tension of water-gaseous space proceeded parallel with
that of water-adsorbent; and there was nothing to indicate that
this was the case. A " negative adsorption " which should,
according to the above theory, occur with electrolytes, has never
been observed at the surface of separation between salt solutions
and solid adsorbing material (H a g g 1 u n d , H., 1910, 64, 294),,
Quite recently Arrhenius (Medd. Nobel-Inst., 1911, 2,
7) has subjected the experimental results of Miss Frances
Homfray, A. Titoff and G. C. Schmidt to
calculation.
As regards the influence of the quantity of the adsorbing
material, it is the magnitude of its surface which is of
the first importance ; for one and the same preparation the quantity
of substance adsorbed is, under similar conditions, proportional
to the active surface. Of especial importance is the experimental
result of the investigations of G. C. Schmidt (Zeitschr.
f physikal. Chem., 1910, 74, 689), namely, that the quantity
adsorbed increases only to a maximum, no matter how high the
concentration of the surrounding solution rises. This maximum
is proportional to the amount of the adsorbing medium and
varies with its nature.
76 GENERAL CHEMISTRY OF THE ENZYMES
G. C. Schmidt has investigated the simplest mathe-
matical formulation of his results, namely,
dx *
where s represents the maximum adsorption, x the quantity
adsorbed, and c the concentration of the surrounding solution.
But the integral equation derived from the above expression
is not in agreement with the observations. On the other hand,
the formula deduced theoretically by Arrhenius,
7 dx _(sx)
K^J ,
dc x
holds generally for adsorption phenomena at low temperatures.
This formula, on integration, gives
logio -0-4343- =7- c.
s x s k
Here x represents the amount condensed on 1 grm. of the
adsorbing medium (charcoal), s the maximum value of this
amount, c the osmotic pressure of the solute (or the pressure
of the surrounding gas which is adsorbed), and k a constant.
Since the value of s is determined directly from the observations,
the formula contains only one arbitrary constant, whilst that
previously in general use, namely,
- = kc n ,
m
contains two, k and n. In spite of this, however, the new
formula agrees much better with the observations.
Another empirical formula for the adsorption-equilibrium
embracing a wider region was given by Freundlich (Zeitschr.
f . physikal. Chem., 1907, 57, 385) :
v i a ^ i a \
-log =X = a(-J
m a x \v /
In this expression a indicates the total quantity of solute and
v the volume of the liquid. The magnitude X is independent
PHYSICAL PROPERTIES OF THE ENZYMES 77
of the quantity of adsorbing substance, but is a function of
a and v, or, more strictly, of the ratio between them; a and n
are magnitudes depending only on the temperature and on the
nature of the solute.
The adsorption-equilibrium determined by the given formula
must be completely reversible and independent of the
path by which it is reached. The adsorbing medium charged
with adsorbed substance must give up the latter to the pure
solvent until a new equilibrium is attained.
This reversible adsorption is often followed by a consequent
phenomenon the fixing of the adsorbed substance brought
about partly by a change of this substance (which may become
insoluble, for instance) and partly by a chemical reaction with
the adsorbent a reaction which, in many cases, leads to the
destruction or denaturation of the adsorbed material. Many
colouring matters are fixed from true solutions, but this more
stable union takes place especially with colloids, in particular
with proteins, toxines and enzymes. This fixation is n o n -
reversible. With the toxines, the sum-total of the phe-
nomena of antitoxine-formation is highly involved and has given
rise to keen controversies. With enzymes, quite analogous
processes are known, e.g., the combination of trypsin and anti-
trypsin, and the fixing of various enzymes by charcoal ( S .
G . H e d i n , H., 1907, 50, 497), to which reference will be
made later.
It must here be mentioned that all adsorption phenomena
are by no means to be attributed to one and the same cause.
Besides the mechanical adsorptions already mentioned,
there exist a large number of adsorption phenomena caused
by chemical transformations, and this is especially the
case with acid and basic dyes, which are not adsorbed mechanically,
but combined chemically, by animal fibres. L. Michaelis
has repeatedly pointed out the chemical nature of many adsorp-
tion processes.
As Michaelis rightly stated (Oppenheimer's
Handbuch, II, 1, 390), of all known substances, charcoal and
cellulose are the only ones with which mechanical adsorption
occurs. " Almost all other substances known as adsorbents,
such as silicic acid, kieselguhr, kaolin, arsenic sulphide, mastic,
ferric hydroxide, clay and zirconium hydroxide, have practically
78 GENERAL CHEMISTRY OF THE ENZYMES
no mechanical adsorptive power and, in general, do not adsorb
electro-indifferent substances like alcohol, acetone and sugars.
They adsorb only substances which can occur in the form of
ions or of electrically-charged suspensions, the adsorption taking
place only in accordance with their electrical charge. Thus
silicic acid adsorbs only such substances as migrate towards
the cathode, and ferric hydroxide only those migrating to the
anode." With these substances, then, it is always a question of
a neutralisation between an acid and a basic compound at the sur-
face of the adsorbing medium. According to M i c h a e 1 i s
and R o n a (Biochem. Z., 1908, 15, 196), even adsorption by
charcoal is not always purely mechanical, but sometimes takes
place by means of electrical forces.
If two substances are adsorbed from a solution by an adsorp-
tion medium, they may replace one another (Michaelis).
This fact is evidently closely related toG. C. Schmidt's
discovery that the adsorption reaches a maximum. This phe-
nomenon plays a part in the experiments of H e d i n (H.,
1909, 63, 143), who found that the retardation of rennet-action
by charcoal is counteracted by other substances, e.g., serum,
white of egg, etc.
Colloids. The adsorption phenomena of greatest interest
in the study of the enzymes are those in which colloids take
part. On the one hand, these substances can, in the solid or
gelatinised condition, function as adsorption media, and, on
the other, they are themselves largely adsorbed by solid sub-
stances.
In the following considerations, we may limit ourselves to
one of the two large groups into which colloids are divided,
namely, the so-called emulsion-colloids or emulsoids, and may
omit any description of the suspension-colloids or suspensoids
which play virtually no part in enzymic solutions or in investiga-
tions of these.
While the suspensoids, to which, for example, colloidal metals
belong, are classed with the true macroscopic suspensions, the
emulsoids are so closely related to the crystalloids that no sharp
limit can be drawn between them. With a number of classes
of bodies, increase of molecular weight is accompanied by the
appearance of a tendency to form complexes and thus pass into
PHYSICAL PROPERTIES OF THE ENZYMES 79
the colloidal state. Good examples of this are presented by
the condensation products of glucose dextrin and starch
E. Fischer's polypeptides, and, according toF.Krafft's
investigations (Chem. Ber., 1895, 28, 2566; 1896, 29, 1328)
especially the fatty acids. Whilst the lower fatty acids exhibit
normal ionisation and normal osmotic pressure, sodium laurate,
Ci2H2s02Na, for example, occurs principally in doubled molecules
and sodium oleate, CisHssCbNa, in 20% solution, produces
no measurable elevation of the boiling point and thus behaves
as a substance of infinitely large molecular weight (Krafft).
The variation in properties is hence continuous from the emulsion-
colloids to the crystalloids. The osmotic pressure and the
magnitudes related to it are very small, even on the basis of
the simplest possible molecular formula, and become still smaller
with the increasing tendency to complex-formation accompanying
increase of molecular weight. The values then often fall within
those due to inefficient methods of purification or within
the unavoidable limits of error, or else are of little significance
owing to the material employed being chemically ill-defined.
But where highly-condensed substances, such as starch, glycogen,
etc., can be obtained in a state of considerable purity, the
depressions of freezing point and elevations of boiling point
indicate molecular weights of at least 100,000, these values
being only minimal ones.
As regards the molecular weights of the enzymes with the
exception of the oxydases of Medicago which were inves-
tigated by the author and B o 1 i n (H., 1909, 61, 1) nothing
certain is as yet known. Great care should be exercised in
drawing conclusions concerning the molecular magnitudes of
enzymes from the diffusion experiments of R. 0. Herzog
and Kasarnowski (Zeitschr. f. Elektrochem., 1907, 13,
527; Biochem. Z., 1908. 11, 172) on commercial enzyme-prepa-
rations, partly on account of the very small enzyme-contents
of these preparations and partly owing to the considerable
weakening to which they must have been subjected during the
investigations. With one of their purest invertase preparations,
Euler and Kullberg (H., 1911, 73, 335) have made
diffusion experiments, the results being calculated by means
of the author's formula, D\/M = const. (Wied. Ann., 1897,
63, 273). For the coefficient of diffusion at 17 the value 0-037
80 GENERAL CHEMISTRY OF THE ENZYMES
was obtained and this gave with considerable extrapolation,
it is true the molecular weight as 27,000. This number repre-
sents a minimal value and holds for neutral solution.
With substances of such great molecular magnitudes,
Brownian movement begins to become visible. This move-
ment is shown by small particles suspended or dissolved in a
colloidal state in a solvent and consists of a continuous irregular
motion, which increases in rapidity with the fineness of the
suspended substance and with diminution of the internal friction
of the liquid.
This motion is to be regarded as an expression of the general
molecular motion of matter, as has been assumed by the kinetic
theory of heat since the middle of last century. Brownian
movement has been thoroughly studied in the case of suspension-
colloids, but not with emulsion-colloids.
In the ultramicroscope of Siedentopf and Z s i g -
m o n d y the emulsoids mostly show only a cone of diffused
light, i.e., the particles are generally too small to be perceived
ultramicroscopically as discrete forms.
Particles visible in the microscope have diameters down to about
2.10~ 5 cm. (microns). As submicrons are known those
particles which are perceptible only in the ultramicroscope, whilst
those the existence of which can only be perceived indirectly, even in
the ultramicroscope, are termed amicrons and have diameters of
from 5.10- 7 to 1.10~ 7 cm.
Everything goes to indicate that colloidal solutions are to
be regarded as mixtures of larger and smaller molecular aggregates,
which are able to change into one another with greater or less
rapidity. The most active chemically must always be the
smallest and therefore the really dissolved parts, which explains
why, in chemical transformations, colloidal substances follow
the laws of reaction derived theoretically for dissolved
substances.
The surface-tension of water is very considerably depressed
by emulsion-colloids, as is shown by qualitative observation.
According to Quincke (Wied. Ann., 1888, [iiil, 35, 580),
the value of a for a 10% tannic acid solution is 29% less than
that for water; the surface-tension of a dilute gelatine solution
is 28% less. Consequently these substances exhibit a marked
PHYSICAL PROPERTIES OF THE ENZYMES 81
tendency to concentrate at the surfaces of the solutions and are
strongly adsorbed.
As regards the first phenomenon, the accumulation of the
colloid at the surface, this is exercised in an especially striking
manner in the formation of solid membranes of peptone at the
surface of gelatinous peptone, either with or without some
chemical change.
Just as clearly is the concentration of emulsion-colloids at the surface
of aqueous solutions seen if the liquid is shaken and the composition
of the foam examined. Further, the great " head-retaining " properties
of many solutions, e.g., of albumins, constitute an indication of accu-
mulation of these substances.
SOLID, NEUTRAL ADSORPTION-MEDIA
Charcoal has often been used as an adsorbent for
enzymes. Thus, S . G . H e d i n (Bio-chemical Journ., 1906,
1, 484; 1907, 2, 81, 112; H., 1907, 50, 497) showed that trypsin
is adsorbed by animal charcoal; if a sufficient quantity of the
latter is employed, the adsorption is complete. Also, a t
first it is reversible. But later the process which has been
already mentioned and is not uncommon with adsorbed, organic
substances, viz., fixing, takes place. Fixing is a relatively
slow process and the amount of trypsin fixed increases with the
amount of animal charcoal and with rise of temperature. Whilst,
therefore, water is no longer able to extract the fixed trypsin
from the charcoal, casein is able to do so and, the higher the
temperature, the more completely is this the case. Charcoal
and talc act in a similar manner towards rennet (H e d i n, H.,
1909, 60, 364), which can also be removed from the charcoal by
the substrate.
According to the same author, the a- and g-proteases occurring
in ox-spleen are likewise taken up by animal charcoal in similar
proportions.
In agreement with H e d i n 's results are those of E.
Buchner andF. Klatte (Biochem. Z., 1908, 9, 436),
who found that trypsin is adsorbed from dilute solution by threads
of silk, wool and cotton, strips of linen, paper and agar-agar
and by asbestos and glass-wool.
82 GENERAL CHEMISTRY OF THE ENZYMES
Lipase can be completely removed from either an alkaline
or an acid solution by means of charcoal or kaolin (L. M i-
c h a e 1 i s and P. R o n a , Biochem. Z., 1907, 4, 11; L. M i-
c h a e 1 i s, ibid., 1908, 7, 488. See also L. M i c h a e 1 i s and
M. Ehrenreich, ibid., 1908, 10, 283). ,
Especially noteworthy is the fact that, in the adsorption
of colloids by charcoal, etc., the colloids exert a reciprocal influence,
and that even crystalloids, such as glucose, may diminish the
capacity of charcoal to take up other crystalloids, presumably
by altering the surface-tension.
On this fact depends the phenomenon, studied by H e d i n
(H., 1909, 63, 143), namely, that the retardation of rennet-action
produced by charcoal is prevented by various substances.
With these neutral adsorption media, which are generally
employed as powders, must be classed those which adsorb when
in the form of solid layers, such as cellulose as filter-paper (on
this depend G r u s s 's investigations [Bot. Ber., 1908, 26a,
191 and 1909, 27, 313] on the capillary analysis of enzymes),
and the materials of the various filter-candles, e.g., the Chamber-
land-filter. Their behaviour towards enzymes is characterised
principally by their ability or inability to retain the enzymes
when solutions of the latter are filtered. Here, too, the molec-
ular magnitude or the size of the particles of the substance to be
filtered comes into play, the filter acting not only as an adsorp-
tion medium but directly as a 'sieve; with emulsion-colloids,
however, the adsorption is usually the more important process.
Of great interest are the investigations of H o 1 d e r e r (C. R. r
1909, 149, 1153; 1910, 150, 230, 285 and 790), who showed that
the permeability of the Chamberland-filter for enzymes depends
on the concentration of the hydrogen ions in the solution. If
the solution is neutral towards phenolphthalein (OH' = 10~ 6 ),
no adsorption takes place, the filter being permeable; but if the
liquid is neutral to methyl orange, the filter-candle is impermeable.
This is found to be the case with invertase, catalase, pepsin and
emulsin. The following data show the behaviour of the enzymes
towards the most common filtering materials.
PHYSICAL PROPERTIES OF THE ENZYMES 83
Chamberland-filter
The following are retained :
Lipases of various origins (Fermi and Pernossi, Ann.
Inst. Pasteur, 1889, 3, 531).
Yeast-invertase (Fermi and Pernossi, ibid.)-
Zymase (B u c h n e r, Zymasegarung, Munich, 1903).
Pepsin and trypsin.
The following pass through :
Maltase (Croft Hill, Journ. Chem. Soc., 1898, 73, 636).
Stomach-steapsin (V o 1 h a r d , Zeitschr. f. klin. Med., 1901,
42, 414).
Liver-aldehydase (J a c o b y, H., 1900, 30, 135).
Proteinases of malt (Fernbach and Hubert, C. R., 1900,
130, 1783; 131, 293).
Parchment
These are retained:
Pepsin (Hammarsten, Maly's Jahresber., 1874, 3, 160).
Peroxydase (E u 1 e r and B o 1 i n , H., 1909, 61, 82.)
These pass through :
Invertin, amylase, and, slowly, emulsin, trypsin and pepsin
(Chodschajew, Arch, de Physiol., 1898, 30, 241).
Rennet and pepsin pass through unstretched amnion-membrane
(J a c o b y, Biochem. Z., 1906, 1, 53).
Rennet, invertin and catalase pass through intestinal membrane
(Vandevelde, Biochem. Z., 1906, 1, 408).
Rennet, invertin and catalase do not pass through cellulose
walls (thimbles from L e u n e , Paris) (Vandevelde,
loc. cit.).
Collodion-membranes
The following is retained :
Pepsin (S t r a d a , Ann. Inst. Pasteur, 1908, 22, 982).
The following pass through:
Emulsin and lactase (B i e r r y and Schaeffer, Soc.
Biol., 1907, 62, 723).
Trypsin partially: after activation with kinase, completely
(S t r a d a , 1 o c. cit.).
84 GENERAL CHEMISTRY OF THE ENZYMES
Since colloids in the sol condition do not, in general, diffuse
through animal membranes and colloidal skins, very different
membranes may be employed as colloid-filters.
In the gradation of the colloid-content, the permeability of a filter
may be varied at will by employing a substratum of cellulose, etc., so
that the solution may be fractionally filtered. On this principle B e c h -
hold constructed his ultra-filter, in which, as colloids, acetic collodion
and gelatine are especially used (Zeitschr. f. physikal. Chem., 1907, 60,
257; 1908, 64, 328).
Fibrin flocks have proved effectual for the adsorption of
many enzymes, such as rennet, pepsin (J a c o b y, Biochem.
Z., 1907, 4, 21) and trypsin (B u c h n e r and K 1 a 1 1 e, Bio-
chem. Z., 1908, 9, 436). Numerous other coagulated proteins
also exhibit marked adsorptive properties. (See also B a y 1 i s s ,
Adsorption in its relation to enzyme action, Kolloid. Zeitschr.,
1908, 3, 224.)
Solid acid or basic constituents. The chief
of these are, on the one hand, the hydroxides of ferric iron,
aluminium and magnesium, and certain of the so-called colloid
metals (Rona; Deleano, Biochem. Z., 1909, 19, 266), and,
on the other, silicic acid. In this case, the principal action is a
chemical union and not mechanical adsorption; this is shown
by the selective adsorption of these sols, acid substances being
vigorously adsorbed by the metallic hydroxides and basic ones
by silicic acid; the same regularity appears in the reciprocal
coagulation of the colloids.
Similar influences govern the adsorption of enzymes. As
M i c h a e 1 i s found (Biochem. Z., 1907, 7, 488), electro-
negative colloid solutions give with, say, invertin no precipita-
tion, whilst the hydroxides of iron and aluminium completely
adsorb this enzyme. Analogous behaviour is shown by pepsin
(Biochem. Z., 1908, 10, 283). On the other hand, the adsorp-
tion of amylase and saliva-diastase (ptyalin) depends on the
reaction of the medium. Zymase appears to be a neutral sub-
stance (M i c h a e 1 i s and Rona) and is consequently not
adsorbed by ferric hydroxide; but the co-enzyme of zymase
seems to be readily adsorbed by ferric hydroxide (R e s e n-
s c h e c k, Biochem. Z., 1908, 15, 1).
To the acid adsorption media belongs also kaolin, which
PHYSICAL PKOPERTIES OF THE ENZYMES 85
hence adsorbs mainly basic substances. Since the rule, that
acid and basic adsorption media adsorb respectively basic and
acid substances, has proved generally valid, we are able, from
observations on adsorption, to draw conclusions concerning
the electro-chemical nature or the charge of adsorbable substances,
in particular of the enzymes. These conclusions are remarkably
well confirmed by other facts.
Electric transference. Under the influence of
a difference of electric potential, emulsion-colloids migrate in
the same manner as particles suspended in water; this effect
is known as cataphoresis. But, whilst suspended particles
migrate, as a rule, to the positive pole and themselves assume a
negative charge, (according to C o e h n ' s rule, this behaviour
depends on the fact that the dielectric constant of these particles
is less than that of water), emulsion-colloids migrate partly to
the positive and partly to the negative pole. This is com-
prehensible if it is borne in mind that emulsion-colloids have,
as a rule, basic or acid properties, or as amphoteric electrolytes
may exhibit the one or the other character according to the
medium in which they exist. As with the ions, the charge which
they assume on ionisation determines the direction of migration.
Further, the velocity of migration is not appreciably different
from those of dissolved ions; according to Whitney and
Blake, for gelatine particles it has the value
25.10 ~ 5 cm./sec.
for 1 volt /cm., the corresponding value for sodium ions being
43.10~. On the other hand, in the cataphoresis of emulsion-
colloids there appear various marked disturbances, caused partly
by the migration of the particles from the two electrodes and their
precipitation in the medium as oppositely charged sols.
How great is the dependence of the electric transference
of proteins on the reaction of the medium is best seen from
Pauli's researches (Hofm. Beitr., 1906, 7, 531). Albumin
poor in electrolytes shows no motion under a pressure of 250
volts, whilst even inO -01 N-hydrochloric acid it assumes an electro-
positive, and in dilute alkali a negative character.
P a u 1 i and Handovsky (Biochem. Z., 1909, 18, 340)
and also M i c h a e 1 i s have recently subjected these questions
to a fresh and thorough investigation.
86 GENERAL CHEMISTRY OF THE ENZYMES
The most important results concerning the electric trans-
ference of enzymes are due to M i c h a e 1 i s. He found firstly
(Biochem. Z., 1909, 16, 81) that, independently of the reaction
of the medium, invertin migrates distinctly to the anode
and is hence decidedly acid in nature. Pepsin also
exhibits a strong negative character as, in neutral and even in
markedly acid solution, it migrates solely to the anode. Shortly
afterwards (Biochem. Z., 1909, 17, 831), it was also found possible,
with a concentration of hydrochloric acid exceeding 5 VN, to
obtain electric transference of pepsin to the cathode, agreement
with the adsorption analysis being therefore complete. On the
other hand, trypsin and diastase behave also in the electric field
as amphoteric substances, migrating to the anode or cathode
according to the reaction of the solution; diastase is, however,
more strongly positive and trypsin more strongly negative.
Michaelis (Biochem. Z., 1909, 19, 181; 1910, 28, 1)
has determined the " relative acidity'' of certain
enzymes. If
X a [undissociated albumin] = [H + ] [ Alb ~ ]
and
X 6 [undissociated albumin] = [OH ~ ] [ Alb + ] ,
then, in an iso-electric state, i.e., when equal members of positive
and negative albumin-ions are present,
K & [OH-]'
This quotient assumes the following values:
Malt-amylase, 1 Trypsin, 10 5 -10 8
Serum-albumin, 10 2 - 10 3 Pepsin, 5. 10 9
Yeast-invertase, oo .
The relation between the " iso-electric constant," /, and the
relative acidity, R, is expressed by the equation
*^W
where k w is the dissociation constant of water.
For a very pure pepsin, Pekelharing and Ringer
have recently (H., 1910, 75, 282) established the iso-electric
point.
PHYSICAL PROPEKTIES OF THE ENZYMES 87
Bierry, V. Henri and Schaeffer have also carried
out experiments on the transference of enzymes with dialysed
enzyme solutions (Soc. BioL, 1907, 63, 226; Biochem. Z., 1909,
16, 473). The enzymes investigated were: amylases of animal
and vegetable origin, invertin from yeast and from Helix
p o m a t i a, emulsin from almonds and from Helix pomatia,
lactase from Helix pomatia, rennet (H a n s e n 's) and
catalase from the liver. Only one of these enzymes, namely,
pancreas-amylase, migrated to the cathode, all the rest going to
the anode.
Even in 0-01N-sodium chloride solution, albumin assumes
an electro-positive character, whilst in dilute alkali it shows
electro-negative behaviour. According to P a u 1 i 's original
results, albumin poor in electrolytes shows no migration; but
more detailed investigations by P a u 1 i and Handovsky
(Biochem. Z., 1909, 18, 340) and, especially, by M i c h a e 1 i s
(ibid., 1909, 19, 181) show that neutral albumin or albumin at
the iso-electric point with an acidity of [H] = about 10 ~ 6 does
not remain stationary but migrates to both the anode and the
cathode at the same time.
Literature: Michaelis, Dynamik der Oberflachen,
Dresden, 1909.
The precipitating action of salts on the colloids has been
studied in great detail, especially as regards the influence of
alkali salts on the proteins.
The fundamental investigations on this subject and the
application of precipitation with salts to the fractionation of
mixtures of proteins are due to Hofmeister, who, as early
as 1887, arranged the salts in the order of their precipitating
actions. His work has been considerably extended in more
recent times by W o. P a u 1 i (Hofm. Beitr., 1902, 3, 225; 1903,
5, 27; 1905, 6, 233; 1906, 7, 531; Biochem. Z., 1909, 17, 235,
etc.).
It has been found that the precipitating properties of the salts
are composed additively of the actions of the cathions and anions.
The following series begins with the ion showing the greatest
precipitating, or the least dissolving power :
S0 4 , HP0 4 , CH 3 C0 2 , Cl, N0 3 , Br, I, CNS
Li, Na, K, NH 4 .
88 GENERAL CHEMISTRY OF THE ENZYMES
With the exception of LiCl, this series agrees perfectly with
the one given below which was obtained by the author
(Zeitschr. f. physikal. Chem., 1899, 31, 360 and 1904, 49, 303)
for the salting-out of non-electrolytes ; the table gives the mean,
relative, molecular depression of solubility (\ w \ s ): L, where
\ w is the solubility of the non-electrolyte in water and L its solubility
in a salt solution of normal concentration.
NH 4 NO 3 ........ KC1 ........... 0-23 iZnSO* ......... 0-31
KI ............. 0-02 iBaCl 2 ......... 0-24 |K 2 SO 4 ......... 0-32
KBr ........... 0-05 |CaCl 2 ......... 0-24 |Na 2 SO 4 ........ 0-35
KNO 3 .......... 0-08 NaCl ........... 0-25 Na 2 CO 3 ........ 0-36
NaNO 3 ......... 0-10 f(NH 4 ) 2 SO 4 ...... 0-29 NaOH ......... 0-36
LiCl ........... 0-21 |MgSO 4 ......... 0-31
It is therefore beyond doubt that the same phenomenon
is being dealt with in the two cases. It will be especially seen
that ammonium and magnesium sulphates, which are most
frequently used for the precipitation of proteins and also of
enzymes, are likewise active towards crystalloids.
The remarkable fact that the rapidity with which the electro-
lyte is added influences the completeness of the precipitation, has
been subjected to detailed study by Freundlich and
by H 6 b e r.
If small quantities of an acid or an alkali are added to the
protein solution, the precipitating actions of the salts are modified
and the order of the ions changed (Posternak, Ann. Inst.
Pasteur, 1901, 15, 85, 169, 570). The precipitation of proteins
by the salts of the alkaline earth metals also exhibits peculiarities,
which have been studied by P a u 1 i (Hofm. Beitr., 1903, 5, 27).
Unlike the precipitations produced by alkali metals or magnesium,
those effected by salts of the alkaline earths are irreversible ;
they differ however, distinctly from those brought about by
heavy metals. Further, in strongly acid solutions, especially
on addition of alkali salts, irreversible precipitations occur.
Especially marked is the effect of addition of acids on the
action of neutral salts on protein When a little acid is added
to carefully-dialysed serum-albumin, not only does the latter
become capable of migrating to the negative pole, but its coagulabil-
ity by heat and by alcohol is impaired. At the same- time its
viscosity undergoes considerable increase. Excess of acid,
however, restores the precipitability by alcohol and lowers the
PHYSICAL PROPERTIES OF THE ENZYMES 89
viscosity again. Addition of any neutral salt has the same effect
as excess of acid in restoring the coagulability of acid-albumin
by heat or alcohol and in diminishing the viscosity.
The emulsion-colloids are distinguished by the considerably
greater internal friction of their solutions from the pseudo-
solutions of the suspension-colloids, which often possess vis-
cosities only slightly higher than that of the pure medium. Very
small additions of salts or, more especially, of acids and bases,
produce marked changes in the viscosity of proteins and the con-
clusion must be drawn that protein- ions give rise to greater
internal friction than amphoteric protein.
The course of proteolysis has often been followed by measuring the
viscosity without, however, the parallelism between the composition of
the solution and its viscosity being sufficiently clearly proved.
Jellies. Many colloid-containing liquids which, to indi-
cate their consistency, are termed sols, solidify on addition
of salts, alcohol, etc., to an apparently homogeneous mass of
peculiar semi-solid consistency a so-called jelly. The best
examples are silicic acid and alumina among the suspension-
colloids and agar-agar and gelatine among the emulsion-colloids.
The homogeneity is, however, only apparent. Although by
no means in all cases, yet in many it can be seen under the micro-
scope that the solidification is accompanied by a de-mixing.
The course of this change has been followed microscopically by
Hardy. But no sharp limit exists between sols and gels and
P a u 1 i was right when he emphasised the fact that all grada-
tions exist between solid and liquid jellies, and that a jelly is
nothing but a thick sol (Biochem. Z., 1909, 18, 367).
According to P a u 1 i (Hofm. Beitr., 1902, 2, 1), salts or ions
exert the same relative influence on precipitation as on gelatinisa-
tion, so that the series, 864 Cl CNS, given above holds also
for the melting- or solidifying-point of gelatine. A corre-
sponding parallelism had formerly been observed by H o f-
m e i s t e r between precipitation and swelling.
CHAPTER III
ACTIVATORS (CO-ENZYMES), PARALYSORS AND
POISONS
FOR the occurrence of enzyme reactions, activators or
co-enzymes are undoubtedly of greater and more general impor-
tance than has been, until quite recently, supposed. In many
cases, enzymic processes do not take place without the help of
specific co-enzymes, which, indeed, always exert a considerable
influence on the course of the reaction; so that those chemical
substances which effect the activation or inactivation of the
enzymes must be studied qualitatively and quantitatively in
as complete a manner as possible. Sorensen and also
Hudson have recently emphasised the marked influence
of acids and bases, or of the concentrations of H + or OH~, on
enzyme action, and, by very complete investigations on invertase,
catalase and pepsin, have obtained numerical expression of this
influence. As regards the less general but none the less important
influence of neutral salts and non-electrolytes, no such com-
prehensive study has been made, so that a resume of the
numerous qualitative data must suffice.
The term co-enzyme has been applied to a number of sub-
stances which take part in enzymic reactions. But this name,
which has come into very general use, is not quite suitable,
since it characterises the substances as enzymes, whilst it refers
partly to inorganic and partly to organic, thermostable bodies
of known compositions. It is therefore best to use the term
" activator " for all substances which, specifically or otherwise,
participate with an enzyme in the acceleration of a reaction.
Following the ordinary conception and method of nomenclature
a distinction must be drawn between those bodies which convert
" zymogens " into the active state kinases and those which,
on the other hand, intensify enzyme actions. It may be, however,
90
ACTIVATORS, PARALYSORS AND POISONS 91
as will be mentioned later, that kinases do not act in an essen-
tially different manner from other activators.
KINASES OF UNKNOWN COMPOSITION
As is well known, tryptase, as it occurs in the pancreatic
juice, is activated by a constituent of the intestinal liquid
enterokinase 1 (P a w 1 o w and collaborators, 1900). Although
enterokinase is regarded by various investigators, especially
B a y 1 i s s and Starling (Journ. of Physiol., 1904, 32, 129)
and Z u n z , as an enzyme, Hamburger and H e k m a
(J. de Physiol. et Pathol. gen., 1902 and 1904) have adduced
important evidence in support of the view that the formation
of trypsin does not consist in a new enzyme action on trypsinogen,
but that a definite quantity of enterokinase can activate only
a certain amount of pepsinogen.
The preparation of pure enterokinase was attempted by
Delezenne (Soc. Biol., 1901, 53; 1902, 54). It can be
precipitated from intestinal juice by calcium phosphate or alcohol.
Also flocculent fibrin and red blood-corpuscles adsorb the kinase,
the former quantitatively. Unlike most activators, enterokinase
is n o t stable to heat, as, according to Hamburger and
H e km a (loc. cit.), it is destroyed at 67 in less than 3 hours;
but B i e r r y and Henri (Soc. Biol., 1902, 54, 667) assert
that it retains its activity after being heated to 120 for 20
minutes.
Enterokinase dissolves in 90% alcohol (Cohnheim, Arch.
Sci. Biol., St. Petersburg, 1904, 9, Suppl., 112).
Kinases which activate trypsinogen were found by H o n -
g a r d y (Arch, internat. de Physiol., 1906, 3, 360) in milk and
by Delezenne in leucocytes, bacteria and fungi and also
in fibrin (Soc. Biol., 1903, 55, 27 and 132).
According to Morawitz (Hofm. Beitr., 1903, 4, 381;
1904, 5, 133) and others (for the literature, see Buckmaster,
Science Progress, 1907, 2, 51), a kinase of unknown character
plays a part in the formation ofthrombin; it converts
thrombogen into a-prothrombin, which, in its turn, is changed
into thrombin by lime.
1 For its preparation, see B a y 1 i s s , Journ. of Physiol., 1904, 30, 80.
92 GENERAL CHEMISTRY OF THE ENZYMES
Thrombokinase is not stable to heat. According to investiga-
tions by E. W. A. Walker (Proceedings of the Physiol.
Soc., Dec. 16, 1905, see Journ. of Physiol., 1905, 33, xxi) coagu-
lated oxalate-plasma, which has been heated at 50 for 2
hours, coagulates n o t on addition of calcium chloride alone,
but when blood or fresh tissue-extract is added at the same time.
So that a thrombogen would be stable at 50, whilst thrombo-
kinase is destroyed at this temperature.
Walker found that saliva-amylase which had been
inactivated by heating at 50-55 could be re-activated by blood
and he therefore regarded this as the mutual action of a thermo-
stable enzymogen and a kinase sensitive to heat.
Further investigation of these phenomena is to be desired.
In most other cases e.g., in that of the esterases in which the
existence of " kinases " has been assumed from the results of
experiments with artificially inactivated enzymes, it is the action
of thermostable activators which has been observed.
That Rosenheim succeeded in separating the lipase of
the pancreas from an activator by filtration, has been already
mentioned (p. 10).
The composition of the organic activator s t i m u 1 i n,
mentioned by Danilewski and more closely examined by
Schapirow, is also unknown.
Cohnheim has recently found that an extract can be
obtained from the boiled pancreas of the cat which strongly
activates the glycolytic enzyme of the muscles. Nothing is,
however, known concerning the chemical nature of this interest-
ing activator. It is precipitated by phosphotungstic acid
(Hall, Amer. Journ. of Physiol., 1907, 18, 283).
SPECIAL ORGANIC ACTIVATORS
In the case of pancreas-lipase, the chemical nature of an
organic activator has recently been established. According
to the observations ofNencki, Pawlow and Bruno,
Rachford, Magnus, and Loevenhart (Journ. of
Biol. Chem., 1907, 2, 391), bile intensifies the hydrolysis of
fats by pancreatic juice; still greater effects were found by
D o n a t h (Hofm. Beitr., 1907, 10, 390) to be produced by salts
of the bile acids. These salts are contained in the co-enzyme,
ACTIVATORS, PARALYSORS AND POISONS 93
resistant to boiling, obtained by R. M a g n u s (H., 1902, 42,
149) from the liver. Lecithin has no action (Kalaboukoff
and T e r r o i n e, Soc. Biol., 1907, 63, 617) or only a slight one
(Loevenhart and Sou d e r, Journ. of Biol. Chem., 1907,
2, 415). A similar intensifying action is produced by the sodium
salts of the synthetic glycocholic and taurocholic acids. The
co-enzyme can be separated from liver-lipase by dialysis. It
must be emphasised that these substances accelerate the action
of pancreas-lipase specifically and have no action on the lipase
of the stomach (L a q u e u r, Hofm. Beitr., 1906, 8, 281) or on
that of the intestines (Boldyreff, Zentralbl, f. Physiol.,
1904, 18, 460; H., 1907, 50, 394).
Of some importance is the observation of O. Rosenheim
and Shaw-Mackenzie (Journ. of Physiol., 1910, 40)
that substances which exert a hsemolytic action, such as alcohol,
soaps, saponins, digitoxin, increase the action of pancreas-
lipase. Such activation is annulled by cholesterol. The hydro-
lytic action of pancreas-lipase is augmented also by blood-
serum.
The action of the amylases is also intensified by bile salts
(Wohlgemuth, Biochem. Z., 1909, 21, 447). The action
of these salts on the amylase of the pancreas is, as was pointed
out by Buglia (Biochem. Z., 1910, 25, 239), independent of
the concentration of the enzyme.
That bile contains also an activator for trypsin has been
long known (Rachford, Journ. of Physiol., 1899, 25, 165;
Delezenne, Soc. Biol., 1902, 54, 283; von Fiirth and
Schtitz, Hofm. Beitr., 1906, 9, 28; Wohlgemuth,
Biochem. Z., 1906, 2, 264).
It is, however, doubtful whether bile salts represent a specific
kinase, as it may be that they influence the
condition of solution of the substrate. Men-
tion must be made of D o n a t h 's view that bile salts do
not activate the ready-formed lipase, but accelerate the conver-
sion of the lipase-zymogen into the enzymic state (Hofm. Beitr.,
1907, 10, 390).
Amino-acids are noteworthy as activators of amyl-
ase (see Chapter IV; also Ford and G u t h r i e , Journ.
Chem. Soc., 1906, 89, 76; Journ. Inst. Brewing, 1908, 14, 61).
Pancreatic juice is also activated by amino-acids. W o h 1 g e-
94 GENERAL CHEMISTRY OF THE ENZYMES
m u t h (Biochem. Z., 1906, 2, 264) observed this effect with
glycocoll, alanine and other representatives of this group.
According to R e i c h e 1 and S p i r o (Hofm. Beitr., 1905,
7, 504), lecithin accelerates the action of rennet. Of much
greater importance is the activation to which z y m a s e is
subjected under the influence of lecithin and other organic
compounds of phosphorus. The investigations of Harden,
Young, and B u c h n e r and Meisenheimer have
shown that these phosphorus compounds constitute the active
constituent in boiled pressed yeast-juice (compare p. 54 and
Chapter IV).
ACIDS, BASES AND NEUTRAL SALTS
Between the actions of purely specific activators or catalysts
and the general actions of acids and bases a definite limit can
hardly be drawn. Thus, according to H o y e r ' s investigations
(H., 1906, 50, 414), the action of lactic acid on the lipase of the
castor-oil seed can also be produced by other acids, the action
being, however, greater than corresponds with the degree of
dissociation of this specific " seed-acid." Similar relations are
observed with pepsin.
Apart from the activation of the zymogens, acids and bases
can influence enzymic reactions in two different ways, which
must be clearly distinguished : firstly, the velocity of
the reaction is changed and reaches a well-defined max-
imum for a certain concentration of the hydrogen-ions; secondly,
acids and bases influence the stability or the decom-
position of the enzyme itself, the stability also
exhibiting a maximum for a certain concentration of the H 4 "
or OH ~ ions.
Pepsin requires the presence of a free acid as an absolutely
necessary activator. Pepsin solutions are, indeed, proteolytically
active only when they contain positive pepsin-ions. In the
organism it is the hydrochloric acid which converts the pepsin-
forming secretion of the mucous membrane of the stomach
L a n g 1 e y 's " pepsinogen " or pro-pepsin into the active
enzyme.
Numerous investigations have been made on the replacemene
of the hydrochloric acid ; of the older ones, those of P f 1 e i -
ACTIVATORS, PARALYSORS AND POISONS 95
d e r e r (Pfliig. Arch., 1897, 66, 605), von Moraczewski,
H a h n (Virch. Arch., 1894, 137, 597) and S j 6 q u i s t (Skand.
Arch. f.'Physiol., 1895, 5, 277) may be mentioned. The following
tables, from a paper by L a r i n (Biochem. Zentralbl., 1905, 1,
484), and from that of Sjoquist, give the acids arranged in the
order of magnitude of their accelerating action.
Larin Sjoquist
1. Hydrochloric acid 7. Lactic acid 1. Hydrochloric acid
2. Oxalic acid 8. Formic acid 2. Phosphoric acid
3. Nitric acid 9. Malic acid 3. Sulphuric acid
4. Sulphuric acid 10. Acetic acid 4. Lactic acid
5. Tartaric acid 11. Butyric acid
6. Citric acid 12. Valeric acid
The relative actions of the acids are also dependent on the
nature of the digested protein (Berg and G r i e s, Journ.
of Biol. Chem., 1907, 2, 489).
Attempts have naturally often been made to connect the
accelerating actions of the acids with their strengths (affinity
constants or degrees of dissociation). To the complicated nature
of the reaction is due the fact that, between these two magnitudes
no quantitative relation, but at most an approximate cor-
respondence, has been found. A large part of the acid must
form salts with the digesting protein and it appears that it is
just these salts which are the cause of peptic decomposition.
The hydrolysis of the protein salt, i.e., the hydrochloride, must
diminish and the concentration of the salt for a given quantity
of protein increase as the strength of the acid present increases.
Since the hydrolysis is governed by the general condition of
equilibrium
T2C 2 X Y 3 C 3 = YiCi X T4C 4
protein acid salt water
where
y 2 and C 2 are the degree of dissociation and the concentration of the protein
T3 ;: c 3 acid
* ; C * " " " salt
Y A- " water,
it is at once clear on what magnitudes the velocity of pepsin-
digestion would depend and in what manner, if the concentration
of the protein salt were the sole determining factor. But first
of all account must be taken of the action of the acid on the enzyme
96 GENERAL CHEMISTRY OF THE ENZYMES
molecule, which is thereby converted from the inactive (zymogen)
into the active condition, the acid presumably remaining com-
bined with the enzyme during the whole course of the digestion.
Especially striking is the slight activity of sulphuric acid
as shown in the above series; this is possibly to be attributed
to the harmful influence which Griitzner (Pfleiderer,
Pflug. Arch., 1897, 66, 605) found to be exerted by sulphates
even in very small amounts. On the other hand, the very strong
activating action of oxalic acid (W roblewski and others)
must be noticed. The position assigned by S j 6 q u i s t to
phosphoric acid is possibly related to the activating influence
often found to be exerted by phosphates.
Sorensen has made a thorough investigation of the
influence of the concentration of the hydrogen ions on the velocity
of digestion; this will be referred to in Chapter IV. It appears
that the optimal action takes place in solutions the concentra-
tion of which with regard to hydrogen ions is about 0-06-normal.
The concentration of the free hydrochloric acid of the gastric
juice was measured in 1889 by F. A. Hofmann (Zentralbl. f.
klin. Med., 1891, 11) by the physico-chemical inversion method.
There are, however, objections to the use of this method for such
measurements (compare Sorensen, Biochem. Z., 1909, 21,
144).
As regards the quantitative determination of the influence
of acids and bases, it is undoubtedly best to investigate the rela-
tion between the velocity of the reaction and the
concentration of the hydrogen ions in the
solution. As Sorensen has shown, the concentration of the
hydrogen ions is most conveniently measured either electro-
metrically or colorimetrically by means of indicators.
Whether the activation of a zymogen by acids is a process
differing from the acceleration of the action of an enzyme already
in the active state has not yet been clearly established. In so
far as the a u t h o r 's experiments go, no such difference exists
and in what follows these two effects will not be treated separately.
Reynolds Green (Proc. Roy. Soc., 1890, 48, 370)
assumed that an acid is necessary for the activation of the lipase-
zymogen of plants, and the subsequent lipolysis also depends on
the presence of dilute acid.
From the results of his experiments on the influence of acids
ACTIVATOKS, PAKALYSORS AND POISONS 97
on the splitting of fats by Ricinus-lipase, H o y e r (Chem. Ber.,
1904, 37, 1436) drew the conclusion that, for a given amount
of fat or enzyme a definite, absolute quantity of acid is necessary
for obtaining the optimal effect. Sulphuric, oxalic, formic,
acetic and butyric acids are about equal in their capacity for
initiating the enzyme action. Armstrong and O r m e r o d,
whose experimental numbers are quoted in the next chapter,
also found no connection between the activating actions of dif-
ferent acids and their dissociation constants. From the fact
that, for a constant quantity of seeds, a definite minimal amount
of acid is required for the maximum fat-splitting action, H o y e r
concluded that the acid reacts chemically with the seeds during
the decomposition of the fat.
V o 1 h a r d (Zeitschr. f. klin. Med., 1901, 42, 414 and 43,
397) has carried out a series of interesting experiments on the
sensitiveness of gastric lipase to acid and alkali. Gastric juice
hydrolyses fat in both neutral and acid solutions, a concentra-
tion corresponding with 0-1-normal hydrochloric acid being
required to diminish the action appreciably; on the other hand,
the juice is extremely sensitive to minimal quantities of sodium
hydroxide. The glycerine extract is, however, very
sensitive towards hydrochloric acid and much more resistant
to alkali. It may hence be concluded that the mucous membrane
of the stomach contains a lipasogen which, in its behaviour
towards acids and alkalis, differs from the stomach lipase (the
so-called steapsin) itself.
Blood-lipase, investigated by R o n a (Biochem. Z.,
1911, 33, 413), exhibits its optimal activity with a H + con-
centration of 1.10~ 7 0-26.10" 8 , i.e., with an approximately
neutral reaction.
According to L i n t n e r and others, the action of malt
diastase is accelerated only by excessively small quantities
of weak acids. Ford finds that the optimal action occurs in
neutral solution. Concentrations of acid as low as 0-001%
of hydrochloric acid, cause retardation (Effront, Cole).
But with pancreas-diastase, rather higher concen-
trations of hydrogen ions about 0-001-normal are necessary
for the activity to reach its maximum.
Saliva-amylase (ptyalin) was first examined
in its relations to acidity and alkalinity by Hammarsten.
98
GENERAL CHEMISTRY OF THE ENZYMES
Numerous subsequent investigations have been made, with vary-
ing results (Chittenden, L a n g 1 e y) . It is active in
faintly alkaline and more so in neutral solution, while dialysed
ptyalin acts on dialysed starch still better in an extremely faintly
acid solution (Cole, Journ. of Physiol., 1903, 30, 202, 281).
But even 0-001% of hydrochloric acid exerts a retarding action.
According to F o a 's measurements, saliva is approximately
neutral.
Inulinase also exhibits its optimal activity in a solution
having a very slight acid reaction (0-0001-normal HC1). Even
1-5% soda solution completely destroys the enzyme
(Bourquelot) .
Concerning the optimal acidity of invertase we have very
detailed information. Apart from the older work of O ' S u 1 -
1 i v a n and T o m p s o n and of Cole, two important series
of experiments have recently been made by Hudson and by
Sorensen.
The following curve (Fig. 2) is taken from H u d s o n 's
work; it refers to the temperature 32 and gives the total
601
20
.002 .004 .006
Concentration of H Cl
FlG. 2.
.008
.010
concentration of the hydrochloric acid in solution, i.e., the sum
of the free and combined (with traces of protein) acid.
ACTIVATORS, PARALYSORS AND POISONS
99
Hudson has proposed the following theory in explanation
of the influence of acids and bases on the activity of invertase
(Journ. Amer. Chem. Soc., 1910, 32, 1220). Starting from the
fact that invertase behaves as an amphoteric electrolyte and is
hence capable of combining with both acids and bases, he regards
the activity of the enzyme solution as proportional to the amount
of enzyme which is not so combined. The values of the activity
calculated in this way are found to correspond with the exper-
imental numbers.
Sorensen (Biochem. Z., 1909, 21, 144) found that the
optimal proportion of sulphuric acid varies
widely for different invertase solutions and, as would be expected,
increases with the nitrogen-content of the enzyme solution;
a similar relation would doubtless be found in the case of other
strong acids. On the other hand, the optimal concen-
tration of the free hydrogen ions was found
to be the same to within 0-00003 (p# = 4-4 4-6) in all the series
of experiments, quite independently of the proportion of proteins,
etc., in the invertase solution. The position of the optimum
may be seen from Fig. 3. The three series of experiments were
made with sulphuric acid at a temperature of 52.
8.5', 4.0 4.5 5.0 5.5 6.0 6.5
Exponent of the Hydrogen-ion Concentration
FIG. 3.
The concentration of the hydrogen ions influences also the
course of the reaction, i.e., the constancy of the values of k
100
GENERAL CHEMISTRY OF THE ENZYMES
calculated from the formula: k = -
(S 6 r e n s e n);
further reference will be made to this in Chapter IV.
As regards the influence of acidity on the destruction of
the invertase, E u 1 e r and .af U g g 1 a s showed that at 50
the stability is a maximum when the concentration of the hydro-
gen ions is 10~ 6 . At lower temperatures this optimum is,
naturally, not so well denned, as is shown by curves given by
Hudson and Paine (Journ. Amer. Chem. Soc., 1910, 32,
779). The numbers given by these investigators are as follows:
Concentration
(grm.-mols. per
litre) normal HC1.
Rate of destruction
of invertase
foX 1000
Concentration
(grm.-mols.per
litre).
Rate of destruction
of invertase,
foX 1000.
0-05
365
Distilled water
0-04
96
Normal NaOH
0-03
42
0-01
3
0-02
4
0-02
11
0-015
1
0-03
50
0-01
0-04
245 .
The rate of destruction, fo, was calculated from the activity A.
The destruction follows the formula for unimolecular reactions,
1 A
namely, log -r- = 2, where A is the activity of the invertase
t A. x
at the beginning of the destruction aiid x is the activity after
the destruction has proceeded for t minutes.
L a c t a s e. According to B i e r r y and S a 1 a z a r (C.R.,
1904, 139, 381) the optimal action takes place with an acidity
of 0-001 N-HC1; 0-01 N-acid has a retarding action.
Pectinase (Bertrand's pectase) shows no action at
all in 0-1% hydrochloric acid solution.
Among the proteolytic enzymes there are some especially
from plants which, as already mentioned, show their optimal
activity in a slightly acid solution. This is the case, according
to Wei s (H., 1900, 31, 79) and Lintner (Zeitschr. f. d.
gesamt. Brauwesen, 1902, 25, 365), with the enzyme of malt,
and, according to V i n e s (Annals of Bot., 1897, 11, 563; 1898,
12, 545; 1901, 15, 563; 1902, 16, 1; 1903, 17, 237, 597; 1904,
18, 289) with numerous plant-extracts containing ereptase and
with yeast-extract. The autolysis of the substance of germinat-
ing plants also proceeds best in acid solution (Butkewitsch).
ACTIVATORS, PARALYSORS AND POISONS 101
E ni u 1 s i n shows its optimal action in neutral solu-
tion, as A u 1 d has recently shown by exact experiments
(Journ. Chem. Soc., 1908, 93, 1251). For the decomposition of
salicin by emulsin, Vulquin and Martini* (Soc. BioL,
1911, 70, 763) give the optimal concentration of hydrogen ions
asO-36.10~ 4 0-41. 10~ 4 .
P a p a i n, according to older statements, acts best in neutral
solution; it is weakened by alkali, the activity being restored
by hydrochloric acid (H., 1907, 51, 488).
Similarly, the animal body contains, in addition to peptase,
proteolytic enzymes which exhibit their action in acid solution.
Among these are the proteolytic enzymes of the lymphatic glands,
kidneys and spleen which were discovered by H e d i n and
Rowland (H., 1901, 32, 341) and are retarded by alkali.
Numerous researches on the autolytic enzymes
are in agreement in indicating that autolysis takes place only
in acid solution or, at any rate, that it proceeds much more
rapidly in acid than in faintly alkaline solution (S c h w i e n i n g,
Virch. Arch., 1894, 136, 444; Hildebrandt, Hofm. Beitr.,
1904, 5, 463; von Drjewezki, Biochem. Z., 1906, 1,
299) . A detailed investigation of the influence of acids and alkalis
on autolysis is due to H e d i n.
Within certain limits of concentration, boric and salicylic
acids cause increase of the autolysis of the liver over that occurring
in chloroform-water. With the optimal concentration, the
following proportions of the total nitrogen pass into solution
(Y o s h i m o t o, H., 1909, 58, 341).
In chloroform- water 21 6%
In 1% boric acid solution 40-8%
In half -saturated salicylic acid solution 47-4%
Similar results were obtained by K i k k o j i (H., 1909,
63, 109).
According to Sachs (H., 1905, 46, 337) the nucleases act
best in faintly acid solution.
Pancreatic and intestinal juices, which act
preferably with a neutral or alkaline solution, are not quite
so sensitive to acids as the gastric juice is to alkalis. In aqueous
solution pure tryptase is rendered inactive by a 0-01-JV con-
centration of hydrogen ions. But presence of the substrate
102 GENERAL CHEMISTRY OF THE ENZYMES
diminishes the sensitiveness and 0-02% of lactic acid even
accelerates tryptic digestion (Lindberger). Hence tryptase
and ereptase hydrolyse proteins not only in alkaline or neutral
solutions, bufc in certain cases also in slightly acid solutions.
But it is found that even free carbonic acid retards the digestion.
With trypsin, the origin of the enzyme also causes irregularities
in this respect; such irregularities have led Pollak (Hofm.
Beitr., 1904, 6, 95) to assume the existence of a separate enzyme,
giutinase.
Just as the optimal acidity is given by the concentration
of the hydrogen ions in solution, so the optimal alkalinity is
given by the concentration of the free hydroxyl ions. From
the results of D i e t z e (Dissertation, Leipzig, 1900), K a n i t z
(H., 1902, 37, 75) has calculated that tryptic digestion is most
rapid when the solution has a concentration normal
with respect to hydroxyl ions.
These values, which were obtained for fibrin-digestion, cannot,
however, be applied immediately to all trypsin actions.
According to Kudo (Biochem. Z., 1909, 15, 473), tryptase-
digestion is retarded by an alkali-concentration of 0-0118%
or 0-003-normal. The reversible retarding action is accompanied
by an irreversible action, which destroys the tryptase.
In experiments on the velocity of decomposition of dipeptides
by pancreatin and by ereptase, the author (H., 1907, 57,
213) obtained very low results. Ereptase from pig-intestine
showed a marked alkalinity-optimum, as is seen from the following
figures obtained with glycylglycine :
0-1 N-glycylglycine. 5 grms. powdered ereptase per 100 c.c.
Concentration of alkali 0-04 0-05 0-075 0-10
Reaction constant, k X 1000 <0-05 7-0 6-2 2-6 0-2
With erepsin from germinating peas, the optimum was not so
sharp (Arkiv for Kemi, 1907, 2, No. 39).
Concentration of alkali 0-025 0-05 0-10
Reaction constant, k X 10000 0-7 10-3 9-0 5-7
When allowance is made for the salt-formation between the
alkali and the glycylglycine, the dissociation constant of which
as an acid amounts to 1-8X10" 8 , the first of these tables gives
ACTIVATORS, PARALYSORS AND POISONS 103
as the optimal concentration of the hydroxyl ions, -00002-normal.
(Combination of the erepsin with alkali is here disregarded.)
Abderhalden andKoelker (H., 1908, 54, 363)
have carried out similar experiments, some of the results of which
(series B, p. 380) are given below:
(a) 1-5 mol. NaOH (6) 1-0 mol. NaOH (c) Without NaOH, as
(calculated on the amount of dipeptide taken) control.
4-0 c.c. of a ^j mol. 4-0 c.c. of a ^ mol. 4-0 c.c. of a ^ mol.
solution of glycyl- solution of glycyl- solution of glycyl-
Z-tyrosine. Z-tyrosine. Z-tyrosine.
0*6 c.c. pancreatic 0-6 c.c. pancreatic 0-6 c.c. pancreatic
juice. juice. juice
0-07 c.c. intestinal 0-07 c.c. intestinal 0-07 c.c. intestinal
juice juice juice
55 c.c. N-NaOH 37 c.c. N-NaOH 1 6 c.c. water
Time in * "^ C<C ' water 1 "23 c.c. water
Minutes. Rotations.
6
+0-80
+0-73
+0-59
15
+0-81
+0-75
+0-57
41
+0-80
+0-64
+0-48
174
+0-76
+0-60
+0-40
260
+0-73
+0-54
+0-38
378
+0-58
+0-43
+0-23
1428
+0-49
-J-0-31
-f-0-09
These results show that even small quantities of alkali retard
the hydrolysis of glycyl-Z-tyrosine by pancreatic juice + intestinal
juice.
A number of investigations on the influence of alkalinity on the
decomposition of proteins and peptones by erepsin have been
carried out by Vernon (Journ. of PhysioL, 1903, 30, 330;
1904, 32, 33). He distinguishes two ereptases pancreatic and
intestinal both of which are accelerated by 4-1 2% of sodium
carbonate, the intestinal ereptase being at the same time irre-
versibly destroyed. The erepsins of different animals exhibit
varying sensitiveness towards alkali; the protective action of
the proteins, studied by V e r n o n (cf. p. 115) may here be the
determining factor.
Also the investigations of B a y 1 i s s (Arch. Sci. Biol. St.
Petersburg, 1904, 11, 261, Supplement) on casein indicate a
smaller optimal alkali-concentration than D i e t z e 's exper-
iments with fibrin.
We have as yet no clear conception concerning the mode
of action of the alkali in tryptic digestion. During the reaction
104 GENERAL CHEMISTRY OF THE ENZYMES
free alkali must disappear, since carboxyl groups are rendered
free by the division of the protein-molecule into amino-acids.
It might then be expected that this diminution of the hydroxyl
ions would be rendered apparent in the course of the reaction.
The obvious step is to test the applicability of one of the formulae
holding for negative auto-catalyses processes in which the
catalyst is used up by the reaction itself. It is found, however,
that such formulae do not correspond with the experimental
numbers; on the contrary, in the experiments of Taylor
and B a y 1 i s s, the coefficient k of the theoretical formula
1 7 a
k ln
t ax
remains comparatively constant. With the help of a gas chain,
T.B.Robertson and Schmidt (Journ. of Biol. Chem.,
1908, 5, 31) have recently studied the law according to which the
hydroxyl ions diminish in concentration during the digestion.
They find that this diminution may be expressed by a unimolec-
ular formula if the OH-concentration is greater than 10 ~ 6 and
by a bimolecular formula if this concentration lies between 10 ~ 6
and 10 ~ 7 . Definite conclusions in regard to the part played by
the OH-ions in the digestion cannot be drawn from these results.
Hexosephosphatese, the enzyme that effects the
esterification of the hexoses with phosphoric acid acts only in
neutral or alkaline solution (E u 1 e r and Kullberg, H., 1911,
74, 15).
Chymase (chymosin) is converted from the state
of zymogen into the active condition by acids ( H a m m a r -
s t e n, 1872) which, according to their efficiency in this respect,
are arranged in the following order (equimolecular proportions):
HC1, HNO 3 , H 2 S0 4 , lactic, acetic, H 3 P0 4 (L 6 r c h e r, Pfliig.
Arch., 1898, 69, 183). After the rennet is activated, it functions
in either neutral or alkaline solution. The differences between
rennets of various origins have been clearly indicated by a series
of investigations by G e r b e r (C. R., 1907-1910, 145-150;
see references on p. 48).
The action of vegetable rennases, which at all temperatures clot
raw milk less easily than boiled, is retarded by small quantities of
alkali and accelerated by larger quantities of acid. Those which act
on fresh milk with difficulty only at high temperatures are retarded by
ACTIVATORS, PARALYSORS AND POISONS 105
acids having a higher basicity than two and also by quite small pro-
portions of dibasic acids, larger proportions of which have an accelerating
action; all other acids exert an intensifying effect. But those chymosins
which clot raw milk more readily than boiled, are accelerated in their
action by all acids.
With certain (calciphile) vegetable rennets, e.g., that from the sap
of Madura aurantiaca, the clotting of both raw and boiled
milk is accelerated. In presence of basiphile rennet, the clotting of
raw milk is only slightly hastened by small doses of HC1 and is con-
siderably retarded by medium amounts of the acid; the action on boiled
milk is in all cases accelerated, but to a much less extent than with the
really calciphile rennases, such as chymosin from calf's stomach.
The concentration of the H'-ions in milk has recently been measured
electrometrically by van Dam (H., 1908, 58, 295) and found to be
14-0 32 X 10~ 6 ; the results obtained indicate that the time of clotting
is inversely proportional to this concentration.
Parachymase (parachymosin) seems to be much
more resistant to acids but much more easily destroyed by alkali,
than chymosin (Bang, Pfliig. Arch., 1900, 79, 425).
Sera showing slight t h r o in b i n action are activated by
either acids or alkalis (Arch. f. klin. Med., 79 and 80).
Z y m a s e-f er mentation is accelerated by small quan-
tities of alkali (B u c h n e r and R a p p , Chem. Ber., 1897,
30, 2668) and retarded by slight amounts of acid.
The remarkable sensitiveness of 1 a c c a s e to acid, which
was determined quantitatively by Bertrand, is shown
by the numbers given in the next chapter.
Peroxydases are also paralysed by acids, of which
larger quantities are required than in the case of laccase. The
paralysing effect of acids is approximately proportional to their
degree of dissociation (Bertrand and Rozenband,
C. R., 1909, 148, 297).
The influence of acids and alkalis on blood-catalases of various
origins was first studied in detail byJacobson (H., 1892,
16, 340). The author has compared the behaviour of the
catalases from fat and from Boletus s c a b e r (Hofm.
Beitr., 1905, 7, 1).
Senter (Zeitschr. f. physikal. Chem., 1903, 44, 257)
found that acids cause considerable retardation of the action of
catalase, without injuring the enzyme permanently. The length
of the incubation period the time during which the enzyme is
106 GENERAL CHEMISTRY OF THE ENZYMES
in contact with the acid before the hydrogen peroxide is added
has no substantial influence on the reaction. That very low
concentrations of acid have a large effect is shown by the following
figures :
Concentration of acid Velocity constant
1/10,000-normal HC1 0-0011
1/20,000-normal HC1 0-0075
I/ oo -normal HC1 0-0100
With hydrochloric and nitric acids the retarding action,
according to S e n t e r , varies very nearly proportionally with the
third power of the acid-concentration. But Faitelowitz
states that the " poisoning " of milk-cat alase by hydrochloric
acid is approximately proportional to the concentration of the
acid.
In connection with the action of acids and bases, mention
must be made of that of acid salts, of which the acid phos-
phates, carbonates, citrates, etc., have more especially to be con-
sidered. In this case, also, the hydrogen ions usually constitute
the active component. Acid salts, or mixtures of neutral salts
with the corresponding (weak) acids, maintain the concentration
of the hydrogen ions in enzyme reactions constant within fairly
narrow limits, since in their presence small quantities of acids
or bases can only cause a slight change in the ionic equilibrium
of the solution. Such acid salts or mixtures of salts are appro-
priately termed " buffers." As will be readily understood,
amino-acids act in the same manner as acid salts, i.e., as buffers.
Activation by Salts. As has been pointed out by
Delezenne and E. Z u n z (Soc. Biol., 1906, 59, 477 and 60,
1070), calcium and magnesium salts exert a very marked activat-
ing and accelerating influence on tryptic digestion . [The harmful
effect of calcium chloride, found by Malfitano (C.R.,
1905, 141, 912) with the protease of splenic fever is perhaps due
to the optinmm concentration of calcium being exceeded.]
It is assumed by Pawl ow, by Bayliss and Star-
ling (Journ. of Physiol., 1905, 32, 129), and by Zunz that
the transformation of pro-enzymes by kinases or by calcium salts
represents a catalytic reaction. In support of this view E .
Zunz has made the following experiments :
ACTIVATORS, PARALYSORS AND POISONS 107
After inactive pancreatic juice had been left for 10-12 hours
in an incubator with calcium chloride or nitrate and the calcium
precipitated by ammonium oxalate, it was found to have a pro-
teolytic action. Under the same conditions the juice was not
activated in 1-2 hours. Analogous results were obtained with
magnesium salts.
When added in the form of soluble salts, sodium, potassium,
ammonium, zinc, beryllium, aluminium, cobalt, nickel, iron,
manganese, uranium and copper had no effect. With salts of
caesium, rubidium and lithium, an activating influence was shown,
but not regularly. For the numerous detailed results obtained
by Z u n z with regard to the activation of pancreatic juice,
reference should be made to his complete monograph " Recherches
sur Tactivation du sue pancreatique " (Brussels, 1906-1907).
a-Pro-thrombase is converted by calcium salts into thrombase
(M o r a w i t z, Hofm. Beitr., 1903, 4, 381; 1904, 5, 133). Also
pectinase, the vegetable clotting enzyme which transforms
pectin into pectinic acid, functions only in presence of calcium
(or barium or strontium) salts (Bertrand and M a 1 1 e v r e ,
C.R., 1894, 119, 1012; 1895, 120, 110 and 121, 726).
As, in addition to tryptase, ereptase (A b d e i h a 1 d e n,
Caemmerer and Pinkussohn, H., 1909, 59, 293) and
pancreatic lipase are intensified in their action by calcium chloride
(Pottevin, C. R., 1903, 136, 767; Kanitz, H., 1905, 46,
482), it can no longer be asserted that calcium is a strictly specific
activator. There is, however, no doubt that, in the cases named,
calcium cannot be replaced by the alkali metals.
That the well-known discoveries of Jacques Loeb
(Vorlesungen liber die Dynamik der Lebenserscheinungen, Leipzig,
1906) on the specific action of calcium salts are related to these
phenomena can scarcely be doubted.
On the other hand, purely chemical reactions are also known
in which calcium acts as a specific catalyst. Thus, O . L o e w
(Chem. Ber., 1888, 21, 270) found that lime is an especially
suitable agent for the condensation of formaldehyde to sugar.
The author has observed the same to be the case with the
formation of formate (Chem. Ber., 1905, 38, 2551).
A specific action, similar to that of calcium, appears to be
exerted in certain cases by magnesium. The important
part played by magnesium in plant life has been referred to by
108 GENERAL CHEMISTRY OF THE ENZYMES
Willstatter (Lieb. Ann., 1906, 350, 48) in a very interest-
ing paper.
Among the metals of biological importance as activators,
manganese must also be numbered. As was first established
by Bertrand, this metal is an essential constituent of the
oxydases. It occurs in company with hydroxy-acids, with
which, in the oxydases, it is combined. As manganese is known
to be a powerful oxidising catalyst, its function in the oxydases
is readily explained from a chemical standpoint.
The accelerating action of manganese on the decomposition
of hydrogen peroxide by catalases is also related to these
effects.
More remarkable is the fact that manganese salts hasten
enzymic hydrolyses. Thus, as H o y e r (Chem. ZentralbL,
1905, II, 582) found, the splitting of fat by vegetable Upases is
favoured by small quantities of manganese sulphate, whilst the
diastatic enzymes of serum and of pancreatic juice were shown
by A . G i g o n and T. Rosenberg (Skand. Arch, f . Physiol.,
1908, 20, 423) to be activated energetically by the same salt,
even in the concentration 0-001%.
According to K a y s e r and Marchand (C. R., 1907,
144, 574, 714; 1907, 145, 343; 1910, 151, 816) and also Fern-
bach and Lanzenberg, glucose is fermented more
quickly and completely in presence of manganese nitrate (0 1-
5%) than without it.
Iron salts serve as general catalysts for purely chemical
oxidations, and, just as they accelerate the decomposition of
hydrogen peroxide by colloidal platinum, they intensify the action
of the catalases, when used either alone or together with man-
ganese salts. They also exert a specific accelerating influence
on the action of the tyrosinases (Durham, Proc. Roy. Soc.,
1904, 74, 310; Bach, Chem. Ber., 1910, 43,364). Whether
the undoubtedly important role of iron salts in the organism is
played in conjunction with the enzymes, or whether it is prefer-
ably independent, cannot yet be decided.
According to B a c h (Chem. Ber., 1910, 43, 366), aluminium
salts intensify the action of tyrosinase even more than manganese
salts do. Less marked, but still appreciable, are the effects of
calcium and magnesium salts.
It may here be mentioned that hydrogen peroxide increases
ACTIVATORS, PARALYSORS AND POISONS 109
the action of the digestive enzymes (Vandevelde, Hofm.
Beitr., 1904, 5, 558).
Among the most noteworthy of inorganic activators are the
alkali phosphates. Apart from the action of primary
and secondary phosphates as " buffers ", the phosphates exert
a marked, specific influence on certain enzyme reactions.
Mention must first of all be made of zymase-fermentation for
which, as explained elsewhere, the presence of a phosphate is
necessary. Further, ammonium and calcium monophosphates
accelerate diastatic action (E f f r o n t), whilst for the action of
ptyalin, phosphates are absolutely necessary (Roger, Soc.
Biol., 1908, 65, 374.) The same is the case with liver-diastase
(see later). This effect possibly explains the following obser-
vation made by R o g e r (Soc. Biol., 1907, 62, 833, 1021, 1070) :
Human saliva is inactivated by heating for 10-15 minutes
at 85-100, but if a small quantity of fresh saliva is subsequently
added, the mixture has a much greater saccharifying action than
the added saliva alone. This observation has recently been
extended and completed by B a n g (Biochem. Z., 1911, 32, 417).
As has been shown by Harden and Young, the phos-
phates play an extremely important part in fermentation. These
actions are described in detail elsewhere (Chapters I and IV).
Rennetic action is also favoured by small quantities of mono-
sodium phosphate (G e r b e r , Soc. Biol., 1908, 64, 1176)
possibly owing to the alteration effected in the acidity. The same
cause may, perhaps, explain the activation of laccase by disodium
phosphate in certain oxidations (J.Wolff, C. R., 1909,
149, 467).
In connection with these intensifications by phosphoric acid,
it may be mentioned that Chittenden observed an accel-
eration of peptic digestion byarsenious acid. Accord-
ing to I z a r , autolysis is sometimes hastened by arsenic.
Also in their effect on alcoholic fermentation, arsenates and
arsenites correspond to some extent with the phosphates, as is
shown by recent important results obtained by Harden and
Young and referred to at length in Chapter IV.
Alkali Salts. The salts of the alkalis are partly accel-
erating and partly inhibiting in their action.
The salt which has been most thoroughly investigated is
sodium chloride. According to Osborne, Bierry and
L10 GENERAL CHEMISTRY OF THE ENZYMES
3 chaffer (Soc. Biol.,1907,62,723), Cole (Journ.of Physiol.,
1903, 30, 202, 281), Wohlgemuth (Biochem. Z., 1908, 9,
10), it facilitates the actions of diastase, maltase and ptyalin.
But the majority of the enzymes, e.g., invertase, peptase, tryptase
and the zymases and catalases (see, for instance, Lockemann,
T h i e s and W i c h e r n , H., 1909, 58, 390) are retarded by
sodium chloride.
Amylase is accelerated, often considerably, by small quantities
of the chlorides, nitrates, sulphates, phosphates, vanadates and
alums of the alkali metals (L i n t n e r , Journ. prakt. Chem.,
1887, [2], 36, 481; E f f r o n t , C. R., 1892, 115, 1324; G r ii s s).
Ptyalin is slightly accelerated by potassium iodide (N e i 1 s o n
and Terry, Amer. Journ. of Physiol., 1908, 22, 43).
Cole came to the conclusion that anions facilitate and
cations weaken the action of amylase, the effects increasing
with the electro-affinity (this magnitude is evidently meant by
Cole, who uses the somewhat indefinite expression " actinising
power ") of the ions.
The amylolytic action of pancreatin is also accelerated by a
number of salts (P r e t i , Biochem. Z., 1907, 4, 1) if these are
added in dilute solution.
Inhibiting effects on amylase are, however, produced by
calcium and barium chlorides and by larger quantities of the
sulphates, phosphates and alums of the alkali metals.
According to F. Kriiger, NaCl, KC1, NH 4 C1, CaCl 2
and MgCl2, in equivalent proportions exert equal retarding effects,
so that it must be concluded that the inhibiting action of the
anion predominates. This recalls the concordant results of
J. Schiitz (Hofm. Beitr., 1904, 5, 406) and Levites
(H., 1906, 48, 187) which indicate that peptic digestion is
retarded principally by the anions.
Kudo found that the digestive action of pancreatin is,
in general, weakened by alkali salts, sodium chloride producing
a rather greater effect than the nitrate or nitrite; the influence
of potassium salts is less than that of sodium salts.
Sodium and potassium sulphates retard the rennetic action
of animal chymosin in proportion to their quantity (if the clotting
effect is determined with fresh milk), whilst the coagulating action
of vegetable rennet is increased by small doses, and diminished
by larger ones, of these salts.
ACTIVATORS, PARALYSORS AND POISONS 111
This different behaviour of vegetable and animal rennet
towards neutral sulphates and towards Na2HPC>4 and K2HPO4,
G e r b e r explains as due to the precipitation of lime by these
salts, lime being less necessary with the vegetable than with
the animal enzymes. By small quantities of acid sulphates, such
as KHSO4, both animal and vegetable rennets are accelerated.
Concerning the action of activators in general, it may be said
that:
As far as the " kinase " of tryptase is concerned, this can
be regarded, on the one hand, as a catalyst of the reaction tryp-
sinogen trypsin and, on the other, as functioning like the activat-
ing acids.
It is highly desirable that a more extended series of exper-
iments should be made to decide if the conversion of pro-enzyme
into enzyme is a reversible process.
There still remains the possibility of a chemical reaction
occurring between zymogen and kinase and in order to obtain
information on this question, the manner in which the activation
varies with the time would have to be studied more closely.
That the process is a relatively rapid one has been shown by
P a w 1 o w and his collaborators.
In one way or another, the zymogen is often activated
" spontaneously/' In most cases the substrate or some foreign
substance succeeding it yields the activator as the result of a slow
reaction of some kind. In an interesting investigation H o y e r
(loc. cit.) observed the appearance of lactic acid as such an
activating substance. If the activator is a normal product of
the substrate under the given conditions, the well-known case
of auto-catalysis presents itself. A typical example
of such a reaction is the spontaneous decomposition of ethyl
acetate in aqueous solution; in this instance the liberated acetic
acid is the catalyst which accelerates the subsequent hydrolysis
in proportion to the concentration of its hydrogen ions.
Meanwhile there are no grounds for making an essential
distinction between the activation of the zymogen with initiation
of the reaction and the action of acids, alkalis and many salts in
accelerating the reaction.
The action of the latter substances often termed co-enzymes
rests undoubtedly on the reversible formation of compounds
of these activators, partly with the substrate and partly with the
112 GENERAL CHEMISTRY OF THE ENZYMES
enzyme. In most cases it is a salt-formation which takes place
(E u 1 e r , Hofm. Beitr., 1905, 7, 1); this occurs instantaneously,
so that the " incubation periods " of the accelerating alkalis,
e.g., with catalase, or those of the inhibiting acids are without
influence of course, only so long as disturbing secondary reactions,
decomposing chemically the enzyme or substrate, are avoided.
In many cases the minimal concentration determining the
optimum of acid- or alkali-action must correspond exactly with
the quantity of acid or alkali necessary for the neutralisation
of the solution (cf. Cole, Journ. of PhysioL, 1903, 30, 202).
The sensitiveness of the enzymes towards acids and alkalis
is, indeed, very great but is quite conceivable if the very small
concentrations in which the enzymes themselves are present in
solution are considered. More or less complete analogies are
found with many well-known catalytic processes. Thus, hydro-
quinone in a solution containing 0-001 normal-manganese sul-
phate undergoes oxidation very slowly; but if sufficient alkali
is added to combine with the majority of the sulphuric acid and
thus to liberate manganese hydroxide, the oxidation is enormously
accelerated. The manganese sulphate corresponds with the
enzyme in its inactive state, the alkali with the activator. In
hydrolytic changes, e.g., the enzymic inversion of cane-sugar,
the chemical reaction presumably consists in the activating acid
liberating the enzyme which in neutral solution is present as a
salt and so bringing it into the active condition (cf. E u 1 e r
and B. af U g g 1 a s , H., 1910, 65, 124).
As well as to salt-formation, an important part in the activa-
tion of enzymes must be attributed to the formation of com-
plex compounds. It is in this way that the specific properties of
phosphoric acid and of calcium and manganese are exerted,
as described above. With manganese the capacity to form com-
plex compounds with hydroxylic bodies has been long known,
and with calcium, physico-chemical investigations have rendered
necessary the assumption that it also yields such complexes.
The reactivity of phosphoric acid with polyhydric alcohols has
often been studied qualitatively, and quantitative experiments
would be of biological interest.
In the description of pepsin action on p. 95, it was mentioned
that hydrochloric acid not only acts on the enzyme but also
accelerates the digestion by forming salts with the proteins.
ACTIVATORS, PARALYSORS AND POISONS 113
Similarly, it is doubtless the alkali salts of peptones and
peptides which constitute the active molecules of tryptic diges-
tion. This complete alteration of the reactivity of a substance
by salt-formation has many chemical analogies: nitrous acid
diazotises, whilst nitrites do not ; in alkaline solution polyphenols
are oxidised by the oxygen of the air with great readiness, but
in acid solution only with difficulty. The velocity of decomposi-
tion of hydrogen peroxide is influenced by the acids or alkalis
present in a similar manner, no matter whether the reaction is
brought about by " catalases " or by inorganic oxides (colloidal
platinum, ferric hydroxide).
A similar argument can be applied also in other cases, e.g.,
that of invertase-action, where it may be assumed that the
compound (cane sugar-mineral acid), which represents the active
molecule in the process of inversion, is resolved catalytically by
the enzyme into glucose, fructose and free acid.
Being amphoteric electrolytes and hence capable of forming
salts with either 'acids or alkalis, the proteins, peptones and
amino-acids often exert indirectly an accelerating, though seldom
a powerful, action; as the author has already emphasised
(Ergeb. der Physiol.; 1907, 6), they regulate' the concentration
of the free acid or base. 1
Finally, those cases must be considered where the activator
represents the common solvent, that is, the con-
necting link between enzyme and substrate, where the
two alone cannot form a homogeneous system. The activation
of the Upases by the bile acids is possibly to be explained
in this way. The purely chemical (not enzymic) splitting of
fats by naphthalene-stearosulphonic acid has thus been inter-
preted by T w i t c h e 1 .
The causes underlying the actions of neutral salts
are also very varied.
These actions are partly due to simple chemical transposi-
tions between the electrolytes present and the consequent altera-
tion of the acidity or alkalinity. So that sodium sulphate,
salicylate and phosphate hinder peptic digestion (P a w 1 o w,
Danilewski), the strong hydrochloric acid being replaced
1 In many instances the addition of proteins to an enzyme solution miti-
gates the harmful action of tryptic ferments on the enzyme. Beneficial
effects of another type are also observed, e.g., with diastase, ptyalin, etc.
114 GENEKAL CHEMISTRY OF THE ENZYMES
by the weaker, less active acid of the salt. With higher con-
centrations of the salts, the influence of dissociation comes into
play, sodium chloride diminishing the electrolytic dissociation
of the protein hydrochloride. Further those influences come
into action which produce the " neutral salt action " in non-
enzymic hydrolyses and which vary widely in different reactions.
The actions of small quantities of neutral salts, such as
NaCl, KC1, etc., would indeed seldom be observable if the salt
were added to enzyme solutions previously free
from electrolytes. Many investigations indicate that
the small quantities, of salts occurring in the organs with the
enzymes are essential for the activity of the latter; if these small
amounts of salts are removed by dialysis, the enzyme action,
for example, of amylase, ceases.
Very large quantities of salt have a coagulating action and
precipitate enzymes more or less directly from enzyme solutions,
which usually contain colloidal substances. Yet even quite
considerable concentrations of salts are often without harmful
effect; thus the activity of ptyalin solutions is not weakened by
large proportions of magnesium and ammonium sulphates 1
(Patten and Stiles, Amer. Journ. of Physiol., 1906,
17, 26).
In addition to the reversible influence on the time-
course of enzyme reactions, acids, alkalis and salts exert also an
influence on the irreversible changes of the enzyme-
substance. These two actions are essentially different. The
latter of the two has been studied in the case of invertin by
Fernbach (Recherches sur la sucrase, Thesis, Paris, 1890).
This investigator found that dissolved invertin, exposed to the
oxygen of the air, gradually undergoes oxidation and becomes
permanently inactive; this oxidation occurs far more rapidly
in alkaline than in acid solution. (Cf . also Hudson and
Paine, Journ. Amer. Chem. Soc., 1910, 32, 774).
1 It is regarded as unnecessary to mention the many cases in which
smaller proportions of salts have no marked effect,
ACTIVATORS, PARALYSORS AND POISONS 115
PROTECTIVE AGENTS
Lastly, the influence of neutral salts on enzyme reactions
makes itself felt in an indirect way, a beneficial effect being pro-
duced by the destruction, alteration or removal of substances
which may be classed together as inhibiting agents.
These causes play a part in the interesting biological
phenomena accompanying fertilisation described some years
ago by Jacques Loeb.
The favourable influence of certain other substances on
enzyme solutions is also often due to the counteraction
of the inhibiting action of the paralysors.
In this way we may regard the acid salts often termed "buffers "
and the proteins too as protective agents. The latter may,
for instance, unite with part of the alkali in the case of tryptic
digestion and thus protect the enzyme, especially at relatively
high temperatures, from destruction (Vernon, Journ. ofPhysiol.,
1904, 31, 346). Also soda, calcium carbonate, etc., which are
able to protect yeast from the poisonous effect of various substances,
do so in virtue of their neutralising action (Henneberg,
Centralbl. f. Bakt., 1908, II, 20, 225).
INHIBITING AGENTS (PARALYSORS)
The term " poison," which has recently been largely used for
all substances which delay or prevent catalytic reactions, is not
justifiable in the light of recent knowledge. The expression
poisoning must therefore be reserved for the disturbance of the
life functions by paralysors.
Concerning the chemistry of the action of paralysors we are
almost completely in the dark, but these bodies are of con-
siderable interest in enzymology since they are indispensable
as sterilising agents in all protracted experiments. The influence
which paralysors, such as chloroform etc., may at times exert on
enzymic decompositions is shown, for example, by E. F i s c h e r's
observations on the hydrolysis of glucosides by yeast-enzymes
(Chem. Ber., 1895, 28, 1436).
Just as with the action of activators, that of poisons and other
inhibiting substances is dependent on the concentration of the
enzyme and the purity of the solution. It is found that the
116 GENERAL CHEMISTRY OF THE ENZYMES
injurious action of poisons on enzyme solutions increases as the
concentration of the enzymes diminishes; this behaviour seems
to indicate that an addition of the poison to the enzyme takes
place.
Inorganic Salts
NaF. According to A r t h u s and H u b e r (C. R., 1892,
115, 839) this salt is without influence on the soluble enzymes,
but kills bacteria. Its effect on lipase is greater than that of
any other antiseptic (Loevenhart, Journ. of Biol. Chem.,
1907, 2, 391 ; K a s 1 1 e and Loevenhart, Amer. Chem.
Journ., 1900, 24, 491). Chymosin is injured by it, but not
stronger solutions of trypsin (Kaufmann, H., 1903, 39,
434). The action of erepsin on dipeptides is partly retarded,
partly accelerated by sodium fluoride (Abderhalden,
Caemmerer and Pinkussohn, H., 1909,59, 293).
According toVandevelde it has no effect on pepsin and
trypsin. B u c h n e r states that ammonium fluoride annuls
the action of zymase. The influence of fluorides on tjirombin
has been thoroughly investigated by B o r d e t and G e n g o u
(Ann. Inst. Pasteur, 1904, 18, 98) and that on diastases by
Ef front .
HgCl2i even in 00005N-solution has a harmful effect on
catalase. In 0-001% solution, it is poisonous to amylase and
still more so to urease. It injures ptyalin or trypsin in 0-005%
solution, but its action on erepsin is much less marked (E u 1 e r ,
Arkiv for Kemi, 1907, 2, No. 39). Invertin is also weakened by
it, but to a relatively small extent (D u c 1 a u x) .
Hg(CN)2: exerts a decided inhibiting action, although less
than that of HgCl2, on catalase (F a i t e 1 o w i t z).
B(OH)s. D u c 1 a u x found that this retards the action of
chymosin, but the more recent results of A g u 1 h o n (C. R.,
1909, 148, 1340) indicate an accelerating effect. According to
the latter author, the enzymes which hydrolyse carbohydrates,
glucosides and proteins act, without alteration, in cold saturated
boric acid solution; catalase is somewhat retarded.
As2Os. As Buchner has shown by an extended series
of experiments, arsenic is i n j u r i o u s to cell-free fermentation,
but proteins and sugar act as protecting agents against this
ACTIVATORS, PARALYSORS AND POISONS 117
poison. Amylase (K j e 1 d a h 1) and pepsin (Asher)
are harmfully affected by arsenic salts.
EbS: has an injurious influence on catalase but is without
action on pepsin, trypsin, diastase and emulsin (Fermi and
P e r n o s s i , Zeitschr. f. Hygiene, 1894, 18, 83).
Os (ozone). Whilst hydrogen peroxide accelerates many
enzyme actions and, so far as is known, has a slow destructive
action only on catalase, ozone has a harmful influence, as has
been shown by S i g m u n d for most of the more important
enzymes, by K a s 1 1 e especially for lipase (Journ. Chem.
Soc., Abs., 1906, i, 615) and by Buchner and H o f m a n n
for zymase (Biochem. Z., 1907, 4, 215).
H202: according to Vandevelde (Hofm. Beitr., 1904,
5, 558), most enzymes are beneficially affected, only catalase
being retarded.
Of salts which have a more specific injurious action, mention
may be made of the following:
Alkali sulphates retard peptic' digestion, as was found by
Grutzner (Pfleiderer, Pfliig. Arch., 1897, 66, 605)
(with trypsin there is either slight retardation or no effect at all) .
CaCl2 has an especially marked weakening action on invertin
(D u c 1 a u x) .
Iron salts are injurious to pepsin (Asher).
Potassium permanganate strongly inhibits lipase (K a s 1 1 e
and Loevenhart).
Nitrates and chlorates are stated to be intense catalase-
poisons.
For certain other substances which hinder the action of
blood-catalase, S e n t e r (Zeitschr. f. physikal. Chem., 1905,
51, 673) gives the following concentrations as necessary to diminish
the velocity of reaction to one-half its original value:
Paralysor Grm.-mol. per litre
I 2 in KI 1/50,000
Hydroxylamine hydrochloride 1/80,000
KNO 3 1/40,000
KC1O 3 1/40,000
For the catalase from frog's muscle, C. G. Santesson
(Skand. Arch. f. Physiol., 1909, 23, 99) has recently obtained
similar results.
118 GENERAL CHEMISTRY OF THE ENZYMES
Inorganic colloids (gold, platinum, silver, arsenic, copper,
mercury, bismuth) do not accelerate pepsin, but in high concen-
trations rather inhibit it (Pinkussohn, Biochem. Z., 1908,
8, 387).
Organic Poisons and Inhibiting Agents
Chloroform. The original statement made by M ii n t z ,
that chloroform injures only the micro-organisms but not the
enzymes, has had to be considerably modified in recent times.
Chloroform injures maltase (according to L i n t n e r and
Krober), amylase, ptyalin, yeast-glucase, pepsin, rennin and
urease, but has no, or but slight action on trypsin, erepsin, invertin
(Fischer), Aspergillus-maltase (H e r i s s e y) or zymase.
For the literature on this subject see K a u f m a n n (H., 1903,
39, 434).
Chloral completely destroys oxydase (from L e p i o t a
americana) (Kastle and Loevenhart, Chem.
Zentralbl., 1906, 77, i, 1554) but injures myrosin only slightly.
Formaldehyde. In 40% concentration, this does
not destroy Lepiota-oxydase (Kastle and Loevenhart,
1 o c . c i t .) but it has an injurious effect on chymosin and amy-
lase. In 1% solution, it is without action on erepsin (E u 1 e r ,
loc. cit.). Zymase is harmfully influenced by formaldehyde but
pepsin only by concentrated solutions.
G 1 y c e r o 1 has an appreciable inhibiting influence on
rennet-action (R e i c h e 1 and S p i r o , Hofm. Beitr., 1905,
7, 485).
Toluene. This hydrocarbon, which E. Fischer
introduced for the sterilisation of enzyme solutions, is harmless
in the great majority of cases, but urease is weakened by it.
Phenol: exerts a deleterious action on pepsin, amylase and
catalase.
Cresols: harmless for liver-butyrase (Kastle, Chem.
Zentralbl., 1906, 77, i, 1555).
Thymol: injurious to oxydases (Kastle and Loeven-
hart) and saliva-diastase (Schlesinger, Pugliese)
and markedly so to zymase (B u c h n e r) and chymosin
(Freudenreich). It does not weaken the action of the more
ACTIVATORS, PARALYbORS AND POISONS 119
concentrated trypsin solutions (Kaufmann, H., 1903, 39,
434) or that of yeast-maltase (E . Fischer).
Maltose retards peptic digestion (Sailer and F a r r) .
Salicylic acid: stated to have a weakening effect
on pepsin and trypsin and also on lipase. Autolytic action
and that of xanthine-oxydase (B u r i a n , H., 1905, 43, 494)
are, however, accelerated.
Hydrocyanic acid. Whilst this acid has proved
itself an extremely powerful poison towards catalase, its deleterious
action on other enzymes is considerably weaker and sometimes
very faint. Zymase is, indeed, completely inactivated, but this
action is reversible (B u c h n e r) . Also, according to F u 1 d
and S p i r o , chymosin is not injured and the same is the case
with pepsin. The decomposition of polypeptides by erepsin is
accelerated by small, but inhibited by larger, quantities of
potassium cyanide (Abderhalden, Caemmerer and
Pinkussohn, H., 1909, 59, 293). In 1% solution, hydro-
cyanic acid weakens but does not destroy the proteolytic enzyme
of yeast-juice (G e r e t and H a h n , Chem. Ber., 1898, 31, 202).
The sensitiveness of catalase towards hydrocyanic acid is
shown by Senter's results which, with those obtained with
certain other organic poisons are given below. 1 The concen-
trations required to diminish the velocity of reaction to one-half
are:
Paralysor. Grm.-mol. per litre.
HCN 1/1000000
Phenylhydrazine 1/20000
Aniline 1/400
A 1 k a 1 oi d s. The older literature on this subject is given
by N a s s e (Pflug. Arch., 1875, 11, 159) and in the work of
1 Faitelowit2 has arranged various paralysors, according to the
concentrations in which they affect milk-catalase, in the following order:
i n
HCN H 2 C 2 O 4
KCN HNO 3
KCNS Ba(NO 3 ) 2
HgCl 2 HC1
H 2 S CHsCOOH
Hg(CN) 2
120 GENERAL CHEMISTRY OF THE ENZYMES
Chittenden, to whom a very thorough investigation is due.
In general, alkaloids retard enzyme action, but not very
strongly (Chittenden; Gockel); cf. also L a q u e u r
(Arch. f. exp. Path., 1906, 55, 240) who studies especially the
effect of quinine on enzymes. According to the latter author,
the autolytic enzyme and blood-oxydase are the most strongly
inhibited by quinine. A s h e r observed a deleterious action
of quinine preparations on peptic digestion. Oxydases appear
to be especially sensitive to alkaloids (R o s e n f e 1 d) . Hor-
denine sulphate retards peptic and tryptic digestion (Camus),
but not the action of maltase, invertase or lipase.
Of practical importance for the technique of enzymology
are the investigations dealing with the action of alcohol
on enzymes :
Small quantities of alcohol have an accelerating influence
on lipase (G i z e 1 1 , Zentralbl. f. Physiol., 1905, 19, 769, 851)
but in other cases a more or less complete inhibiting action, e.g.,
on trypsin (Gizelt, loc. cit.), rennet (R e i c h e 1 and
S p i r o , Hofm. Beitr., 1905, 7, 485) and diastase. Only tyro-
sinase is able to act in 50% methyl-ethyl alcohol. Inhibition
by alcohol is almost always reversible.
Even very large quantities of alcohol are withstood for a
time by all enzymes, as the ordinary precipitation methods
show. If the alcohol is removed after precipitation of the enzyme,
the latter resumes its activity (Schondorff and V i c t o r o w ,
Pflug. Arch., 1907, 116, 495).
Like the activators, inhibiting agents exert their action by
combining partly with the substrate, partly with the enzyme
and partly with the activator. Especially often where the
paralysors are acids or bases must these actions play a part.
Also, salts of the heavy metals alter the state of solution of
colloidal substrates such as proteins, starch, etc.
The behaviour exhibited by antiseptic and narcotic media,
such as toluene, thymol and chloroform, towards living
c e 1 1 s is determined principally by the behaviour and the altera-
tions of the lipoidal plasma-skin. The plasma undergoes change
only after the penetration of the plasma-skin by the narcotic.
The enzymic actions of living cells are influenced in various
ways. By toluene or chloroform, fermentation, for example,
is interrupted, although zymase itself as is shown by experi-
ACTIVATORS, PARALYSORS AND POISONS
121
ments with press yeast-juice is not injured by chloroform.
On the other hand, yeast-cells invert cane-sugar equally quickly
in absence or presence of chloroform or toluene.
H. Euler and Beth af Ugglas (H., 1911, 70, 279)
have arrived at the result, that the activity of those enzymes
which are combined with the plasma in
the living cell, e.g., zymase, is annulled by narcotics,
whilst the enzymes occurring free in the cells, as, for example,
invertase, are not influenced by these substances.
Euler and K u 1 1 b e r g have investigated the connection
between the sensitiveness to poisons, extractability, and relative
quantities of the enzymes of a beer-yeast called " H " from a
Stockholm brewery (H., 1911, 73, 85), the results being given
in the following table :
Zymase.
Monilia-
invertase.
Maltase.
Beer-yeast
invertase.
Living
yeast
Relative velocity
of reaction in8%
sugar solution
Extractability . . .
Action of poisons:
Chloroform
Thymol
1
inhibited
completely
ditto
1 (to 2)
inhibited
completely
ditto
1 (to 2)
inhibited
completely
considerable
170
slight
scarcely any
weakening
no weaken-
Toluene
Weakening on
drying
ditto
20 : 1
ditto
25 : 1
weakening
considerable
weakening
ing
ditto
2 : 1
Dried
yeast
Extraetability . . .
Action of poisons :
Chloroform. . .
Toluene
very slight
weakened .
weakened
very slight
weakened
very in-
complete
weakened
about 20%
not weak-
ened
not weak-
ened
Concerning the chemical compounds presumably formed in
the poisoning of the free enzymes we have no data, since we
do not even know the nature of the reacting enzymic substance.
These chemical combinations often appear to be only loose
ones; the ability of the enzyme to regain wholly or partially
its original activity after removal of the paralysor, is especially
marked with " poisoning " by hydrocyanic acid.
122 GENERAL CHEMISTRY OF THE ENZYMES
If the paralysor is present in only very small quantities,
it may be destroyed by any oxidising agent present or by other
enzymes without external action. To this must be attributed
the spontaneous re-activation which is known as " recovery."
Analogies to known chemical processes are here also not
lacking. A comparison has often been drawn between the actions
exerted by paralysors on catalase and on B r e d i g ; s so-called
" colloidal metals " which are, indeed oxides and other oxidis-
ing catalysts. But even S e n t e r ' s thorough experiments,
referred to above, are not able to explain to what chemical changes
the active oxygen, the action of which is here in question, is
subjected by the paralysors. Besides, the " poisonings '" with
catalase and colloidal platinum follow by no means parallel
courses. The principal result of the interesting contributions of
H 6 b e r (Pfliig. Arch., 1900, 82, 631) and of Loevenhart
and K a s 1 1 e (Amer. Chem. Journ., 1903, 29, 397) to' this sub-
ject may be summed up in the sentence: " That the effect of any
particular substance on the catalyser can be explained, in the
majority of cases at least, upon purely chemical grounds."
In many respects the anti-bodies correspond with the inhibit-
ing substances here considered and the limits of the term anti-
bodies are determined by characteristics somewhat similar to
those which define the limits of the enzymes in the wider field
of the catalysts. A number of substances which retard enzymic
reactions in the normal organism should also be
termed inhibiting substances in contradistinction to the anti-
enzymes, which the organism forms as protective substances
after the introduction of foreign enzymes.
A whole group of inhibition phenomena are to be attributed
to the adsorption of enzymes. To such phenomena belongs
especially the inhibition of trypsin, rennet and invertase by
charcoal, studied by S. G. H e d i n and by A. Eriksson.
H e d i n showed that the retardations caused by white of egg and
serum-albumin are analogous to those produced by charcoal,
that is, they are due to adsorption phenomena. To this group
belong therefore all the non-specific inhibiting effects of serum
which were formerly ascribed to anti-bodies. H e d i n has
collected the literature on this subject in the ninth yearly
volume of the Ergebnisse der Physiologic (1910) (cf. H., 1911,
72, 313).
ACTIVATORS, PARALYSORS AND POISONS 123
The data are, however, too incomplete to permit of a critical
classification of these inhibiting substances, so that a resume
of all substances referred to in the literature as anti-enzymes
will be given in a later chapter.
Reference must finally be made to those inhibiting effects
observed when heated enzyme solutions are added to the active
enzymes.
Such retardation has been observed:
With tryptase by Pollak (Hofm. Beitr., 1904, 6, 95)
in the digestion of gelatine.
With peptase by Schwarz (Hofm. Beitr., 1905, 6, 524),
according to whom the inhibitory substance resists the action of
heat and exists ready-formed in fresh solutions of the enzyme.
With peptase, rennet and taka-diastase by Cramer and
Beam (Proceedings of the Physiol. Soc., June 2, 1906, see
Journ. of Physiol., 1906, 34, xxxvi; Biochem. J., 1907, 2, 174),
who heated the enzyme solutions to 50-60; at 100 the inhibit-
ing substance is destroyed.
With invertase by A. Eriksson (H., 1911, 72, 330), who
assumes the existence of an inhibiting agent, resistant to heat,
in invertase solutions.
Here belong also the results obtained by P o r t e r (Biochem.
Z., 1910, 25, 301) with peptase, tryptase, rennet, lipase, saliva-
amylase, amygdalase and taka-diastase. In contact with collo-
dion membranes these enzymes, with the exception of taka-
diastase, lose their activity, all of them except saliva-amylase
then exhibiting a retarding action on the corresponding enzymes.
CHAPTER IV
CHEMICAL DYNAMICS OF ENZYME REACTIONS
THE relations required by chemical dynamics for the simplest
cases of catalytic reactions are found to be fulfilled by enzymic
processes to very varying extents. In some cases the time-law
for unimolecular reactions and the proportionality between veloc-
ity of reaction and amount of catalyst are closely followed.
But in the majority of instances the experimental data are in
agreement with the doctrine of reaction only within a limited
region of concentration, and not a few reactions will be described
for which no simple theoretical representation has yet been
found possible.
The arrangement of the various enzymic processes according
to the mathematical expressions which they follow is apparently
the simplest method to adopt. But this can only be carried
out at the expense of brevity and clearness since, as already
mentioned, the kinetics of the action of one and the same enzyme
may be altered completely by change of the external conditions.
Separate treatment will therefore be given for each of the more
important enzymes, beginning with the hydrolysing enzymes
and passing on to the fermentation enzymes, oxydases and
catalases.
The opportunity must not be neglected of emphasising the
necessity of a critical examination of the numerical data of the
quantitative results given below. Even in the most favourable
cases, where a chemically individual substrate has been employed,
the solutions dealt with not only contained a chemically unknown
catalyst in unknown concentration, but were also contaminated
with the foreign constituents of the enzyme preparation and, in-
deed, with substances which, sometimes even in minimal amounts,
might exert a deciding influence on the course of the reaction to
be observed. In short, one is in the doubtful position of making
124
CHEMICAL DYNAMICS OF ENZYME REACTIONS 125
quantitative observations on a system insufficiently investigated
qualitatively.
It might then be asked: Are quantitative results obtained
with enzyme solutions of any value at all and is any detailed
treatment of them advisable? If the observations made on a
preparation are not very comprehensive and if the experimental
conditions are varied only inconsiderably, the value of a physico-
chemical investigation of an enzyme is, indeed, small. But
our knowledge of enzymes has been widely extended by a number
of studies of the kinetics of reaction, which, especially when
considered as a whole, have elucidated the general relations of
enzyme action and have furnished valuable aid in the elabora-
tion of enzymological methods.
Before the experimental results are examined in detail, it
will be well to consider the theoretical principles to be applied
in judging these results.
THEORETICAL PRINCIPLES OF ENZYMIC DYNAMICS
A knowledge of the law of mass action may be assumed.
This states that tlie active mass of a substance is proportional
to its osmotic pressure and hence (within rather narrower limits)
to its concentration. If a single type of molecule A with a con-
centration C A is changed by a chemical reaction into new sub-
stances, without the concentration of any other kind of mole-
cule present being appreciably altered, then for given external
conditions of temperature, pressure and medium the quantity
of substance dC A changed in every interval of time dt per unit
of volume will be given by the equation:
(la)
where k f is a constant, termed the velocity constant
(also reaction constant) of the process.
This constant k r retains and on this emphasis must be laid
its value, undiminished, no matter how far the reaction has pro-
ceeded or what initial concentrations may be chosen.
On the other hand, the velocity v, that is, the amount
of substance changed per unit of time and in unit volume, is
126 GENERAL CHEMISTRY OF THE ENZYMES
dependent on (proportional to) the concentration C A . It assumes
the same numerical value as k', if C A = 1, i.e., if the substance
transformed amounts to one grm.-mol. per litre and is in some
way maintained at this concentration.
This simplest equation allows, therefore, of the representa-
tion of a chemical process which proceeds (practically) completely
in one direction.
Integration of (la) gives the constant for so-called u n i m o-
lecular reactions:
A/=.Zn or fc = .l og -- . . . (16)
t ax t & a x'
if we indicate decimal logarithms by log and hence make
/b = 0-4343 A:'. 1
An example of an enzyme reaction in which molecules of
only one kind undergo change, is the decomposition of hydrogen
1 If a reaction proceeds so that equimolecular quantities of t w o sub-
stances combine to form the product of the reaction, i.e., according to the
scheme,
A +-><?,
then, if the initial concentrations are a and 6, and x denotes the amounts
of these two substances (and hence the concentration of C) changed after
time t, the velocity at any moment is proportional to the concentrations of
the two reacting substances; thus,
or, if the two substances have originally the same comcentration, a,
-*<-*).
This, the simplest case of a so-called bimolecular reaction,
corresponds with the integral
* = --.
at ax'
Enzymic processes proceeding according to the equation for bimolecular
reactions, are as yet unknown.
CHEMICAL DYNAMICS OF ENZYME REACTIONS 127
peroxide by catalase; the tables given on p. 216 show that the
values of k are constant within the limits of experimental error.
The above equation is also confirmed in numerous cases
where, in addition to a dissolved substrate, water takes part
in the reaction. Since the water is usually present in large
excess compared with the dissolved substance, its concentration
may be regarded as constant; so that here also only molecules of
a single kind undergo change. Indeed the first example
of the validity of the unimolecular reaction law was that of the
inversion of cane-sugar by acids (W i 1 h e 1 m y , 1850). Hud-
son's recent measurements, the results of which are given
on p. 160, show that the same law holds for the hydrolysis of
this sugar by invertase.
It may here be pointed out that the law of mass action, on
which the whole of chemical kinetics depends, may be derived
from the two fundamental laws of thermodynamics, and is
hence independent of our present molecular kinetic conceptions.
That the law of mass action underlies all chemical processes,
cannot therefore be doubted; the only question is as to when and
how far the assumptions, according to which it can be expressed
in the above simple form, are valid. If we find deviations from
the simple formulae to which the law of mass action leads, we
have to enquire which of the assumptions are not fulfilled under
the experimental conditions chosen.
CATALYSIS
The hydrolysis of cane-sugar, as is well known, proceeds
with extreme slowness in pure water; the velocity is considerably
increased only when the solution contains an acid (or an enzyme,
invertase), in addition to the sugar. So far as we can determine
by titration, the concentration of the added acid does not change
during the hydrolysis. Also the law for unimolecular reactions
holds equally well for a solution containing either 0-001 or 0-1
grm.-mol. of acid per litre, the sole change (so long as we remain
within the region of dilute aqueous solutions) being in the nu-
merical value of the reaction constant k. A catalyst is there-
fore, as mentioned at the outset, to be defined asasubstance
which, without being consumed in the reac-
tion, alters the velocity with which a
128 GENERAL CHEMISTRY OF THE ENZYMES
reaction attains its position of equilib-
rium. 1
In 1884 Arrhenius made the important discovery, that
the catalytic action of different acids runs parallel with their
conductivity or, more accurately, with the amount dissociated.
This law, which has been often confirmed and is of the widest signifi-
cance, can be expressed by saying that different acids catalyse hydrolytic
reactions in proportion to the concentration of the hydrogen-ions of their
solutions. This fact is often stated in the literature in such a way as
to imply that the hydrogen-ions alone are the catalysing agent and that
they function as a kind of contact-substance. But such a representation
by no means corresponds with the chemical facts. Rather must it be
supposed that, by the catalysing acid the concentration of the ions effect-
ing the reaction is increased (E u 1 e r , Zeitschr. f. physikal. Chem.,
1901, 36, 681)
The supposition that combination of the catalyst with the
substrate yields the molecules which carry on the reaction, has
already received general acceptance in enzymology. Of the
authors who, on the basis of their own investigations, have expressed
themselves in this sense, mention need only be made of : K a s 1 1 e
and Loevenhart, Bach, Hanriot, A. Brown, H.
Brown and Glendinning, Bodenstein, Henri,
Medwedew, Hedin, Armstrong and B a y 1 i s s .
In many enzymic reactions, compounds between enzyme
and substrate seem to occur to a far greater extent than is the
case in catalytic hydrolyses by acids; yet in no instance has it
been determined what proportions of the total quantities of
enzyme and substrate present combine during the reaction.
It would lead too far to indicate the reasons which have
caused the various investigators to assume a combination of
enzyme with substrate. We shall only indicate briefly the
mathematical formulation of the hypothesis in question.
In his researches on the inversion of cane-sugar, Henri found
that the reaction constants of the first order increased considerably
1 This holds for all those cases where a substance does not catalyse its
own transformation, as is, for example, the case with the formation of
lactones from y- and 8-hydroxy-acids, the hydro xy-acid here accelerating
the lactone-formation proportionately with the amount of its dissociated
portion (auto- catalysis).
CHEMICAL DYNAMICS OF ENZYME REACTIONS 129
(cf. p. 158), and, in order to obtain a mathematical expression of his
results, he employed (Zeitschr. f. physikal. Chem., 1902, 39, 194) a
method given by s t w a 1 d (Lehrbuch der allgem. Chemie, II, 2, 265).
If a reaction is accelerated by a substance having the concentration
p, the expression k(a x) is to be multiplied by (1 +ep), e being a constant.
For the case in which the product of the reaction is the accelerating
agent, the Velocity will hence increase in the ratio 1 :(l+e j, i.e.,
in proportion to the transformed part of the substrate. The equation
therefore becomes :
f-for(l+.|)(a^) (Ic)
On integration, this gives:
.... (Id)
or
Calculation of several series of experiments on the action of invertase
gave, for the new constant e, values approaching 1. Thus, if e = l,
the equation becomes
or
Shortly afterwards Bodenstein subjected Henri's numbers
to a re-examination, making the assumption that both the cane-sugar a
and its hydrolytic products weaken the invertase, the former more
strongly than the invert-sugar. From this he derived the formula:
... (3)
(Cf. Henri, Lois gene"rales de Faction des diastases, Paris, 1903,
p. 77 et seq.)
Here i indicates the quantity of invert-sugar previously added, E
denotes the quantity of enzyme and m and n are constants expressing
the specific weakening of the invertase by cane-sugar and invert-sugar
respectively. Bodenstein chose for these constants the values
130 GENERAL CHEMISTRY OF THE ENZYMES
m=2 and n = l, thus indicating the stronger action of the cane-sugar.
If the solution originally contains no reaction-product (i =0), his formula
simplifies to the following:
While this equation corresponds satisfactorily with observations on
moderately dilute solutions, the numbers obtained by Henri with
dilute solutions are not in agreement with it.
Henri has therefore deduced another expression on the assumption
that both the cane-sugar and the invert-sugar (especially the fructose)
combine with the enzyme (Lois generates, p. 85 et seq., and C. R., 1902,
135, 916).
Of the original quantity of substance a let x molecules be hydrolysed,
so that a x molecules remain. Further, let the quantity of enzyme
be E and X the quantity of it which is free and Z and Y the amounts
which, at the time t, are combined with the cane-sugar and invert-sugar
respectively.
Between enzyme and substrate on the one hand, and enzyme and
products of reaction on the other, equilibria must set in in accordance
with the law of mass action. Hence for these two equilibria the follow-
ing relations hold (in the case where 1 mol. of enzyme unites with 1 mol.
of substrate)
X(a-x}= .Z, (5)
m '
and
**=i-r, ........ (6)
where m and n are equilibrium constants.
Further, for the total quantity of enzyme
E=X + Y+Z ......... (7)
From these Henri calculated the quantity of free enzyme X
and that of the compound sugar-enzyme Z:
E
X=
l+m(a-x)+nx'
and
Z __
l+m(a-x)+nx'
Only two assumptions can now be made:
CHEMICAL DYNAMICS OF ENZYME REACTIONS 131
1. The free portion of the enzyme acts on the
sugar; in this case the velocity is proportional to the quantity of
free enzyme and to the quantity of cane-sugar, i.e., to X and to ax.
Hence
^ = const. X(a-x) (10)
If X is substituted in accordance with Eq. (8), this gives
dx const. E(a x)
dt~l+m(a-x)+nx
2. If, on the other hand, the reaction is effected by the
complex, enzyme-sugar, the velocity will be proportional
to the concentration of these molecules, i.e., to Z. From
7= const. Z,
is obtained, from Eq. (9),
dx const. m.E(a x)
dt ~ 1 +m(a x) +nx '
(12)
So that both assumptions lead to one and the same expression for
the velocity of reaction.
On the supposition that complexes are formed consisting of 1 mol.
enzyme, p mols. of substrate and q mols. of the various products of the
reaction, Arrhenius (Immunochemistry, p. 60) gives a still more
general form for the above expression.
For every single such molecule a formula is obtained of the form:
..... (13)
where
If we assume that sZ is small compared with a and x, we obtain
Z=K f X(a
so that
If the first term of the denominator, 1, is small with respect to the
terms under the summation sign, Bodenstein's formula is obtained
if we make a = 1 and assume that there are two terms under the s, one
in which p = I and q =0 and a second in which p =0 and q = l.
132 GENERAL CHEMISTRY OF THE ENZYMES
In a preliminary communication published two years later, Henri
(Zeitschr. f. physikal. Chem., 1905, 51, 19) outlines a new theory of
enzyme-action which takes account of the colloidal condition of enzymes.
According to the law of distribution, the dissolved body, sugar for
example, must be distributed between the aqueous solution and the
colloid, the velocity of reaction being determined by the concentration
of the sugar in the colloid. In this way Henri explains the similarity
between the ordinary adsorption curves and those representing the
influence of the concentration of the cane-sugar on the velocity of inver-
sion by invertase.
None of these formulae and theories have been confirmed in a manner
free from objection.
As has been mentioned previously, the hydrolysis of* cane-
sugar, esters and other similar substances is accelerated by acids,
the acceleration being proportional to the concentration of the
hydrogen-ions in the solution. If, as is usually done, strong acids
are used in very low concentrations, there is approximate pro-
portionality between the concentration of
the catalyst and the velocity of reaction,
since the electrolytic dissociation of the acid is virtually complete.
Such proportionality between concentration of the catalyst and
the velocity of reaction is found to hold in numerous enzyme
reactions within quite wide limits of concentration. This is the
case, for example, with the actions of the lipases (p. 146 et seq.),
catalases (p. 216), invertase (p. 160) and erepsin (p. 189).
An exception to this very simple relation has, however, been
known for a long time. The amounts of protein
digested by pepsin in a definite time are
proportional, not directly to the quantities
of pepsin, but to the square roots of these.
This is the law which was enunciated byEmil Schiitz in
1885 (H., 1885, 9, 577).
The validity of this rule has, indeed, often even in recent
times been contested. But the reliability of the numerous
experiments, made by different methods (cf. p. 176 et seq.)
and confirming S c h ii t z 's numbers we are referring now
exclusively to pepsin action cannot be doubted. Hence for
peptic digestion, at any rate in the first third of the reaction,
S c h ii t z 's rule is obeyed, and we are met with the problem
of explaining this experimental relation on the basis of the doc-
trine of chemical dynamics.
CHEMICAL DYNAMICS OF ENZYME REACTIONS 133
Arrhenius (Medd. Nobel Inst., 1908, 1, No. 9) has
deduced S c h ii t z 's rule theoretically in the following way :
In order to ascertain the circumstances which condition
S c h ii t z 's rule, we proceed as follows : If the amount of
substance transformed is indicated by x and the time by t, the
rule states that :
x = KiVT or x 2 = ^ 2 t ...... (15)
On differentiation this gives:
or:
dx_*l\_ , }
dt~ 2 x'
So that, for S c h ii t z ' s rule to hold, it is a necessary
and, as may easily be seen, sufficient condition that the velocity
of reaction shall be inversely proportional to the quantity of
substance transformed, i.e., to x. Since the rule is only obeyed
during the initial stages of the reaction, this proportionality must
only be assumed for the first part of the change.
Such proportionality may be brought about as follows: The
velocity of reaction in the case when only one molecule of each
of the reacting bodies goes into the product of the reaction is
proportional to the product of the concentrations of the reacting
substances. At the beginning of the reaction, such small
quantities of the bodies are transformed that generally their
total quantities may, with sufficient accuracy, be regarded as
constant. It is this that limits the validity of Schiitz's rule
to the beginning of the reaction. Now the active quantity
(M) of one of the reacting bodies must be inversely proportional
to the amount changed, i.e., to the quantity of new product
(x), so that:
,, const. ,,
M = -- or MX = const.
x
This is evidently the case if a chemical equilibrium is set up
between the new product and one of the reacting bodies, on the
one hand, and a compound of them in almost constant quantity,
on the other. Such a case is already known in the saponifying
action of ammonia on an ester, e.g., ethyl acetate. The one
134 GENERAL CHEMISTRY OF THE ENZYMES
reacting body here is the ion OH and the quantity of this ion
(Mon) is diminished by the NH4-ions (amount, z N H 4 ) of the
ammonium acetate formed during the reaction according to the
equation :
4 = Cl (N N H 4 OH + N N H 3 ) ,
where N N H 4 oH and N N H 3 denote the amounts of the OTUOH-
and NHs-molecules respectively. The volume remains constant
during the reaction and the last-named quantity may be regarded
as constant, so long as the beginning of the reaction is alone
considered.
A closer investigation of this case by Arrhenius has
revealed distinctly an analogy to the course of peptic digestion.
Arrhenius considered a system in which ammonia in an
initial quantity A acts on ethyl acetate, and he assumed, for
the sake of simplicity, that the amount of ethyl acetate is so
large that it is not changed appreciably by the reaction but may
be regarded as constant (P); that is, the ethyl acetate must
be present in considerable excess. If now x mols. of ammonium
acetate which with the high dilution that we assume may be
considered as completely decomposed into NEU- and CHsCC^-
ions are formed at the time t, (ax) mols. of ammonia will be
present at the same time. Owing to the slight dissociation of
ammonia, only a very small fraction of it is changed into NH4-
and OH -ions, so that it is sufficiently exact to denote the amount
of non-dissociated ammonia by (ax). Even in presence of a very
small quantity of ammonium salt, the NH4-ions from the ammonia
may be neglected in comparison with those formed by the salt,
and the amount of NH4-ions may be taken as x. On this assump-
tion the first moments of the reaction must be disregarded, as
then no ammonium salt or but very little is present. The con-
centration of the hydroxyl-ions, q, is then determined by the
following equation:
q.x = K 2 (a-x),
where K% denotes the dissociation constant of the ammonia.
The velocity of reaction on hydrolysis is now proportional
to the quantity of hydroxyl-ions (q) and to the amount of ethyl
acetate (P), that is:
CHEMICAL DYNAMICS OF ENZYME REACTIONS 135
where #2 and x are constants and xP may be regarded as a new
constant, since the value of P does not change in any single
experiment
On integration, the last equation gives:
F(x)=aln- x =
a x
(18)
The following table, taken from that given byArrhenius,
contains the result of a duplicated experiment in which a given
quantity of ammonia acted on 0-66-normal ethyl acetate at
14-8.
Mean value of fcP = 21.
Percentage of ammonia converted.
1
cP.
Observed. 1
Calculated.
1
17-5
19-4
17-4
2
25-5
25-2
22-0
3
30-7
30-6
21-2
5
38-5
38-5
20-9
10
51-2
51-3
20-9
15
59-6
59-7
20-9
22
67-5
68-0
20-6
30
74-5
74-7
21-2
50
84-8
85-0
20-8
70
91-1
90-7
21-6
100
95-3
95-3
21. 1
1 Mean of two experiments.
As will be seen, formula (18) is in excellent agreement with
the observed results.
The analogy between the hydrolysis of esters by ammonia
and the digestion of protein by pepsin is, according to A r r -
h e n i u s , as follows :
In the decomposition of protein, albumoses and peptones are
formed. Most of the pepsin is fixed by the products of the
reaction, so that the following equilibrium is set up:
[Free pepsin] X [products of reaction] = const, [combined pepsin].
The quantity of free pepsin is, approximately, inversely
proportional to the amount of the reaction products, x. This
holds as soon as so much reaction-product is formed that the
136 GENERAL CHEMISTRY OF THE ENZYMES
greater part of it is not combined with pepsin, whilst the greater
part of the latter is combined.
Further, the amount of free pepsin is evidently proportional
to the quantity of pepsin employed. Hence, if we indicate the
concentration of the enzyme (pepsin) by [E], the unaltered pro-
tein by (ax) and the products of digestion by x, we obtain:
(19)
and consequently, if we express the amount of hydrolytic products
as fractions of 1000:
1000(7nlOOO-Zn unaltered protein) digested protein = K [E]t. (20)
In reality, peptic digestion corresponds remarkably well
with Arrhenius's formula and it is because the latter holds
over wider concentration-limits of enzyme and substrate that the
work of Arrhenius on Schiitz's rule has been here re-
ferred to at length.
Whether, indeed, the above considerations take account of
all the facts and factors essential to peptic digestion, e.g., the
combination of hydrochloric acid (cf . Jastrowitz, Biochem.
Z., 1907, 2, 157), cannot at present be easily decided. In par-
ticular it is still uncertain what proportion of the pepsin com-
bines, under definite conditions, with the substrate or reaction-
products and what proportion with the hydrochloric acid. All
that can be said is that the spatial configuration of the participat-
ing substances plays a very real part. This can be seen from
the influence which additions of optically active bodies exert on
digestive processes.
It would be of interest to ascertain the concentrations of
hydrochloric acid for which pepsin-digestion is proportional to
the concentration of the enzyme.
The opportunity must be taken here of pointing out that,
with many enzymic reactions, the chemical change is composed
of several separate processes which have not yet been studied
singly; this is the case, for example, with the hydrolysis of pro-
teins or starch by proteinases or amylases. In such complex
reactions as these it cannot, of course, be expected that the laws
of chemical dynamics will be apparent in their simplest form.
CHEMICAL DYNAMICS OF ENZYME REACTIONS 137
In addition to the above processes, the fermentation of bioses
by yeasts may be mentioned; it is here assumed that the first
phase consists of a hydrolysis of the bioses (cf . E u 1 e r and
Lundeqvist, H., 1911, 72, 103).
Finally, as has been done by C . E n g 1 e r and R . O . H e r -
z o g (H., 1909, 59, 327), enzymic oxidations may be represented
as coupled or induction reactions. If M, N and
P are three substances capable of reacting as follows:
M+N = (MN) react.
P-\-N do not react.
M+ N+P = (MN) + (BN) react.
(where the bracketed letters indicate the substances reacting
with one another), the reaction is a coupled or induced one.
Schilow (Zeitschr. f. physikal. Chem., 1903, 42, 641; cf.
also Luther and Schilow, Zeitschr. f. physikal. Chem.,
1903, 46, 777) terms the substance N, taking part in both reactions,
the actor, M the inductor and P the acceptor.
According to E n g 1 e r and H e r z o g , the
oxydase functions as inductor,
oxygen actor,
oxidised substance acceptor.
This conception opens up a number of interesting points.
REVERSIBLE REACTIONS
Influence of the Products of Reaction.
Up to the present, hydrolytic enzyme reactions have been treated
as though they proceeded completely in one direction or, more
accurately, as though the opposite synthetical reaction were so
inconsiderable as to be negligible. We know that the hydrolysis
of cane-sugar by acids the classic reaction of chemical dynamics
is practically complete under ordinary conditions and we
should at first expect the same to occur also with enzymic
hydrolyses. But recent researches of Y. Osaka (Journ.
138 GENERAL CHEMISTRY OF THE ENZYMES
Coll. of Science, Tokyo, 1908, 25, 1) have shown that, even
with cane-sugar, if sufficiently concentrated solutions are employed,
the equilibrium is apparent just as it usually is with the esters
of organic acids. The change which such a reversible system
undergoes with lapse of time is the difference between two opposite
actions.
For instance, the velocity of hydrolysis of the
ester, v i is given by the equation :
vi = ki [ester],
if, as usual, we indicate the concentration by [ ]. The
velocity of formation of the ester V2 is expressed by:
V2 = ^[acid] [alcohol],
and the actual resultant velocity in a system not in equilibrium
and containing ester, acid, alcohol and water, is:
v = vi 1>2 = fci [ester] /^[acid] [alcohol]
or, if we start from a pure ester solution of the concentration
a and indicate the amount changed in time t by x:
= v = vi V2 = ki(a x)k2X 2 ..... (21)
The ratio between the two velocity constants is, as was shown
byvan't Hoff (compare Chapter VI), the constant of
chemical equilibrium, so that
ki__ [ester]
&2 [acid] [alcohol]'
When the constant of the " reverse reaction " is not vanish-
ingly small, the time course of the total reaction v, as can be
easily seen, is changed if the product of the reaction x is previously
added to the system, and, in general, the progress
of a reversible reaction must be retarded
by addition of the products of the reaction.
When ki and k% have been experimentally determined, it is
easy to calculate how v changes with increasing additions of x.
CHEMICAL DYNAMICS OF ENZYME REACTIONS 139
For dilute solutions and inorganic catalysts these relations have
been completely worked out. 1
With enzyme reactions another circumstance, which has to
be considered, complicates matters to some extent. We have
already discussed the assumption, now generally accepted, that
the enzymes form compounds with the substrate and with the
products of reaction. In no case do we know in what meas-
ure an enzyme enters into such combination, but we assume
that these compounds exert considerable influence on the time
course, so that the velocity of an enzymichydrolysis is altered
by addition of the products of the reaction to the system, not
only according to Eq. (21), but also by combination (inactiva-
tion) of the catalyst.
This influence has not only been observed qualitatively,
but has, in many cases, been measured.
Of the experiments showing the retarding influence of the
hydrolytic products, that quoted by W . K ii h n e (Lehrbuch der
physiol. Chem., 1866, p. 39) is one of the earliest: If a digestion
solution, filtered from excess of undigested fibrin, is placed in a
dialyser, most of the peptones diffuse into water, whilst the
pepsin remains behind. When the pepsin solution is restored
to its original volume by evaporation and to its initial acidity,
it dissolves almost exactly as much fibrin as was previously
dissolved. It is consequently the peptones which hinder the
digestion.
We may also recall the investigation, already mentioned,
of Tammann (H., 1891, 16, 271) which showed in a con-
vincing manner that the products of hydrolysis generally influence
the completeness of enzymic reactions; but, as he says, " not
only by removal and destruction of the decomposition products
can an enzyme reaction be rendered complete, as the same end
is attained by repeated additions of enzyme."
Of Tammann's numerous experiments, the following
may be mentioned :
To different solutions, each containing 0-51 grm. of amygdalin,
were added equal amounts of emulsin and varying quantities
of saturated benzaldehyde solution, all the solutions being then
1 In a modification of this equation devised for reversible enzyme reactions,
B. Moore (1906) makes an attempt to take account of the activity of the
enzyme.
140 GENERAL CHEMISTRY OF THE ENZYMES
made up to 25 c.c. When the final state was reached (at 20 ),
the following quantities of amygdalin were decomposed:
Volume of aldehyde solution. Percentage of amygdalin decomposed.
c.c. 20-3
1 c.c. 18-8
5 c.c. 14-7
10 c.c. 11-31 Precipitation
Solution saturated with benzaldehyde. 5-7J of emulsin.
Hydrocyanic acid has a still more marked action :
25 c.c. of solution at 30 contained 0-5 grm. emulsin and 0-001
grm.-mol. of amygdalin:
Hydrocyanic acid added. Amygdalin decomposed.
0-0000 grm.-mol. 23-7
0-0001 " 18-7
0-0002 " 16-4
,0-0003 " 12-1
The effect of the third product of the decomposition, glucose,
is much less marked, as is shown by the following table, due to
Auld :
Minutes. Grm. of Glucose added. Amygdalin hydrolysed.
30 0-0 13-5%
30 0-2 13-3
30 0-75 11-8
30 1-0 11-6
In the further investigation of this influence it should be
remembered that emulsin consists of several specifically-acting
enzymes.
That the diastatic hydrolysis of starch gives an end point
which is influenced by the sugar formed, is pointed out by
M o r i t z and Glendinning (Journ. Chem. Soc., 1892,
61, 689).
Henri has made numerous experiments on the effect of
added glucose and fructose on the inversion of cane-sugar, the
establishment of an effect of this kind being of importance for
the development of his formula. As has been often pointed out,
it is greatly to be regretted that his experimental numbers are
considerably distorted owing to the mutarotation of glucose,
so that quantitatively they are almost valueless. It does appear,
CHEMICAL DYNAMICS OF ENZYME REACTIONS 141
however, that invert-sugar lessens the velocity of inversion.
The following table is compiled from Henri's numbers
(1 o c . c i t . , p. 202) :
2 c.c. of enzyme solution +50 c.c. 0-2-normal cane-sugar solution.
t
Without
addition.
X
+0-3-normal
invert-sugar.
X
a
a
99
0-138
0-072
215
0-301
0-160
299
0-407
0-224
459
0-594
0-340
586
0-700
0-417
1202
0-927
0-672
The 2-normal cane-sugar solution which is also 3-normal
with respect to invert-sugar was therefore hydrolysed only half
as rapidly as that without invert-sugar.
This retarding influence of invert-sugar seems to be due
exclusively to the fructose, as is shown by the following values of
cc
given by H e n r i .
Time.
0-2 N-cane-sugar.
2 N-cane-sugar.
+ 0-2 N-glucose.
0-2 N-cane-sugar
+ 0-2 N-fructose.
0-2 N-cane-sugar.
+ 0-2 N-invert-
sugar.
75
0-142
0-144 ,
0-123
0-119
184
0-385
0-362
0-317
0-306
275
0-564
0-532
0-457
0-446
445
0-798
0-746
0-672
0-648
605
0-906
0-878
0-799
0-794
As will be seen, the retardation of a reaction by the hydro-
lytic products is decidedly specific. That this is the case was
also brought out very clearly in a table given by E. F. A r m -
strong (Proc. Roy. Soc., 1904, 73, 516) to show the effect of
the hexoses in delaying the hydrolysis of sugars. Still more
striking examples of the specificity of such retardations will
doubtless be obtained from the investigation of the polypeptides
which has recently been taken in hand.
142
GENERAL CHEMISTRY OF THE ENZYMES
K ii h n e 's experiment on the influence of the decomposi-
tion products on the peptic digestion of fibrin has already been
quoted. B a y 1 i s s has made experiments on the retardation
of the tryptic digestion of casein by albumoses, peptones and
amino-acids (Arch. Sci. Biol. St. Petersburg, 1904, 11, Supple-
ment, pp. 261 et seq.), his results showing that the amino-acids
glycine and leucine were examined are the most active in
this respect.
In investigating the hydrolysis of dipeptides (glycylglycine)
by erepsin E u 1 e r also made experiments on the influence of
the amino-acids (glycine) (H., 1907, 61, 213). In this case it
was found that addition of glycine has only a subordinate effect.
0- 10 N-glycylglycine; 0-04 N-NaOH.
0-05 N-glycylglycine; 0-10 N-glycine;
0-04N-NaOH.
Minutes.
1000(a-a;).
1000&.
Minutes.
1000(o -x).
lOOOfc.
955
480
8
852
6-25
10
414
6-40
16
766
6-00
18
372
6-20
25
678
5-95
27
329
6-08
0-20 N-glycylglycine; 0-05N-NaOH.
0-10 N-glycylglycine; 0-2 N-glycine;
O-OSN-NaOH.
1860
_
900
_
6
1692
6-9
6
829
5-9
12
1545
6-7
12
767
5-8
20
1376
6-55
30
1210
6-2
It is here, of course, essential that the relation between the
NaOH and the acid present (dipeptide+amino-acid) is not
appreciably changed by the addition of glycine.
Special mention must further be made of the work of
Abderhalden and Gigon (H., 1907, 53, 251) on the
hydrolysis of glycyl-Z-tyrosine. In this case the tyrosine in the
solution produces a considerable retardation. Similar effects
are produced by the previous addition of active amino-acids,
especially of those occurring in nature : d-alanine, Z-serine, Meucine,
d-glutaminic acid, Z-phenylalanine, d-tryptophane and Z-diamino-
trihydroxydodecanic acid.
CHEMICAL DYNAMICS OF ENZYME REACTIONS 143
The order of magnitude of the retardations is shown by
the following results :
0-1 grm. glycyl-Z-tyrosine +
1 c.c. pressed yeast-juice.
0-1 grm. glycyW-tyrosine+1 c c. pressed yeast- juice
+ 0-05 grm. d-glutaminic acid.
Rotation corrected
Minutes.
Rotation.
Minutes.
Rotation.
for that of the
glutaminic acid.
+0-70
+0-70
+0-55
9
+0-51
7
+0-55
+0-47
23
+0-20
19
+0-54
+0-46
34
+0-00
29
+0-54
+0-46
49
+0-53
+0-45
79
+0-53
+0-45
105
+0-47
4-0-39
On the other hand, glycine, Z-alanine and d-leucine exert
no retarding action, whilst with the racemic compounds, such as
dZ-alanine, the effects are small. From the specific influences
which the hydrolytic products thus show towards the digestion
of polypeptides, it must again be concluded that the hydrolysing
enzyme enters into direct union with these protein decomposi-
tion products. At about the same time C h o d a t (Arch.
Sci. phys. nat., 1907, 26, 112) made similar measurements with
the anhydrides of Z-tyrosine and glycyl-Z-tyrosine. In extremely
high dilutions, the amino-acids have an accelerating
action. Abderhalden and G i g o n also point out the
difference between digestion in vitro, where the decomposi-
tion products of the proteins have a considerable retarding
influence, and digestion in the intestinal canal, where these inhibit-
ing products are rapidly removed by resorption.
In other words: In the organism we have (in certain periods)
so-called stationary states, in which the substance, acted on by
the enzyme and then removed from the sphere of action of the
latter by diffusion or some other method, is continually replaced
by an equivalent quantity of fresh starting material. Such cases
of stationary chemical processes, for example, with the unimolec-
ular reaction A +B+C, can be represented by the following
scheme.
Entry of A : *>
C o
x grm.-mols. !|2
per unit of time.
Field of reaction:
conversion of x
II
grm.-mols. per unit time. ~
Exit of B and C:
x grm.-mols. of
each per unit time.
144 GENERAL CHEMISTRY OF THE ENZYMES
For the given conditions, we have the simple relation :
dx
-j- = const.
at
For such a stationary condition to obtain, it is by no means
necessary that a system should be limited by solid walls. H.
Goldschmidt (Zeitschr. f. physikal. Chem., 1899, 31, 235)
has studied a chemical process in which the above equation is
realised.
If an excess of an ester, slightly soluble in water, is shaken
with a dilute hydrochloric acid solution, in which the ester (in
so far as it is dissolved) is hydrolysed with a certain velocity,
the concentration of the dissolved ester is kept constant merely
by the shaking, since the amounts destroyed by hydrolysis are
continually removed by the diffusion of the solution. If the
velocity of reaction is not very great, the ester disappearing from
the aqueous phase owing to the reaction, can be carried away
.completely by diffusion, so that the concentration of the ester
in the aqueous solution is maintained at a constant value, namely,
that corresponding with saturation.
The usual equation for a unimolecular reaction,
= (a-x}k
becomes
dx _ ,
since
a x = a = const.
A condition for such a reaction is, therefore, that the phase
in which the reaction occurs, is always saturated with reference
to the substrate; this condition may be fulfilled by vigorous
shaking of the heterogeneous system, or by employing the body
to be dissolved in an extremely finely divided state, so that its
surface of contact with the solution is very great. Excessively
laige surfaces of this kind occur particularly in so-called colloidal
solutions.
CHEMICAL DYNAMICS OF ENZYME REACTIONS 145
In this connection, it must be again pointed out that the
acceleration or retardation suffered by an enzyme reaction is
by no means always to be attributed to combination of the
enzyme with the substrate. In many, perhaps even in most,
cases it is a question of the alteration of the concentration of the
activators (co-enzyme, etc.), either by the reaction products and
the substrate acting in a reversible manner on these substances
or by the latter being changed or destroyed by secondary reactions.
The opportunity has already been taken of pointing out that the
deviation from the theoretical course of the reaction which is
termed S c h ii t z 's rule might, according to the data as yet
to hand concerning peptic digestion, very well be caused by the
gradual fixing of the hydrochloric acid by the albumoses and
peptones formed, the acid being withdrawn from both the sub-
strate and the pepsin. It is to be hoped that digestion exper-
iments carried out in vitro and with a constant concentration
of hydrochloric acid, may decide this not unimportant question.
According to theory, all reactions, even if they apparently
proceed to completion, are finally arrested at a position of equilib-
rium. Chemical reactions often go so far in one direction that,
under ordinary conditions, the equilibrium cannot be detected
analytically; only in very concentrated solutions is the equilib-
rium apparent. The manner in which the equilibrium of a rever-
sible reaction A^2B depends on the velocities of the two opposed
processes A 2B and A< 2B, and the way in which natural equilib-
ria and the final states of enzymic reactions are connected, will
be described in Chapter VI.
This short resume of the theory of enzymic dynamics may
now be brought to an end. It will be seen later how the exper-
imental data agree with the requirements of theory.
146
GENERAL CHEMISTRY OF THE ENZYMES
H. EXPERIMENTAL DATA ON THE COURSE OF
ENZYME REACTIONS
ESTERASES AND LIPASES
With esterases, that is, with enzymes which hydrolyse lower
esters but not neutral fats, K a s 1 1 e and Loevenhart
(Amer.Chem. Journ., 1900, 24, 491) and Kastle, Johnston
and E 1 v o v e (ibid, 1904, 31, 521) have carried out a number
of experiments; they used turbid aqueous extracts of pig's
liver and pancreas filtered through cloth.
Tubes containing 4 c.c. water, 0-1 c.c. toluene, and 0-26 c.c.
ethyl butyrate were heated for 5 minutes at 40. One c.c. of a
10% extract was then added and after a definite time the solu-
tion titrated with N/20-KOH solution:
Minutes.
X
k. ID*
K.E
5
(6-53)
1354
(0-45)
10
(8-66)
907
(0-40)
15
8-53
597
0-26
20
9-54
500
0-24
25
10-67
500
0-24
30
(9-41)
329
(0-16)
60
17-32
316
0-28
120
25-35
244
0-32
180
28-36
184
0-28
The constant k, calculated from the equation of a reaction
of the first order, diminishes regularly and very considerably.
Also interpolation shows that the errors of observation, pre-
sumably owing to the difficulty of pipetting small volumes of the
ester exactly, were very large; the values given in brackets fall
quite away from the curve. For the other numbers, the values
of K.E calculated from formula (18)
a In
a x
EXPERIMENTAL DATA OF ENZYME REACTIONS 147
show satisfactory agreement. This formula was deduced (pp.
133-135) on the assumption that the concentration of the free
enzyme is inversely proportional to that of the products of the
reaction. The same formula is obtained on the supposition
highly probable in this case at least that the activity
and not the concentration of the enzyme is inversely
proportional to the reaction products, chief among these being
the acid. As was found by K a s 1 1 e and Loevenhart,
the lipase of the pancreas is very sensitive to acids. It would
doubtless be of value to ascertain if the formula for unimolec-
ular reactions does not hold with good approximation if a cer-
tain quantity of strong acid were previously added in order to
depress the action of the products of the reaction.
With a moderate degree of accuracy the_ above figures and
those given below agree with the formula x/\/t = k.
The measurements made by S t a d e on an emulsion of egg-
yolk and neutralised gastric juice (Hofm. Beitr., 1902, 3, 291)
have been calculated according to the above formula by
Arrhenius and found to agree closely with it, as is seen from
the following table :
Hours.
x (observed).
x (calculated).
2
0-204
0-186
K# = 10
4
0-256
0-257
6
0-298
0-308
8
0-353
0-348
10
0-376
0-383
25
0-495
0-552
29
0-515
0-582
31
0-554
0-596
35
0-609
0-620
75
0-775
0-784
Further, Engel (Hofm. Beitr., 1905, 7, 77), in a careful
investigation with emulsion of egg-yolk and pancreatin on the
lines of Volhard's and S t a d e ' s experiments, arrived
at the result that S c h u t z 's rule holds for the enzymic saponifica-
tion of fats ; that is, for a constant period of digestion, the amounts
of digestion are in the ratio of the square roots of the quantities
of enzyme, and for the same quantity of enzyme the products
148
GENERAL CHEMISTRY OF THE ENZYMES
of digestion are proportional to the square roots of the times of
digestion. Hence, for one and the same juice, the equation
must hold. This is found to be the case.
Pancreatin.
4 hours.
9 hours.
25 hours.
x (obs.)
x (calc.)
VEi
x (obs.)
x (calc.)
X
V~Ei
x (obs.)
x (calc.)
X
VTsi
0-04grm.
0-09 "
0-16 "
17-6
20-9
35-2
16-8
24-5
31-6
4-4
3-5
4-4
18-4
36-3
48-4
24-5
35-0
44-6
3-1
4-0
4-0
35-0
58-2
72-1
38-3
53-0
65-0
3-5 15s
3-8L1
3.6(|
The values of x (calc.) have been obtained by means of A r r-
h e n i u s 's formula, the constant K being taken as 1. Although
the experimental errors are considerable, it is clear that the
numbers follow S c h ii t z ' s rule.
The experimental data obtained by Z e 1 1 n e r (Monatsh.
f . Chemie, 1905, 26, 727) with lipase from fly agaric (A m a n i t a
m u s c a r i a) have been subjected to calculation by K a n i t z ,
who found that the results vary, as in two series of experiments
T*
the quotient y was constant and in another series the expression
x
vl-
E u 1 e r (Hofm. Beitr., 1905, 7, 1) hydrolysed ethylbutyrate in
aqueous solution with esterases (from the fatty tissues of the
pig) and found the law for unimolecular reactions to hold.
Minutes.
t
Vol. of baryta solution
used in the titration
(C.C.).
x
Vol. of baryta solution
not used.
a x
Unimolecular reaction
constant.
fc.10*
2-70
2
0-3
2-40
256
6
0-75
1-95
235
9
1-05
1-65
237
16
1-65
1-05
250
From his observations on the lipase of Lactarius
sanguifluus, Rouge (Centralbl. f. Bakt., 1907, II,
EXPERIMENTAL DATA OF ENZYME REACTIONS 149
18, 403, 587) also drew the conclusion that, in dilute solutions,
the action of the enzyme is directly proportional to its amount.
Experiments with the true lipases all relate to systems with
limiting surfaces perceptible macroscopically (suspensions), since
most of the lipases are insoluble in water. Glycerine extracts
showing lipolytic activity can be obtained from the pancreatic
glands of the pig (A. K a n i t z , H., 1905, 46, 482). This author
followed the course of hydrolysis of olive and castor oils.
Into each of a series of test-tubes were placed 10 c.c. of olive oil,
3-9 c.c. 0-lN-sodium hydroxide, 0-25 c.c.N-calcium chloride, and 1 c.c.
of lipase extract, the contents of the tubes being titrated after t minutes.
The numbers of c.c. of 0-lN-sodium hydroxide used are given under x.
X
X
t
X
t
vT
0-0
_
70
9-2
0-131
1-10
140
12-3
0-088
1-04
288
19-0
0-065
1-12
405
23-1
0-057
1-15
1455
34-2
0-023
0-90
The time-law of the vegetable lipases was first investigated
by Connstein, Hoyer and Wartenberg (Chem.
Ber., 1903, 35, 3988), who established the essential fact that
considerable quantities of free acid are necessary for the lipases to
exhibit their action. If, as suggested by S i g m u n d (Monatsh.
f. Chemie, 1890, 11, 272), powdered castor-oil seeds are ground
with water and left for 24 hours at about 40, small quantities
of acid are detectable by titration. Later, however, the quan-
tity of acid formed rises suddenly. This " jump " occurs after
2-3 days at 35-40 or after 4-6 days at 15-20.
For instance, 5 grms. of castor-oil seeds were pounded with
5 grms. of 1% chloral hydrate solution and the mass kept at 16.
Immediately. After 2 days.
Per cent of ricinoleic acid found 1 3
4 days. 6 days. 8 days.
52 59 59
This is a case of autocatalysis, in which one of the products
of the reaction exercises an accelerating action. As to the most
150
GENERAL CHEMISTRY OF THE ENZYMES
favourable concentration of acid, information is given by the
following results, obtained with sulphuric acid:
Normality of the acid 0-02 0-05 0-10 0-12 0-2 0-5
Percentage hydrolysed in 18 hours ... 25 80 86 84 86 13
In addition to the seed-lipases, only pepsin acts in such strongly
acid solutions . According to the authors named above, the idea
that the acid transforms a pro-enzyme (zymogen) present in the
seeds into an active enzyme, is untenable, since when the seeds
are treated for a long time with acid and the latter then removed,
they exhibit no alteration; in fact they show as little fat-splitting
action in neutral solution as before treatment, and they also
become active in presence of acid.
In their examination of the influence of various organic acids
on Ricinus-lipase, H. E. Armstrong and O r m e r o d (Proc.
Roy. Soc., 1906, 78, 378) obtained the following numbers:
Concentration of the acid.
0-01 N.
0-02 N.
0-10 N.
0-50 N.
k. 10*.
Acetic acid
5-45
14-9
14-6
13-6
1-8
Succinic acid
2-80
14-6
15-4
12-2
6
Citric acid
7-25
15-3
14-7
1-1
82
Tartaric acid
6-95
15-4
14-2
97
Here also there occurs a rather flat maximum. No relation
is evident between the strength of the acid the dissociation
constants are given in the last column and the extent to which
the reaction is accelerated or the enzyme activated.
The nature of the added acid seems, indeed, to have but
slight influence, and this suggests the idea that the action consists
of a liberation of the true activator, itself presumably a weak
acid.
In order to ascertain the influence of the quantity of the
enzyme on the velocity of hydrolysis, quantities of 0-5 grm. of
the Ricinus-seeds were mixed well with 5, 10, 15, 20, 25, and 50
grms. respectively of castor oil and with similar amounts of 2%
acetic acid. Assuming that the active mass of the lipase is
proportional to the total quantity, and calculating the results
obtained after a certain time by means of the formula previously
deduced (p. 136) :
1000
EXPERIMENTAL DATA OF ENZYME REACTIONS 151
the following numbers are obtained, these agreeing satisfactorily
with the observed values excepting in the case of the largest
proportions of enzyme:
Action of 0-5 grm.
castor-oil
seeds on
After 1 day,
After 2 days,
grms. of oil. '
Solution.
x (obs.)
x (calc.)
x (obs.)
x (calc.)
50
50
49
49
49
59
25
25
60
65
74
74
20
20
71
69
80
78
15
15
77
75
87
84
10
10
81
83
86
91
5
5
89
94
92
98
K =
186
K
= 300
The time-course of the reaction is shown by a number of the
experiments carried out by these authors, among them Nos.
26, 28, 38, and 46. The last two were carried out by pounding
5 grms. of castor-oil seeds with 6 5 grms. of castor-oil and either
4 grms. of 1 N-sulphuric acid (No. 46) or 4 grms. of acetic acid
(No. 38). The results were as follows:
EXPERIMENT 46.
EXPERIMENT 38.
0-10 N-sulphuric acid.
0-10 N-acetic acid.
0-40 N-acetic acid.
t (mins.)
x (obs.)
x (calc.)
t (hours)
* (obs.)
x (calc.)
t (hours)
x (obs.)
x (calc.)
15
12
20
1
52
48-6
1
65
63-9
30
20
27
2
65
62-8
2
86
78-8
45
30
32
3
70
71-5
3
84
86-5
00
33
36
4
72
77-6
4
84
91-2
90
41
43
24
80
99-5
24
91
99-9
150
54
53
*E= 180
K.i = 380
210
59
59
330
68
69
1620
81
97
With the longer times, the deviations of the observed numbers
from the calculated ones are considerable and on this account
Arrhenius supposes equilibrium to be set up in these cases.
N i c 1 o u x (Soc. Biol., 1902, 54, 840) has also made a thorough
investigation of Ricinus-lipase or, as he terms it, the lipolytic
action of the cytoplasm of Ricinus-seeds. N i c 1 o u x like-
152 GENERAL CHEMISTRY OF THE ENZYMES
wise found his preparation to be insoluble in water; he emulsified
the cytoplasm in the oil mostly cottonseed oil to be hydro-
lysed and added dilute acetic acid. The following numbers were
obtained at 18:
t (minutes) ....... 30 45 60 90 127 150 210 450
Percent, hydrolysed 23-6 33-1 40-4 54-8 67-0 73-2 85-5 94-4
K E = lOOOZn -- x 1-10 1-58 1-89 2-73 3-45 3-89 5-12 4-31
1000 -x
100 100
k= - log - 0-388 0-387 0-375 0-382 0-392 0-381 0-399 0.278
t 100-x
It is evident that the observed numbers do not follow
S c h u t z 's rule; on the contrary, the course of the hydrolysis
agrees moderately well with the formula for unimolecular reactions.
A thorough series of experiments with Ricinus-lipase has
recently been carried out by Jalander (Biochem. Z., 1911, 36, 435).
For short times (up to 60 minutes) be found proportionality with
enzyme-concentrations of 1-4 per 1000. For more protracted
action this proportionality disappears, S c h ii t z ' s rule, x:VE =
const, then holding approximately.
Bodenstein and Dietz (Zeitschr. f. Elektrochem.,
1906, 12,605; Dietz, H., 1907, 52, 279) have also studied a
heterogeneous system. Pancreatic lipase, in the shape of shavings
of the tissue of the pancreatic glands of the pig, was emulsified
with amyl alcohol containing water and butyric acid or amyl
butyrate in solution.
One would expect the velocity equation,
~TT = ^1 * ^ acid " C alcohol K>2 ' C ester ' C water
at
to be fulfilled, all the concentrations relating to the enzyme
phase. The concentrations of water and alcohol are approx-
imately constant, since only very small quantities of these dis-
appear during the process. On the other hand, the substances
must be divided between enzyme and liquid according to
Nernst's law of distribution, i.e., C en zyme = a. C so i u tion. The
proportionality factors, like the constant concentrations of
alcohol and water, become included in the constants, so that
-- = ki Cacld &2 Cester = &1 (a x) k 2 X.
EXPERIMENTAL DATA OF ENZYME REACTIONS 153
If but little water is dissolved in the amyl alcohol, the reverse
reaction is very slight and the process goes on according to the
simple equation:.
dx ,
as is shown by the following table :
a = 10 normal
4% H 2 0.
2% H 2 O.
t (hours).
Titre of 5 c.c.
k
t (hours).
Titre of 5 c.c.
k
0-00
13-55
0-00
13-55
-
2-00
12-75
0-013
2-50
13-15
0-0064
5-53
11-90
0-010
9-57
11-80
0-0062
10-33
10-35
0-012
14-35
10-95
0-0064
15-17
9-15
0-012
24-45
9-45
0-0063
25-20
7-05
0-012
31-88
8-45
0-0063
32-58
5-85
0-012
47-89
6-55
0-0065
48-83
4-25
0-012
55-27
5-90
0-0064
55-92
3-90
0-011
76-67
4-60
0-0060
77-73
3-00
0-010
100-42
3-25
0-0062
308-00
1-50
308-00
1-10
On passing to higher contents of water, a final condition
attainable from either side is set up and the velocities of the two
opposed reactions become measureable. Such a case is presented
by the following tables.
Formation of ester.
Hydrolysis of ester.
t (hours).
Titre of 5 c.c.
ki
t (hours).
Titre of 5 c.c.
h
0-00
13-40
0-00
0-00
1-98
12-60
0-014
3-63
0-75
0-0075
4-00
11-82
0-014
7-60
1-45
0-0077
6-98
10-80
0-014
16-77
2-35
0-0064
11-55
9-10
0-016
24-05
2-95
0-0070
14-98
8-40
0-016
89-30
4-20
25-10
6-55
0-016
96-95
4-25
Mean 0-015
Mean 0-0072
154
GENERAL CHEMISTRY OF THE ENZYMES
Both constants are approximately doubled if the quantity
of enzyme is doubled:
Quantity of enzyme. ^ Constants^
1 0-015 0-0072
2 0-028 0-014
A . E . Taylor, who hydrolysed triacetin, the acetic ester
of glycerine, with powdered Ricinus-seeds, found the course of
the reaction to be as with unimolecular reactions. He gives the
following results for experiments in which 0-5, 1, and 2% solu-
tions of triacetin were employed. The constants k refer to 18.
t (hours).
4
8
16
24
28
32
40
48
0-5%
x (obs.)
0-096
0-162
0-287
0-418
0-489
0-477
0-623
0-652
k
109
96
92
98
104
88
106
96
1-0
x (obs.)
0-083
0-174
0-338
0-418
0-488
0-542
0-609
0-655
k
94
104
112
98
104
106
102
96
2-0
x (obs.)
0-098
0-174
0-323
0-431
0-502
0-485
0-595
0-636
k
112
104
106
102
108
90
98
91
How it happens that in all these experiments the quantity of
fat hydrolysed can be calculated by means of the very simple
law holding for homogeneous" systems, is not very easy to under-
stand. With the hydrolysis of fats and of triacetin this is all
the more remarkable, since this hydrolysis takes place in three
stages, which doubtless occur with different velocities.
New experiments with esterase from pig's liver have been made
by G. Peirce (Journ. Amer. Chem. Soc., 1910, 32, 1517).
The principal results are as follows:
(1) In a solution of given volume and given acidity, the time
required for the hydrolysis of a definite quantity of ethyl butyrate
is inversely proportional to the concentration of the enzyme.
Under similar conditions of acidity, each particle of enzyme
hydrolyses the same absolute amount of ester per instant, no
matter what the concentration of the enzyme.
(2) With a given concentration of enzyme, the time taken
to hydrolyse a given amount of ethyl butyrate is dependent on
the acid-concentration but not on the ester-concentration, pro-
EXPEKIMENTAL DATA OF ENZYME REACTIONS 155
vided this is above N/200. In other words, for each concentra-
tion of acid a given amount of enzyme hydrolyses very nearly the
same amount of ethyl butyrate over a wide range of ester-con-
centration.
(3) This phenomenon can be brought into conformity with
the law of mass action by assuming that the enzyme and the ester
form an intermediate compound, which, in concentrations of
the ester above N/200 contains most of the enzyme.
AMYLASE
The chemical process of the hydrolysis of starch presumably
takes place in several phases, in which the dextrins formed as
intermediate products are broken down; amylase may con-
sequently be composed of several enzymes.
In experiments with diastase (amylase), the difficulties
attending the varying constitution of the enzyme are supplemented
by the complication introduced by a non-individual substrate.
As has been shown, more especially by Maquenne's inves-
tigations, starch consists in reality of two components, namely,
80-85% of amylose and 15-20% of amylopectin. A m y 1 o s e
forms no paste and in solution is coloured an intense blue by
iodine; only in solution is it attacked by malt-diastase. There
exist a series of condensation products increasingly soluble in
water, amylose being the last of these; the lower members more
especially are largely contained in " soluble starch." In the
saccharification of pure amylose maltose alone is formed. Amylo-
pectin is a gelatinous substance which is insoluble in water and
alkalis and gives dextrin as well as maltose (?) on hydrolysis.
It is not yet certain, but is probable that this is brought about
by a special amylopectinase and not by. the amylase. In any
case, in quantitative experiments on amylase the purest possible
amylose or, at any rate, " soluble starch " should be employed.
For the technical determination of diastase, potato starch is
treated according to L i n t n e r 's method (see Appendix).
As regards the hydrolysis curves, the experiments made by
different authors do not agree especially well. The earliest of these
experiments, carried out by H . Brown and G 1 e n d i n -
n i n g (Journ. Chem. Soc., 1902, 81, 388) with malt-extract do
156
GENERAL CHEMISTRY OF THE ENZYMES
not correspond with the simple logarithmic curve.
are expressed better by the formula
1 , a+x
The results
as is shown by the following numbers.
3% solution of starch with 0-25 c.c. malt-extract per 100 c.c.:
Minutes.
X
fc.10 5
fctf.10 5
Temperature, 51-52.
10
0-1084
498
'472
20
0-2250
553
497
40
0-4350
620
506
60
0-6150
690
518
80
0-7385
728
514
100
0-8150
732
495
120
0-8800
762
497
140
0-9220
791
497
160
0-9500
813
492
3% solution of starch with 1 c.c. malt-extract per 100 c.c.
10
0-081
366
352
Temperature, 21
20
0-163
386
357
40
0-308
399
345
60
0-440
419
341
70
0-506
437
345
Henri's experiments (Lois generates etc.), on the other
hand, follow the logarithmic law; whether the method employed
(determination of the change of reducing power) is free from
objection depends on the purity of the amylase used.
Also with saliva-diastase, A . E . Taylor obtained the
following values:
Substrate 0-25%
t (minutes.)
30
45
60
75
90
120
150
180
fc(X10 6 )
490
465
455
470
465
455
460
455
Substrate 0-5%
k (X10 6 )
430
420
390
415
405
395
430
410
Substrate 0-75%
k (X10 6 )
390
370
385
390
380
370
365
370
As Taylor emphasised, the values of k vary considerably
with the concentration of the substrate.
In the course of a valuable investigation on pancreas-amylase,
Kendall and Sherman (Journ. Amer. Chem. Soc., 1910,
EXPERIMENTAL DATA OF ENZYME REACTIONS 157
32, 1087) found: (1) that the initial speed of conversion for a
constant amount of enzyme was the same for different concentra-
tions of starch; (2) that the speed of the reaction diminished
the more rapidly, the smaller the initial concentration of the starch.
For the formula deduced by these investigators to express
the course of the reaction, their original paper must be consulted.
As regards the influence of the concentration of the amylase,
Henri found, with a vegetable preparation, proportionality
between the concentration of the enzyme and the quantity of
starch hydrolysed per unit of time.
Taylor arrived at the same result with saliva-diastase.
Brown and Glendinning (Journ. Chem. Soc., 1902,
81, 381), however, found the velocity of reaction to be propor-
tional to the square-root of the concentration of the saliva-diastase,
and Klempin (Biochem. Z., 1908, 10, 206), from experiments
with oat-diastase, concluded that S c h ii t z 's rule, E^/i= K,
holds for this enzyme.
P a w 1 o w 's experiments (Arbeit der Verdauungsdrlisen,
Wiesbaden, 1898), which were carried out with M e 1 1 's capillary
tubes and are of interest in themselves, hardly give any informa-
tion concerning the course of the saccharification of starch, since
they dealt with the liquefaction of starch-paste and since also
the velocity of diffusion exerts a determining influence on the
velocity of the process.
A large number of experiments were made by Mdlle.
P h i 1 o c h e (Journ. de Chim. physique, 1908, 6, 213, 355) with
" Merck " diastase and taka-diastase, starch and glycogen
being used as substrates.
The constants of the formula for unimolecular reactions di-
minish rapidly as the reaction proceeds. The following numbers
were obtained with 1% starch solution, the concentration of the
diastase being 1 : 20,000.
Minutes.
X
1 a
a
t ax'
21
0-06
123
51
0-11
100
113
0-15
62
224
0-26
58
390
0-36
50
158 GENERAL CHEMISTRY OF THE ENZYMES
The influence of the concentration of the starch on the amount
of maltose formed in 60 minutes is shown by the following table:
Concentration of starch (%). Quantity of maltose (60 mins.).
1 0-24
1-5 0-30
2 0-338
2-5 0-397
3 0-397
Activators and Inhibitors. The following substances
favour the hydrolysis of starch: vanadium and aluminium salts, ammo-
nium and calcium phosphates, asparagine, ammo-acids, proteins and
picric acid (Ef front, Enzymes and their Applications, p. 117;
Soc: Biol., 1904, 57, 234; Allg. Brauer- und Hopfenzeitung, 1905, 45).
The accelerations are sometimes very considerable; thus an addition of
0-05 grm. of asparagine to 100 c.c. of a starch solution containing
amylase increases the velocity sevenfold. Carbon dioxide also has an
accelerating action, especially when under increased pressure (D e t m e r ,
Mtiller-Thurgau).
The optimum activity of vegetable diastases occurs with a slight
excess of hydrogen-ions (small quantities of organic acids); hydroxyl-
ions, even in very small concentration, retard the hydrolysis, but the
inactivation is annulled immediately the alkali is neutralised. The
optimum temperature (measured by K j e 1 d a h 1 by the reducing
power of the hydrolytic products) is 63.
INVERTASE
On the hydrolysis of cane-sugar by invertase numerous
quantitative investigations have been carried out. Of these,
besides the earlier work of K j e 1 d a h 1 (Medd. fra. Carlsberg
Lab., 1881), mention must first be made of the researches of
T a m m a n n (Zeitschr. f. physikal. Chem., 1889, 3, 25) and of
those executed almost simultaneously by O ' S u 1 1 i v a n and
T o m p s o n (Journ. Chem. Soc., 1890, 57, 834). These investiga-
tors showed first that the reaction is a catalytic one and O ' S u 1 -
1 i v a n and T o m p s o n found it to be unimolecular. This
result was contested later by D u c 1 a u x (Traite de Micro-
biologie, Vol. 2, 129), while Henri (Zeitschr. f. physikal.
Chem., 1902, 39, 194) also arrived at a formula differing from
the unimolecular one, namely, the expression (compare p. 129),
1 a+x
EXPERIMENTAL DATA OF ENZYME REACTIONS 159
which corresponded moderately well with his experimental data.
Still later Bodenstein deduced the complicated formula
mentioned on p. 129, and finally Henri (Theses, p. 92)
arrived at the following equation:
a 1 . 1 i a
J +- f log -_-,
which he obtained by integration of the differential Eq. (12)
of p. 131. As has been already stated, it has recently been found
that the experimental data of Henri and his collaborators
are considerably influenced by the mutarotation of glucose
and therefore give no definite information as to the time-course
of the hydrolysis of cane-sugar; further, in these experiments
the concentration of the hydrogen-ions was not defined. Hud-
son (Journ. Amer. Chem. Soc., 1908, 30, 1160, 1564) has per-
formed a valuable service not only in demonstrating the reliabil-
ity of O ' S u 1 1 i v a n and Tompson's data, but also in
continuing and considerably extending the investigations of
these workers.
O ' S u 1 1 i v a n and T o m p s o n had rightly recognised that
glucose formed from cane-sugar by inversion appears firstly in a
mutarotating condition and that therefore the optical activity
of a solution which has been inverted by enzyme affords no measure
of the progress of the reaction. To the test-portions removed
after definite times from their solutions they hence added a little
alkali, which annuls the mutarotation almost instantaneously.
Their results show that experiments carried out in this way
correspond with the unimolecular formula, but that, if the pre-
caution mentioned is not taken, the constant increases con-
siderably. Their numbers are as follows :
Rotation.
1 . a
k =ln .
x ax
Minutes.
Without alkali.
With alkali.
Without alkali.
With alkali.
74-5
69-4
37
57-9
37-6
0-0021
0-0046
152
0-6
-20-4
0-0037
0-0074
268
-19-1
-24-8
0-0041
0-0058
00
-27-8
-27-8
160
GENERAL CHEMISTRY OF THE ENZYMES
The constancy of the figures in the final column leaves some-
thing to be desired and Hudson's numbers may well be given
here:
Rotation.
10*-^. log -^-.
t a-x
Minutes.
Without alkali.
With alkali.
^Without alkali.
With alkali.
24-50
24-50
30
16-85
14-27
396
558
60
10-95
7-90
399
530
90
4-75
3-00
464
539
110
1-95
0-80
482
534
130
-0-55
-1-49
511
559
150
-2-20
-2-40
522
533
00
-7-47
-7-47
As is seen from this and other tables given by Hudson,
the values of k are constant.
According to Sorensen (Biochem. Z., 1909, 21, 131)
and Michaelis and Davidsohn (Biochem. Z., 1911,
35, 386), however, this constancy of the reaction-coefficient is
observed only with a certain concentration of the hydrogen-ions.
With different H'-concentrations, Sorensen obtained the
following results:
Temperature 52
H'=0-2.10- 6
H' = 0-1.10- 3
H-=0-2.10~ 3
t
k
t
k
/
k
2
2
2
17
91
17
127
17
53-6
32
103
32
127
32
39-3
47
111
47
132
47
26-1
62
127
62
135
62
18-2
92
147
92
149
92
15-3
122
230
122
126
122
11-2
While, therefore, with H'-concentration of 0-2.10 6 , the coefficient
k increases, with H* =0- 1.10~ 3 it remains constant
and with H'=0-2.10~ 3 it diminishes. Michaelis and
Davidsohn explain this behaviour on the assumption that
EXPERIMENTAL DATA OF ENZYME REACTIONS 161
the constancy of the values of k is brought about by the com-
pensating effect of the destruction of the enzyme on the increasing
values of the coefficient. Considering the high temperature, 52,
chosen for Sorensen's experiments, this view is most prob-
ably accurate, and at lower temperatures, and higher concentra-
tions of hydrogen-ions (H' = 10~ 3 ) M i c h a e 1 i s and David-
s o h n found increasing values for k.
That other experimenters have been unable to obtain agreement
with the law of mass action, cannot therefore, be due solely to non-
removal of the mutarotation. Thus, H. E. Armstrong and
Glover, in comparing the actions of invertase on cane-sugar and
on raffinose (Proc. Roy. Soc., 1908, 80, 312), rendered the sugar solutions
alkaline before reading them in the polarimeter, but still obtained no
greater constancy, as is shown by the figures in the left-hand part of
the following table:
34-2 grms. cane-sugar
+4 c.c. invertase-extract per 1000 c.c.
59-4 grms. raffinose
+4 c.c. invertase-extract per 1000 c.c.
Minutes.
Percentage
hydrolysed.
&.10 5 .
Percentage
hydrolysed.
fc.10 5 .
0-0
0-0
5
8-3
753
1-8
157
15
25-9
868
5-9
176
25
39-5
865
13-4
249
40
62-4
1062
20-1
243
60
78-2
1102
29-3
251
95
01-1
1106
41-6
246
140
93-7
859
53-4
237
200
95-1
656
66-6
238
260
96-0
537
77-9
252
oo
100-0
These tables show that invertase hydrolyses cane-sugar into fructose
and glucose about four times as rapidly as this enzyme decomposes
raffinose into fructose and melibiose.
Our knowledge of the other conditions governing the action
of invertase is due to the work ofO'Sullivan and T o m p -
son and of H u d s o n, confirmation of which has been supplied
by Taylor (Journ. of Biol. Chem., 1909, 5, 405). The pro-
portionality found by the first-named authors to exist between
the velocity of inversion and the concentration of the enzyme
is completely confirmed by H u d s o n 's results.
162
GENERAL CHEMISTRY OF THE ENZYMES
O ' S u 1 1 i v a n and T o m p s o n give the following table
(1 o c . c i t . , p. 848) :
Temp.
Invertase
prepara-
N-H 2 SO4.
Time readings.
Reading
in 2 d.m.
Minutes taken to
reach zero rotation.
tion, grms.
tube.
Beginning.
End.
A (obs.).
B (calc.).
15-5
0-15
0-00187
11 40
4 41
- 2-0
283-0
291
15-5
0-45
0-0031
3 00
4 40
- 1-8
94-8
96-3
15-5
1-50
0-0050
11 56
12 26
+ 1-0
30-7
29-1
56-5
0-0345
0-00025
11 00
12 43
+ 16-5
157-6
157-1
56-5
0-0722
0-000375
11 22
12 15
+ 13-5
74-8
75-1
The third column gives the acidity of the solution, the con-
centrations of acid used having been found by preliminary
experiments to be the most favourable to the velocity of reaction.
In the last column but one, marked A, are given the times elapsing
before the rotation falls to and in the last column, B, the times
which would be necessary for this to occur on the assumption
of proportionality between concentration of enzyme and velocity
of reaction.
Also with change of the concentration of sugar, the require-
ments of theory seem to be satisfied within very wide limits by
O' S u 1 1 i v a n and Tompson's results, that is, in equal
times one and the same quantity of enzyme hydrolyses equal
proportions of the sugar, no matter what the concentration of the
latter may be.
On the other hand, A . J . Brown (Journ. Chem. Soc., 1902, 81,
373) gives numbers indicating that a given quantity of enzyme inverts
the same absolute amount of sugar in a definite time:
Grms. of cane-sugar
per 100 c.c.
Grms. of cane-sugar inverted
in 60 minutes.
Percentage of cane-sugar
inverted in 60 minutes.
4-89
1-230
25-2
9-85
1-355
13-8
19-91
1-355
6-8
29-96
1-235
4-1
40-02
1-076
2-7
But in dilute sugar solutions containing relatively large amounts of
enzyme, the action of a given quantity of enzyme is, according to
Brown, proportional to the concentration of the sugar.
EXPERIMENTAL DATA OF ENZYME REACTIONS 163
Grms. of cane-sugar
per 100 c.c.
Grms. of cane-sugar
inverted in 60 minutes.
; lO'fc^log^-.
t ax
(2-0)
(0-308)
(132)
1-0
0-249
219
0-5
0-129
239
0-25
0-060
228
Hudson collects his results in the following table :
Influence of the Concentration of Inver-
tase on the Velocity of Inversion at 30
Percentage of cane-sugar inverted with the
Concentration
of the
Minutes.
Product.
following amounts of cane-sugar per litre.
invertase,
E.
t
E.t.
A c c grms.
90-9^:
litre
o_o grms.
litre
litre
2-00
15
30
73-2
45-3
11-2
2-00
30
60
93-0
74-2
22-0
1-50
20
30
73-2
44-8
11-2
1-50
40
60
92-8
74-5
22-7
1-00
30
30
72-9
45-3
11-5
1-00
60
60
93-0
74-7
22-3
0-50
60
30
72-9
45-2
11-4
0-50
120
60
92-7
74-5
22-6
0-25
120
30
73-1
45-2
10-9
0-25
240
60
92-7
74-7
21-9
The results are therefore as follows :
1. Proportionality exists between the amount of sugar inverted
per unit of time and concentration of the enzyme.
2. The concentration functions are, in addition, dependent
on the relative quantities of substrate and enzyme. So
long as the enzyme is not present in large excess the relative
amount of hydrolysis diminishes as the amount of substrate is
increased.
With his erroneous method, Henri observed a relation indicated
by the following table. The number of milligrams of sugar inverted
after the first minute in a c-normal cane-sugar solution is denoted by n:
0-58
0-01
1-41
0-025
2-40
0-05
2-96 4-65
0-10 0-25
5-04
0-50
4-45 2-82
1-00 1-50
1-15
2-00
Apart from the fact that Hudson' s investigation has led
again to a method which is free from objection, the observation
164
GENERAL CHEMISTRY OF THE ENZYMES
that cane-sugar gives rise first of all to a-glucose possesses on
little interest. (Its bearing on the enzymic synthesis of disac-
charides will be considered later.) From the work referred to
we may take the following extract:
From cane-sugar, not only glucose but also fructose is formed
in a labile (characterised by high rotatory power) modification.
But the disappearance of the mutarotation measured at 30
in the case of fructose (constant of reaction k = 186) proceeds
about 11 times as fast as with glucose (k = 0-0167). Dissolved
invertase-preparations have no influence on these velocity con-
stants. Hence, in a cane-sugar solution undergoing enzymic
hydrolysis, the difference between the apparent and the actual
degree of hydrolysis depends almost entirely on the alteration
of the rotation of the glucose. If by means of a very active
invertase, a cane-sugar solution can be inverted almost instan-
taneously, all further alteration in the rotation of the solution
must be attributed almost wholly to the mutarotation of the glu-
cose and the velocity of this change must be nearly coincident with
the fall of rotation occurring with pure glucose. This has now
been actually confirmed.
Use was made of an invertase solution so active that 72%
of the cane-sugar was inverted within half a minute. The final
rotation was determined after addition of a little sodium hydroxide
solution:
Minutes.
Rotation without
alkali.
10' . a
-log
t ax
Minutes.
103 , a
log
t ax
33-50
3
11-88
99
4
7-32
99
5
4-77
93
9
- 0-35
72
10
- 1-35
69
13
- 3-57
63
Commencement
16
- 4-90
57
3
32-3
19
- 6-03
54
6
33-4
23
- 7-15
50
10
33-6
29
- 7-92
44
16
28-7
30
- 8-22
45
17 .
30-7
00
-10-22
31 '7
EXPERIMENTAL DATA OF ENZYME REACTIONS 165
In an equally concentrated solution of invertase the velocity
constant of the disappearance of the mutarotation of glucose
was found to be /b.!0 3 = 29-9, which is in good agreement with
the value, 31-7, given above.
Hudson's excellent investigations have been treated at
length, because the results obtained with invertase are of impor-
tance to the consideration of the enzymic decomposition of other
disaccharides and of the glucosides, where the mutarotation of
the hexoses makes itself felt to a greater or less extent as a source
of error.
Worthy of notice is the influence of the products of the reac-
tion on the course of inversion. As is shown by the experiments
ofE.F. Armstrong (Proc. Roy. Soc., B, 1904, 73, 500),
Barendrecht (Zeitschr. f. physikal. Chem., 1904, 49, 456)
and also Henri (loc. cit.), fructose retards inversion to a much
greater extent than glucose does. According to Baren-
drecht, galactose also exercises a retarding influence, which
is, however, less than that of fructose.
The marked acceleration of enzymic inversion by acids has
already been mentioned.
It was found by O'Sullivan and T o m p s o n that invertase
is very sensitive to small amounts of acid, and this observation has
been confirmed by Hudson (loc. cit.). According to the work of
the latter, invertase shows its optimum activity in about 0006 normal
hydrochloric acid. Sorensen, who made a very thorough inves-
tigation of the influence of acidity on enzyme actions (Biochem. Z.,
1909, 21, 131) found the optimum concentration of the hydrogen-ions
for invertase to be W~ 4 ' 4 10 ~ 4 ' 6 . Very weak acids, like carbonic
acid, produce corresponding accelerations.
The slightest excess of OH-ions brings the reaction. to a standstill.
The enzyme is almost entirely uninfluenced by antiseptics such as
toluene and chloroform.
The temperature-optimum is given as 50-60. The inactivation
constant, & t !0 3 , of invertase from a yeast of Frohberg type was found
to have the value 4 at 60 and with a H'-concentration of 10 ~ 6 in
aqueous solution (a f U g g 1 a s , H., 1910, 65, 124).
The velocity of inversion by living yeast has been
studied byEuler andS.Kullberg (H., 1911, 71, 24).
In this case the system is macro-heterogeneous, so that the
velocity of diffusion should exert an influence on the course of the
process.
166
GENERAL CHEMISTRY OF THE ENZYMES
Here also the inversion corresponds with the formula for
unimoleeular reactions.
Temperature, 20.
Inversion mixture.
Time
(mins.).
Rotation.
A-x
&.1O>.
la
0-25grm. fresh distillery yeast
+20 c.c. 20% cane-sugar solu-
tion +5 c.c. H2O + 1 c.c. chlo- i
reform; the reaction was
stopped by 5 c.c. 0-4N-NaOH
solution
17
25
34
00
7-15
6-25
5-80
5-25
-2-29
9-44
8-54
8-09
7-54
26
27
29
7-15
9-44
16
Same as above, but without ad-
dition of chloroform
17
25
6-20
5-84
8-49
8-13
27
26
36
5-30
7-59
26
00
-2-29
It will be seen that chloroform exerts no influence on inversion
by living yeast. Between 20 and 30 , the effect of the tem-
perature is the same as with inversion by dissolved enzyme.
As is well known, invertase attacks the trisaccharide, raffinose,
decomposing the cane-sugar group present. This hydrolysis
proceeds more slowly than that of free cane-sugar; it has been
investigated by H. E. Armstrong and Glover (Proc.
Roy. Soc., 1908, 80, 317).
MALTASE
As with emulsin, so also with maltase, E.F.Armstrong
(Proc. Roy. Soc., 1904, 73, 508) found the reaction-constants of
the first order to diminish considerably.
Maltose, 5%.
Hours.
X
k. 10*
Hours.
X
k.W*
1
7.3
329
1
4-7
209
2
13-9
325
3
11-7
180
4
24-4
304
5
17-8
170
7-25
31-7
229
23
23-9
52
23
35-2
82
28
25-0
45
47
31-4
35
Maltose, 10%
EXPERIMENTAL DATA OF ENZYME REACTIONS 167
In striking contrast to these are the numbers of Henri
and Mdlle. P h i 1 o c h e (Soc. Biol., 1904, 57, 171), of H e n r i
and of Terroine (Archivio di Fisologia, 1904, 2, 1), who
found that the constants k for a unimolecular reaction at first
rise, whilst the constants kn for a definite initial concentration
remain comparatively constant.
Terroine (loc. cit., p. 4).
Maltose, 4%.
P h i 1 o c h e (loc. cit., p. 6).
Maltose, 4%.
Minutes.
fc.lO*.
2k H .W 5 .
Minutes.
X
a,
k. 105.
50
88
167
63
0-176
134
112
86
156
120
0-312
135
175
103
170
181
0-441
139
230
127
202
241
0-588
163
349
119
176
363
0-753
167
470
134
184
480
0-824
157
588
122
166
600
0-859
142
780
113
148
750
0-869
117
903
106
148
930
0-900
107
The constants vary, however, with the dilution of the mal-
tase, so that Henri first employed formula (3), p. 129, for the
calculation (m = 3, w=l), but as the agreement with the exper-
imental data was not satisfactory, he proposed the introduction
of new constants into this formula.
Further, H e r z o g (Zeitschr. f. allg. Physiol., 1904, 4,
177) obtained different results, which he calculated according
to formula (le), p. 129, with variable coefficients.
Maltose, 3-67%. =1-12.
Maltose, 3-217%. e=2.
Minutes.
X
a
k
*i(l-e)10.
Minutes.
X
a
t
&i(l-e)io.
30
0-128
195
27
20
0-066
149
16
60
0-215
175
23
40
0-138
161
19
120
0-328
144
22
80
0-193
116
15
180
0-415
127
22
120
0-248
103
14
240
0-471
115
20
180
0-312
90
15
370
0-567
76
21
240
0-358
80
15
590
0-631
73
20
620
0-687
81
22
168
GENERAL CHEMISTRY OF THE ENZYMES
It is very probable that the action of maltase, like invertase-
action, depends on the concentration of the H'-ions present.
It is most desirable that a new investigation should be made in
which this influence is considered; more simple laws of reaction
would then probably be found to hold.
According to Croft Hill and to Lintner and K r 6 b e r
(Chem. Ber., 1895, 28, 1050) the optimum temperature is 40. A s p e r -
g i 1 1 u s - maltase is stated to be only slightly sensitive towards chloro-
form (H 6 r i s s e y , Soc. Biol, 1896, 48, 915). In working with yeast-
maltase Fischer recommends the use of toluene.
LACTASE
Quantitative measurements have been made by E . F . A r m -
strong (Proc. Roy. Soc., 1904, 73, 506).
In the various series of experiments, the reaction-constants
of the first order diminish considerably, but not regularly. As
examples, the following tables may be given-
Two GRMS. MILK-SUGAR PER 100 c.c.
I.
II.
100 c.c. enzyme-extract.
40 c.c. enzyme-extract.
Hours.
X
fc.lO<
Hours.
X
.10*
1
22-1
1085
0-33
3-2
423
2
31-2
812
0-66
6-4
430
3
38-9
713
1
9-6
438
4
45-8
665
1-5
13-2
410
5
51-5
629
2
16-4
389
6
56-6
664
3
20-8
338
10
69-0
509
5
25-2
252
17
84-2
471
23
47-6
122
23
92-4
461
100
89-6
82
The constants are evidently dependent on the concentration
ratio, enzyme : substrate. With a relatively large amount of
enzyme, the constant diminishes continuously, but if less enzyme
is present, equal amounts of sugar are at first hydrolysed in
equal intervals of time.
The following table shows the influence of the concentration
of lactase on the velocity of hydrolysis of a 5% milk-sugar solu-
tion; the quantities of sugar hydrolysed are given in percentages:
EXPERIMENTAL DATA OF ENZYME REACTIONS 169
C.c. Lactase.
1 -5 hour.
20 hours.
25 hours.
45 hours.
68 hours.
1-0
0-15
2-2
2-6
3-9
4-8
2-5
0-4
5-8
6-8
10-2
12-6
10.
1-6
23-3
38-6
48-5
20.
3-2
45-8 54-5
1
The quantities hydrolysed are approximately proportional
to the enzyme-concentrations, so long as these are not too high.
As is shown by the next table , very small amounts of enzyme
are able to hydrolyse only small amounts of sugar; their activity
then ceases, indicating that the products of hydrolysis, glucose
and galactose, combine with the enzyme and so withdraw it
from the reaction with the substrate.
5% Milk-sugar solution; amounts hydrolysed in percentages.
c.c. Lactase.
24 hours.
144 hours.
0-66
2-3
2-3
1-0
3-2
3-5
2-0
6-3
7-4
5-0
15-4
34-0
If the quantity of milk-sugar is varied, it is found that, with
large proportions of enzyme, the amount hydrolysed in unit
time is proportional to the concentration of the sugar, so that the
values of k are equal.
PERCENTAGES OF MILK-SUGAR HYDROLYSED
Milk-sugar
per 100 c.c.
Hydrolysed
after 3 hours.
k. 10*.
1-0 grm.
0-5 "
0-185
0-098
296
298
0-2 "
0-0416
337 .
For comparatively large amounts of substrate the percentage
of sugar hydrolysed is inversely proportional to its concentration,
so that in unit time equal absolute amounts of sugar are decom-
posed, no matter what the concentration.
170
GENERAL CHEMISTRY OF THE ENZYMES
According to H. E.Armstrong, E. F. Armstrong
and H o r t o n , emulsin contains a gluco-lactase. In a series of
experiments carried out by Armstrong (Proc. Roy. Soc.,
1904, 73, 507) with milk-sugar and emulsin, the values of k
fell rapidly.
2 grms. lactose
2 grms. lactose \ per 100 c.c.
-2 grm. emulsin /*"
0-4 grm. emulsin ^^
Minutes.
X
&.10*.
X
vr
Minutes.
X
k. 10*.
X
~VT
0-5
3-2
282
4.5
1-0
4-8
214
4-8
1-0
4-9
218
4-9
2-0
6-4
143
4-5
2-0
7-5
169
5-3
3-0
7-6
114
4-4
4-5
9-0
91
4-2
4-5
9-4
95
4-4
6-0
10-0
91
4-1
6-0
10-6
81
4-3
23-0
19-7
41
4-1
23-0
30-5
69
2-0
29-0
22-0
37
4-1
29-0
35-0
64
2-0
48-0
29-0
31
4-2
48-0
47-8
59
2-2
53-0
30-7
30
4-2
53-0
50-0
57
2-2
144-0
62-2
29
5-2
144-0
84-0
55
7-0
264-0
77-5
24
4-8
No simple relation between the time and amount of reaction
is at first evident from these figures, and the tables given in
this paper indicate none between the velocity of reaction and the
concentration of enzyme. The diminution of the constant may
be explained by the retarding influence of the products formed
by the reaction.
Later investigations (Proc. Roy. Soc., 1908, 80, 326) show
good agreement with a unimolecular reaction, if relatively large
quantities of enzyme (almond-extract) are employed.
5% Milk-sugar solution
Hours.
40 c.c. enzyme-solution.
60 c.c. enzyme-solution.
X
fc.KM.
X
&.10*.
2
15-2
358
18-5
444
3
18-5
296
22-7
373
5
21-1
251
36-5
394
7
33-5
253
44-9
380
9
42-2
264
52-5
360
10
44-0
252
53-5
332
24
73-4
240
76-0
258
EXPERIMENTAL DATA OF ENZYME REACTIONS 171
As is seen from the third and fifth columns, there is here
also no proportionality between concentration of enzyme and
velocity. Only relatively small amounts of enzyme have hydro-
lysing actions proportional to their concentrations, as is shown
by the following figures:
Amount of enzyme 10 20 40 60 c.c.
Velocity constant, /c.10 4 107 212 279 385
Like invertase and maltase, lactase exhibits its optimal activity in
faintly acid solution: according to B i e r r y and S a 1 a z a r (C. R.,
1904, 139, 381), with 0-002-0-004% of HC1, which corresponds with a
concentration of hydrogen-ions of about 10~ 3 . Lactic acid is stated
by Bokorny (Maly's Jahrb., 1903, 33) to exert a specific accelerating
influence.
ENZYMES OF EMULSIN
1. (i-G lucosidase
The first quantitative investigation of emulsin was made
byTammann (H., 1891, 16, 298 et seq.), who examined
the action of this enzyme on amygdalin, salicin, arbutin and
coniferin. As the following comparison of the calculated and
observed values of (ax) shows, at 25 the process appears to be
unimolecular :
DECOMPOSITION OF SALICIN BY EMULSIN
t (hours).
a x
ft
found.
calc.
1
87
88
0-061
3
68
67
0-057
5
42
52
0-075
8
35
35
0-058
12
24
21
0-052
26
9
3
0-040
Important progress is marked by the work of Hudson
and Paine (Journ. Amer. Chem. Soc., 1909, 31, 1242) on the
decomposition of salicin. These authors paid attention to the
facts that the hydrolysis of salicin yields ^-glucose and that the
reaction is extremely sensitive towards hydrogen and hydroxyl-
172
GENERAL CHEMISTRY OF THE ENZYMES
Ions. The following numbers show that, under suitable exper-
imental conditions, satisfactory constancy of the velocity constant
k of the first order is obtained.
Minutes.
Specific rotation
(alkaline solution).
IWk.
-62-0
Temperature, 30
10
OT: * O
360
20
-48-7
330
Concentration of
30
-41-6
353
salicin,
35
-39-5
339
5%
85
-15-8
374
145
+ 2-9
350
00
+32-2
~~~
The influence of acids and bases is indicated by the following
table:
Concentration of
NaOH.
Activity of the
emulsin.
1
Concentration of
HC1.
Activity of the
emulsin.
0-005
0-00027
222
0-0009
138
0-0005
225
0-0005
195
0-0018
242
0-00009
222
0-005
255
0-009
206
The optimal activity is hence shown with 0-005 grm.-mols.
of HC1 per litre.
A u 1 d ' s experiments (Journ. Chem. Soc., 1908, 93, 1251)
on the hydrolysis of salicin by an enzyme (phaseolunatase)
present in Phaseolus lunatus also seem to indicate
constancy of the values of k (Table III).
Hours.
X
k
Hydrolysis of salicin by phaseolunatase
at 39-5
0-5
1
15-8
30-9
141
145
2-0
3-0
59-0
6*7
156
147
H e r z o g (K. Akad. v. Wetensch., Amsterdam, Sitzungsber.,
1903, and Zeitschr. f. allg. PhysioL, 1904, 4, 163) has likewise
made experiments on the decomposition of salicin by emulsin:
EXPERIMENTAL DATA OF ENZYME REACTIONS 173
Minutes.
X
a
fc.10 5 .
10< . o - (X
k H=~ lOS ^C
Temp. 25.
24
0-174
346
15
54
0-354
351
16
Salicin solution,
86
0-450
302
' 14
0-07 N.
210
0-691
243
13
270
0-775
239
14
s=0-6
371
0-847 .
219
14
2. Amygdalase and Hydroxynitrilase
The investigations of Armstrong, on the one hand, and
Rosenthaler, on the other, indicate that the name
amygdalase should be given to that enzyme which hydrolyses
amygdalin into mandelonitrile glucoside and glucose. A g-
glucosidase present in " emulsin " then decomposes the mandelo-
nitrile glucoside further into glucose and mandelonitrile, and the
latter product is finally broken down into benzaldehyde and
hydrocyanic acid by the hydroxynitrilase. So that three enzymes
take part in the hydrolysis of amygdalin. It can, therefore,
hardly be expected that the formation of the final products
should correspond with a simple reaction-formula.
The first investigation of the system amygdalin-emulsin
was made by T a m m a n n ; certain of his experiments on the
retardation of the reaction by the products formed have already
been referred to in the preceding section (p. 139).
Auld (Journ. Chem. Soc., 1908, 93, 1251) has recently
made a very thorough investigation of the hydrolysis of amygdalin.
He followed the reaction by titrating the liberated hydrocyanic
acid with iodine and found increasing values for the unimolec-
ular constant:
300 c.c. 2% amygdalin solution+15 c.c. 2% emulsin solution.
Minutes.
X
fc.10 5 .
10
6-1
255
80
45-2
295
Temperature 40
100
54-9
309
150
75-3
347
1360
93-3
(x indicates the quantities of amygdalin decomposed, in percentages.)
174
GENERAL CHEMISTRY OF THE ENZYMES
H . E . and E. F. Armstrong and H o r t o n (Proc.
Roy. Soc., 1908, 80, 330) give their results in the form of the
following curves (Fig. 4) :
The curve representing the glucose formed is not coincident
with that showing the hydrocyanic acid liberated, which would
be understandable if these two substances were set free in two
different reactions effected by two different enzymes. These
authors have therefore done right in not calculating the reaction-
HYDROLYSIS OF AMYGDALIN
BY EMU LSI NAT 25
10 12 14
Time in Hours
FiG. 4.
constants for the complex of reactions comprised in the hydro-
lysis of amygdalin.
Similar results are given by A u 1 d (loc. cit., p. 1268) for the
temperature 41.
Quantity of enzyme 2 3 4 6 12 25 50 c.c.
Percent, hydrolysis in 10 mins. 3-7 6-05 8-7 12-5 17-2 21-6 24-1
If dilute amygdalin solutions are employed, the constants
are, as they should be theoretically, independent of the concen-
tration of amygdalin (A u 1 d , loc. cit., p. 1270).
Hydrocyanic acid and glucose produce retarding effects.
EXPERIMENTAL DATA OF ENZYME REACTIONS 175
Mention must finally be made of Henri and L a 1 o u ' s
polarimetric experiments (Soc. Biol., 1903, 55, 868) on the
simultaneous decomposition of amygdalin and salicin.
t
minutes.
2%
salicin.
2-5%.
amygdalin.
2% salicin
+2-5%
amygdalin
4%
salicin.
1-25%
amygdalin.
46
0-67
0-97
1-05
1-08
0-90
130
1-58
2-38
3-63 2-25
1-57
268
2-32
3-15
4-22
3-45
1-56
GO
3-15
3-17
6-32 6-30
1-59
It will be seen that the hydrolysis of the mixture takeb place
much more slowly than that of the two constituents separately.
This fact also indicates combination of enzyme and substrate.
The enzyme is moderately resistant to chloroform and toluene and
its optimum temperature is given as 45.
PROTEOLYTIC ENZYMES
The first accurate experiments on the time-course of the
decomposition of protein by pepsin are due toE. Schiitz
(H., 1885, 9, 577). They were carried out with solutions of
globulin-free egg-albumin (about 1 grm. per 10 c.c.), to which
were added 5 c.c. of 5% hydrochloric acid and a pepsin solution
of definite strength; the solutions were then diluted to 100 c.c.
and kept at 37-5 for 16 hours. The albumin was then removed
from the solutions and the amounts of peptone formed determined
by means of the optical rotations. In this way S c h ii t z found
the velocity of digestion to be proportional to the square-root
of the concentration of the pepsin. The results of the first of
the three series of experiments are given here :
Quantity of
Rotation of the peptone in minutes.
pepsin.
Observed (mean).
Calculated.
1
7-3
7-4
2
9-75
10-4
3
12-8
12-7
4
14-8
14-7
5
16-5
16-4
6
18-45
18-9
176
. GENERAL CHEMISTRY OF THE ENZYMES
Mention must also be made of the experiments ofBorissow
with trypsin and of Samojloff with pepsin, these exper-
imenters also arriving at the relation x = K^/Et. (Dissertation,
St. Petersburg, 1901; Arch, des Sci. BioL, 1893, 2, 699; see
P a w 1 o w , Arbeit der Verdauungsdriisen) ; there was here no
intention of obtaining a representation of the chemical dynamics
of enzyme action and, owing to the experimental methods employed
in these investigations, no conclusions concerning this can be
drawn. The same may be said of Walther's researches
(Arch, des Sci. BioL, 1899, 7, 15).
Very extensive numerical data on the digestion of protein
by pepsin were given in 1895 by J. S j 6 q v i s t (Skand. Arch,
f. PhysioL, 1895, 5, 317), who followed the course of the digestion
by measuring the electrical conductivity. Every 100 c.c. of
solution, 0-05N with reference to hydrochloric acid, contained
2-23 grins, of albumin (almost freed from salts by dialysis)
and also 2-5, 5, 10 or 20 c.c. of pepsin solution. The con-
ductivity of these solutions fell during the experiment from the
initial value [i = 188-4 (old units) to a final value of about 83-4.
The amount of albumin acted on, x, was taken as proportional
to the fall A in the conductivity. The following tables contain
the observed values of A at 37, together with the corresponding
2-5 c.c. of pepsin solution per 100 c.c.
Hours.
Conductivity,
M
Change of
conductivity,
A
X
*4
k -
H'S /
Vt
188-4
_
0-5
1
.
2
177-3
11-1
10-5
2-97
7-45
4
171-1
17-3
16-41
3-78
8-21
6
167-4
21-0
19-93
3-81
8-13
8
164-5
(23-9)
22-68
3-77
8-02
9
163-1
25-3
24-00
3-82
7-90
12
159-9
28-5
27-04
3-70
7-70
16
156-4
(32-0)
30-36
3-62
7-59
20
152-9
35-5
33-68
3-70
7-53
32
146-2
42-2
40-04
3-40
7-08
48
139-8
(48-6)
45-06
3-20
6-50
64
135-0
(53-4)
50-78
3-13
6-34
96
127-9
60-5
57-41
2-80
5-87
00
3-49
EXPERIMENTAL DATA OF ENZYME REACTIONS 177
proportional values of x, the calculated values of the constant
K A of the A r r h e n i u s formula (18) or (20) and, finally, the
values of the constant of S c h ii t z 's formula x = K s -\/t.
5 c.c. of pepsin solution per 100 c.c.
Hours.
Conductivity,
M
Change of
conductivity,
A
X
KA
X
KS = -7=-
Vt
188.4
_
_
0-5
1
178-2
10-2
9-68
4-93
9-8
2
172-8
15-6
14-80
6-09
10-5
4
164-7
23-7
22-49
7-49
11-2
6
159-5
28-9
27-42
7-70
11-2
8
155-5
(32-9)
31-22
7-62
11-0
9
153-5
(34-9)
33-11
7-88
11-0
12
149-4
39-0
36-58
7-47
10-6
16
145-2
(43-2)
40-15
8-25
10-0
20
141-0
47-4
44-98
7-39
10-0
32
133-1
(55-3)
52-47
6-85
9-3
48
126-2
(62-2)
59-02
6-30
8-5
64
121-4
(67-0)
63-58
5-85
7-9
96
114-4
74-0
70-21
5-21
7-3
00
6-84
10 c.c. of pepsin solution per 100 c.c.
Hours.
Conductivity,
M
Change of
conductivity,
A
X
KA
X
KS= ,
Vt
188-4
0-5
179-2
9-2
8-73
8-04
12-35
1
174-2
14-2
13-47
10-34
13-47
2
165-9
22-5
21-35
13-40
15-10
4
154-8
33-6
31-88
16-30
15-94
6
148-0
40-4
38-34
16-65
16-01
8
143-2
(45-2)
42-88
16-42
15-31
9
140-8
47-6
45-14
16-51
15-05
12
136-1
52-3
49-62
14-09
14-32
16
130-9
(57-5)
54-56
15-23
13-64
20
125-7
62-7
59-50
15-44
13-30
32
119-4
69-0
65-46
12-90
11-60
48
113-1
75-3
71-45
11-25
10-31
64
109-1
(79-3)
75-25
10-08
9-41
96
101-8
86-6
82-17
9-60
8-39
00
13-30
178
GENERAL CHEMISTRY OF THE ENZYMES
20 c.c. of pepsin solution per 100 c.c.
Hours.
Conductivity,
M
Change of
conductivity,
A
X
KA
X
KS = 7=
Vt
188-4
'0.5
176-0
12-4
11-77
15-0
16-64
1
167-8
20-6
19-55
22-2
19-55
2
157-9
30-3
28-75
25-9
20-33
4
144-5
43-9
41-66
30-6
20-83
6
137-2
51-2
48-58
29-7
19-83
8
133-0
(55-4)
52-57
25-1
18-54
9
130-1
58-3
55-52
28-0
18-44
12
125-8
62-6
59-40
25-6
17-15
16
121-6
(66-8)
63-34
25-1
15-81
20
117-3
71-7
68-03
22-5
15-21
32
109-8
78-6
74-58
19-5
13-18
48
102-3
86-1
81-70
18-3
11-79
64
97-4
(91-0)
86-34
14-4
10-49
96
91-2
97-2
92-24
18-0
9-41
oo
22-7
S j 6 q v i s t 's observations hence indicate that the relation
x = K s \/t holds moderately well during the first half of the reaction.
Calculation of the constants for the various concentrations
of enzyme E shows that these are approximately in the propor-
tions, A/0^2 : VoT : VtTo^ : Vo-025.
K S .^ 7-6 10-4 14-4 18-2
VE 0-500 0-707 1-00 1-42
Quotient K S : VE 15-2 14-7 14-4 12-8
So that, for small and equal values of t, S c h ii t z 's rule
holds, i.e., the quantity of substance transformed is inversely
proportional to the square-root of the concentration of the enzyme.
The proportionality between the amount of albumin hydrolysed
and the square-root of Et (enzyme-concentration X time) is
much more general, as is shown by the following table in which
Arrhenius (Immunochemistry, p. 67) has collected to-
gether the diminutions of conductivity given by S j 6 q v i s t.
Et= 0-05 0-1 0-2 0-4 0-8 1-6 3-2 4-8 6-4 9-6
# = 0-025 11-1 17-3 23-9 32-0 42-2 53-4
0-05 10-2 15-6 23-7 32-9 43-2 55-3 67-0 74-0
0-01 9-2 14-2 22-5 33-6 45-2 57-5 69-0 75-3 79-3 86-6
0-02 12-4 20-6 30-3 43-7 55-4 66-8 73-6 78-6 86-1
Mean. . . . 10-2
Calculated 11
14-9
15-6
22-7
22
32-2
31-1
43-8
44
55-4 67-6 74-3 79-0
EXPERIMENTAL DATA OF ENZYME REACTIONS 179
Further investigation of peptic digestion is due to Julius
Schutz (H., 1900, 30, 1), who coagulated the undigested
protein remaining after 15 hours and determined the nitrogen in
the filtered liquids by K j e 1 d a h 1 ' s method. At a tem-
perature of 38, he obtained the following quantities of hydrolysed
protein ( bs.) the values calculated from Schiitz's rule
being given in the final column.
Quantity of pepsin.
10 4 .Zobs.
10 4 .Z ca ic.
1
212
213
4
471
426
9
652
639
16
799
852
25
935
1065
36
1031
1278
In the same year E . Schutz and H u p p e r t (Pfliig.
Arch., 1900, 80, 470) gave further data concerning peptic diges-
tion. The decomposition products termed secondary albumoses
of the protein were determined polarimetrically. " The quan-
tities of secondary albumoses formed are proportional to the
square-roots of the times." Further, the quantities of digested
protein, the sum-totals of the intermediate products and the
amounts of secondary albumoses, are in the same ratios as the
amounts of protein employed, namely, 1:2:3:4.
E . Schutz and H u p p e r t also investigated the influence
of hydrochloric acid. Under certain conditions, secondary
albumoses are formed in proportion to the quantity of protein,
to the square-root of the time, and to the concentrations of pepsin
and acid. The conditions for this rule to hold are a moderately
rapid reaction and a concentration of acid not exceeding 2%.
If we denote the amount of secondary albumoses by S, that
of albumin by A, the time by t, the concentration of hydrochloric
acid by s, and the quantity of pepsin by P, the velocity with
which the secondary albumoses are formed is expressed by
It must be stated that objections have been raised by
Sjoqvist (loc. cit.) to the methods used in these experi-
ments.
180
GENERAL CHEMISTRY OF THE ENZYMES
Gross (Berl. klin. Wochens., 1908, 45, 643) expressed
the view that S c h (i t z 's rule does not hold for peptic diges-
tion, but that the amount of digestion is proportional directly
to the quantity of enzyme and inversely to the time of digestion.
His experiments were carried out as follows: Increasing amounts
of pepsin were added to constant quantities of an acid solution
of casein, observation being then made in each case of the time
when the whole of the casein was digested, i.e., when no turbidity
was produced by addition of saturated sodium acetate solution.
The observations were made at intervals of 10-20 seconds, the
temperature being 40.
The following series of results may be quoted :
Casein solution, 50 c.c.
Casein solution, 50 c.c.
Gastric juice,
c.c.
Digestion complete
in minutes.
Griibler pepsin,
(0-1%) c.c.
Digestion complete
in minutes.
1-0
52-7
1-0
64-0
2-0
25-0
2-0
31-7
4-0
12-2
4-0
16-7
8-0
6-25
In order to test Gross's results, Kurt Meyer (Berl.
klin. Wochens., 1908, 45, 1485) made a number of experiments
by F u 1 d ' s edestin method. 1 The quantity of pepsin in any
tube was four times, and that of protein twice, that in the pre-
ceding tube. M e y e r collects his results in two tables, of
which one is given here:
1% edestin solu-
1% pepsin solution (G r u b 1 e r) in 0-03 HC1.
tion in 0-03 HC1.
c.c.
0-0025 c.c.
0-01 c.c.
0-04 c.c.
0-16 c.c.
0-1
4.
_
0-2
+
0-4
-f.
_
0-8
-f
1-6
3-2
1 The method was so modified that the amounts of digestion could be
obtained in one series of experiments; thus, series with increasing quantities
of edestin and similar series with increasing quantities of pepsin were carried
out.
EXPERIMENTAL DATA OF ENZYME REACTIONS 181
Incipient turbidity is indicated by +.
No exact idea can be formed of the magnitude of the exper r
imental error is these investigations. K . Meyer himself,
however, draws the conclusion that S c h ii t z ' s rule is valid
for peptic digestion.
0-05 c.c. gastric juice.
Quantity of casein,
c.c.
Time of digestion
in minutes.
Quantity of casein,
c.c.
Time of digestion
in minutes.
5
6-3
10
13-3
6
7-5
12
14-3
7
8-7
14
17-0
8
10-0
16
21-2
For the sake of completeness it may be mentioned that S p r i g g s
(Journ. of PhysioL, 1902, 35, 465) followed the course of pepsin-action
by measurements of the viscosity with s t w a 1 d ' s viscosimeter.
Against these experiments the objection may, however, be raised that
the relation of viscosity to the degree of protein hydrolysis is not suffi-
ciently known, so that no safe conclusions can be drawn from the results
of these measurements.
W e i s (Medd. fra Carlsberg Lab., 1903, 5, 127) has made a
very thorough investigation of the action of vegetable proteases.
He found that peptic action proceeds relatively rapidly, whilst
the tryptic decomposition of the albumoses is more gradual;
the two actions can, to some extent, be separated. From the
results obtained, which are extremely difficult to deal with in
detail, it is to be concluded that, for the proteolysis of vegetable
protein at any rate within a certain region of concentration
S c h u t z ' s rule appears to hold. According to the results
given on p. 176, etc., the exponent of the enzyme -concentration
increases, with increasing dilution of the enzyme, from 0-5
(Schiitz 's rule) to 1.
In the separate series of experiments, the amounts of sub-
stance transformed are proportional to the square-roots of the
times. From the table on p. 183 of the above paper we extract
the following numbers, the values of x:\/T being given in addi-
tion.
182
GENERAL CHEMISTRY OF THE ENZYMES
Amount of change.
Amount of change.
Hours.
Mgrms. N.
Ratio^_
Hours.
Mgrms. N.
Ratio.
/
t
X
x : -\t
t
X
x : Vt
1
5-34
5-34
I
5-80
5-80
2
8-42
5-96
2
8-54
6-04
3
9-82
5-67
3
(12-00)
5-50
4
11-92
5-96
4
11-34
5-67
5
12-98
5-81
5
12-94
5-79
6
13-70
5-59
6
13-32
5-44
9
17-22
5-74
9
14-20
4-73
The author has also calculated the results of the experiments
in which W e i s varied the concentration a of the substrate (pro-
tein). The third column gives the constants k for unimolecular
reactions; the fourth, the product ka and the fifth, the constant
K S of the formula x^/a = K s .
AFTER 5 HOURS l
Protein
Amount of N trans-
concentra-
tion.
formed as percentage
of total N.
7 1 1
k= -log
t ax
k.a.lQG
K s
a
X
1%
36-2
0-00065
65
36-2
2
25-9
0-00043
86
36-6
3
20-3
0-00033
100
35-2
4
16-0
0-00025
100
32-0
5
13-2
0-00020
102
29-5
AFTER 2 HOURS
Protein
Amount of N trans-
concentra-
tion.
formed as percentage
of total N.
, 1 , a
k= log
t ax
fc.a.lO"
K s
a
X
1%
22-0
0-00090
90
22-0
2
17-0
0-00067
134
24-0
3
13-1
0-00051
153
22-7
4
9-1
0-00035
140
18-2
5
7-9
0-00030
150
17-7
1 The numbers in the two tables were obtained in two separate series of experiments
md are hence not comparable.
EXPERIMENTAL DATA OF ENZYME REACTIONS 183
It will be seen that the amount of protein changed diminishes
with increasing values of a. Arrhenius (Immunochemistry,
p. 85) explains this as follows: When, for example, 10% of the
protein is digested, the absolute amount of the products of the
reaction is doubled if the initial concentration a is doubled. But
the velocity of reaction is inversely proportional to the absolute
quantity of the products and, therefore, also to the initial con-
centration. According to what was stated on p. 133, the amount
of change, expressed as a percentage of the total amount of pro-
tein, is hence approximately inversely proportional to the square-
root of a, as is shown by the fifth columns of the two tables given
above.
The work of W e i s on vegetable proteinases deals also with
the trypsins, that is, with those proteolytic enzymes which act
in alkaline solution.
From the results of experiments carried out virtually by
M e 1 1 ' s method, H.M.Vernon (Journ. of Physiol., 1901,
26, 421) has drawn the conclusion that the digestion of fibrin
by trypsin follows S c h ii t z ' s rule if the times of digestion
are corrected for the destruction of the trypsin in the soda
solution.
Amount of enzyme-
extract E in c.c.
Time of digestion
t in minutes.
Corrected time of
digestion in minutes.
txVE.
2
11-8
11-14
16-2
1
17-7
16-25
16-3
0-5
26-8
23-62
16-7
0-25
36-1
30-57
15-3
0-125
80-4
57-32
20-3
0-0625
176-0
93-57
23-4
As has long been known qualitatively, the concentration of
the acid present in peptic digestions exerts a marked influence
on the time-course of the reaction. This influence is expressed
quantitatively in Schiitz's formula mentioned on p. 179,
according to which with certain definite conditions of the enzyme-
and substrate-concentrations the velocity of reaction is pro-
portional to the square-root of the concentration of the hydro-
chloric acid.
184 GENERAL CHEMISTRY OF THE ENZYMES
The optimal concentration of the hydrogen-ions has been
the subject of a recent and thorough investigation bySorensen
(Biochem. Z., 1909, 21, 288). In this work the concentration
of the H-ions was determined electrometrically, the progress of
the reaction being measured by the amounts of protein pre-
ciptable after different times by stannous chloride or tannic acid.
The results are given in the following table, the " exponents of
the hydrogen-ions," PH, being given in the first column; by this
Sorensen understands the logarithm to base 10 of the recip-
rocal of the normality-factor of the solution as regards hydrogen-
ions. The concentration of these ions is given also in the ord-
inary form (column 2).
It will be seen that the acidity-optimum increases with the
duration of the peptic action.
The influence of the hydrochloric acid must be explained, as
already mentioned, by the protein hydrochloride being more
readily acted on than the free protein. Further the enzyme
itself may be in the form of a salt, this pepsin hydrochloride
showing increased activity; this possibility has recently been
emphasised by J. L o e b (Biochem. Z., 1909, 19, 534) but we
have no definite indications on this question. As the author has
pointed out (Ergeb. der Physiol., 1907, 6), the " pepsin-hydro-
chloric acid " the existence of which has been so often assumed-
can mean nothing but a pepsin salt of hydrochloric acid.
The older investigations ofBorissow on tryptic digestion
have already been mentioned.
L . P o 1 1 a k (Hofm. Beitr., 1904, 6, 95) also employed
M e 1 1 ' s method to examine a tryptic enzyme, g 1 u t i n a s e ,
which he isolated, and which acts on gelatine. He arrived at the
result that the action of this enzyme does not correspond exactly
with S c h ii t z ' s rule, which, however, it approaches far more
closely than does the mixture of enzymes of an ordinary pancreas
infusion or Griibler's trypsin.
V. Henri and Larguier des Bancels have inves-
tigated the action of trypsin on gelatine (C. R., 1902, 136, 1581).
By regarding, as S j 6 q v i s t did, the change of conductivity
as proportional to the progress of the reaction, they obtained
confirmation of the formula
a-x
EXPERIMENTAL DATA OF ENZYME REACTIONS 185
JS
3
00
"#
I <M O5 ?O CO O
00 00 00 l^- 00
1 1 T ( 1 t 1 1
a
1.
?
5
O CD (M CO Cfl (M
rH rJH cq 00 CO CO
10 O CO O ^ IO
1
Si
1?
*- J_
c^
S ? | S
C^J CO CO CO rH CO
11
I*
*l
ej d
CD
iO 00 CO (N CO (N
(N iO T^ (N 00 "5
O5 O5 O O 00 <N
rH i-H
II
II
a
co
<M O CO (N O 00
O CD Tf CO <M *
CO CO t^ l^ CO i-H
Increase
1C '
& % S 8 8 |
CO -^ 10 O Tt< O
>>
^2
05
*
00 (N C^> GO (N
<M CO O5 O TH
^ ^ 8 ^ 2
!
s,g
SO
d O T(H OO
* oo O5 o 1
00 00 00 t^
<N (N ca (N
II
g^
a-*,
CO
O <M Tjn O (M 00
CO l>- t^ Tt< * iO
iO l> 00 CO CO
(N (N (M <N <N I-H
II
ta "**
'3 S3
00
g 2 S S
^ ^ ^ &
a!
SI
*
? 9 ? 9 ?
O5 rH 4l C^ CO
rH (N (M C<I rH
si
s
o}
? ? ? ? ? ?
CO Tj< 1^ 00 t^ (N
ai
5|
.
o
-
S(N CO * Q O
CO <N (N O 00
00 O5 <N O tO O
rH rH rH 1
I
O CO O O 00 00
CO CO Oi rH O t^.
h
a d
r" -H (N (N n C
1 1 1 1 1 1
o o o o ' o o
11
11
b
t^
rH rH CO (M 10 00
if
s*
&
CO O5 (N CO CO OJ
l> C5 (N CO (M O
O O rH rH (N ^
186 GENERAL CHEMISTRY OF THE ENZYMES
Since, however, the final value of the process is not observed
(or, at any rate, not given) and the observations are only spread
over the very short period of about an hour, the real decomposi-
tion of protein cannot have proceeded very far and these exper-
iments tell little about the course of tryptic digestion. As has
been calculated by Arrhenius, the conductivity is approx-
imately proportional to the square-root of the time of digestion.
/ (minutes) 10 20 30 40 50
Conductivity (mean) 27-3 44-0 53-0 58-7 65-7
7-37\A 29-3 41-5 50-0 58-7 68-8
Of wider scope are the experiments of B a y 1 i s s (Arch.
Sci. Biol. St. Petersburg, 1904, 11, Supplement), in which also the
conductivity method was employed. The substrate used was
partly caseinate in faintly ammoniacal solution and partly gelatine.
The experimental results are given mostly in the graphic form.
It is found that the constants for unimolecular reactions diminish
considerably with lapse of time, this being attributed to the retard-
ing effect of the products of the reaction. The numbers obtained
by B a y 1 i s s are indeed in good agreement with the formula,
aln- x = KEt.
a x
With concentrations of casein up to about 4%, the velocity of
digestion is proportional to the concentration of the substrate; in
4-8% solutions, the velocity is independent of the concentration
of the substrate, whilst with more than 8%, inverse proportionality
sets in. As regards the relation between the velocity and the
concentration of the enzyme, approximate proportionality exists
during the first quarter of the reaction; but even in the second
quarter, the velocity of reaction is considerably less than would
correspond with the concentration of the trypsin (loc. cit., p. 26).
Using V o 1 h a r d ' s method for estimating pepsin and
trypsin which will be referred to in the Appendix W. L 6 h -
lein (Hofm. Beitr., 1905, 7, 120) arrived at the result that
tryptic digestion does not follow S c h u t z ' s rule. . F a u -
b e 1' s results (Hofm. Beitr., 1907, 10, 35) point to the same
conclusion.
The measurements which have as yet been made do not
indicate, with the certainty that might be desired, the con-
EXPERIMENTAL DATA OF ENZYME REACTIONS 187
ditions for simple proportionality between velocity of reaction
and the concentration of the enzyme. But far more often than in
experiments with pepsin have the conditions been such that the
relations :
Amount of digested protein = const.
and
Amount of digested protein = const. \/T,
have been found to hold only over a very limited range.
But, as with pepsin (cf. p. 179), it appears to be quite general
that the same quantity a of protein is digested if the amonut
of enzyme is made to vary inversely with the time, i.e.
x = const. f(E.t) ....... (22)
This rule evidently holds only for those cases in which the
proportionality between x and Et is direct. This occurs with
undisturbed unimolecular catalytic reactions, so long as the
products of the reaction x are small in amount compared with
the substrate a. Further, according to the measurements of
Bayliss, Hedin and others, this rule is obeyed, in the case
of trypsin, with small quantities of enzyme and also with
conditions so chosen that
x = const. \/Et.
This rule is contained in the widely-used formula (18), as is
shown by the derivation of the latter.
Formula (22) evidently means that the progress of the diges-
tion depends only on the quantity of protein
already hydrolysed, no matter whether this has been
obtained by the action of much enzyme for a short time or by
that of less enzyme for a longer time.
This relation has been confirmed by H e d i n (Journ. of Physiol.,
1905, 32, 468; ibid, 1906, 34, 370; H., 1908, 57, 471). One of the
tables from the last of these papers is given below.
The amount of casein digested was measured by the quantity of
nitrogen not precipitated by tannic acid. The values given in the
various columns under E.t indicate the numbers of tenths of a c.c. of
normal acid required to neutralise the ammonia obtained by K j e 1 -
188 GENERAL CHEMISTRY OF THE ENZYMES
d a h 1 ' s method from equal volumes of the filtrate from the tannin
precipitate :
Et...
1
.. 1
5
8
2-0
10-35
2
12
5
95
5-0
20-15
7-5
24-50
10
[26
95
15-0
31-0
20-0
34-05
2.. . .
3....
4
. . 5
.. 5
6
85
70
10
10-75
10 85
10-75
13
13
13
-25
35
15
20-40
20-30
19-90
24-65
24-35
23-80
L *^
27
27
26
8
95
31-7
31-05
30-75
33-75
33-55
As regards the investigation of the kinetics of the digestive
enzymes, the difficulties which beset all enzymic problems are
here supplemented by others due to the complicated nature of the
proteins. Observations made by any of the physico-chemical
methods, whether optical or electrical, represent the effect of
a large number of decompositions proceeding simultaneously,
and it is, as the author has repeatedly emphasised, surprising
that simple direct relations are so often found. The processes
of decomposition would hence become decidedly more apparent
if the dipeptides, which F i s c h e r 's work has rendered
accessible, were employed as substrates. The first experiments
in this direction were made by E u 1 e r (H., 1907, 51, 213) on
glycylglycine, not however with the trypsin of the pancreas,
but with the proteolytic enzyme of the walls of the intestines,
the so-called erepsin.
The course of the reaction was followed by the change in
conductivity of an alkaline solution of glycylglycine. The final
conductivity, obtainable by complete hydrolysis of the glycyl-
glycine, was determined beforehand with glycine solutions of
corresponding concentration. In the possibility of a certain
knowledge of this final value seems to lie one of the principal
advantages of using dipeptides as substrates in tryptic and ereptic
decompositions. It was also found that the diminution of the
conductivity is proportional to the diminution of the concentra-
tion of the dipeptide. 1
The first result obtained was that the velocity of hydrolysis
of glycylglycine depends greatly on the alkalinity of the erepsin
solution. 2
1 It is to be noted that the existence of such proportionality, which forms
the basis of further calculations, was not shown in previous investigations
where tryptic or peptic digestion of protein was followed by means of the
conductivity.
2 The alkalinity is expressed as the normality of the total soda added.
The actual alkalinity, that is, the concentration of the free base, is very much
smaller, since the greater part of the soda is used up in neutralising the
glycylglycine.
EXPERIMENTAL DATA OF ENZYME REACTIONS 189
N/10-glycylglycine. 5 grms. powdered erepsin per 100 c.c.
Concentration of alkali 0-04 0-05 0-075
Reaction constant X 1000 <0-05 7-0 6-2 2-6
0-10
0-2
Different preparations of the enzyme showed varying sensitive-
ness towards alkali.
It was next ascertained that the decomposition of glycylglycine
is a reaction of the first order and that, under favourable con-
ditions, the corresponding velocity coefficients k remain constant
until the reaction is half completed. 1
N/10-glycylglycine. 5 grms. powdered erepsin per 100 c.c.
0-04 N-NaOH.
b.
0-05N-NaOH.
Minutes.
1000 (a x).
1000/fc.
Minutes.
1000(o -x).
lOOOfc.
920
935
7
819
7-20
10
806
6-46
15
721
7-08
17
739
6-00
22
649
6-88
25
654
6-18
30
579
6-70
30
622
5-90
In most cases a considerable fall in the velocity occurs even
after about half an hour. This depends principally on destruction
of the erepsin, the rapidity of the destruction increasing with the
amount of free alkali in the solution.
Within certain limits the velocity of reaction is almost independ-
ent of the concentration of the dipeptide. This independency
holds, however, only for certain relations between the concen-
trations of the enzyme and substrate. With small amounts of
enzyme, the velocity of reaction rises with increase of the con-
centration of the glycylglycine.
0-1 N-glycylglycine; 0-04 N-NaOH.
1000/c=0-35
. 2 N-glycylglycine; . 05 N-NaOH.
1000/0 = 0-55
Since, as has already been mentioned, the concentration of
free alkali has here also a considerable influence on the velocity of
reaction, the influence of the concentration of the substrate is
very difficult to explain. The author concludes, from the results
1 As the behaviour of erepsin, like that of other enzymes, depends on the
absolute activity of the solution, it is advisable, in order to make differene
results comparable, to establish a standard action. A normal preparation
might be taken as one which decomposes glycylglycine or an optically
active dipeptide, in 5% solution, to the extent of one-half in 1 hour.
190
GENERAL CHEMISTRY OF THE ENZYMES
of these experiments, that only the alkali salt of the
d i p e p t i d e undergoes hydrolysis (Arkiv for Kemi, 1907, 2,
No. 39).
As regards the effect of the concentration of enzyme, it may
be said that, in most of the conditions of concentration examined,
the velocities of reaction were proportional to the enzyme-con-
centrations ; the Schiitz-Borissow law did not hold in
any instance. The following numbers serve as examples:
Concentration of erepsin 5 4 3 2
1000/c: 6-5 5-4 4-3 2-8
With low concentrations of the enzyme, especially with feeble
preparations of pancreatin, k increases far more rapidly than the
concentration of the enzyme.
With relatively high concentrations of enzyme, deviations from
proportionality in the sense of S c h ii t z's rule do, indeed, occur;
but the experimental errors are so great in these reactions, which
take place in a few minutes, that conclusions cannot be drawn
from the results.
If optically active polypeptides are employed as substrates,
the enzymic hydrolysis can often be followed polarimetrically.
Such measurements have been carried out byAbderhalden
and his collaborators.
The following results, obtained with d-alanyl-d-alanine,
indicate the time-law of this reaction (Abderhalden and
Koelker, H., 1907, 51, 294; Abderhalden and
M i c h a e 1 i s , H., 1907, 52, 326) :
0-45 grm. dipeptide+6 c.c. pressed juice.
0-45 grm. dipeptide +4 c.c. pressed juice
+2 c.c. physiological salt solution.
Min.
Rotation
X
10"z
k.W*.
Min.
Rotation.
X
10"z
&.10*.
t
t
-1-21
-1-31
3
-0-96
0:39
130
453
3
-I'll
0-18
60
192
7
-0-74
0-59
84
390
7
-0-98
0-37
53
183
11
-0-51
0-84
71
382
10
-0-81
0-54
54
202
18
-0-20
1-15
64
380
16
-0-56
0-79
49
214
20
-0-16
1-19
59
373
25
-0-21
1-14
46
268
27
+0-01
1-36
50
451
30
-0-09
1-26
42
294
34
+0-05
1-40
41
430
35
+0-02
1-37
359
40
+0-07
1-42
35
43
+0-07
1-42
55
+0-10
1-45
54
+0-08
1-43
65
+0-10
1-45
EXPERIMENTAL DATA OF ENZYME REACTIONS 191
0-45 grm. dipeptide+3 c.c. pressed juice +3 c.c. physiological salt solution.
Minutes.
Rotation.
X
IWx
t
fc.10 4
o-o
-1-31
5-0
-1-16
0-19
380
125
6-5
-1-09
0-26
400
132
7-5
-1-05
0-30
400
134
16-0
-0-76
0-59
369
142
22-0
-0-54
0-81
368
161
28-0
-0-32
1-03
368
192
30-0
-0-25
1-10
314
209
38-0
-0-09
1-28
332
232
45-0
+0-01
1-36
265
60-0
+0-07
1-42
~
0-6 grm.d-Alanyl-d-alaninein 7 -6 c.c. pressed
juice +0-4 c.c. physiological salt solution.
0-6 grm. d-Alanyl-d-alanine +5-7 c.c. pressed
juice +3 -3 c.c. physiological salt solution.
Min.
Rotation
X
X
~t
&.10*
Min.
Rotation.
X
X
T
fc.10*
5
12
19
26
30
-1-30
-1-08
-0-85
-0-59
-0-23
-0-09
22
45
71
107
121
44
38
38
40
40
148
140
162
241
289
7
13
26
37
50
-1-23
-1-09
-0-91
-0-46
-0-11
21
39
84
119
140
30
30
32
32
28
60
92
144
216
40
30
It will be seen that the theoretical formula, k = -log - ,
t a x
leads to reaction-coefficients which show a continuous rise, the
amount of this increasing with diminution of the amount of enzyme
for a constant amount of substrate.
On the other hand, with relatively small amounts of enzyme,
x
the value of the expression is strikingly constant, and this
appears to indicate that the change proceeds independently of
the amount of substrate still to be decomposed. But it is more
probable that here, as is always the case, the velocity of reaction,
dx : dt, is proportional to the quantity of substrate present, and
that the reaction, as it proceeds, is subjected to some secondary
acceleration.
192
GENERAL CHEMISTRY OF THE ENZYMES
The velocity of reaction is approximately proportional to
the amount of enzyme:
40 : 7-6 = 5-4,
30 : 5-7 = 5-4.
This proportionality is much less sharp with d-alanylglycine,
with which Abderhalden and K o e 1 k e r (H., 1908, 55,
416) have experimented; the two following tables appear on p.
422 of this paper :
2 c.c. Dipeptide solution =0-001 normal.
-5 c c. pressed yeast juice. 4 c.c. water.
2 c.c. Dipeptide solution =0-001 normal.
2-0 c.c. pressed yeast juice. 2-5 c.c. water.
Minutes.
Rotation.
i
*.10*.
Minutes.
Rotation.
ft. 10'.
12
+0-85(extrap.)
0-80
22
12
+0-85 (extrap.)
0-70
70
31
0-71
25
31
0-54
64
62
0-63
22
62
0-38
56
86
0-53
24
86
0-28
56
118
0-49
23
118
0-19
55
190
0-36
20
Here, therefore, the velocity of reaction increases considerably
more slowly than the concentration of the enzyme, but there
is no indication of the validity of S c h ii t z 's law.
With reference to the effect of the concentration of the sub-
strate, Abderhalden andKoelker (H., 1908, 54, 363)
have made experiments with active material, the following table
giving some of their results :
(rfr-mol.)
( 2 BB-mol.)
(sfo-mol.)
3-0 c.c. d-Alanyl-rf-
alanine solution,
1 -0 c.c. pressed yeast
juice. 2-0 c.c. water.
4-0 c.c. d-Alanyl-d-
alanine solution,
1 -0 c.c. pressed yeast
juice. 1 -0 c.c. water.
5-0 c.c. d-Alanyl-d-
alanine solution,
1 -0 c.c. pressed yeast
juice.
Minutes.
Angle.
Angle.
Angle.
4
-1-36
-1-83
-2-15
12
-1-31
-1-75
-2-10
32
-1-17
-1-58
-1-97
61
-0-95
-1-31
-1-73
92
-0-67
-0-97
-1-43
128
-0-42
-0-69
-1-16
167
-0-18
-0-38
-0-86
190
-0-08
-0-23
-0-69
238
+0-03
+0-01
-0-35
308
+0-05
+0-13
+0-02
357
+0-05
+0-12
+0-15
377
+0-11
+0-18
+0-29
EXPERIMENTAL DATA OF ENZYME REACTIONS 193
The dipeptide employed in these experiments was, as these
investigators state, not free from the racemic compound, so that
the course of the reaction and the calculations become more
complicated. It can, however, be seen from the above numbers
that the amounts of dipeptide hydrolysed in a certain time are,
as a first approximation, independent of the initial concentration
of the dipeptide. The velocity constants calculated according
to the unimolecular formula would, therefore, diminish considerably
with increasing concentration of the dipeptide instead of being
independent of this concentration, as theoretically they should
be. Within a certain region of concentration or with a certain
ratio between the concentrations of substrate and enzyme, this
relation is found in the case of most enzymes. It has already
been shown, by experiments made by the author, how greatly
the degree and character of the reaction change with the alkalinity
of the solution, and confirmation of this result is afforded by the
investigations of Abderhalden and K o e 1 k e r (loc. cit.,
p. 378). To obtain definite results in any study of the kinetics
of trypsin and erepsin, careful attention rrust be paid to the
alkali-content of the solutions. If, as the above measurements
indicate, it is the alkali salts of the dipeptides or proteins
which undergo hydrolysis and thus function as the " active
molecules," the concentration of the substrate is not merely that of
the dipeptide or protein but depends also on that of the alkali
added; hence no general simple relations for the velocity of
reaction will be found if the concentration of the dipeptide or
protein alone is varied. It is of more value to ascertain how
the concentration of the alkali must be altered at the same
time for the theoretical requirement independence of the
reaction constant on the concentration of the substrate to be
fulfilled.
Abderhalden and Koelker (H., 1908, 55, 416)
have examined also the course of the reaction in the case of tri- and
tetra-peptides. Study of such higher polypeptides not only
serves to characterise the polypeptides, with which the velocity
of hydrolysis is a characteristic constant, but they are also of
interest in indicating the course of the reaction when, as happens
with the true proteins, several hydrolyses take place at the same
time. As examples are given the results of two series of exper-
iments on d-alanylgly cylglycine ; since the separate observations
194
GENERAL CHEMISTRY OF THE ENZYMES
are subject to considerable errors, the results given here have
been interpolated graphically.
3-32 c.c. Peptide solution =0-002 mol.
4-98 c.c. Peptide solution =0-003 mol.
1 -0 c.c. pressed yeast juice. 2 18 c.c. H2O.
1-0 c.c. pressed yeast juice. 0-52 c.c. HzO.
Minutes.
Angle (interp.)
k. 10*.
Minutes.
Angle (interp.)
k.10*.
1-70
2-55
20
1-15
85
20
1-80
76
40
0-70
96
40
1-20
82
60
0-41
103
60
0-80
83
80
0-20
116
80
0-55
83
120
0-26
82
It will be seen that the constants calculated from the unimo-
lecular formula and given in the third column, do not increase
very greatly with the time.
A dynamic investigation of the vegetable ereptases
discovered by V i n e s (Annals of Bot., 1910, 24, 213), would be
of considerable interest.
At about the same time as the author, A . E . Taylor car-
ried out experiments on alkaline proteolysis, using a chemically
definite substrate in order to render the results more definite.
To this end he prepared protamine sulphate from the salmon
by K o s s e 1 ' s method and hydrolysed it with G r u b 1 e r ' s
pancreatin. Unfortunately the results of these researches are
given only very briefly (Berkeley: On Fermentation, 1907,
p. 152). The numbers do, however, show that the law x = n\/t
does not hold for trypsin. The results of each series of exper-
iments can be calculated by the formula for unimolecular reac-
tions: but the constants vary if at different times the concentra-
tion of the substrate is altered. Further, under constant external
conditions, the velocity of protamine digestion is simply and
directly proportional to the concentration of the trypsin. These
results were obtained with the optimal concentration of hydroxyl-
ions.
Protamine, 0-100 grm. in 50 c.c. Temperature 40 C
Quantity of trypsin, grm.
Mean time of digestion, t
Quantity of trypsin X
0-008 0-006 0-004 0-003 0-002 0-001 0-0005
37 50 76-5 103 156 313 657
296 300 306 309 312 313 328
EXPERIMENTAL DATA OF ENZYME REACTIONS 195
Here also, then, the relation x = K S \/E does not hold.
In connection with the above investigations on the action of
proteolytic enzymes in vitro, reference may be made to the
relations deduced by Arrhenius (H., 1909, 63, 323)
from the results of the numerous experiments made by E . S .
London on digestion in the stomach of the dog.
For the experimental details reference must be made to
London's original papers (H., 1905, 45, 381; 46, 209; 1906,
47, 368; 49, 324; 50, 125; 1907, 61, 241, 468; 52, 482; 53, 148,
240, 246, 326, 334, 356, 403, 429; 54, 80; 1908, 55, 447;
56, 378-416, 512-553; 57, 113, 529; 1909, 60, 191-283; 61,
69; 62, 443-464; 1910, 65, 189-218; 68, 346-380). The follow-
ing cases are to be distinguished:
1. Digestion of different quantities of
meat introduced by the mouth. The fact, estab-
lished by L o n d o n , that the amount digested in a given time
is not proportional to the quantity of nutriment taken, is explained
as follows:
" The first 100 grms. of meat lie close to the stomach-wall
in a layer of uniform thickness. Within this lies another layer of
100 grms. of meat, this being rather thicker, as its surface dimin-
ishes approximately as the cross-section of a cone as the apex
is approached. Further layers of 100 grms. follow, the thickness
continually increasing. Into these layers the gastric juice diffuses
from the mucous membrane. The quantity of gastric juice
in each layer diminishes considerably and the diminution increases
in extent as the layers lie farther away from the mucous membrane.
This is conditioned partly by their increasing thickness and partly
by the laws of diffusion, which require such increasing diminution
even for layers of equal thickness. Hence it comes about that
the innermost layers are not perceptibly digested and the amount
digested in a given time increases, as the amount of food taken
is augmented, only to a maximum."
As far as digestion is concerned, every layer is independent
on every other and the total quantity digested is the sum of those
digested in the various layers. On the simple assumption that
the digestion in the outermost layer is always the same, the
jmmber of layers which may lie inside has been calculated by
Arrhenius from London's results by means of an
196
GENERAL CHEMISTRY OF THE ENZYMES
empirical formula. The latter gives the quantities of food
digested in a certain time in the various layers and corresponds
very well with the experimental figures.
With the aid of this formula Arrhenius has prepared a
table showing the time-course of the digestion which he represents
also graphically (Fig. 5). 1
icoo
DIGESTION OF DIFFERENT QUANTITIES
OF MEAT, 100-1000 GRAMS.
\
400
200
\\
\
10 11
1 From C h i g i n and Lobassow's results (Dissertation, St.
Petersburg, 1896), H e r z o g (Zeitschr. f. allg. Physiol., 1904, 4, 163) has
attempted to calculate the time-course of the secretion of gastric juice.
Determinations were made of the quantities of pepsin (measured by the
number of c.c. of gastric juice and its specific digestive power) secreted in a
blind sac in the fundus part of the stomach, when food was introduced into
the stomach. According to Herzog's calculations, the secretion of
pepsin follows the formula for unimolecular reactions.
With introduction of solid food the values of k obtained are moderately
constant. But in what degree the measurement and calculation of the
quantities of pepsin by the Russian workers are free from objection, cannot
well be judged. C h i g i n and Lobassow's results have recently
been referred to in a paper by Arrhenius (H., 1909, 63, 323).
EXPERIMENTAL DATA OF ENZYME REACTIONS 197
From the curve for 1000 grms. the quantities remaining
undigested after a certain number of hours have been calculated,
the results being as follows:
Time in hours: 1 2 3. 4 5 6 7 8 9101111-5
Amount undigested: 1000 875 750 627 507 390 276 180 100 51 24 7 3
Difference per hour: 125 125 123 120 117 114 96 80 49 27 17
It will therefore be seen that the quan-
tity digested is at first very nearly pro-
portional to the time, but gradually di-
minishes later.
This apparent contradiction ofSchiitz's rule depends on
the fact that in vivo the products of digestion are continually
removed, whilst in vitro they remain in the solution and
hence retard the reaction.
The above table leads to a very important rule. It will
be seen that for practically complete digestion of 1000 grms. of
meat 11-5 hours are necessary. The time t required to digest
another quantity M is calculated by subtracting from 11-5 hours
the time taken to digest (1000-M) grms. of meat when the
initial quantity is 1000 grms. In this way the times b s . given
below for varying values of M have been obtained.
M
W
kale.
(kbs. kale.)
1000
11-50
10-81
+0-69
900
10-70
10-26
-fO-44
800
9-90
9-67
+0-23
700
9-09
9-05
+0-04
600
8-27
8-38
-0-11
500
7-44
7-65
-0-21
400
6-60
6-84
-0-24
300
5-73
5-92
-0-19
200
4-74
4-84
-0-10
100
3-45
3-42
+0-03
50
2-40
2-42
-0-02
25
1-55
1-71
-0-16
Besides the values of obs ., those of ca i c . are also given, these
being calculated from the formula
198 GENERAL CHEMISTRY OF THE ENZYMES
Hence, the time required for the virtually complete digestion of
flesh-food introduced per o s is very nearly proportional to the
square-root of the quantity of food.
This square-root law is repeated in numerous series of experi-
ments and also with different nutriment. It holds
not only for ordinary meat but, as was shown byArrhenius,
for the digestion of gliadin (London, H., 1908, 56, 394),
of the albumin of hens' eggs (London, H., 1908, 56, 405)
and of bread (London, H., 1906, 49, 359), and is therefore
of the greatest importance for calculating the digestion periods
of solid foods.
2. Digestion of meat introduced directly
into the stomach by a fistula. If the nutriment
is taken per o s , a reflex secretion of gastric juice occurs, so
that the digestion is greatly accelerated. When, however, the
introduction of food takes place through a fistula, the secretion
of gastric juice produced by the stimulating action of the meat
on the stomach-wall is at first gradual and increases with the time;
indeed, as the following calculation shows, the quantity of gastric
juice in the stomach is about proportional to the time elapsing
since the introduction of the meat.
Thus, if the digestion is proportional to the quantity of gastric
juice present and the latter to the time, the amount digested per
unit of time, , is proportional to the time t and to the quantity
at
of meat (M x) still present (M being the amount initially
added), or, mathematically:
dt
from which follows:
London and Pewsner's results, given below, have been
calculated according to this formula.
EXPERIMENTAL DATA OF ENZYME REACTIONS 199
A. Feeding of dog per fistulam. Eyes and nose covered.
t (hours)
Undigested
quantity (Mx)
(M-x) C aic.
, Difference.
100
100
2
84
84-7
-0-7
4
56
51-5
+4-5
6
20
22-5
-2-5
8
7
7-0
9
3-5
-3-5
B. Feeding per fistulam. Eyes and nose uncovered.
t (hours).
Undigested
quantity (Mx).
(M-a) ca i c .
Difference.
100
100
2
84
83-7
+0-3
4
53
49-1
+3-9
6
18
20-1
-2-1
8
5
5-8
-0-8
9
2-7
-2-7
In the experiment A, the constant had the value k = 0-0180
and in B, k = 0-0193.
3. If we now ask how the total course of the digestion
is to be represented (i.e., not only as regards the time necessary
for local digestion), it may in general be stated that, in so far as
small quantities of food (the layer which, according to the above
assumption, is in immediate contact with the stomach-wall)
are concerned, digestion follows the law holding for unimolecular
reactions. This is shown by the following examples :
Digestion of boiled protein, 200 grma.
Digestion of meat, 200 grms.
t
(100 -z)
(100-aOcalc.
Diff.
t
(100-3)
(lOO-z)calc.
Diff.
100
100
100
100
1
70
65
+5
1
60
56
+4
2
32
42
-10
2
31
32
-1
3
28
27
+ 1
3
15
18
-3
4
18
18
4
5
10
-5
5
15
12
+3
5
6
6
6
4
7
-3
(7
5
-5)
200 GENERAL CHEMISTRY OF THE ENZYMES
By (a x) is denoted the quantity of undigested protein still
in the stomach at the time t. The calculated figures are obtained
by means of the formula:
7 1 100
fc=-7-log
t & 100-ar
The same formula holds for the digestion of dissolved
carbohydrates (amylodextrin) .
4. The resorplioii of a sugar solution in the intestines also follows
the unimolecular law.
If a solution of glucose is introduced into the intestine, the latter
gives up water to it if the solution is concentrated, but takes up
water from a dilute solution. The quantity of water yielded to the
solution reaches a maximum, amounting to 900 c.c., corresponding with
the capacity of the body to give up water From London's results,
Arrhenius has calculated the concentration of the solution when
no water is taken up o give out by the intestine, this being 10 5%
of glucose. As was to be expected, it is found that the amount of water
taken from the intestine is proportional to the excess of the concentra-
tion over 10-5%. The law followed is hence expressed by the equation:
where W is the amount of water given up by the intestine and (C 10-5)
represents the excess of the concentration over 10 5%.
RENNET (CHYMOSIN)
By the action of rennet, casein is converted into paracasein
(compare p. 45); whether at the same time some component,
possibly an albumose, is split from the casein molecule is a dis-
puted question. On account of this supposed partial hydrolysis,
and of the occurrence of pepsin and chymosin together, the latter
is classed with the true proteolytic enzymes.
It must here be borne in mind that the clotting of milk by
rennet consists of two processes (Hammarsten) : chymosin
only accelerates the conversion of casein into paracasein, a
change which proceeds without calcium salts. These salts are
necessary only for the precipitation of the curd (para-
casein) for which, indeed, no rennet is required.
It would therefore be expected that the time which clotting
takes to begin is made up of two periods: (1), that necessary for
EXPERIMENTAL DATA OF ENZYME REACTIONS 201
the casein to be transformed, almost completely, into paracasein
time of conversion, and (2), that necessary for the formation
of a visible clot time of separation. Such an assumption was
advanced by Fuld (Hofm. Beitr., 1902, 2, 169). According
to this author, the time of separation amounts to several days
or to a few minutes or even less, in dependence on the temperature
of the paracasein solution, and is so inappreciable in comparison
with the long period of conversion of the experiments referred
to above, that it may be unhesitatingly neglected. On the other
hand, R e i c h e 1 and S p i r o , in their most recent investiga-
tion, regard such an assumption as that of F u 1 d as unjustified,
and express the view that, with reference to its time-course, the
whole clotting process must be considered as a single process
(Hofm. Beitr., 1906, 8, 15).
According to Bang (Skand. Arch. f. Physiol., 1911, 25, 105),
the clotting of milk by rennet is an extremely complicated process,
in which the calcium salts present are distributed between organic
acids, lactalbumin and lactoglobulin, whilst, on the other hand, the
casein is distributed among the basic constituents of the solution.
The clotting process obeys the law, [E].t = const., or
The product of the enzyme-concentra-
tion [#] a n d time of clotting t is constant.
This relation was first observed by Segelcke and S t o r c h
(Ugeskrift for Landmaend, 1870) and was subsequently fully
confirmed by H a n s e n and S o x h 1 e t (Milchzeitung, 1877).
The most complete investigations of the action of rennet are
due to F u 1 d and to R e i c h e 1 and S p i r o .
At 40, E . F u 1 d (Hofm. Beitr., 1902, 2, 172) obtained the
following numbers, which proved the exact and general validity
of the time-law even with small amounts of rennet:
Quantity of rennet.
Time of clotting, t, in seconds.
Product.
0-4
6
24
0-4
6-6
26
0-2
13
26
0-1
25
25
0-8
6
48
0-4
17
44
0-2
22
44
0-1
45
45
202
GENERAL CHEMISTRY OF THE ENZYMES
According to C. G e r b e r (Soc. BioL, 1907, 63, 575), who has
recently made a thorough study of the clotting process, it is essen-
tial, when working with the rennet of commercial pepsin, to
employ temperatures between 25 and 30 so that the enzyme
shall be under normal conditions. Within these limits of tem-
perature, G e r b e r finds that the law of Segelcke arid
S t o r c h holds closely for parachymosin.
Drops of parachymosin #....1 2 3 46 7 8 9 10
Time of clotting, t 29-66' 14-75' 10-30' 7-60' 5-50' 4-50' 3-66' 3-16' 2-92'
E.t 29-6 29-5 31 30-3 33 31-5 29-3 28-4 29-2
M a d s e n has also investigated the duration of milk-clotting,
the method used being similar to that employed with pepsin
action (see Arrhenius, Immunochemistry, p. 72).
He adds, for instance, varying quantities of rennet to equal amounts
of milk in test-tubes, places the mixtures in a water-bath at a definite
temperature, takes them out after time t, cools them quickly and deter-
mines the smallest quantity of rennet L which has produced clotting.
COAGULATION OF MILK BY RENNET SOLUTIONS OF DIFFERENT CONCENTRATIONS
AT 36-55
Minutes.
L.
Lt.
Minutes.
L.
Lt.
4
0-08
0-32
35
0-007
0-25
6
0-05
0-30
50
0-005
0-25
9
0-033
0-30
70
0-004
0-28
11
0-024
0-26
80
0-0032
0-26
12
0-019
0-23
100
0-0028
0-28
14
0-0175
0-25
120
0-0025
0-30
20
0-013
0-26
180
0-00185
0-33
25
0-010
0-25
240
0-0017
0-41
30
0-007
0-21
Bang (Pflug. Arch., 1900, 79, 425) found that the relation,
[E] t = const., did not hold for parachymosin.
By a series of careful experiments R e i c h e 1 and S p i r o
(Hofm. Beitr., 1905, 7, 485) showed that the relation between
quantity of rennet L and period of clotting t can be expressed
generally by the formula:
L n .t = const.
EXPERIMENTAL DATA OF ENZYME REACTIONS 203
The exponent n of L changes with the nature and amount
of the other substances in the solution, and in a liquid of the com-
position of milk it assumes exactly the value 1, so that the law
holds in its simplest form,
L.t = const.
In the following experiments the solutions were prepared by
dilution with whey, the clotting action of which was negligible
in comparison with that of the enzyme added.
Milk.
c.c. of 35%
rennet diluted
with whey.
Whey.
Time of clotting t in seconds.
Whey I.
Whey II.
Whey III.
0-5
0-5
4-0
22
22
33
1-0
0-5
3-5
17
22-5
27
1-5
0-5
3-0
18
27
28
2-0
0-5
2-5
17
25-5
26-5
2-5
0-5
2-0
18
24-5
25-5
3-0
0-5
1-5
16
26
26
3-5
0-5
1-0
16
26
24
4-0
0-5
0-5
15
25
26
4-5
0-5
~
16
24
27
As will be seen from these results, the constant Lt or, since
L is the same in all cases, the time of clotting, is independent
of the concentration of milk (casein) in the
solution from 20% up to 90 %.
Replacement of the whey by 0-9% sodium chloride solution
resulted in constancy of the time of clotting for more dilute as
well as for more concentrated solutions.
More extended investigations then showed that the difference
between the times of clotting for dilute and concentrated milks
was approximately proportional to the difference between the
dilutions. Hence, if V denotes the volume of the diluted milk,
M that of the undiluted milk, and t and t' the times of clotting
in the two media, then:
M
V-M
const.
204
GENERAL CHEMISTRY OF THE ENZYMES
This is shown by an extract from Table VII of R e i c h e 1
and S p i r o ' s paper.
Rennet.
Milk.
Whey.
Time.
-<'> (T^T)
1-0
0-2
8-8
110
1-60
1-0
0-6
8-4
50
1-81
1-0
1-0
8-0
39
1-93
1-0
2-0
7-0
28-6
1-75
1-0
4-0
5-0
24
1-60
1-0
8-0
1-0
22
1-60
The influence of the Ca-ions is also expressed by a simple and
important relation,
Time t for amount of rennet.
Constant, Ca" Xt for amount of
Milk,
CaClj,
/ _
rennet.
c.c.
u /oo
1-0
0-5
0-25
1-0
0-5
0-25
8-0
95
48
24
57-0
28-8
14-4
8-0
0-05
88-6
45-6
23
57-6
29-6
15-0
8-0
0-1
79
41-6
22
55-3
29-1
15-4
8-0
0-2
66-4
36
19
53-1
28-8
15-2
8-0
0-5
48
26-4
14
52-8
29-0
15-4
8-0
1-0
30
18-2
10-6
48-0
29-1
17-0
8-0
2-0
17
11
6-8
44-2
28-6
17-7
8-0
5-0
10
7-4
5-4
56-0
41-4
30-2
8-0
10-0
13
9-2
6-2
137-8
97-5
65-7
8-0
20-0
22
15
8-6
453-2
309-0
177-2
The values of the constant, which show little change up to about
0-02% of calcium, are calculated on the assumption that the Ca-ions
in the milk amount to 0-6%o; about 15% of the total calcium of
the milk must then be in an ionised condition.
Further, as was shown in a subsequent paper (Hofm. Beitr.,
1906, 8, 15) the rennet-action a is, in general (not merely at the
clotting point), directly proportional to the quantity of enzyme
L and to the time t, that is
= L.t. const.
EXPERIMENTAL DATA OF ENZYME REACTIONS 205
The more recent assertion of H.Kottlitz (Arch, intern, de
Physiol., 1907, 5, 140) that S c h ii t z ' s rule holds also for rennetic
action in no way diminishes the importance of R e i c h e 1 and S p i r o ' s
results.
Several attempts have been made to estimate the value of the
time-laws determined, on the one hand, for the action of rennet
and, on the other, for that of pepsin, as criteria for the disputed
identity of chymosin and pepsin.
The discussion of this question would not be in place in a
chapter dealing with the chemical dynamics of the enzymes;
but it may at least be asserted that, especially since H a m -
marsten's comprehensive investigation (H., 1908, 56, 18),
it is no longer possible to regard the clotting of milk simply as a
peptic action. The influence of concentration, and also that of
temperature, are so divergent in the cases of clotting and diges-
tion, that it must at the least be assumed that two different
enzymic groups are united in one molecule. This assumption,
so long as the preparation of the enzymes in a pure state is not
achieved, can be neither refuted nor proved, and is indeed, under
present circumstances, of subordinate importance.
FIBRIN-FERMENT
E . F u 1 d (Hofm. Beitr., 1902, 2, 514) mixed the plasma of
bird-blood with the enzyme solution (obtained by extracting
muscle with 0-8% sodium chloride solution). The velocity
of clotting increased more slowly than the concentration of the
enzyme, the results agreeing approximately with S c h ii t z ' s
rule but more accurately with the expression :
^ \ 0-585
aj '
where vi and v% are the velocities of clotting and E\ and E% the
concentrations of the enzyme. For protracted clotting periods,
that is, for low enzyme-concentrations, this relation fails, the
duration of clotting then showing disproportionate increase.
It has been shown, especially by M a r t i n (Journ. of Physiol.,
1905, 32, 207), that the rule obeyed by chymosin and numerous
other enzymes that the same amount of action occurs for equal
206
GENERAL CHEMISTRY OF THE ENZYMES
values of the product E.t, no matter what the values of E and
t holds also for the fibrin-ferment.
ZYMASE
Although the " zymase " of pressed yeast-juice is accompanied
by an extremely large quantity of various other substances and
is further removed from a state of purity than is the case with
any other enzyme, yet in recent years, especially by the work of
Harden and Young, important information has been
obtained concerning the chemistry of alcoholic fermentation.
If the final products, alcohol and carbon dioxide, of pressed
yeast-juice containing sugar are studied quantitatively, as has
been done by the author (H., 1905, 44, 53), the following
results are obtained:
The expression log == k gives moderately constant
numbers during the first half of the reaction, but subsequently the
value of k exhibits considerable diminution. To this effect, two
principal causes contribute, firstly, the separation of protein
substances which carry down part of the fermentation enzymes,
and secondly, alteration of one of the activators of the zymase;
this activator consisting of organic compounds of phosphoric
acid is attacked by the lipase of the yeast-juice, the latter
gradually becoming impoverished in the " co-enzyme " which
is of s\ich great importance to alcoholic fermentation.
The quantity of carbon dioxide evolved per unit of time was
determined partly by the loss in weight of the solution and partly
volumetrically.
4 grms. glucose in 20 c.c. of yeast-juice.
Minutes.
x (grms. CO 2 ).
(ax).
fc.10 4 .
1-800
80
0-078
722
2-4
315
0-299
501
2-51
379
0-360
440
2-56
505
0-460
340
2-54
1024
0-779
021
2-41
1180
0-810
0-990
(2-20)
1544
0-899
0-901
(1-95)
2119
0-955
0-845
(1-55)
EXPERIMENTAL DATA OF ENZYME REACTIONS 207
For about 18 hours, the velocity of reaction is here moderately
constant. This is, however, a favourable case and under other
conditions, as is shown by the following table, the pressed juice
remains unchanged only for 6-8 hours.
The influence of the concentration of the substrate is also
shown by the following results.
20 c.c. juice +8 grms. of glucose in 20 c.c. of solution.
Minutes. : x(grms.)
(o-*).
k. 10*.
Minutes.
x(grms.)
(a-x).
fc.105.
3-909
3-909
3-900
3-900
161
0-078
3-822
5-44
160
0-0815
3-8185
5-38
260
0-120
3-780
5-24
257
0-1195
3-7805
5-24
358
0-161
3-739
5-11
355
0-159
3-741
5-05
404
0-181
3-719
5-10
404
0-180
3-720
5-10
5-21
5-19
20 c.c. juice +2 grms. of glucose in 20 c.c. of solution.
Minutes.
x(grms.)
(a-x).
fc.10*.
Minutes.
x(grms.)
(a-x).
k.lW.
0-977
0-977
0-968
0-962
167
0-0855
0-8825
2-40
153
0-081
0-881
2-50
240
0-123
0-845
2-46
260
0-123
0-839
2-27
332
0-152
0-816
2-29
331
0-182
0-780
2-75
2-38
2-51
In addition to these series of experiments, three others were
carried out, also with concentrations of sugar in the ratio 4:1.
The constants obtained were as follows:
i
fci . 10 5
:*,.
10 5
No.
4.
5-2
: 24
4
= 1
: 4
7
11
5.
2-5
: 12
= 1
: 4
8
11
7.
15-0
: 75
= 1
: 5
"
6.
20-0
: 97
1
: 4
85
It will be seen that the velocities are not, as they should be
according to theory, independent of the concentration of the
substrate; also they are not inversely proportional to the initial
208 GENERAL CHEMISTRY OF THE ENZYMES
concentration a, so that ka is not a constant magnitude but, in
the region of concentration investigated, increases as a decreases.
In each of the four series of experiments, ai : a2 = 4 : 1.
ftiai.10 6 ' & 2 a 2 .10 6
No. 4. 208 244
" 5. 100 120
" 7. 600 750
" 6. 800 990
Whilst, in general, the velocity of reaction increases either
proportionally to, or slower than, the concentration of enzyme,
the velocity of fermentation increases more rapidly than the
concentration of the pressed juice but always slower than its
square.
If the exponents n are calculated according to the equation
In k\ In k%
1 7 >
In ci ln 2
it is found that, for a constant sugar-content, n increases with
diminution of k, that is, with diminution of the concentration
of the zymase:
ki . 10 6 n
100 1-29
86 1-33
35 1-52
12 1-67
The numbers appear to indicate that, with very high fer-
mentative activity, proportionality between the concentration
of the pressed juice and the velocity of fermentation is attained.
Finally, if pressed juice containing sugar is diluted, that is,
the volume increased while the absolute amounts of the juice and
sugar remain constant, the following relations are found:
f Concentration"32 : 52 = 1:1-63 f 20: 30: 50 = 1:1-5 :2-5
) [Velocity 192:282=1:1-47 () { 120 : 188 : 315 = 1: 1-57: 2-63
The mean of these two experiments indicates, within the limits
of concentration employed, approximate proportionality between
concentration and velocity.
EXPERIMENTAL DATA OF ENZYME REACTIONS 209
Prior to these experiments with pressed yeast-juice, A b e r s o n
Rec. Trav. Chim. Pays-Bas, 1903, 22, 78) had studied the course
of the alcoholic fermentation of glucose by living yeast-cells.
It must however be emphasised that Aberson's observa-
tions were made polarimetrically, so that he measured the amount
of sugar disappearing during the reaction. But new measurements
made by E u 1 e r and Johansson (H., 1912, 76, 347) with
living yeast show that the diminution in rotation of a fermenting
sugar solution is by no means proportional to the evolution of
carbon dioxide. This is indicated by the following numbers:
CO 2 evolved.
Change in rotation.
Concen-i ^
Dif-
No.
Temp.
tration
of
Sugar.
yeast in
25 c.c.
solution
Time.
Min.
Grms.
Per
cent.
A
Degrees.
Per
cent.
B
fer-
ence.
B-A
(1
30
10%
0-5
125
0-0936
7-7
5-37-4-67=0-70
13 1
5-4
12
30
10
0-5
181
0-1492
12-2
5-32-4-46 = 0-86
16 2
4-0
3
30
10
1
42
0-0644
5-3
5-33-4-80=0-53
10-0
4-7
4
30
10
1
63
0-1160
9-5
5-33-4-48 = 0-85
16-0
6-5
5
30
10
1
232
0-4918
40-3
5-33-2-84 = 2-49
47-0
6-7
6
30
10
1
406
0-7256
59-4
5-33-1-85 = 3-48
65-9
6-5
The cause of this difference has not been fully elucidated.
Owing to this circumstance, Aberson's experiments
give information concerning only the first phase of alcoholic
fermentation, namely, the transformation of the sugar. The
results of his numerous experiments do not correspond with the
expected law, but the values given in the last column of the fol-
lowing table which is taken from that given on p. 97 (1 o c .
c i t . ) are far more nearly constant.
Minutes.
Rotation of
the glucose
solution.
1 . a
fc= log
t ax
1 a+x
KH log
t ax
34-1
Temperature, 29-3.
31
33-0
45-9
90-0
Volume, 600 c.c.
91
30-9
47-0
90-0
Amount of yeast :
125
29-7
48-0
90-0
mins. : 0-288grm.
213
26-7
50-6
90-0
dry yeast per 50 c.c.
306
23-5
51-2
91-0
514 mins. ': 0-294
393
20-9
54-1
90-0
grm. dry yeast per
514
17-7
55-4
90-3
50 c.c.
210
GENERAL CHEMISTRY OF THE ENZYMES
In the fermentation of similar sugar solutions with varying
quantities of yeast, A b e r s o n obtained proportionality between
the velocity of reaction and the amount of yeast (1 o c . c i t .,
p. 84):
Grms. yeast
k
60
271
20
93-4
Grms. yeast
k
25
165
15
104
SI at or (Journ. Chem. Soc., 1906, 89, 128), who measured
volumetrically the carbon dioxide evolved, also found propor-
tionality between the velocity of fermentation and the quantity
of yeast added. He showed likewise that the initial velocity
of fermentation is independent except with very dilute solu-
tions of the sugar-content.
The fermentation experiments of H e r z o g (H., 1902, 37,
149) and G r i g o r i e w (H., 1904, 42, 299) also refer to hetero-
geneous systems.
The course of fermentation with permanent yeast (which,
however, contained glycogen) is shown by the following table from
H e r z o g ' s paper.
Concentration, 1-136 grms. glucose (a = l) and 1-2 grms. zymase (perma-
nent yeast) in 100 c.c. Temperature 24-5.
t
a x
10 6 a
10 6 a+x
. '*8
t ax
. '*S
t ax
120
0-961
144
141
240
0-931
129
133
1200
0-687
137
117
1440
0-627
140
118
1740
0-560
145
117
2690
0-403
146
111
3000
0-365
146
108
4140
0-271
137
99
The exponent n of the enzyme-concentration which shows
the increase of the velocity of the reaction, is greater in the exper-
iments with permanent yeast than in those with the pressed
juice. The following table indicates the behaviour of living
yeast, acetone-permanent yeast and yeast-juice from a dynamical
point of view.
EXPERIMENTAL DATA OF ENZYME REACTIONS 211
Reaction constant.
Living yeast.
Acetone permanent yeast,
Zymase solution
(pressed juice).
k =
Influence of the
amount of yeast
or concentration
of zymase
log ki log kz
1 a+x
1 l a 1 1 a+x
1 a
. Jog
t a x
1
k . a i n -
creases
with in-
crease
of a
log or log
t a-x t a-x
2 (Herzog)
log
t a-x
(loc. cit., p. 62)
1-67-1-29
log ci - log c 2
Influence of the
concentration of
sugar, a
k.a increases with diminution of a , within the
region' of concentration examined.
As has been already stated, B u c h n e r and M e i s e n -
h e i m e r arrived at the conclusion that zymase using the
word in its wider sense is a mixture of at least two enzymes,
namely, zymase in a restricted sense which decomposes fermentable
hexoses into lactic acid, and lactacidase, which breaks down the
lactic acid into alcohol and carbon dioxide. There has been
a considerable amount of discussion concerning the constituent
chemical processes which take part in the transformation of
sugar into alcohol and carbonic acid. Since grave objections
have been advanced to the intermediate formation of lactic
acid (compare the critical review byBuchner and M e i s e n -
h e i m e r in Landw. Jahrbiicher, 1909, 38, Erganz.-Band 5,
265), nothing final can be said as regards the number 'and
nature of the participating enzymes.
That a " co-enzyme " plays an important part in fermenta-
tion must now be considered an established fact. The same is
the case with the observation that addition of phosphates to
pressed yeast-juice containing glucose not only accelerates the
fermentation but also increases the total fermenting power or
the amounts of alcohol and carbon dioxide which can be formed
from a given quantity of sugar by the pressed juice (Harden
and Young, Proc. Roy. Soc., 1905, 77, 405; 1906, 78, 369;
1908, 80, 299).
These investigators have studied quantitatively the accelera-
tion of fermentation by phosphates. Of their results, which are
212
GENERAL CHEMISTRY OF THE ENZYMES
expressed in the form of tables and curves,the following may be
cited (Proc. Roy. Soc., 1908, 80, 307):
Time after
addition,
in minutes.
Carbon dioxide evolved in preceding 5 minutes, with n c.c. of 0-3
molar potassium phosphate solution added.
n = c.c.
n = 10 c.c.
n = 15 c.c.
5
4-0
11-1
7-7
10
3-2
16-0
9-7
15
4-2
20-2
12-1
20
3-6
22-4
16-1
25
4-3
17-4
18-4
30
3-6
6-6
19-4
35
4-3
4-6
20-4
40
3-2
4-7
16-7
45
4-5
12-7
50
4-2
6-0
55
~
4-1
4-0
It is evident that a marked optimum of phosphate-con-
centration exists, the velocity of fermentation diminishing if this
is exceeded.
The results of another series of experiments are shown graph-
ically in the curves given below (Fig. 6). The concentrations of
the solutions were as follows :
Curve A : 25 c.c. pressed juice + 5 c.c. phosphate +15 c.c. bicarbonate
" B : 25 c.c. +10 c.c. +10 c.c.
" C : 25 c.c. +15 c.c. + 5 c.c.
" D : 25 c.c. +20 c.c. + c.c.
" These results suggest that the phosphate is capable of form-
ing two or more different unstable associations with the fermenting
complex. One of these, formed with low concentrations of the
phosphate, has the composition most favourable for the decom-
position of sugar, whilst the others, formed with high concen-
trations of phosphate, contain more of the latter, probably
associated in such a way with the fermenting complex as to ren-
der the latter partially or wholly incapable of effecting the decom-
position of the sugar molecule."
The influence of arsenates and arsenites on the evolution of
carbon dioxide produced by yeast-juice has also been studied
EXPERIMENTAL DATA OF ENZYME REACTIONS 213
in detail by H a r d e n and Young (Proc. Roy. Soc., B, 1911,
83, 451).
" A striking feature of the effect of the addition of a phos-
phate to yeast-juice is that the marked acceleration only con-
tinues until an amount of carbon dioxide has been evolved which
is chemically equivalent to the phosphate added. Moreover,
at the close of this period of enhanced fermentation, the added
phosphate is no longer present in a form precipitable by magnesium
citrate mixture, but has become converted into a hexosephosphate.
5 10 15 20 25 30
50 60 70
.Time in Minutes
FIG. 6.
Neither of these phenomena occurs when an arsenate is substituted
for the phosphate. The enhanced rate of fermentation continues
long after an equivalent of carbon dioxide has been evolved and
no organic combination of arsenic is formed.
The sharp contrast between the actions of arsenate and phos-
phate is clearly shown, when the effects of equivalent amounts
of phosphate and arsenate on the same sample of yeast-juice
are directly compared, as is done in the following experiments."
214
GENERAL CHEMISTRY OF THE ENZYMES
20 c.c. Yeast-juice
+5 c.c. 0-3 mol. phosphate.
+5 c.c. 0-3 mol. arsenate.
+0-75 c.c. 0-3 mol. arsenate
Time.
Total
Rate per
5 minutes.
Total.
Rate per
5 minutes.
Total.
Rate per
5 minutes.
5
7-1
7-1
11 4
11-4
21-7
21-7
10
19-8
12-7
26-5
15-1
46
24-3
15
36 1
16-3
43
16-5
71
25
20
43-8
7-7
59
16
95-3
24-3
25
45-7
1-9
75
16-3
119-8
24-5
30
47-5
1-8
"
Formation of the Phosphoric Ester
Contrary to the opinion of I wan of f (H., 1907, 50, 281;
Centralbl. f. Bakt., 1909, II, 24, 1), Harden and Young
(Centralbl. f. Bakt., 1911, II, 26, 178) regard the formation of
the ester as due, not to a special enzyme, but to the zymase.
According to E u 1 e r and K u 1 1 b e r g ' s and E u 1 e r and
O h 1 s e n ' s experiments with the extract of dried yeast (H., 1911,
74, 15, 1912, 76, 468), the enzymic formation of the phosphoric
ester can, however, be separated from the other fermentative
processes, and the author (H., 1911, 74, 13) hence suggests
the name phosphatese for this synthesising enzyme.
If glucose is fermented by pressed yeast-juice in presence of
phosphates, the latter very soon become combined organically.
This also occurs with extract of Munich yeast, no matter whether
this be dried at 40 or, as von Lebedew suggests, at 25-35;
in both cases, as was described by Harden and Young,
fermentation and formation of phosphate proceed together.
But if another race of yeast with a lower fermenting power
is dried at 40 in a vacuum and then extracted in the usual manner,
no formation of phosphoric ester occurs with glucose (or mannose
or fructose) (Table a, below) . If, however, the glucose is partially
fermented beforehand in absence of living yeast, organic combina-
tion of the phosphate takes place (Table 6) (E u 1 e r and O h 1 -
sen, Biochem. Z., 1911, 36, 313).
EXPERIMENTAL DATA OF ENZYME REACTIONS 215
TABLE a.
25 c.c. yeast-extract.
20 c.c. glucose solution (20%).
10 c.c. 5% Na 2 HP0 4 .
Minutes.
Grms. of Mg 2 P 2 Or
from 10 c.c. of the
mixture.
150
269
0-0399
0-0394
0-0394
TABLE 6.
25 c.c. yeast-extract.
20 c.c. fermenting glucose solution (2Q%).
10 c.c. 5% Na 2 HP04.
Minutes.
Grms. of Mg 2 P 2 O7
from 10 c.c. of the
mixture.
0-0414
74
0-0134
264
0-0000
Like other enzymes, the phosphatese may be precipitated from
the aqueous yeast-extract by means of alcohol, a considerable
proportion of its activity remaining after this treatment.
It is remarkable that the synthetic enzymic action of the
extract is greatly increased by heating it at 40 for half an hour.
CATALASES
The decomposition of hydrogen peroxide effected by the action
of unknown constituents termed, after O . L o e w , catalases
of the animal and vegetable body, is so easily followed quanti-
tatively, that this process has formed the subject of a large
number of investigations and is to-day one of the best-
known enzymic changes. Especially from the fatty tissues of
animals can solutions be prepared which, beyond their ability
to decompose hydrogen peroxide, exhibit few enzymic activities;
particularly small are the amounts of organic matter present in
these liquids, which must therefore be comparatively pure. Also
blood-serum, the organs of plants, etc., by precipitation with
alcohol and suitable treatment of the precipitates, yield prepara-
tions which, per unit of weight, rapidly decompose hydrogen
peroxide (O . L o e w , Rep. U. S. Dep. of Agric., 1901, No.
68; S enter, Zeitschr. f. physikal. Chem., 1903, 44, 257 and
Proc. Roy. Soc., 1904, 74, 201; E. J. Lesser, Zeitschr. f.
Biol., 1906, 48, 1; L. van Italie, Soc. Biol., 1906, 0, 150;
K a s 1 1 e and Loevenhart, Amer. Chem. Journ., 1903,
29,563; L.von Liebermann, Pfliig. Arch., 1903, 104,
176 and Chem. Ber., 1905, 38, 1524).
Catalase follows the theoretically simplest formulae and
relations with an approximation shown with few other enzymes,
216
GENERAL CHEMISTRY OF THE ENZYMES
possibly because the simple nature of the chemical process con-
ditions especially simple relations, or possibly owing to the
exclusion of any considerable complication by the relatively
high degree of purity of the enzyme. The most important general
conclusions arrived at are as follows :
The enzymic decomposition of hydrogen peroxide in neutral
or acid solution is a reaction of the first order.
In dilute solutions of the peroxide, decomposition follows the
law of mass action exactly. Numerous physico-chemical measure-
ments of the action of catalases have been carried out, the fol-
lowing series of numbers being given by G. Senter and by
H. Euler :
Blood-catalase. Senter, loc. cit.,
p. 282. jJs-molar H2O2-solution.
Fungus-catalase. Euler, Hofm. Beitr.,
1905, 7, 1. 2 Vmolar H 2 O 2 -solution.
Minutes.
a x
fc.10*.
T=0.
Minutes.
a x.
k.lW.
r = i5.
46-1
8-0
5
37-1
190
6
6-9
107
10
29-8
192
12
5-8
116
20
19-6
190
19
5-0
107
30
12-3
193
55
2-5
100
50
5
194
I s s a e w (H., 1904, 42, 102) also obtained very constant
values of k with dilute solutions of hydrogen peroxide; with
more concentrated solutions (0-01 N and above) the constants
diminish.
Catalases are extremely sensitive preparations of different
origins to varying degrees towards higher concentrations of
hydrogen peroxide. Thus, blood-cat alase is appreciably destroyed
(perhaps oxidised) in about 0-01 N-H2O2, this .action being
naturally more rapid at higher than at low temperatures, the
values of k being greatly diminished even at 30. It is hence
advisable to employ low temperatures in working with catalase.
According to Waentig and Steche (H., 1911, 72, 226),
this injurious action is observable even at in about N/80-
hydrogen peroxide solution.
The reaction constants are, as they should be theoretically,
nearly independent of the concentration of the
peroxide, equal percentage amounts being decomposed per
EXPEKIMENTAL DATA OF ENZYME REACTIONS 217
unit of time by equal quantities of enzyme. Senter (loc.
c i t . , p. 283) gives the following resume:
CH 2 0,
/c.10 4
CH 2 o 2
fc.10 3
Cn 2 o 2
fc.10 3
290*
120
i N
175
Io^ N
192
1 N
122
AT
188
1
225
1100 N
460
(The two values of k in each of these pairs are comparable, one with the
other, but not with those of the other pairs.)
The following results, given by Senter, indicate the
influence of the concentration of the enzyme:
Concentrations, E, in the proportions 3 6 8 24
fc.10 4 28 58 72 230
k : C 9-3 9-7 9-0 9-6
Within the limits of experimental error the velocities of
reaction in very dilute (^iir-niolar) solutions of hydrogen peroxide
are therefore proportional to the concentrations of the enzyme.
With stronger peroxide solutions, Senter found deviation from
this rule. While fc.10 4 is about 100 in weak catalase solutions,
its value in presence of the threefold quantity of enzyme is approx-
imately 360. That, under these conditions, the velocity of reac-
tion increases more rapidly than the concentration of the enzyme,
is confirmed by experiments made by Bach, who investigated
the dynamics of the catalases with a preparation from beef-fat
(Chem. Ber., 1905, 38, 1878).
With N/210-peroxide solutions, I s s a e w observed deviation
from proportionality in the opposite direction, that is, a slower increase
of the velocity than of the enzyme-concentration. But the yeast-catalase
he employed must, like all yeast-enzymes, have been very impure.
As was shown originally by L o e w , catalases are extremely
sensitive to acids. The action of acid has also been the subject of
numerous quantitative investigations, which show that the enzyme
is not permanently changed by acids, neutralisation of these being
accompanied by return of the catalytic activity.
218
GENEKAL CHEMISTRY OF THE ENZYMES
100 c.c. of catalase solution were poisoned, mixed with 100 c.c.
of H2O2 solution and 25 c.c. then titrated; concentration of
H2(>2 in the mixture 0-005 -normal.
snVu norm. HC1.
Tffs0o norm. HC1.
Without addition.
Minutes
CH 2 2
/c.10 4
Minutes
CH 2 2
/c.10 4
Minutes
Cn 2 o 2
fc.10 4
27-9
27-9
27-9
70i
25-3
6
15*
25-6
26
5|
19-8
278
136
23-0
6
35|
22-4
27
15
10-5
275
195
20-9
6
66^
18-7
26
25i
5-8
258
1305
3-0
6
185
10-1
24
Yuggg-norm. HOI.
Incubation period, 2 hours; then a small excess
of NaOH added, and afterwards the H 2 O 2 .
Without addition.
Minutes.
CH O!
fc.10 4
Minutes.
CH 2 O 2
fc.10 4
28-7
28-7
5|
15*
23-8
18-5
121
113
5
25-2
16-9
120
121
26i
14-9
86
31*
11-9
127
41*
11-7
70
53
6-5
120
As is shown by the latter table, the catalase is not changed
by the dilute hydrochloric acid solution, even after two hours.
The incubation period, that is, the time during which the enzyme
remains in contact with the acid, has been repeatedly shown
to be without influence on the subsequent activity of the enzyme.
The catalytic power of catalases is also reduced by quite
small amounts of baryta.
Temperature, 10 TTO norm. Ba(OH) 2 . ah norm. Ba(OH) 2 . Without
Catalase from fatty tissue Incubation period, Incubation period, addition
(E u 1 e r , Hofm. Beitr., 15 minutes.
1905,7,12) fc.!0 3 = 40
40 minutes.
A;.10 3 =40
of baryta.
/c.10 3 =60
This sensitiveness towards alkali varies considerably with catalases
of different origins.
A very slight increase of the alkalinity, however, appears to
Increase the velocity of the decomposition to some extent. Thus,
the author found (1 o c . c i t . ) that the velocity of reaction of
catalase from Boletus scaber is doubled by suspending
pure magnesium hydroxide in the solution.
EXPERIMENTAL DATA OF ENZYME REACTIONS 219
As is brought out most clearly by'Sorensen's results
'(Biochem. Z., 1909, 21, 131), the optimal activity of catalase
is always exhibited in almost neutral solution.
The actions of acids and bases on this enzyme depend, in
all probability, on the formation of salts by these electrolytes
with the catalase.
Bach (Chem. Ber., 1905, 38, 1878) has also investigated
catalase quantitatively, his results in general agreeing with those
required by theory.
OXYDASES
Substances of unknown constitution and composition, formed
in the animal and vegetable kingdoms and capable of initiating
oxidation changes, have been designated oxydases, generally
without any definite proof of their mode of action; their sensitive-
ness to heat was, however, established and they were then classed
with the enzymes. That these substances effected catalytic
acceleration of oxidation processes was seldom, and could indeed
only with difficulty be, proved, especially when isolated con-
stituents of an organ or juice were not examined. On the other
hand, many other enzymes are not catalysts in the strictest sense
of the word, so that no limit could easily be drawn. The wide
distribution of oxydases in vegetable and animal organisms ren-
ders it probable that these substances perform an important
function in all life-processes; but what this function is, what
reactions the oxydases bring about in the living animal or plant,
still remains unknown. The members of this class of bodies
which have as yet been obtained exhibit a somewhat limited
sphere of action.
Aldehydases
Medwedew has made a very complete study of the oxidising
agent of the liver, with reference to its action on salicylic aldehyde
(Pfliig. Arch., 1896, 65, 249; 1899, 74, 193; 1900, 81, 540 and
1904, 103, 403).
As regards the final state or equilibrium, the following results
were obtained. Case 1 : relatively high concentration of salicylic
aldehyde in neutral-acid solution. The concentration of the
oxidation product (salicylic acid) is inversely proportional to the
square-root of the amount of substance to be oxidised and approx-
220 GENERAL CHEMISTRY OF THE ENZYMES
imately proportional to the square of the concentration of the
aldehydase.
Case 2: relatively high concentration of salicylic aldehyde in
neutral-alkaline solution. One and the same quantity of the
oxydase gives at the end of the reaction, that is, on complete
exhaustion of the oxidising power, one and the same amount of
acid, no matter what the concentration of the aldehyde.
In relation to the velocity it was found : (a) If to the quantity
of oxydase m an excess of aldehyde a is added, the velocity of
oxidation is proportional to the square-root of the concentration
of aldehyde. In Medwedew's opinion, liver-oxydase is
inactivated by the oxidation. But this must, in reality, only
be a question of the consumption of an oxidising agent obtained
from the liver.
(b) If the concentration of aldehyde is less than that which
the oxydase present is able to oxidise, the velocity of oxidation
is proportional to the square of the aldehyde-concentration, so
that dx : dt = k(ax) 2 , where x is the concentration of the alde-
hyde changed up to time t and a the initial concentration.
How far these relations are quantitatively reproducible
and hence are independent of the non-controllable composition
of the liver-extract, and how far this interpretation of the numbers
obtained is fitting, remain undecided. Doubts have, however,
been expressed on these questions (compare Bach's remarks,
Chem. Ber., 1905, 38, 3791) ; but D o n y - H e n a u 1 1 and
van Duuren's experiments have led to different results
(Bull. Acad. roy. Belgique, 1907, 577).
Slowtzpff's observation (H., 1900, 31, 227) that " potato-
laccase " oxidises paraphenylenediamine solution with a velocity
proportional to the square-root of the quantity of this " laccase,"
also appears to the author to be insufficiently proved. What
Slowtzoff investigated must have been a very impure
mixture of a peroxydase and a substance Allied toMedicago-
laccase. The author obtained the latter component from potatoes
by precipitation with alcohol. The nitrogen-content of the
first precipitate amounted to 2 84% ; after solution of the prepa-
ration, treatment with animal charcoal and further precipitation
with alcohol, the proportion of nitrogen present fell to 1-6%,
whilst the ability to accelerate the oxidation of hydroquinone
solutions in presence of manganese salts remained undiminished.
EXPERIMENTAL DATA OF ENZYME REACTIONS 221
As regards the " laccases," the investigations of E u 1 e r
and B o 1 i n (H., 1908, 57, 80; 1909, 61, 1 and 72) show that the
oxydase of Rhus vernicifera and Rhus succe-
d a n e a differs considerably from those of Medicago
s a t i v a , etc., which, according to Bertrand, are also,
termed laccases. As Bertrand himself showed, the Rhus-
preparations are rich in manganese, whilst this is not the case
with laccases of the Medicago -type. The former alone
turn guaiaconic acid solutions blue directly and redden solutions
of guaiacol. This effect of Rhus -laccase and also the power
to transfer molecular oxygen to phenols (hydroquinone, pyrogallol)
are destroyed by heating the solution to 100 for a short time,
whilst Medicago -laccase is unchanged by heating.
In the case of Rhus -laccase, Bertrand (Bull. Soc.
Chim., 1897, [iii], 17, 619) established approximate proportionality
between the manganese-content and the catalytic activity.
The extraordinary sensitiveness of this preparation to acid is
shown by the following measurements (Ann. Inst. Pasteur, 1907,
21, 673).
The oxidation of guaiacol to tetraguaiacoquinone was measured.
To a 2% guaiacol solution were added a little (0 1 grm. per litre) laccase
(from Rhus succedanea) and sufficient sulphuric acid to give
the mixture the acidity shown in the table. The numbers represent
the intensities of the red colour measured in a colorimeter produced
after 5 hours by the tetraguaiacoquinone formed.
Normality of the acid.
Serie3 1.
Series 2.
1
100
100
100
73.4
48-8
20-3
100
90-9
75-2
60-4
60-4
60-4
500,000
1
400,000
1
200,000
1
100,000
1
50,000
222
GENERAL CHEMISTRY OF THE ENZYMES
Oxydases of the M e d i c a g o -type, in presence of neutral
manganese salts, accelerate the transference of molecular oxygen
to the poly phenols.
A representation of the course followed by this action is
given by the following experiments :
50 c.c. of a solution, 0-2-normal as regards hydroquinone and
0-001 -equivalent normal as regards manganese acetate, and
containing a weighed quantity of Medicago -oxydase, were
shaken in a glass vessel from which the air had been replaced
by pure oxygen. The oxidation of the hydroquinone to quinone
or quinhydrone, produced by the manganese and oxydase, was
measured by the diminution in the volume of oxygen in the vessel.
0-2 grm. oxydase per 50 c.c.
1 grm. oxydase per 50 c.c.
Minutes.
Oxygen absorbed
(c.c.).
Minutes.
Oxygen absorbed
(c.c.).
5
1-8
5
1-0
10
2-3
10
1-7
15
3-0
15
2-2
20
4-1
20
2-5
30
5-9
30
3-1
It has been shown that Medicago -oxydase is a mixture
of calcium salts of organic mono- and poly-basic hydroxy-acids,
among which are glycollic, citric, malic and mesoxalic acids.
The catalytic action of these pure salts corresponds closely with
that of Medicago -oxydase, as is shown by the following
numbers obtained under the conditions described above.
0-2 grm. calcium oxalate per 50 c.c.
1 grm. calcium glycollate ]
0-05 grm. calcium malate ^ per 50 c.c.
0-05 grm. calcium mesoxalate J
Minutes.
Oxygen absorbed
(c.c.).
Minutes.
Oxygen absorbed
(c.c.)
5
1-8
5
2-3
10
2-9
10
3-4
20
4-5
15
4-1
30
5-7
20
4-8
30
5-9
As is well known, the oxidation of polyphenols, either alone
or, more markedly, in presence of manganese salts, is considerably
EXPERIMENTAL DATA OF ENZYME REACTIONS 223
increased by small quantities of alkali, so that especial care must
be taken as regards the neutrality of the oxydase and of the calcium
salts. In the above investigation they were absolutely neutral.
Since the salts of all the aliphatic hydroxy-acids appear to be
more or less active in this direction, oxydases of this group must
be of very frequent occurrence in the vegetable kingdom.
The indophenol reaction, which was first employed by
Ehrlich in 1885 and consists in the formation of indophenol
from a-naphthol and ^-phenylenediamine, has been used recently
by V e r n o n (Journ. of PhysioL, 1911, 42, 402) as the basis of
a quantitative method. With the help of his method, this author
has investigated quantitatively numerous oxidation phenomena
induced by tissues.
According to the concentration of the substrate (naphthol
and diamine), the amount of indophenol formed was found to be
proportional either to the square of the quantity of enzyme, or to
this quantity itself or to its square root.
PEROXYDASES
Presumably still more widespread and consequently of more
general action are those substances of the animal and vegetable
body which activate 'peroxides, including hydrogen peroxide,
i.e., transfer the peroxide-oxygen to other substances; these are
termed peroxydases.
The changes which the peroxydases themselves undergo,
during this transference of oxygen from the peroxides to sub-
stances like a-guaiaconic acid, are unknown, and it has often been
doubted whether the peroxydases should really be regarded as
catalysts and as enzymes. Our conception of the latter, in
particular, is so indefinite, that at the present time, when so little
is known concerning the exact chemical nature of the peroxydases,
discussion of this question is of little value.
Owing more especially to the work of J. Wolff and E. d e
S t o e c k 1 i n (C. R., 1911, 153, 139), substances such as potas-
sium ferrithiocyanate, K 3 Fe(CNS) 6 , are however known which
are not of organic origin and yet behave like the peroxydases. 1
1 It appears to be established that, in most biological oxidations which are
regarded as enzymic, the true oxidising agent is peroxydic in character.
This view was advanced almost simultaneously by C h o d a t and Bach
224 GENERAL CHEMISTRY OF THE ENZYMES
We shall proceed at once to a consideration of the numerical
relations due to C h o d a t and Bach and their pupils
characterising the action of these enzymes.
In the experiments which will first be described (Chem.
Ber., 1904, 37, 1342), definite quantities of peroxydase (from
horseradish), hydrogen peroxide and pyrogallol were mixed in
aqueous solution, . the purpurogallin formed in 24 hours being
collected on a tared filter, washed with 100 c.c. of water, and
dried at 110 until of constant weight. Pyrogallol is not appre-
ciably attacked by either peroxydase or hydrogen peroxide alone.
Three series of experiments were carried out :
A. With variation only of the amount of peroxydase.
B. With variation only of the amount of hydrogen peroxide.
C. With variation only of the amount of pyrogallol.
Temperature, 15-17. Volume of the mixture, 35 c.c.
A
HYDROGEN PEROXIDE, 0-10 GRM.; PYROGALLOL, 1 GRM.
Amount of peroxydase, in grm. :
0-01 0-02 0-03 0-04 0-05 0-06 0-07 0-08 0-09 0-10
Purpurogallin formed, in grm.:
0-021 0-042 0-066 0-083 0-102 0-123 0-145 0-166 0-167 0-162
B
PEROXYDASE, 0-10 GRM.; PYROGALLOL, 1 GRM.
Hvdrogen peroxide, in grm.:
0-01 0-02 0-03 0-04 0-05 0-06 0-07 0-08 0-09 0-10
Purpurogallin, in grm.:
0-205 0-42 0-60 0-78 0-99 0-121 0-142 0-168 0-168 0-165
Experiments A show very clearly that with a constant amount
(in excess) of hydrogen peroxide, the yields of purpurogallin are
exactly proportional to the quantities of peroxydase employed;
whilst, with varying amounts of hydrogen peroxide and a constant
quantity (in excess) of peroxydase, the amount of change is, as
experiments B show, proportional to the former.
An excess of peroxydase or of hydrogen peroxide is without
influence on the oxidising capacity of the system peroxydase-
hydrogen peroxide. From these results Bach and C h o d a t
(Chem. Ber., 1902, 35, 1275, 2487, 3943; 1903, 36, 600, 606, 1756) and by
K as tie and Loevenhart (Amer. Chem. Journ., 1901, 26, 539).
The application of these noteworthy theories is often hindered by the un-
certainty of their chemical foundations, so that the theoretical results need
not be repeated here.
EXPERIMENTAL DATA OF ENZYME REACTIONS 225
drew the conclusion that peroxydase and hydrogen peroxide take
part in the reaction always in constant relations. They arrived
thus at the hypothesis which had been previously advanced
by K a s 1 1 e and Loevenhart (Amer. Chem. Journ., 1901,
26, 593), namely, that the peroxydase forms with the hydrogen
peroxide a definite compound exhibiting more energetic oxidising
properties than the peroxide alone. The result obtained by the
two first-named investigators that excess of hydrogen peroxide
does not affect the change indicates that the peroxydase
is not used up during the course of the
oxidation.
c
PEROXYDASE, 0-10 GRM.; HYDROGEN PEROXIDE, 0-10 GRM.
Pyrogallol, in grm. 1-5 2 3 4
Purpurogallin, in grm. 0-205 0-203 0-208 0-202
From the results of experiments C it is seen that the concentra-
tion of the pyrogallol is without influence on the magnitude of
the change.
Since the formation of purpurogallin and the procedure
described above are not suitable for the determinination of the
time-law of peroxydase-action, Bach and C h o d a t (Chem.
Ber., 1904, 37, 2434) chose for this purpose the oxidation of
hydrogen iodide.
Five c.c. of a solution, g^^-normal with respect to acetic
acid and hydrogen peroxide, were added to 45 c.c. of a solution
containing ^^-equivalent of potassium iodide and varying
quantities of peroxydase. The iodine separated after a certain
time was estimated by titration with thiosulphate.
1-25
-EQUIVALENT PEROXYDASE *
luU.uUU
Minutes 1 2 4 6 8 10 12 20
c.c. thiosulphate. 1-3 2-4 4-4 6-1 7-1 7-9 8-1 8-4
PARALLEL EXPERIMENT WITHOUT PEROXYDASE
Minutes 2 4 6 8 10 12 20
c.c. thiosulphate 0-25 0-45 0-6 0-8 0-9 1-1 1-6
2-5.10~ 5 -EQUIVALENT PEROXYDASE
Minutes 1 2 4 6 8 10 20
c.c. thiosulphate 2-4 4-7 8-4 10-2 11-7 11-8 12-3
* The peroxydase-preparation was the same as irf the above experiments. It activated
exactly its own weight of hydrogen peroxide, or, according to Bach and C h o d a t ' s
mode of expression, it had the activating power 1.
226 GENERAL CHEMISTRY OF THE ENZYMES
5 . 10~~ 5 -EQUIVALENT PEROXYDASE
Minutes '. . . 1 2 46 8 10 20
c.c. thiosulphate 4-9 8-9 14-0 14-7 15-0 13-1 15-4
10.10~ 5 -EQUIVALENT PEROXYDASE
Minutes 1 2 4 6 8 10 20
c.c. thiosulphate 10-1 15-7 17-0 17 1 17-2 17-2 17-4
15.10~ 5 -EQUIVALENT PEROXYDASE
Minutes 1246 8 10 20
c.c. thiosulphate 15-0 18-6 19-1 19-1 19-2 19-2 19-4
It will be seen first of all that, in the oxidation of hydriodic
acid just as in that of pyrogallol, the peroxydase ultimately loses
its activity, since after some time the procedure of the reaction
becomes exactly that exhibited when no peroxydase is added;
the rate at which the activity of the peroxydase diminishes
increases with the concentration of the peroxide. Hence the
results obtained with different concentrations of peroxydase
are comparable only for those phases of the reaction where the
enzyme still exerts almost its full activity
This is still the case at the end of the first minute, after which
time the magnitudes of the change are exactly
within the limits of experimental error-
proportional to the amounts of peroxydase:
Peroxydase-concentration X 10 5 2-5 5 10 15
Amount of change after 1 minute (c.c.) 2-4 4-9 10-1 15-0
Comparison of the final states reached in the system
hydriodic acid-peroxydase-hydrogen peroxide shows that the
amount of the product of the reaction (iodine) is not, as with the
oxidation of pyrogallol, directly proportional to the quantity
of peroxydase, but increases more slowly than the latter. It
/y
is to be noted that the activating power of the peroxydase, -
a
(where x denotes the amount of hydrogen peroxide
activated by a weight a of peroxydase), is considerably greater
in the oxidation of hydriodic acid by hydrogen peroxide than in
the oxidation of pyrogallol.
These experiments with hydriodic acid were extended by
Bach (Chem. Ber., 1904, 37, 3785), who determined the
EXPERIMENTAL DATA OF ENZYME REACTIONS 227
separation of iodine occurring in 10 minutes at 22 in differently
concentrated mixtures of HI, H202 and horseradish-peroxydase ;
his results are collected in the following table :
Increase of the change measured in c.c. of 0-01 N-thiosulphate.
Peroxy-
dase.
I.
12-5HI.
II.
25HI.
ill.
37-5HI.
IV.
50HI.
V.
75 HI.
VI.
100HI.
A
1-25
3-1
4-4
5-0
5-4
5-3
5-4
B
2-5
5-1
7-1
7-9
8-5
8-6
8-5
C
5-0
7-0
9-5
11-3
13-2
13-6
13-6
D
10-0
9-3
12-9
15-7
17*9
21-4
25-1
E
15-0
10-9
15-9
18-5
21-6
26-0
29-3
F
21-0
10-7
17-6
21-4
25-5
29-1
33-6
G
25-0
10-8
17-8
24-0
28-4
32-6
37-1
Control
0-7
1-4
2-0
2-7
4-1
5-4
The increase in the amount of action in a given time rises
both with the concentration of the peroxydase and with that of the
hydriodic acid and, for each concentration of enzyme and acid,
attains a certain limiting value and then remains constant; it
must therefore be concluded that peroxydase, hydriodic acid
and hydrogen peroxide react together in definite proportions.
It is readily seen from the preceding table that the pro-
duct of the concentrations is (within certain
limits) constant; for instance, DXl = CXlI, etc.
Finally, comparison of the concentrations of hydriodic acid
which correspond with different increments in the change shows
that these increments are almost exactly proportional to the
square-roots of the concentrations of the acid; this is evident
from the following summary:
I.
II.
III.
IV.
V.
VI.
Series D
9-2
12-9
15-7
17-9
21-4
25-1
Calculated
9-2
13-0
15-9
18-6
22-4
25-9
Series E
10-9
15-9
18-5
21-6
26-1
29 3
Calculated
10-9
15-3
18-8
21-8
26-5
30-7
Bach did not, however, obtain such regularity with another
preparation, this giving a different relation, which had not pre-
viously been observed.
228
GENERAL CHEMISTRY OF THE ENZYMES
Increase of the change measured in c.c. 0-01 N-thiosulphate.
Peroxy-
dase.
12-5HI.
II.
25HI.
ill.
37-5HI.
IV.
50HI.
v.
75HI.
VI.
loom.
A
1-25
1-2
2-4
3-3
4-2
4-2
4-1
B
2-50
2-2
4-2
6-1
8-1
8-3
8-2
C
5-0
3-6
6-0
9-4
.12-1
15-2
15-8
D
10-0
4-4
8-3
12-2
14-6
20-7
25-9
E
15-0
5-0
9-6
13-8
18-5
27-4
36-6
F
20-0
5-0
10-1
15-1
20-1
30-2'
41-0
G
25-0
5-1
10-2
15-6
20-4
30-0
40-8
Control
0-7
1-6
2-2
2-9
4-1
5-6
The results of series F and G show that, after the p e r -
oxydase-maximum is reached, the increase
in the amount of t r a n s f o r m a t i o n i s e x a c 1 1 y
proportional to the concentration of the
hydriodic acid.
For the complete utilisation of peroxydase, a definite acidity
(concentration of the hydrogen-ions) of the liquid is necessary,
the nature of the anion of the acid being without influence.
The darkening of many plant-juices owing to enzymic oxida-
tion was found by G r a f e and Weevers to be conditioned
by the presence of catechol and this discovery has recently been
confirmed by Miss Wh el dale (Proc. Roy. Soc., B, 1911, 84,
122). The study of the oxidation of catechol by plant-juices from
a kinetic standpoint would be of great interest.
TYROSINASE
In conjunction with an investigation on melanotic pigments
and the enzyme formation of melanins, 1 O. von Ftirth and
E. Jerusalem (Hofm. Beitr., 1907, 10, 131) studied the
mode of action of tyrosinase. The very complicated relations
found by these authors led Bach (Chem. Ber., 1908, 41, 216,
221), who regarded tyrosinase as a mixture of an oxygenase and
a peroxydase, to establish the conditions of action of this enzyme.
The amounts of melanin formed were determined as follows: The
juice was diluted tenfold with distilled water and 10 c.c. of this solution
1 A step towards the elucidation of the chemistry of tyrosinase action
has been made in a valuable investigation by Abderhalden and
Guggenheim (H., 1907, 54, 331).
EXPERIMENTAL DATA OF ENZYME REACTIONS 229
mixed with 10 c.c. of tyrosine solution (containing 0-05% of tyrosine
and 0-04% of sodium carbonate) and 30 c.c. of water; after 24 hours,
the solution was acidified with 1 c.c. of 10% sulphuric acid and titrated
with 0-002-normal permanganate until the latter was decolorised.
The tyrosinase was extracted from Russula delica.
1. Dependence of melanin-formation on the concentration
of tyrosinase.
Into each of a series of eight beakers were placed 10 c.c. of the
tyrosine solution and a certain quantity of the enzyme solution,
the volume being then made up to 50 c.c. with water.
Permanganate solution used, in c.c.
I.
II.
.III.
IV.
V.
VI.
VII.
VIII.
Volume of enzyme solution
(c.c.)
0-5
1-0
1-5
2-0
5-0
10-0
15-0
20-0
A After 24 hours
10-8
14-2
17-3
19-8
25-8
30-4
33-6
35-8
B. After 48 hours
13-2
16-0
17-8
20-4
25-6
31-2
34-4
35-4
These results show that : (1) the amount of the product of
the reaction increases with that of the enzyme, although more
slowly than the latter, and (2) the rapidity with which the reaction
comes to a standstill increases with the concentration of the
enzyme. In these respects, tyrosinase behaves similarly to horse-
radish-peroxydase (see p. 227).
2. Velocity of reaction and concentration of enzyme: Three
750 c.c. Erlenmeyer flasks were each charged with 100 c.c. of
tyrosine solution, a certain volume of enzyme solution, and water
up to a volume of 500 c.c.
Quantity
Time in 1
of enzyme solution per 500 c.c. .
lours : 1
10 c.c.
c.c.
0-0
20 c.c.
c.c.
1-4
30 c.c.
c.c.
2-8
2
0-0
3-9
5-7
3
1-6
5-8
8-8
4
2-7
7-8
11-1
6
5-5
11-1
16-1
* '
9
9-4
16-3
20-8
14
15-9
19-0
22-3
24
16-0
19-9
2-8
Volume of permanganate solution
required.
230
GENERAL CHEMISTRY OF THE ENZYMES
A reaction constant can scarcely be calculated with any
degree of certainty, since the position of the end-value is doubtful.
But if the times corresponding with equal amounts of change are
compared, inverse proportionality between the amount of enzyme
and the time of reaction is clearly evident: the product of the
quantity of enzyme and the time is hence constant. That the
initial and final stages of the reaction must be disregarded is
explained by the slower initiation of the reaction with low than
with higher concentrations of enzyme, whilst these higher con-
centrations lead to more rapid inactivation of the enzyme.
3. Velocity of reaction and concentration of substrate:
Three flasks, containing 25, 50 or 75 c.c. of the tyrosine and
30 c.c. of enzyme solution diluted to 500 c.c.
Volume of permanganate
solution required.
Volume of tyrosine solution, in c.c
After 1 hour
25
1-0
3-1
4-8
7-0
8-4
50
1-9
6-3
9-4
12-4
12-6
75
3-0
9-2
13-9
14-7
16-2
1 ' 3 hours
" 5 "
" 8 "
11 24 "
With a constant concentration of enzyme, the quantity of
melanin formed per unit of time is apart from the final stages
of the reaction proportional to the amount of tyrosine present.
Tyrosinase hence corresponds well with the law of mass action.
OXIDATION OF XANTHINE
In conclusion, it may be pointed out that B u r i a n (H.,
1905, 43, 497) has investigated dynamically the oxidation of
xanthine to uric acid. This reaction is, however, not one of pure
oxidation, so that mention of this paper must suffice.
CHAPTER V
INFLUENCE OF TEMPERATURE AND RADIATION ON
ENZYMIC REACTIONS
TEMPERATURE influences chemical systems in two ways:
It is, first of all, one of the factors which determine the posi-
tion of equilibrium between the substances taking part in a rever-
sible reaction. The degree to which the equilibrium changes
with the temperature in any case is closely related to the heat-
change of the reaction. If the equilibrium constant of a reaction
is indicated, as before, by K, while U denotes the total heat-
change determined calorimetrically, T the absolute temperature
and 72 the gas-constant, van't Hoff's fundamental thermo-
chemical law states that :
U
dT ' R.T 2 '
If, therefore, the heat-change accompanying any reaction is
small and this is the case with most enzymic processes t h e
equilibrium is only slightly dependent on
the temperature.
A much greater alteration with temperature is shown by
the velocity with which a system proceeds towards its
equilibrium or final position. In most cases, a rise of temperature
of 10 doubles or trebles this velocity a phenomenon to which
van't Hoff first directed attention. With non-enzymic reac-
tions, indeed, still higher temperature-coefficients are observed.
From the abundant experimental data, the following figures,
referring to reactions of biological interest, may be quoted:
Author. Reaction. Jemp^ *?10 ^
Price, Svenska Vet. Akad. Forh, 1899 . . Ethyl acetate +H 2 O 28-50 2 4 17,390
E u 1 e r , Chem. Ber., 1905, 38, 2551 ..... Formaldehyde +NaOH 50-85 3 6 24,900
S p o h r , Z. physikal. Chem., 1888, 2, 194 . Inversion of cane-sugar 25-55 3-6 25,600
231
232 GENERAL CHEMISTRY OF THE ENZYMES
k
The values of the quotient, ^ 10 , hold only for a certain
hi
interval of temperature, the increase of the velocity of reaction
per degree diminishing with rise of temperature. On the other
hand, the constant [L retains its value over a very wide range of
temperature. This constant is given by the formula derived
theoretically byArrhenius and found to be generally valid :
/* (T,-TA
k 2 = k l eR\ T lTz ) t (23);
it represents therefore an exact expression for the dependence
of the Velocity of reaction on the temperature. In this equation,
ki and 2 denote the reaction-constants at the absolute tem-
peratures TI and T2, while R is the constant of the gas-laws and
e the base of the natural system of logarithms.
An influence as great as on the velocity of chemical reactions
is exerted by the temperature on the vapour pressure of liquids
and on the equilibria of certain dissociations. In the latter
case this is explainable on the assumption that organic reactions
are also effected by ions. The constant [L gives the heat of
transformation accompanying the conversion of the participating
molecules from the " normal " into the " active " state, and
hence corresponds with the sum of the heats of dissociation of the
substances taking part. If, for instance, the inversion of cane-
sugar is considered, [i is the sum of the heat of dissociation of
water, t/idi ss . and that t/2diss.> f cane-sugar (the latter taken as
a base). The heat of dissociation of water is 13,450 Cals.; the
value of E/2diss. is still unknown, but it is known that with such
extremely weak electrolytes as cane-sugar must be, the heats of
dissociation are, in general, approximately of the same magnitude
as that of water. On this assumption for t/2diss.> ^ would be
26,900, while calculation of the experimental results according
to A r r h e n i u s 's formula leads to the value 25,600 (E u 1 e r ,
Zeitschr. f. physikal. Chem., 1904, 47, 353). The influence of
temperature on the compound, substrate-catalyst (i.e., the
hydrochloride, sulphate, etc., of the cane-sugar) is here apparently
neglected. But, since the dissociation of strong acids and of
salts changes comparatively slightly with the temperature,
this influence is determined principally by the heats of dissociation
of the cane-sugar and the water.
INFLUENCE OF TEMPERATURE AND RADIATION 233
On the velocity of enzyme reactions, temperature has a
twofold influence : that just referred to and, in addition,
an action on the activity of the catalysing enzyme, which becomes
more rapidly destroyed or permanently inactivated as the tem-
perature rises.
The processes resulting from these two actions were first
treated, theoretically and experimentally, by Tammann
(Zeitschr. f. physikal. Chem., 1895, 18, 426).
The simplest assumption is that the enzyme is inactivated
in aqueous solution by a unimolecular reaction, independently
of whatever else is in the solution. So that, if E is the initial
concentration of the enzyme and y the concentration at time t,
then:
hence
E-y = E.e~ k E, . . , .... (24)
where e is the base of the natural system of logarithms, and
If further, a denotes as usual the original concentration of
the substrate, which undergoes decomposition according to
an equation of the first order, it follows that
v = ~ = k(a-x)(E-y), ...... (26)
that is, the velocity with which the reaction proceeds at time t
must be equal to the product of the amounts of enzyme and sub-
strate still present.
Substitution in the last equation of the value of E y from
Eq. (24) gives, as Tammann showed, the integral equation:
(27)
234
GENERAL CHEMISTRY OF THE ENZYMES
From Eq. (27), T a m m a n n calculated the "false equi-
librium," that is, the final state of the enzymic reaction, and
compared the result with that determined experimentally with the
system emulsin-salicin. The following tables are taken from
those given by Tammann.
To solutions of salicin, previously heated, were added varying quan-
tities of emulsin, 100 c.c. of each of the mixtures containing 3 -007 grms.
of salicin and the amount of enzyme shown in the table.
PERCENTAGE OF SALICIN HYDROLYSED
Emulsin (grin.). 72 hours. 104 hours. 148 hours.
0-250 63-4 65-1 65-4 }
0-125 48-3 50-2 50-4 \ Temp., 65
0-0312 16-4 17-0 16-8 J
Emulsin (grm.).
0-250
0-125
0-0156
45 hours.
(101-2)
97-5
59-3
86 hours.
99-2
97-5
65-7
166 hours.
(100-2)
67-6
Temp., 45 c
Emulsin (grm.). 45 hours. 93 hours. 334 hours.
0-250 96-5 98-0 100.2 )
0-125 96-5 97-5 99-6 [ Temp., 26
0-0156 85-8 92-1 98-0 J
As will be seen, the higher the temperature, the sooner the
reaction comes to a standstill, i.e., the more rapidly the enzyme
is inactivated.
It must particularly be pointed out that a distinction is to
be made between the permanent non-reversible inac-
tivation which every enzyme undergoes, especially at higher tem-
peratures, and the inactivation due to the products of reaction
and disappearing when these are removed.
In the latter case, where the catalysing enzyme is held by the
reaction-products, the reaction retards itself by consuming its
own catalyst. Such catalytic retardation has been treated by
O s t w a 1 d (Lehrbuch, II, 2, 271).
The differential equation for this case,
INFLUENCE OF TEMPERATURE AND RADIATION 235
gives, on integration,
ki(Ak 2 k
As O s t w a 1 d pointed out, k 2 x at the beginning of the
reaction must not be greater than fci, as otherwise the reaction
does not take place. The reaction therefore leads here to a
" false equilibrium " or end-state.
The influence of rise of temperature on an enzymic reaction
is hence two-fold: 1, Acceleration of the reaction and, 2,
inactivation of the enzyme. So that both k and ks increase
as the temperature rises, and when the influence
of temperature on an enzymic reaction is
to be defined, the temperature-coefficient
should be give n a s was done by Tammann
b oth for k and for k E- The temperature-function of
the two magnitudes is best expressed by Arrhenius's
formula.
Owing to the dependence of the stability of the catalyst on
the temperature, the temperature-curves of enzymic reactions
differ in appearance from those of most other chemical processes.
Thus, they show an optimum, the temperature-coefficient at a
certain temperature being zero, owing to the increased velocity
of decomposition of the substrate being exactly compensated
by the increased rate of destruction of the enzyme; further rise
of temperature is then accompanied by decrease of the velocity
of reaction.
It was assumed on p. 233 that the decomposition
of enzymes obeys the formula for unimolecular reactions. 1
This is best ascertained by keeping the enzyme for a certain time
in aqueous solution at the temperature to be investigated, then
mixing it where possible at a lower temperature with the
substrate and calculating the constant k E from the initial velocity.
The values of k E obtained at different temperatures then give
the constant y. of the temperature-formula of Arrhenius
given on p. 232,
1 Whether all enzymes decompose according to this law is still question-
able. According to S e n t e r it is not the case with catalase.
236 GENERAL CHEMISTRY OF THE ENZYMES
Spontaneous decomposition of: ji
Emulsin in 0-5% solution 45,000 T a m m a n n
Rennet in 2% solution l 90,000 ]
Trypsin in 2% solution 62,034 V M a d s e n and W a 1 b u m *
Pepsin in 2% solution 2 75,600 J
Yeast-invertase 72,000 E u 1 e r and a f U g g 1 a s
Dry emulsin 26,300 T a m m a n n
For yeast-invertase, E u 1 e r and K u 1 1 b e r g (H., 1911,
71, 134) have shown that (JL is independent of the impurities
arising from the yeast and hence represents a well-defined constant.
Mention must also be made of the measurements made by
N i c 1 o u x with lipase (Soc. Biol., 1904, 56, 701, 839, 868), the
value obtained for [L being about 26,000.
S e n t e r (Zeitschr. f. physikal. Chem., 1903, 44, 257) states
that the destruction of blood-catalase at 55 is about 6-7 times
as rapid as at 45; [L is about 50,000.
Special emphasis must be laid on the high values of \L com-
pared with those of the corresponding constants for 'other reac-
tions, such as those given on p. 231. The result obtained with
dry emulsin shows the slight sensitiveness to heat of dry enzymes
relatively to that shown in the dissolved state. Similar observa-
tions of a qualitative character have often been made with other
enzymes. The numerical values obtained under these conditions
for [L are, as is easily understood, dependent in a high degree on
1 Merely by shaking for a few minutes, even at room-temperature, enzyme
solutions are rapidly inactivated. This action was observed almost simul-
taneously by Signe and Sigval Schmidt-Nielsen with rennet,
Abderhalden and Guggenheim with tyrosinase, and S h a k 1 e e
and M e 1 1 z e r with pepsin.
As was formerly assumed by the author, inactivation by shaking or
denaturation and skin-formation are due partly to a surface-action and partly
to the influence of atmospheric oxygen; with many colloidal solutions, shaking
in the air produces flocculation (S. and S. Schmidt-Nielsen, H.,
1910, 68, 317).
"According to Shaklee (Zentralbl. f. Physiol., 1909, 23, 4), at 37
pepsin loses its activity according to the formula for bimolecular reactions
at a x
where a is the original amount of pepsin and x the amount changed (destroyed)
in time t. After 12 days, 86% of the enzyme is destroyed.
3 Calculated by Arrhenius (Immunochemistry, p. 98).
INFLUENCE OF TEMPERATURE AND RADIATION 237
the previous treatment and especially on the moisture-content
of the enzyme-preparations, and they have a real significance
only if the compositions of the preparations are suitably denned.
The velocity of decomposition of dissolved enzymes and
its temperature-coefficient are also largely dependent on the other
substances present in the solution. Small proportions of acids or
bases often influence the stability enormously; thus, for example,
bases occasion a very considerable acceleration of the destruction
of rennet and of trypsin (Arrhenius, Immunochemistry,
p. 88).
A very complete study of the influence of acids and alkalies
on the destruction of invertase has been made by Hudson
and Paine (Journ. Amer. Chem. Soc., 1910, 32, 985), from
whose paper the following figure (p. 238) is taken.
An extract from Table 2 of Hudson and P a i n e ' s
paper shows that at 0-45, the temperature-coefficient of the
destruction by acid or alkali does not differ from that of ordi-
nary reactions. This is very remarkable, for, at the optimal sta-
bility (H'= about 10~ 5 ), the temperature-coefficient of enzyme-
destruction is, at any rate between 55 and 65, extremely high.
It recalls the temperature-coefficients of the denaturation of pro-
teins recently measured by Martin, and it is to be supposed
that with the enzymes it is a case of similar denaturation, i.e.,
of a change of their state of solution. In this connection, an
ultramicroscopical investigation would be of interest.
In many cases, salts produced a marked increase in the stability
of enzymes; thus, according to V e r n o n (Journ. of Physiol.,*
1901, 27, 174), the optimum of pancreas-diastase in a starch
solution containing 2% of sodium chloride is at 50, whilst in
pure aqueous starch solution the optimum temperature is 35.
Apart from this, it is known that many neutral substances,
particularly proteins l and other colloids (Journ. of Physiol., 1904,
31, 346), but more especially the specific substrates and reaction-
products, increase the stability of the enzymes to a marked extent.
This latter fact was pointed out by O ' Sullivan and T o m p -
son (loc. cit.). Biernacki (Zeitschr. f. BioL, 1891,
28, 49) and Vernon (Journ. of Physiol., 1901,27, 269; 1902,
28, 375, 448; 1904, 31, 346) made the same observation in the
1 Non-specifically hydrolysable proteins often act as "buffers."
238
GENERAL CHEMISTRY OF THE -ENZYMES
case of trypsin, 1 and W o h 1 and G 1 i m m (Biochem. Z., 1910,
27, 365) in that of amylase. In other instances, for example,
according to the author's measurements, with invertase, the
protective action of the substrate is slight. The stability of
this enzyme is, however, considerably increased by the presence
.10
.08 .06 .04 .02
Concentration of Acid-<
.02 .04 .06 .08
-> Concentration of Alkali
.10
FIG. 7.
of fructose (Hudson
Soc., 1911, 32, 988).
and Paine, Journ. Amer. Chem.
1 In this connection mention must be made of the observations of W .
Cramer and Beam (Proceedings of the Physiol. Soc., June 2, 1906;
see Journ. of Physiol., 1906, 34, XXXVI), according to whom active pepsin
is retarded by the addition of pepsin solutions inactivated at 60, whereas
preparations inactivated at 100 produce little or no retardation.
INFLUENCE OF TEMPERATURE AND RADIATION 239
In dilute solution diastase keeps better than in more concen-
trated ones (E f f r o n t , Enzymes and their Applications,
1902, p. 56).
The t emper atur e- coef f i c i ent s of the enzy-
mic reactions themselves, that is, the alterations
of the velocity-constants k with the temperature are of the same
order of magnitude as those of other chemical processes. An
attempt should always be made to measure the temperature-
coefficient in a region of temperature where the destruction of
the enzyme comes into consideration as little as possible. In
most of the previous measurements, the distance from the op-
timum is so slight that the constants [i are influenced by the
destruction of the enzyme and are consequently too low. Fur-
ther, as the following examples show, the errors of observation
are generally very large, even in the work of reliable experimenters.
A u 1 d (Journ. Chem. Soc., 1908, 93, 1275) : measurements
on amygdalin-emulsin.
kv, :/Ci5 = 2-37
/c 3 o :fc 20 = l-81
35 : & 25 =2-14
fc 40 :/c 30 =l-68
45
&50
K a s 1 1 e and Loevenhart (Amer. Chem. Journ., ] 900>
24, 501) left tubes containing 4 c.c. of water, 0-1 c.c. of toluene
and 1 c.c. of a 10% liver- or pancreas-extract for 5 minutes in
baths at 40, 30, 20, 10, and - 10, so that they assumed
these temperatures. Ethyl butyrate (0-26 c.c.) was then added
and the solutions titrated after 30 minutes.
Percentage hydrolysed.
By liver-extract.
By pancreas-extract.
40
ll-29(?)
2-82
30
5-96
3-16
20
5-27
2-51
10
3-89
1-88
2-26
1-25
-10
0-70
240 GENERAL CHEMISTRY OF THE ENZYMES
The value obtained with liver-extract at 40 must be due to
an error of experiment. H a n r i o t (C. R., 1897, 124, 778)
obtained similar results with his esterases from serum and pancreas.
Reference must finally be made to a series of experiments
made by C h o d a t (Arch. Sci. phys. nat., 1907, 23, 13) on the
action of tyrosinase on tyrosine.
The second row below gives the times in which the solution
had attained a certain intensity of colour.
Temperature 10 20 30 40 45 50
Time (minutes) 180 100 60 40 30 20 10
Very unreliable quantitatively are the temperatures given by
Tammann (loc. cit.) for invertase and cane-sugar,
Lindner and Krober (Chem. Ber., 1895, 28, 1053) for
maltase, H a n r i o t and Camus (C. R., 1897, 124, 235)
for serum-esterase and monobutyrin, M i q u e 1 (see H e r -
z o g , 1 o c . c i t .) for urease, and by experiments made accord-
ing to M e 1 1 ' s method.
Consequently reference will only be made to the calculation
of these results by H e r z o g (Zeitschr. f. allg. Physiol., 1904,
4, 189).
In the following table (p. 241) are collected the data as yet
obtained concerning the temperature-coefficients of enzyme-
reactions. 1
In other cases the value of [JL changes considerably with
temperature; this is shown, for instance, byMuller-Thur-
gau's results with amylase (Landw. Jahrb., 18.85, 795).
An extended investigation of the temperature coefficients
of alcoholic fermentation by living yeast has been made by
S 1 a t o r , with the following results :
t vt+io : v t
5 5-6
10 3-8
15 2-8
20 2-25
25 1-95
30 1-60
1 Complicated biological processes, such as the assimilation of carbon by
green leaves, are not considered here.
INFLUENCE OF TEMPERATURE AND RADIATION 241
Author.
Substrate and enzyme.
Temp.-
interval.
kt+io
k t
(A
K a s 1 1 e and Loevenhart
(1 o c . c i t .)
Ethyl butyrate, esterase
20-30
1-3
4,650
Tammann (loc. cit.)
K j e 1 d a h 1 (Medd. fra Carlsberg
Lab 1881 335)
Cane-sugar, invertase
Cane-sugar, invertase
20-30
30-40
1-4
1-5
6,000
7;800
O ' S u 1 1 i v a n and T o m p s o n
(loc cit)
Cane-sugar, invertase
40-50
1-4
6,800
S e n t e r (Zeitschr. f. physikal.
Chem., 1903, 44, 257)
Chodat (loc cit )
HzOa, catalase
0-10
20-30
1-5
1-5
6,200
7 200
E u 1 e r and af Ugglas (loc.
cit)
0-20
2-0
11,000
Vernon (Journ. of Physiol.,
1901, 27, 190)
Starch, amylase
20-30
2-0
12,300
Vernon ( ibid. )
Vernon (ibid. 1903, 30, 364). . .
Tammann (loc. cit.)
Vernon (Journ. of Physiol.,
1903, 30, 364)
Taylor (Journ. of Biol. Chem.,
1906, 2 87)
Milk, rennet
Witte's peptone, trypsin
Salicin, emulsin
Witte's peptone, erepsin
Triacetin, lipase
20-30
15-25
15-25
15-25
18-28
2-1
2-3
2-4
2-6
2-6
13,400
14,300
15,000
16,400
16 700
F u 1 d (Hofm. Beitr., 1908, 2, 169)
B a y 1 i s s (Arch. Sci. Biol., St.
Petersburg, 1904, 11, 261, Sup-
plement)
Milk, rennet
Casein, trypsin
30-40
20-7-30-7
3-2
5-3
22,000
37 500
Heterogeneous systems
A b e r s o n (Rec. Trav. Chim.
Pays-Bas, 1903, 22, 100)
Sugar, living yeast
Sugar, permanent yeast
18-28
15-25
2-7
2-8
15,600
18,000
Herzog (H., 1902,37, 160)..
...
In consequence of the foregoing data it must again be
emphasised that the influence of temperature on enzymic reac-
tions is exactly denned only by determination of the inactivation
constant k E at a given temperature and measurement of the
initial velocities of the reaction itself at temperatures at least
20 lower than the above.
The following very instructive numbers, obtained by G e r -
ber (Soc. Biol., 1903, 63, 375), show how the temperature-
coefficient of an enzymic reaction may depend on the quantity
of enzyme present:
242
GENERAL CHEMISTRY OF THE ENZYMES
Concen-
Time of coagulation of milk at different temperatures.
rennet.
25
30
33
36
39
42
45
0-005
0-010
30' 20"
14 45
29' 00"
11 30
( no coag.
\ in 360'
r oo"
1 no coag.
in 360'
(no coag.
in 360'
No
No
0-015
9 40
7 25
4 40
5' 35"
(
coag.
coag.
0-020
7 30
5 00
2 30
3 15
5' 30"
0-025
6 15
3 30
2 05
2 20
2 40
in
0-030
4 40
2 50
1 40
1 30
1 40
360'
in
0-040
3 40
2 20
1 30
1 00
50
0-050
3 00
1 50
1 10
55
40
2' 05"
360'
0-075
2 20
1 20
50
40
30
1 00
0-100
1 40
1 00
40
30
25
35
0' 45"
Very striking is the result of a comparison between the values
of [L for the acceleration of the inversion of cane-sugar by an acid
and by invertase respectively; the data on pp. 231 and 241
give:
Cane-sugar-hydrochloric acid. Cane-sugar-invertase.
H = 25,600 [1 = 11,000
Although enzymic temperature-coefficients had been deter-
mined by very reliable experimenters (Kjeldahl, T a m -
m a n n , O ' Sullivan and Tompson), new investiga-
tions on this point were to be desired. On this account, the
author and Miss B. af Ugglas have made fresh determina-
tions of these coefficients under various experimental conditions
(H., 1910, 65, 124); at the optimum concentration of hydrogen-
ions, it was found that /CSQ : 20 = 1-9 -2-1. This is in agree-
ment with the result obtained by V i s s e r (Zeitschr. f. physikal.
Chem., 1905, 52, 257), namely 2, for the ratio k 2 o : kio.
The first conclusion that could be drawn is that the con-
stant [L includes the heat-change occurring during the forma-
tion of the compound between the cane-sugar and the acid or
invertase. It is also possible that the small rise of temperature
taking place during enzymic inversion may be due to rise of tem-
perature not only irreversibly destroying the invertase but also
reversibly inactivating it; apart from the destroyed portion,
the invertase resumes its original activity at the lower temperature.
Hence, rise of temperature renders the substrates, cane-sugar
INFLUENCE OF TEMPERATURE AND RADIATION 243
and water, more active and the catalysing enzyme less active.
Other reactions, for instance, the hydrolysis of esters, also exhibit
smaller temperature-coefficients for enzymic than for acid catal-
ysis. For non-enzymic reactions, S 1 a t o r (Zeitschr. f .
physikal. Chem., 1903, 45, 547) found temperature-coefficients
varying with the catalyst.
Very ill-defined is the so-called " optimum temperature,"
the position of which depends entirely on the period or phase
of the reaction considered. Indeed, even at the optimal tem-
perature the enzyme undergoes partial destruction during the
reaction, so that if comparison is made of the times taken for the
reaction to proceed to the extent of one-half, the optimum is
apparently lower than if only the first one-fifth of the reaction
is considered. The real initial velocity will, in general, show no
optimum if the time of observation is made short enough. For
practical purposes it may be of interest to know the temperature
at which the reaction proceeds most rapidly, and it would then
be best to consider the times in which, say, 90-95% of the sub-
strate is decomposed. In any case, in giving the optimum tem-
perature, it must be stated for which stage of the reaction it
holds.
Still more indefinite are most of the data on "maximum
temperatures" (temperatures of destruction). Measure-
ments of these temperatures are of value only when the duration
of the experiment and the magnitude of the weakening are deter-
mined. It is therefore advisable to give that temperature at
which the enzyme is weakened, for example, to the extent of one-
half in 30 minutes; still better is it to measure the inactivation
constant ks of the dissolved enzyme at a given temperature.
The presence and concentration of other substances in the
solution may influence both the optimal and the maximal tem-
peratures very considerably; marked alterations of these tem-
peratures are produced especially by acids and bases, so that in
measurements of the stability it is necessary to define the con-
centration of the H'- or OH'-ions. Non-electrolytes so long
as they do not constitute the substrate or a product of the reaction
appear to have little influence, the stability-constant being,
therefore, readily reproducible (cf. the measurements of B. a f
U g g 1 a s and Kullberg, loc. cit.).
The very great differences existing, according toR.Huerre
244 GENERAL CHEMISTRY OF THE ENZYMES
(C. R., 1909, 148, 300), between the maltase of white maize
and that of yellow maize, may be due to the action of a foreign
substance of some kind; but it can, by no means, be denied that
the two sorts of maize may contain different maltases.
For most enzymes the optimal temperature is stated to be
between 37 and 53, and the maximal temperature between 60
and 75.
Many oxydases resist surprisingly high degrees of tem-
perature. That known as Medicago -laccase is especially
stable to heat, as is to be expected from its composition (see p. 62).
Other oxydases are destroyed only at 80-90 (K a s 1 1 e , Chem.
ZentralbL, 1906, 77, i, 1554).
Also peroxydases, at any rate in the natural juices, are only
slightly injured by heating. Thus, a preliminary experiment
with the juice from pressed horseradish showed that heating
for two hours at 60 diminished the activity of the peroxydase
on guaiaconic acid only in the proportion of 7 : 5. For ^ the
very low value, 4000, was obtained.
Apparently still more resistant is myrosin, which, according
to Guignard's experiments (C. R., 1890, 111, 249, 920;
Bull. Soc. Bot. de France, 1894, [3], 418), is not destroyed even
at 81, although a knowledge of the duration of the heating is to
be desired.
Peculiar behaviour towards high temperatures has been
observed byDelezenne, Mouton and P o z e r s k i
(Soc. Biol., 1906, 60, 68, 390) in the case of papain. At tem-
peratures up to 40, papain exerts no digestive action on egg-
albumin or blood-serum. Digestion only occurs, and then very
rapidly, on further heating of the solution. These results were
completely confirmed byJonescu (Biochem. Z., 1906, 2, 177),
who studied the differences between ordinary and " heat-
digestion," while Gerber (Soc. Biol., 1909, 66, 227) has
also remarked the notable resistance of papain to high
temperatures.
Towards low temperatures, enzymes appear to be highly
resistant; thus, Miss White showed that the enzymes of
cereals are not destroyed by exposure to the temperature of
liquid air for two days (Proc. Roy. Soc., ., 1909, 81, 440).
To sum up, the sensitiveness of enzymes to heat is, indeed,
very great, but is not so marked as with the toxines, for which
INFLUENCE OF TEMPERATURE AND RADIATION 245
M a d s e n found the temperature-constants of spontaneous
decomposition in solution to be as high as [L = 198,500.
Enzymic reactions appear to have rather lower temperature-
coefficients than the corresponding non-enzymic catalyses.
INFLUENCE OF RADIATION
The manifold action exerted by light on the processes occurring
in living cells and tissues has naturally given rise to the impres-
sion that enzymes are sensitive to rays of various wave-lengths.
The success of the modern photo-therapeutic methods of
F i n s e n and others, on the one hand, and the knowledge that
toxines undergo very rapid destruction in the light, on the other,
endow this subject with considerable practical and scientific
interest.
1. Light-rays
It may be mentioned firstly that, in general, enzymes do not
appear to exhibit so high a degree of sensitiveness to light as
do the toxines. This observation was, indeed, made some years
ago by O . E m m e r 1 i n g (Chem. Ber., 1901, 34, 3811).
Hertel subjected a number of enzymes and toxines to
the influence of light-rays and observed, among other results,
that trypsin and also diastase and rennet are weakened by rays
having the wave-length 280 ^. His investigations also showed
that the destruction of enzymes requires a longer exposure than
does that of the toxines, the enzymes being therefore decidedly
more photo-stable substances than the toxines (Biol. Zentralbl.,
1907, 27, 510).
Just as in the study of the thermo-lability of the enzymes,
so also in investigating the action of light, a distinction must
be drawn between the destruction of the enzyme by light and
the alteration of the enzymic action under the influence of the
radiation. In so far as the results already obtained indicate,
the former influence predominates, the observed retardations
of enzyme-action being therefore due principally to a partial
annihilation of the enzyme molecule.
In his paper cited above, E m m e r 1 i n g states that, in
absence of air, invertase, lactase, emulsin, amylase, trypsin 1
1 According to Fermi and P e r n o s s i (Zeitschr. f . Hygiene, 1894,
18, 83), pepsin and trypsin are weakened in sunlight.
246 GENERAL CHEMISTRY OF THE ENZYMES
and pepsin are injured but slightly by daylight; yeast maltase
and rennet are rather more sensitive to these conditions. The
activity of the last-named enzyme, in 1% aqueous solution,
was reduced to one-half by diffused sunlight, and to one-third
by direct sunlight, in the course of five days.
D o w n e s and Blunt found this action of light to be
of slight extent and the same result was obtained by F. W e i s
(Medd. fra Carlsberg Lab., 1903, 5, 135) with the proteolytic
enzyme of malt and by Schmidt-Nielsen (Medd. fra
Finsens med. Lysinstitut, 1903) with chymosin. On the other
hand, F. G . Kohl (Beitr. z. Bot. Zentralbl., 1908, 23, 64)
states that invertase is considerably affected even by diffused
daylight. The disagreement between this statement and the
earlier observations is explained by the results of J a m a d a and
Jodlbauer (Biochem. Z., 1908, 8, 61), who showed that the
rays of sunlight which pass through glass are alone capable of
injuring invertase, but to a marked extent only
in presence of oxygen.
With reference to the stability of catalase under the action
of light, a comprehensive investigation was carried out by
Lockemann, Thies and W i c h e r n (H., 1909, 58,
390). The inhibiting action of light on blood-catalase, both
when the blood-solution is kept and during the reaction with
hydrogen peroxide, is greatest with white and least with red light,
blue occupying an intermediate position. 1 Also in this case,
according to Z e 1 1 e r and Jodlbauer (Biochem. Z., 1908,
8, 84), appreciable injury is produced by visible rays only when
oxygen is present; a similar result was arrived at by Z e 1 1 e r
and Jodlbauer, and almost simultaneously by Bach
(Chem. Ber., 1908, 41, 225), with peroxydase.
The results of a large number of investigations are in agree-
ment in indicating that the inhibiting influence of ultra-
violet rays is much greater than that of the visible rays.
The action of these rays was investigated firstly and very com-
1 In absence of catalase, additions of sodium chloride retard the decom-
position of hydrogen peroxide by light. For the sensitiveness to light of
solutions of the peroxide either containing or free from catalase, W o .
O s t w a 1 d (Biochem. Z., 1908, 10, 1) found the influences of different
kinds of light to be in the following order of diminishing magnitude: white,
violet, yellow, dark.
INFLUENCE OF TEMPERATURE AND RADIATION 247
pletely by Reynolds Green (Trans. Roy. Soc., 1897,
188, 167), who showed that violet and ultra-violet rays destroy
diastase, but that the action of this enzyme_is enhanced by visible
rays owing to activation of the zymogen. As was mentioned
above, visible rays have only a slight retarding action on chymosin,
catalase and peroxydase, but these enzymes are rapidly and per-
manently inactivated by ultra-violet radiation (Schmidt-
Nielsen, Zeller and Jodlbauer). Signe and Sigval
Schmidt-Nielsen made a detailed, kinetic study of the
destruction of rennet by ultra-violet light (H., 1908, 58, 235),
whilst shortly beforehand Georges Dreyer and O 1 a v
H a n s s e n (C. R., 1907, 145, 564) showed that the destruc-
tion of enzymes by radiation follows the law for unimolecular
reactions.
Of Schmidt-Nielsen's experiments, which were
made in the Finsen Institute with a mercury-vapour lamp, the
following may be described :
A 1% solution of dry, commercial rennet powder was exposed
to the radiation for a definite period and the time required for
the coagulation of cow's milk subsequently measured; this
time was assumed to be a direct measure of the amount of unaltered
enzyme in the solution.
Temperature,
C.
Exposure to the light,
minutes.
Time of clotting,
minutes.
1000/t.
24-2
8-5
24-2
1-0
23-5
442
24-2
1-5
39-25
443
24-2
2-0
71-0
461
7-7
12-75
1-0
19-5
405
12-80
1-5
34-5
434
12-85
2-0
59-0
442
13-90
2-0
56-0
431
12-95
2-0
54-5
425
As was, indeed, to be expected, these numbers show that
the destruction of rennet by light is undoubtedly a unimolecular
reaction. But what deserves special attention is the extraor-
dinarily small temperature-coefficient of this reaction. That such
248 GENERAL CHEMISTRY OF THE ENZYMES
processes possess low temperature-coefficients has been repeatedly
observed and seems to be a general rule. It may be assumed
in the above case that the temperature of the chymosin-mole-
cules, after exposure to the light, is considerably higher than that
of the surrounding solution and is not very different in the two
series of experiments; the temperature of the solution had therefore
little influence on the thermal condition of the chymosin.
In Schmidt-Nielsen's experiments, the reaction
constant diminished with increase of the concentration. This
is not surprising, since the destruction by heat of enzymes proceeds
more slowly in their concentrated than in their dilute solutions.
Hence, as their concentration increases, enzymes become more
stable to both heat and light. Of the total effect of the radia-
tion of the mercury lamp, 96% is due to rays with the wave-
lengths 220-250 \L[L and only about 0-3% to the visible rays.
Very interesting experiments have been made byvonTap-
p e i n e r and his co-workers on the action of sunlight on dias-
tases and invertase in presence of fluorescent substances (sensi-
tisers). Very small quantities of eosin, Magdala red or quinoline
red are sufficient to cause sunlight which of itself is with action
to exert a marked inhibiting effect.
In diffuse daylight the fermentative power of yeast is destroyed
by fluorescent bodies. With living yeast, only certain fluorescent
substances are active; but with permanent acetone-yeast and,
still more, with yeast-juice, all the fluorescent bodies examined,
such as eosin, methylene blue, fluorescein, dichloranthracene-
disulphonic acid, etc., induce considerable diminution of the
fermentative power (von Tappeiner, Biochem. Z., 1908,
8, 47).
Also on catalase all the fluorescent substances investigated
have a sensitising action, whilst with peroxydase this is the case
only with eosin and Bengal red; in both these instances, the
action only occurs when the ultra-violet rays are, as far as possible,
lacking ( J a m a d a and Jodlbauer, Biochem. Z., 1908,
8, 61 ; Z e 1 1 e r and Jodlbauer, ibid., 84; Karamit-
s a s , Dissertation, Munich, 1907).
Thus the biological action of light is of two kinds (Jodl-
bauer and von Tappeiner, Deut. Arch. f. klin. Med.,
1906, 85, 386): One requiring the presence of oxygen and
accelerated by fluorescent substances, and the other produced
INFLUENCE OF TEMPERATURE AND RADIATION 249
only by ultra-violet rays without any part being played by
oxygen or fluorescent substances.
The results mentioned above show that light exerts actions
of two kinds on enzymes :
(1) A destroying action, corresponding with denaturation
by heat.
(2) An activating effect, due to conversion of the " zymogen "
into the active enzyme.
2. Other Forms of Radiation
By Rontgen rays, enzymes are not weakened. This
was shown by P. F. R i c h t e r and Gerhartz (Berl. klin.
Wochens., 1908, 45, 646) to be the case with chymosin, yeast,
pepsin, pancreatin and papain, while Lockemann, Thies
and W i c h e r n obtained the same result with blood-catalase.
Radium rays and radium emanation, how-
ever, do appear to exert an action on enzymes, although, accord-
ing to W i 1 c o c k (Journ. of Physiol., 1907, 34, 207), tyrosinase
is not affected by radium rays, while Schmidt-Nielsen
found that even a very active preparation of radium has a very
slight effect on chymosin; Henri and Mayer (C. R., 1904,
138, 521) state that invertase, emulsin and trypsin are injured.
Against these negative assertions are arrayed a number of other
positive results.
B e r g e 1 1 and B i c k e 1 (Verhandl. d. Kongr. f. inn. Med.,
Wiesbaden, 1906) first showed that peptic digestion is favoured
by the emanation. N e u b e r g (Verhandl. d. deutsch. path.
Ges., 1904) and Wohlgemuth (ibid) observed accelera-
tion of the autolytic processes by radium radiation, while
Loewenthal and E d e 1 s t e i n (Biochem. Z., 1908, 14,
484) found these processes to be facilitated by the emanation.
Also Loewenthal and Wohlgemuth (Biochem. Z.,
1909, 21, 476) have recently proved that radium emanation is
capable of accelerating the action of the diastatic enzyme of the
blood, liver, saliva or pancreas. " This favourable action is
not always observable immediately; very often retardation
occurs during the first 24 hours, this being gradually neutralised
and then replaced, if the experiment is sufficiently prolonged,
by an acceleration. In other cases, the emanation produced only
250 GENERAL CHEMISTRY OF THE ENZYMES
inhibition, which was not compensated when the duration of the
experiment was extended."
Acceleration of enzyme-action by the emanation has been
established with pepsin and trypsin.
Of importance from a therapeutic standpoint isGudzent's
discovery that the enzyme of purine-metabolism is activated
by radium emanation.
Action ofmesothorium.
The mesothorium bromide discovered by 0. H a h n emits
rays of three kinds: a-, @- and y-rays. Of these, the @- and y-
rays pass without alteration through a mica plate, if this is not
too thick; they are also able to traverse a thin sheet of glass, but
are then weakened to some extent. B i c k e 1 and M i n a m i
(Berl. klin. Wochens., 1911, 48, 1413) found that exposure of
carcinoma, sarcoma and liver to the radiation of mesothorium
bromide the action of emanation or a-rays being excluded
has no influence on the autolytic enzymes. These authors regard
their results as of fundamental importance. If it is a fact that the
P- and y-rays of mesothorium are identical in every respect with
the @- and y-rays of radium, it must be concluded that the activa-
tion of the autolytic enzymes observed as a result of the action
of radium is s o 1 e 1 y an effect of the a-rays or emanation. The
same is probably the case with other enzymes. According to
Minami (Berl. klin. Wochens., 1911, 48, 1798) the - and
y-rays of mesothorium exert a very slight influence on the
digestive enzymes, amylase, pepsin and trypsin. The biological
action of thorium emanation has been studied by B i c k e 1
(Berl. klin. Wochens., 1911, 48, 2107) ; like that of radium emana-
tion, it consists sometimes of a retardation and sometimes of an
activation of enzymic action, and is more intense than that of
g- and y-rays.
In general, it may be stated that the healing action found
by experience to be exerted by radium emanation depends on
the activation of enzymes. The promotion of plant-growth by
the emanation (F a 1 1 a and S c h w a r z , Berl. klin. Wochens.,
1911, 48) is also to be attributed to enzyme-activation.
CHAPTER VI
CHEMICAL STATICS IN ENZYME REACTIONS
THE position of equilibrium of a chemical system is deter-
mined, as is well known, by the law of mass action.
If 1 mol. of acetic acid reacts with 1 mol. of alcohol, so that
1 mol. of ester and 1 mol. of water are formed, then, according
to the law of mass action-
[ester]
[acid] [alcohol]
if the concentrations of the substances in dilute aqueous solution
are indicated by [ ] and K denotes the equilibrium constant.
According tovan't Hoff, chemical equilibrium of the
above reaction is due to the equality of the velocity v\ of ester-
formation and of the velocity V2 of ester-decomposition, so that,
v\ = &i[acid] [alcohol] = V2 = fetester],
and therefore
[ester] _T^_&I
[acid] [alcohol] ~ ~ k 2 '
The position of equilibrium is independent of the rapidity
with which it is reached and also as exact experiments show
independent of the presence and concentration of a catalyst,
in so far as this does not combine to an appreciable extent with
the components of the system.
As was mentioned in Chapter IV (p. 128), a number of
investigators have arrived at the conclusion that enzymic reactions
are effected by means of a compound of the enzyme and the
substrate.
With the non-enzymic hydrolyses which have been as yet
investigated and in which, according to the author's theory, the
251
252 GENERAL CHEMISTRY OF THE ENZYMES
increase of active molecules is due to the formation of a salt of
the substrate with the catalysing acid, the concentration of the
compound substrate-catalyst is so small that no great alteration
occurs in the concentration of the substrate or catalyst, either
during the course of the reaction or when equilibrium has been
attained.
Enzymic decompositions usually differ from those effected
by inorganic catalysts in that their velocity is determined not
only by the absolute concentration of the catalyst (enzyme)
but also, and to a far greater extent, by the concentration-r a t i o
between enzyme and substrate. If the substrate is in excess,
the velocity of reaction is approximately proportional to the
concentration of the enzyme, whilst if excess of the latter is present,
the velocity will be very nearly proportional to the concentration
of the substance; in every case, the velocity of reaction appears
to be proportional to the concentration of the " intermediate
product."
On the other hand, quantitative study of enzyme-reactions
has shown that the products of reaction are also fixed by the
enzyme (Henri, Bodenstein, and others).
The question now to be considered is the relations in the
case of enzymic reactions.
A distinction must here be made between true equilibria
and end-states.
A. Equilibria
The assumption of the existence of the molecules enzyme-
substrate and enzyme-reaction product which bring about the
reaction presumes that the mutual action between these mole-
cules proceeds far more rapidly than those between the free
substrate molecules and their decomposition products. It is,
hence, principally the concentrations of the molecules of the
enzyme-substrate and enzyme-reaction product which condi-
tion the end-state.
The simple assumption may first be made that, in miitMime,
equal numbers of enzyme-substrate and enzyme-reaction pro-
duct molecules take part in the reaction. The velocity constant
ki of the decomposition of the substrate is
CHEMICAL STATICS IN ENZYME REACTIONS 253
proportional to the concentration of the molecules of the enzyme-
substrate compound; or if
_ [enzyme-substrate]
[enzyme] [substrate] '
then
ki=Ki[enzyme] [substrate].
In a similar manner the velocity constant of the forma-
tion of the substrate is expressed by
2 = .^[enzyme] [reaction product] 2 ,
if, as is often the case, 2 mols. of reaction product are formed from
1 mol. of substrate.
According tovan't Hoff, the equilibrium is then given
by the quotients
KI [substrate]
_ _
~
^[reaction product] 2 '
As will be at once seen, the numerical value of this " enzymic "
end-state coincides with that of the natural stable equilibrium as
reached with an inorganic catalyst, only if K\=K%, hence only
in the case where the combinations enzyme-substrate and enzyme-
reaction product are exactly equal. No convincing cause has,
however, yet been suggested for such an assumption; on the
contrary, the results obtained by Henri with invertase bear
the interpretation that this enzyme is combined equally by
cane-sugar, glucose and fructose.
If the simple assumption, that equal numbers of the two
" active " molecules react per unit of time, is abandoned and it
is assumed that n% of enzyme-substrate and m% of enzyme-
reaction product molecules react in equal times, then
_g - Jil _ KI ^[substrate]
&2 J2?w [reaction product] 2 '
and K is identical with the constant of stable equilibrium if
Hence it will in general be expected that enzymes lead to an
end-state different from that given by inorganic catalysts and
254 GENERAL CHEMISTRY OF THE ENZYMES
the first question to be decided is the position of the natural
equilibrium. The natural equilibrium can be determined directly
in many cases, for instance, in the system fatty acid-alcohol-
ester-water. In order to accelerate the attainment of equilibrium,
a strong mineral acid may be employed, since the position of
equilibrium is not altered by such an ideal catalyst. In certain
other cases, an indication of the position of equilibrium may
be obtained from the heat-change of the reaction (van't Hoff,
Sitzungsber. K. Akad. Berlin, 1909, 42, 1065).
As was shown above, the heat-effects of enzymic processes
are mostly very small. For such changes, however, the equi-
librium is of a simple nature. " Optical antipodes which form
no racemic compound present the ideal example, and it has
been shown both theoretically and experimentally that, in the
solid state, the two antipodes are in equilibrium, whilst in the
vaporous, fused and dissolved conditions, they form an inactive
mixture of equal amounts. The relation between the equilib-
rium constant K, i.e., the quotient of the concentrations of the
two antipodes, and the work of transformation E may be expressed
thermodynamically (van't Hoff, Svenska Vet. Akad.
Handl., 1886) by
Hence in this case # = and K l.
What applies strictly to optical antipodes also holds approx-
imately with reactions of small heat-effect and the equilibrium
is not far removed from that corresponding with thermo-neutrality.
How far true enzymic equilibrium may differ from the
natural equilibrium cannot be stated exactly. It will depend
on the proportion of the components of the equilibrium which
combines with the enzyme or if it is assumed that separate
enzymes accelerate the reaction in the two directions two
enzymes to form complex compounds. Since the concentration
of the enzyme is usually very low, the concentrations of the mole-
cules of enzyme-substrate and enzyme-reaction product must also
be low, and the enzymic and natural equilibria will then differ
but little. In other words, if the corresponding enzyme
or mixture of enzymes is added to & system in equilibrium, the
CHEMICAL STATICS IN ENZYME REACTIONS 255
latter undergoes only slight change. This is required by thermo-
dynamics, which also shows that in the case where the catalyst
and the reacting substances do not (practically) unite, no change
in the equilibrium should be produced. Otherwise the equilib-
rium could be altered by alternate removal and introduction
of the catalyst and perpetual motion thus attained.
B. End-states and Stationary States
Starting with a system not in chemical equilibrium, the
natural equilibrium is not necessarily arrived at by addition of
an enzyme-preparation capable of acting on the system. An
end-state differing from the equilibrium will be attained.
1 . If two enzymes exist which catalyse the reaction in opposite
directions. The final state then depends on the relative quan-
tities of these two enzymes.
2. If two enzymes are present, one catalysing the formation
of a compound A of the components by the non-reversible reaction
Enzyme 1
B+C -* A,
and the other using up this compound A according to another
r.on-reversible reaction.
Enzyme 2
A - D+F.
This case may evidently lead to widely varying stationary
states, depending on the relative concentrations of the two
enzymes 1 and 2.
T a m m a n n (H., 1892, 16, 271) gave an account of the
experimental material obtained before 1892 and also of his own
investigations. From the results of the latter he deduced the
important and undoubtedly correct consequence, that the end-
states of enzymic reactions do not coincide with the positions of
stable equilibrium of the reactions.
Concerning the end-states attained under the influence of
lipases, two more recent papers have been published:
256 GENEBAL CHEMISTRY OF THE ENZYMES
Bodenstein and D i e t z (Zeitschr. f. Elektrochem.,
1906, 12, 605) have compared the equilibrium formed between
amyl butyrate, water, amyl alcohol and butyric acid with the
end-state attained by this system under the influence of lipase.
The measurements of the velocity-constants, k\ and 2, with
which the formation and resolution of the ester proceed, have
already been referred to on p. 152. The mean values obtained
were:
ki -0-015 & 2 =
As should theoretically be the case, the quotient of these
two velocity constants was found to be equal to the equilibrium
constant determined directly. 1
Hence
The end-state determined in these two ways showed, however,
considerable and regular deviations from the natural stable equi-
librium. Thus, while the natural equilibrium constant had the
value 1-96, the enzyme experiments gave the following results:
Initial concentration of
the reacting substances. &
0-05 0-45
0-10 0-74
0-20 1-12
The fact that these end-states were reached from both direc-
tions proved that they were not dependent on retardations of
the reaction.
Unfortunately, these data cannot serve for proving the above
relations quantitatively, since the system examined was hetero-
geneous (macro-heterogeneous) .
A. E. Taylor (Journ. of Biol. Chem., 1906, 2, 87) also
worked with Ricinus-lipase in the form of a moderately finely-
divided suspension. The substrate employed was triacetin,
the triglyceride of acetic acid. The natural equilibrium was
investigated with 0-5, 1-0 and 2-0% solutions of the triacetin;
1 Since water and amyl alcohol were present in excess, the constant K
simplifies to
[amyl butyrate]
[butyric acid]
CHEMICAL STATICS IN ENZYME REACTIONS 257
mixtures of equal volumes of these solutions and of normal
sulphuric acid, left for several months, gave the values:
Initial concentration of the ester. Composition of the equilibrated liquid.
0-5% 12% ester, 88% hydrolysed
1 18 " 82
2 22 " 78
For the enzymic end-state, the following numbers were
obtained :
Initial concentration of the eater. Composition at the end-state.
0-5% 14% ester, 86% hydrolysed
1 21 " 79 "
2 30 " 70
From these numbers, Taylor drew the conclusion that
the enzyme does not displace the equilibrium; but the dif-
ferences between these two series of numbers are so large and so
regular that, in the author's opinion, they do not indicate identity
of the natural and enzymic equilibria. Whether such a dif-
ference exists generally and how it depends on the concentrations
of enzyme and substrate are questions of great interest, and exper-
iments in this direction should give valuable results.
Scarcely any other quantitative determinations of enzymic
end-states have been made which are comparable with the natural
equilibria. From Croft Hill's results on the equilibrium
between maltose and glucose, Pomeranz (Wien. Sitzungsber.,
II B, 1902, 111, 554) has, indeed, calculated the equilibrium con-
stants, which are in good agreement. From a qualitative point
of view however, this end-state is by no means clear; maltose
is re-formed either not at all or only in inappreciable amounts,
being replaced by dextrins and isomaltose.
A paper communicated byvan't Hoff, shortly before
his death, to the Berlin Academy of Sciences (Sitzungsber. K.
Akad. Berlin, 1910, 48, 963), treats of the equilibrium of glucosides
in presence of emulsin.
Measurements were made first with the natural glucoside
salicin, in presence of solid salicin and solid saligenin. It was
found that the formation of solid salicin from the solid products
of hydrolysis is accompanied by an expansion in volume of
9-47 c.c. per grm.-molecule. The result was that the hydrolysis
258
GENERAL CHEMISTRY OF THE ENZYMES
of salicin proceeds to practical completion; the equilibrium was,
however, not measurable. [Also V i s s e r (Zeitschr. f. physikal.
Chem., 1905, 52, 257) had previously obtained only indications
of a synthesis of salicin.]
With arbutin and aescillin, the hydrolyses were also virtually
complete.
On the other hand, the system glycerol-glucose-water-glyc-
erolglucoside gives a measurable condition of equilibrium.
Experiments were made with the molecular proportion 1 : 4
between glucose and glycerol and with increasing amounts of
water. If the number of mols. of the latter is expressed by b
and the fraction of the glucose changed by a, then
glucosideX water a (6+ a)
glucose X glycerol ( 1 a) (4 a)
= k.
Formation of glucose still occurred with 6 = 15 and a = 0-38, the
value for k being 2-6. With molecular proportions of glycerol
and glucose, as much as 70% may undergo change.
T a m m a n n has investigated experimentally the dependence
of the end-state on the quantities of the enzyme and of the react-
ing compounds. In the action of emulsin on arbutin and on
coniferin, it was found that the amount of substance hydrolysed
at the end-state increases to a maximum as the quantity of
enzyme increases. This indicates that, with increasing concen-
tration of the enzyme, the number of the active molecules,
enzyme-product of reaction, increases more than that of the
molecules enzyme-substrate.
When constant amounts of emulsin and different amounts of
amygdalin are dissolved in 25 c.c. of water, the following amounts of
amygdalin are decomposed at 40:
Original quantity
of amygdalin.
Amounts hydrolysed
Absolute amounts.
Percentage amounts.
0-51 grm.
1-02 grms.
2-04 "
0-11 grm.
0-15 "
0-24 "
20
15
12
CHEMICAL STATICS IN ENZYME REACTIONS
259
Similar relations are found for the end-states of the system emulsin-
arbutin. The solution contained 0625 grm. of emulsin and the follow-
ing amounts of arbutin at 35:
Original quantity
of arbutin.
Hydrolysed after
48 hours.
72 hours.
0-576 grm.
4-000 grms.
52-3%
44-0
52-3%
44-0
With constant amounts of enzyme, relatively more amygdalin
and arbutin are hydrolysed in dilute than in concentrated solutions;
the same probably holds for the hydrolysis of coniferin by emulsin.
The question now arises : Within what limits is the e n d -
state of an enzymic chemical system variable? That these limits
must be quite wide is shown at once by the above facts. They depend
not only on the concentration, but also on the previous history of the
enzyme.
Of the equilibria of biological importance with which considerable
variations have been observed, that between starch and sugar in the
living plant deserves special mention. The synthesis, but not
the hydrolysis of starch is largely influenced by even trifling variations
of temperature or by narcosis. These phenomena may be explained in
two ways:
(1) The existence may be assumed of two different enzymes, one
responsible for the synthesis and the other for the hydrolysis; this is,
of course, only conceivable on the supposition that different quantities
of the two enzymes combine preferably with the starch or sugar, so
that both starch and sugar participate in two equilibria:
and
[enzyme a][starch] =& m [enzyme a starch]
[enzyme b] [starch] =/c w [enzyme b starch].
If corresponding equilibria hold for the compounds of the two
enzymes with the sugar, it is obvious that there arise two enzymic end-
states, which may assume widely different values. Their relations, one
to the other, depend only on the total concentration of the starch. None
of the facts are contradictory to this hypothesis, for which, however, no
experimental proof is forthcoming. In such a case, indeed, the enzymes
are far removed from ideal catalysts. (2) Or it remains to be tried
as the author has emphasised in another place (Pflanzenchemie, II and
III, p. 237) whether the assumption of a single catalyst or a single
260 GENERAL CHEMISTRY OF THE ENZYMES
equilibrium constant does not meet the case. The very varying ways in
which the opposed processes of building-up and breaking down react
towards external influences, would then be attributed to the different
constitutions of the media in which condensation and hydrolysis
proceed. The catalyst and the reacting substances are then regarded as
distributed between the aqueous cell-sap and the protoplasmic
hydrosol. In the former, owing to the great excess of water present, the
hydrolysis may proceed far; but in the protoplasts, which are rich in
lipoids and proteins, such a relatively small proportion of water dissolves
that, with a given attainable magnitude of the sugar-concentration, the
opposite reversionary changes predominate.
CHAPTER VI I
ENZYMIC SYNTHESES
THE suggestion expressed byvan't Hoff in 1898, that
enzymes are able to effect or accelerate chemical syntheses (Zeitschr.
f. anorg. Chem., 1898, 18, 1), has since then been confirmed by
the results of numerous investigations.
It was in the above year that Croft Hill (Journ. Chem.
Soc., 1898, 73, 634) observed a synthetic action in the case of
y east-malt ase.
Croft Hill found that when yeast-maltase is allowed
to act for a month at 30 on a 40% solution of glucose, the
reducing and rotatory powers of the solution are so altered as to
indicate formation of maltose. Shortly afterwards, however,
E m m e r 1 i n g (Chem. Ber., 1901, 34, 600, 2207) showed that
the effect observed by C r o f t Hill depends on the formation,
not of maltose, but of isomaltose and dextrinous products.
Isomaltose is not again hydrolysed by maltase. Similar behaviour
was noted byE. Fischer and E. F. Armstrong (Chem.
Ber., 1902, 35, 3144) with kephir-lactase, which from galactose
and glucose synthesises not lactose but isolactose, a carbohydrate
not attacked by the lactase. Finally, E.F. Armstrong
(Proc. Roy. Soc., B, 1904, 73, 516) made a number of interesting
observations which extended the discovery of E m m e r 1 i n g
referred to above: the behaviour of emulsin is the opposite
of that of maltase, as it hydrolyses isomaltose but synthesises
glucose to maltose. These results led Armstrong to the
generalisation that " Enzymes build up just those molecules
which they are unable to break down."
This is a problem of fundamental theoretical importance,
If Armstrong's view is correct, it must be assumed thai
those enzymes which give rise to a chemical equilibrium froir
both directions represent mixtures of a synthesising and a hydro-
lysing enzyme. Against the admissibility of this hypothesis
261
262 GENERAL CHEMISTRY OF THE ENZYMES
which is only weakly supported by experiment, no fundamental
objection can be advanced, and we may again consider the facts
favouring such a two-enzyme theory. It has been emphasised,
especially by Bayliss, that the experimental foundation
for this view is indeed rather weak, inasmuch as Croft Hill
made use of ordinary brewers' yeast, which has been shown
by H e n r y and A u 1 d (Proc. Roy. Soc., B, 1905, 76, 568)
to contain " emulsin " and hence a ^-glucosidase. This emulsin
may have occasioned the formation of isomaltose. The forma-
tion of isomaltose and isolactose admits, however, of another
possible interpretation, which has been given by E . F . Arm-
strong (Proc. Roy. Soc., B, 1905, 76> 513).
It has been known since O ' S u 1 1 i v a n and Tompson's
work, and has been confirmed by H u d s o n , that the hydrolysis
of cane-sugar yields a d-glucose distinguished by its high rotatory
power; this sugar, to which T a n r e t to whom we owe a very
complete investigation of this sugar gave the name a-glucose,
passes gradually into e-glucose. This s-glucose appears to con-
sist of a- and ^-glucoses in equilibrium. In aqueous solution there
is little a-glucose and a relatively large proportion of ^-glucose.
On adding to such a solution, an enzyme the synthetic action of
which is to be studied, an excess of the ^-modification is available
and it is to be expected that the synthetic biose will contain the
glucose-residue mainly in a form corresponding with ^-glucose.
The biose corresponding with the oc-hexose should also be formed
in smaller amount at the same time, but this has not been observed.
Nothing, however, is yet known as to what constitutes the dif-
ferences between these modifications.
This view is capable of experimental proof in various ways.
Firstly, it might be expected that the bioses, such as iso-
maltose, synthesised from glucose solutions, should yield ^-glucose
directly, but no such result appears to have been obtained;
this should also be the case with those bioses and glucosides which
undergo the same enzymic hydrolyses. Further, it would be
expected that maltose could be synthesised from a-glucose
i.e., from a freshly-prepared solution of glucose and a very
active enzyme capable of effecting the synthesis, before the change
from a- to ^-modification is complete. 1
1 With reference to certain statements in the literature, it must be pointed
out that originally, the names a-glucose and a-glucoside were not connected.
ENZYMIC SYNTHESES
263
Apart from the facts mentioned above with reference to the
synthesis of maltose and lactose, numerous other statements
have been made relating to enzymic syntheses which are regarded
as pure reversions.
Ethyl butyrate is formed from butyric acid and ethyl alcohol
by the action of pancreas-lipase (K a s 1 1 e and L o e v e n -
hart, Amer. Chem. Journ., 1900, 24, 491).
Glyceryl butyrate (H a n r i o t , C. R., 1901, 132, 212) and
amyl butyrate (Bodenstein and D i e t z , Zeitschr. f .
Elektrochem., 1906, 12, 605) are also formed from their com-
ponents.
An extensive series of experiments on the formation of ester
from methyl alcohol and oleic acid by pancreas-lipase has been
carried out by Pottevin (Bull. Soc. Chim., 1906, 35, 693;
Ann. Inst. Pasteur, 1906, 20, 901). These show, among other
results, that the equilibrium between glycerol and oleic acid is
independent of the quantity of enzyme added.
Quantity of
Percentage of ester formed.
pancreatin employed.
1 day.
2 days.
20 days.
1
8
56
84
2
12
66
82
5
21
66
84
10
43
74
85
The following results, relating to the synthesis of a true
fat, indicate the influence of the amount of water present.
40 grms. of oleic acid + 3 grms. powdered pancreas,
experiment, 20 days. Temperature, 33.
Duration of
Amounts of
Percentage esterified.
Glycerol.
Water.
130 grms.
grms.
3
120
10
77
110
20
64
100
30
51
64
66
20
28
102
5
8
122
264 GENERAL CHEMISTRY OF THE ENZYMES
Syntheses of true fats from various higher fatty acids (of
(ocoanut oil, etc.) and glycerol are described by Welter
cZeitschr. f. angew. Chem., 1911, 24, 385).
Glyceryltriacetate is formed from its components by Ricinus-
lipase (Taylor, Journ. of Biol. Chem., 1906, 2, 87). Cf.
p. 154.
Amygdalin is formed from mandelonitrile glucoside and
glucose (E m m e r 1 i n g , Chem. Ber., 1901, 34, 3810).
Benzaldehydecyanohydrin results from benzaldehyde and
hydrocyanic acid (Rosenthaler, Biochem. Z., 1908, 14,
238; 1909, 19, 186).
Triacetylglucose is given by acetic acid and glucose under
the influence of pancreatin (A c r e e and H i n k i n s , Amer.
Chem. Journ., 1902, 28, 370).
Glycogen is formed in pressed yeast-juice from sugar
(C r e m e r , Chem. Ber., 1899, 32, 2062).
A condensation, the nature of which is not clearly understood,
is produced in invert-sugar solutions by the revertase of
Mucor mucedo, etc. (P a ri t a n e 1 1 i , Atti Real. Accad.
Lincei, 1907, [v], 16, ii, 419; Bot. Ber., 1908, 26a, 494). Also
no definite conclusions can be drawn concerning the action of
the yeast-revertase investigated by Kohl (Beitr. z. bot. Zen-
tralbl., 1908, 23, i, 64).
Reference must also be made here to the studies of
Maquenne (Bull. Soc. Chim., 1906, [iii], 35; lecture) and of
Wolff and Fern bach (C. R., 1903, 137, 718) on the
re-formation of amylose from its decomposition products; men-
tion should also be made ojf the equilibrium attained under the
action of malt-diastase (M o r i t z and Glendinning,
Journ. Chem. Soc., 1892, 61, 689). Possibly the reversion of
starch in plasmolysed vegetable cells, observed by Overton
(Vierteljahrsschr. d. naturf. Ges. in Zurich, 1899, 44, 88), also
represents such an enzyme action.
Kendall and Sherman (Journ. Amer. Chem. Soc.,
1910, 32, 1087) found that a state of equilibrium is also set up in
the decomposition of starch by amylase (pancreas-amylase) . In
1 per cent starch solution, equilibrium is attained independently
of the amounts of salt and alkali present when the amount of
maltose is about 85% of the initial weight of the starch.
The formation of hippuric acid from benzoic acid (benzyl
ENZYMIC SYNTHESES 265
alcohol) and glycine by the action of kidney-extract has been
observed by Abelous and Ribaut (Soc. Biol., 1900,
52, 543), but confirmation of this result is desirable.
As regards the synthesis of protein substances, mention
must first be made of the experiments on plastein-formation,
which must undoubtedly be regarded as syntheses.
D a n i 1 e w s k i established the fact that, in concentrated
solutions of Witte's peptone, rennet produces characteristic
protein precipitates. This phenomenon, " plastein-formation,"
which also occurs under the influence of pepsin preparations,
was further investigated in Danilewski's laboratory and
has since been examined more especially by Russian workers,
for instance, S aw j alow (Centralbl. f. Physiol., 1902, 16,
625) and Okuneff (Dissertation, St. Petersburg, 1895).
Kurajeff (Hofm. Beitr., 1901, 1, 121; 1903, 4, 476) found
papain to possess a similar coagulating property. L a w r o w
and S a 1 a s k i n (H., 1902, 36, 277) showed that the precipita-
tion of concentrated Witte's peptone solutions by gastric juice
occurs with albumoses of all types. Our knowledge of plastein-
formation has recently (H., 1907, 51, 1) been considerably
extended by L a w r o w , according to whom, not only
the albumoses but also substances of the amino-acid type
can be coagulated best in faintly alkaline solution. The
coagulums exhibit the reactions of proteins but contain less
nitrogen than these.
Plastein-formation is favoured by increasing the concen-
trations of the reacting solutions and occurs especially under
conditions which retard the hydrolysis of proteins.
The precipitation of plasteins may possibly consist of a salting-
out process.
Everything seems to indicate that Danilewski's reac-
tion is really a synthesising action 1 of the pepsin or rennet,
although true reversibility of the peptic action, i.e.-, re-forma-
tion of the starting material has not been proved. Rosen-
f e 1 d (Hofm. Beitr., 1906, 9, 215) has shown that the hydrolytic
products of casein-plastein differ, at any rate quantitatively,
from those of casein. This is proved very clearly by a more
recent investigation of Henriques and G j a 1 d b a k (H.,
1 Cf . also R. O. Herzog (H., 1903, 39, 305) and A . Number g
(Hofm. Beitr., 1903, 4, 543).
266 GENERAL CHEMISTRY OF THE ENZYMES
1911, 71, 485), who followed the reaction by means of S 6 r e n -
s e n 's method of titration with formaldehyde.
An interesting pepsin-synthesis from the hydrolytic products
of casein has been communicated by Robertson (Journ.
of Biol. Chem., 1907, 3, 95) who obtained a body which con-
tained 3-17% P2Os and which he termed u paranuclein."
Taylor succeeded in effecting an enzymic synthesis with
trypsin. He hydrolysed protamine sulphate from R o c c u s
1 i n a t u s completely into its components and treated the mix-
ture of amino-acids with trypsin obtained from the liver of the
mollusc Schizothaerus Nuttalii (Journ. of Biol.
Chem., 1907,3,87).
The same author has recently described further experiments
both with glycerine liver-extract of Schizothaerus Nut-
talii and with pancreatin (G r ii b 1 e r ' s) , the substrate
being obtained as before from salmin sulphate. After a lapse of
4 months, the solutions, which proved to be still germ-free, were
diluted with 4 parts of water, acidified with sulphuric acid and
mixed with 3 parts of absolute alcohol; the control solutions
showed no change, whilst the two containing enzyme gave thick
white precipitates. The product was purified by means of its
picrate, after which it was found to have a composition closely
agreeing with that of salmin.
Indications of a synthetic action of trypsin on the hydro-
lytic products of casein were obtained by B a y 1 i s s (Arch.
Sci. Biol. St. Petersburg, 1904, 11, Supplement, 261), while the
number of cases in which retardation of the hydrolysis by the
reaction products has- been observed show that a state of rever-
sible equilibrium is assumed.
The experiments of Beitzke and N e u b e r g (Verh.
d. deutsch. path. Ges., 1905, 160; Virch. Arch., 1906, 183, 169)
are of special interest, both in themselves and in relation to the
representation of enzymic end-states given on p. 253 et seq.
It was found that subcutaneous injection of emulsin (in rabbits)
leads to the formation of anti-emulsin, which is able to synthesise
glucose to maltose or to a disaccharide similar to maltose.
Should further experiments show that our enzyme-prepara-
tions contain in general a resolving and a synthesising con-
stituent, the prevailing views concerning the formation and com-
bination of anti-enzymes may require modification, the deter-
ENZYMIC SYNTHESES 267
mining factor in the equilibrium between enzyme and anti-
enzyme being ascribed to the chemical substrate and the products
it yields.
Anti-enzymes
Like the toxines, many enzymes are able to cause production,
in the living organism, of anti-bodies which retard the actions of
the enzymes. Anti-enzyme action was first observed by
Hildebrandt in 1893.
From their actions, anti-enzymes appear, like enzymes, to be
organic catalysts. They seem to correspond with the enzymes
in physical properties and also in chemical lability, although the
observations in this direction are few and not very definite.
They are, as far as is known, approximately as unstable towards
high temperatures as the enzymes themselves. Among the
most stable anti-enzymes are the anti-lipase of Ricinus which,
according to Bertarelli (Centralbl. f. Bakt., 1905, I, 40,
231), is not weakened at 70 and slowly loses its activity only at
80. The degree of purity of the enzyme appears to be without
influence on the formation of anti-body, and a co-enzyme is
apparently unnecessary to this reaction.
This behaviour corresponds closely with that of the antitoxines,
which are generally injured at 70. Like enzymes, antitoxines are more
stable in the dry state than in solution.
Judging from the results of physico-chemical investigations,
the relations between enzymes and anti-enzymes are essentially
identical with those between toxines and anti-toxines. For
further information on this interesting problem, the monographs
of Arrhenius and of M i c h a e 1 i s should be consulted.
The most important facts concerning anti-enzymes are here
brought together, because the problem of the reversibility of
enzymic reactions is bound up with the action of anti-enzymes.
By the term anti-enzymes, in its stricter meaning, is to be
understood those specifically-acting secretions produced in the
organism, in presence of enzymes, by immunisation. But normal
serum also contains substances which, for instance, annul tryptic
action more or less completely. It is not probable, from the facts
yet known, that these differ essentially from the anti-bodies
268 GENERAL CHEMISTRY OF THE ENZYMES
produced by immunisation, and they will therefore be discussed
with the anti-enzymes. On the other hand, the thermo-stable
and inorganic substances which prevent enzyme action to a greater
or less extent will be termed, after Neuberg's suggestion,
inhibiting agents.
Anti-steapsin was prepared by A. Schlitze (Deut. med.
Wochens., 1904, 30, 308), who, after injecting steapsin, obtained
in two cases rabbit-serum showing strong anti-lipolytic action.
His results have more recently been confirmed by B e i t z k e
and N e u b e r g (Virch. Arch., 1906, 183, 169) and by
B e r t a r e 1 1 i .
The latter author (Centralbl. f. Bakt., 1905, I, 40, 231) was
not able to obtain the anti-enzymes corresponding with the
lipases from ox-liver and ox-blood serum, but by injection of
various vegetable lipases (Ricinus- and nut-lipase) he separated
from dog-serum anti-lipases with specific actions. Thus the
anti-ricinus-lipase influenced neither the serum-lipase, nor the
liver-lipase, nor the nut-lipase.
Anti-emulsin, the first known anti-enzyme, was
discovered by Hildebrandt (Virch. Arch., 1893, 131, 12)
who, like B e i t z k e and N e u b e r g (Virch. Arch., 1906,
183, 169), immunised rabbits and precipitated the anti-body
from the anti-ferment-serum with the globulin fraction. The
synthetic action of this preparation is discussed later.
Immunisation with diastase may perhaps have been observed
by Kussmaul (Arch. f. klin. Med., 14), but his results *are
uncertain. A s c o 1 i ' s work (H., 1904, 43, 156) also led to
no definite result.
Subcutaneous injection of diamalt a commercial enzyme
solution prepared from green malt leads to the production in
rabbit-serum of substances which retard the saccharifying action
of diastase, while the serum originally showed an inverting action
(B r a u n and S c h ii t z e , Med. Klin., 1907, No. 9, quoted
in Biochem. Zentralbl., 1907, 6, 389).
S a i k i prepared an anti-inulinase by injection
(Journ. of Biol. Chem., 1997, 3, 395).
An anti-invertase of slight activity was obtained by
Schtitze and Berg ell (Zeitschr. klin. Med., 1907, 61, 366).
S c h ii t z e (Zeitschr. f. Hygiene, 1904, 48, 457) also prepared an
anti-lactase by injecting ' kephir-lactase under the skin
ENZYMIC SYNTHESES 269
of rabbits or into the breast-muscles of the dog; the anti-body
appears in the serum, which in the normal state did not contain it.
Anti-pepsin. Sachs ^(Fortschr. d. Med., 1902, 20,
425) immunised geese against pepsin, the serum exhibiting
sufficient anti-peptic action to annul 20 times its amount of pepsin.
The anti-pepsin which was discovered by Weinland
(Zeitschr. f. Biol., 1903, 44, 45) and is regarded as a normal
secretion of the gastric mucous membrane, corresponds with the
normal anti-trypsin of blood-serum. It retards peptic digestion
in vitro and doubtless prevents auto-digestion of the mucous
membranes.
The same author has detected anti-enzymes of pepsin and
trypsin in the pressed juice of Ascaris, and, according to
R.O. Herzog (H., 1909, 60, 306), the action of rennet
preparations is also retarded by Ascaris juice.
Unlike these anti-enzymes, an agent which inhibits peptic
action and was found by B 1 u m in gastric juice is stable to heat.
While B e r g e 1 1 and S c h ti t z e tried in vain to obtain an
anti-pancreatin (Zeitschr. f. Hygiene, 1905, 50, 305), J o c h -
m a n n and Kantorowicz, in a recent preliminary com-
munication (Munch, med. Wochens., 1908, 55, 728), refer to
an anti-enzyme to pancreas-trypsin which must be identical
with the anti-body of the leucocyte-enzyme. The same inves-
tigators state that blood contains at least two anti-pepsins, one
of which inhibits the digestion of serum-albumin, being destroyed
at 80-85, while the other prevents the digestion of solidified
hens' egg-albumin, being thermo-stable.
Attempts at immunisation against papain have as yet been
unsuccessful (B e r g e 1 1 and Schiitze, loc. cit.; von
Stenitzer, Biochem. Z., 1908, 9, 382).
Anti-tryptic Paralysors
The experiments of H a h n (Berl. klin. Wochens., 1897,
34, 499) and those made almost simultaneously by P u g 1 i e s e
and Coggi (Boll. Sci. Med., 1897, 8) first established the
fact that normal serum retards tryptic digestion. Fermi
had, however, previously observed that trypsin disappears soon
after injection. Further work on this subject has been done by
A c h a 1 m e (Ann. Inst. Pasteur, 1901, 15, 737), Camus and
270 GENERAL CHEMISTRY OF THE ENZYMES
G 1 e y (Soc. Biol., 1897, 47, 425), Charrin and Levaditi,
S i m n i t z k i and Glaessner (Hofm. Beitr., 1903, 4,
79). This anti-tryptic action is bound up with the serum-
albumin (Landsteiner, Centralbl. f. Bakt., 1900, I, 27,
357; Cathcart, Journ. of Physiol., 1904, 31, 497; H e d i n ,
Journ. of Physiol., 1905, 32, 390). With a number of diseases,
such as diabetes, tuberculosis, etc., increased anti-tryptic activity
of the serum is observed (B r i e g e r and T r e b i n g ,
Berl. klin. Wochens., 1908, 45, 1041).
According to A. D 6 b 1 i n (Zeitschr. f . Immunitatsforsch.
u. exp. Therap., 1910, 4, 229), the anti-trypsin of serum is stable
to heat, which weakens the anti-tryptic action of urine only
slightly. The inhibiting body is not a lipoid but is colloidal in
character.
Delezenne (Soc. BioL, 1903, 55, 112) found that the
retarding action of normal serum is not a direct action on the
proteolytic enzyme, but is to be attributed to neutralisation of
the corresponding kinase. He describes the following experiments:
After preliminary determination of the amount of serum just
necessary to annul the digestive action of a mixture of pancreatic
and intestinal juices, three tubes were each filled with equal
quantities of the substance to be digested, the pancreatic-intestinal
mixture and the corresponding quantity of serum. After the
lapse of some hours, when it was found that no digestion had
occurred in any of the tubes, to one (A) was added an excess of
pancreatic juice and to another (B) an excess of gastric juice,
while C served as control; digestion took place only in B. From
these results Delezenne inferred that, in the digestion with
the pancreatic-intestinal juice only the intestinal juice (the
kinase) was neutralised, and that the serum has no action on the
pancreatic enzyme. A s c o 1 i and B e z z o 1 a (Centralbl. f.
Bakt., 1903, I, 33, 783) arrived at somewhat similar conclusions.
Against this view objections have, however, been advanced.
According to Delezenne, the anti-trypsin would be an anti-
kinase, and. the existence of an anti-trypsinogen might also be
expected. B a y 1 i s s and Starling (Journ. of Physiol.,
1904, 32, 129) were unable to detect such a body in blood-serum
after subcutaneous injection. It was also shown that normal
rabbit-serum possesses, in addition to its anti-tryptic properties,
the ability to neutralise enterokinase, this power being enhanced
ENZYMIC SYNTHESES 271
by injection of enterokinase. On the other hand, " anti-kinase "
produced in serum does not increase its anti-tryptic properties.
These observations have been confirmed and extended by
Zunz (Bull. Acad. Roy. Med. de Belgique, 1905, [4], 19).
Normal blood-serum contains not only anti-trypsin, but
also a proteolytic enzyme, serum-protease, the action of which
is retarded by the anti-trypsin. Serum-protease can be separated
from anti-trypsin by salting-out, the former passing into the
globulin fraction and the latter into the albumin fraction.
That the amount of anti-trypsin in serum can be increased
considerably by injection of trypsin solutions, has been shown
by Achalme (Ann. Inst. Pasteur, 1901, 15, 737) and by
W e i n 1 a n d (Zeitschr. f. Biol., 1902, 44, 1, 45).
As regards the sensitiveness of the " anti-proteolase " to
heat, Vandevelde's experiments (Biochem. Z., 1909, 18,
142) appear to indicate that weakening takes place at 55.
According to F e r m i (Centralbl. f. Bakt., 1909, I, 50, 225),
anti-tryptic action is exhibited by various organic tissues and
by certain protein substances, such as yolk of egg and milk;
casein alone also has an anti-tryptic action.
The clinical significance of the tryptases and anti-tryptases,
which cannot be treated in detail here, will be found discussed
in a comprehensive paper by von Bergmann and Kurt
Meyer (Berl. klin. Wochens., 1908, 45, 1673).
Anti-urease. As was discovered by L . Moll (Hofm.
Beitr., 1902, 2, 344), normal (rabbit-) serum and normal albumin-
free urine always exert a retarding action on urease. This action
is markedly increased by injection of small doses of a urease-
preparation from Micrococcus ureae Pasteuri.
Moll does not regard the anti-bodies of normal and of immu-
nised serum as identical, since the latter loses the excess of its
inhibiting power and hence becomes normal in this respect if
heated for an hour at 65 (but not at 56), whilst the retarding
capacity of normal serum is not altered by heating either for an
hour at 65 or for six hours at 56.
Anti-bodies of clotting enzymes. The
important discovery of Morgenroth, that subcutaneous
injection of rennet produces an anti-rennet in the serum
and milk of the immunised animal (Centralbl. f. Bakt., 1899,
26, 349; 1900, 27, 721), directed attention to these anti-enzymes.
272 GENERAL CHEMISTRY OF THE ENZYMES
As regards anti-rennet itself, Morgenroth and more especially
M a d s e n and W a 1 b u m , and Bashford have inves-
tigated its action quantitatively, whilst F u 1 d and S p i r o (H.,
1900, 31, 132) have made a comprehensive study of the rennetic
and anti-rennetic action of the blood. Arrhenius has
pointed out the analogy existing between the behaviour of the
clotting enzymes and that of certain precipitins towards the
anti-bodies. It is hence unnecessary to describe the equilibrium
phenomena between rennet or the fibrin-ferment and the anti-
bodies.
Emphasis must be laid on the fact discovered by H a m -
m a r s t e n and R 6 d e n as early as 1887 that the normal
serum of various animals contains a substance which inhibits the
action of rennet. This substance is, however, not identical with
that produced by active immunisation (Bashford, Journ.
of Pathol., 1902, 8,52). According to Fuld and Spiro,
the " anti-rennet " contained in horse-blood serum is a pseudo-
globulin which acts by fixing a portion of the calcium ions and
so retarding coagulation; these authors separate the chymosin
and anti-chymosin of normal blood by precipitation with ammo-
nium sulphate.
For the relations between anti-pepsin and anti-rennet, see
J a c o b y (Biochem. Z., 1907, 4, 471).
Anti-fibrin-ferment. Bordet and G e n g o u
(Ann. Inst. Pasteur, 1901, 15, 129) obtained this anti-body in
the following manner: They injected guinea-pigs with normal
rabbit-serum, by which means the guinea-pig-serum acquires
the property of retarding the coagulation of rabbit-blood, i.e.,
the anti-fibrin-ferment is formed. This acts, as these authors
showed, in a somewhat markedly specific manner on the sera
of different animals. This anti-body is not affected by heating
to 55.
The composition of the anti-fibrin-ferment is not definitely
known.
Anti-laccase was thought to have been obtained by
G e s s a r d (Soc. Biol., 1903, 55, 227) in rabbit-serum by injec-
tion of laccase; the shortness of his communication renders
criticism of the work impossible.
The anti-catalase of Battelli and Stern (Soc.
Biol., 1905, 58, 235, 758) behaves like ferric sulphate and should be
ENZYMIC SYNTHESES 273
classed with the inhibiting agents. A true anti-enzyme of catalase
does not appear to exist (D e W a e 1 e and Vandevelde,
Biochem. Z., 1908, 9, 264).
Although anti-enzymes corresponding with vegetable enzymes
have often been produced in the animal body (M a g n u s and
Friedenthal), no appreciable formation of anti-enzymes
in plants has yet been observed.
The most remarkable property of the anti-enzymes, in the
narrow meaning of the word, is the rigid specificity of their
action; this property they possess in common with the anti-
toxines. Whether this is a peculiarity of the anti-bodies formed
as protective agents in the organism, or whether closer investiga-
tion will show that the anti-enzymes act specifically only in the
same sense as do the enzymes, cannot at present be decided.
It can, however, be asserted from the experimental material
at present available, that the specificity of the enzymes does not
differ fundamentally from that of other catalysts.
CHAPTER VIII
SPECIFICITY OF ENZYME ACTION
THE question of the acceleration of one and the same reaction
by different enzyme-preparations does not lend itself to a
critical examination, owing to the impossibility of judging the
physiological purity of the preparations. If therefore it is stated,
for example, that emulsin hydrolyses fats, this can scarcely have
any other meaning than that the preparations considered con-
tain lipases, unless indeed it is shown that the actions on glucosides
and fats always exhibit parallel courses. Meanwhile, the decid-
ing factors in such cases are physiological in nature, whilst
the chemist is concerned more nearly with the other problem,
namely, that of determining what different reactions are always
initiated by one and the same enzyme-preparation and enzyme. 1
The investigations of recent years have rendered it probable
that in many enzyme-preparations in which formerly the presence
of only one enzyme was assumed, a number of different enzymes
exist with more restricted spheres of action. It has, indeed,
been mentioned that emulsin contains at least five enzymes,
and that diastase is presumably composed of a number of enzymes
which effect the degradation of starch in stages.
The term specificity is applied to cases where the action of
an enzyme is exerted only on separate representatives of a larger
class of bodies.
Some of these cases are understood in so far as the course
of the chemical change can be followed. Thus, the well-known
fact that only sugars with six or nine carbon atoms, and not,
for instance, the pentoses, are fermentable, is much less remark-
(Zeitschr. f. physikal. Chem., 1903, 45, 513) has studied the
interesting case in which different, inorganic catalysts (stannic chloride,
iodine chloride, etc.) cause or accelerate catalytically different reactions of
the substrate (chlorine and benzene).
274
SPECIFICITY OF ENZYME ACTION 275
able now that a representation of the intermediate products of
the reaction has been attained.
The specificity of the oxydases, to which attention has been
repeatedly drawn, would most readily, arise as a consequence
of purely chemical facts.
In the cases where the specific nature of the oxydases is
most pronounced, namely, with the phenolases, it may be asserted
that the reactivity of the simple and substituted mono-, di-
and tri-phenols is dependent on the constitution in the same
way when " oxydases," as when non-enzymic manganese com-
pounds, form the oxidising catalysts.
To choose the simplest example: of hydroquinone, pyro-
catechol and resorcinol, the first is oxidised rapidly and the
second considerably more slowly, whilst the last is extremely
resistant to oxidation (cf. BertrandBull. Soc. Chim., 1896,
[iii], 15, 791).
Another case is that of the Upases. From the results of
K a s 1 1 e and Loevenhart's measurements, it is known
that the ester-resolving action of pancreas-extract is by no means
exerted on all esters. Apart from the fact that the true fats
are only very slightly hydrolysed by this extract, enormous
differences are observed between the velocities of hydrolysis of
such closely-allied chemical individuals as ethyl acetate and
ethyl butyrate. A further list of similar differences has been
given by H. E. Armstrong and O r m e r o d . But
with catalytic decompositions, completely analogous behaviour is
shown. For example, according to R. Lowenherz, the
constants of hydrolysis (with hydrochloric acid as catalyst)
of ethyl formate and methyl benzoate are in the ratio of 1-1 :
0-0003, and still larger differences can easily be found. In this
connection, it is to be noted that in enzyme reactions very small
velocities do not show, since the extended duration of the action
results in the enzyme becoming inactive.
Also the results ofH. BierryandGiaja's experiments
(C. R., 1908, 147, 268) on the action of maltases and lactases of
various origins appear to depend on differences of degree, and
not of kind, in the activities; the resolution of lactose, lactobionic
acid and lactosazone is effected by an active preparation, whilst
another preparation which resolves only lactose must be generally
weaker.
276 GENERAL CHEMISTRY OF THE ENZYMES
Fischer andAbderhalden (H., 1905, 46, 52; 1907,
50, 264) have collected a large mass of data concerning the
power possessed by pancreatic juice of decomposing polypeptides-
The behaviour of these substances towards P a w 1 o w ' s pan-
creatic juice is shown in the following table:
Hydroly sable. Non-hydrolysable.
*Alanylglycine 1 Glycylalanine
*Alanylalanine Glycylglycine
*Alanylleucine A Alanylleucine B
*Leucylisoserine Leucylalanine
Glycyl-Z-tyrosine Leucylglycine
*Alanylglycylglycine Aminobutyrylglycine
*Leucylglycylglycine Aminobutyrylaminobutyric acid A
* Glycylleucylalanine Aminobutyrylaminobutyric acid B
* Alanylleucylglycine Aminoisovalerylglycine
Dialanylcystine Glycylphenylalanine
Dileucylcystine Leucylproline
Tetraglycylglycine Diglycylglycine
Triglycylglycine ester (Cur- Triglycylglycine
t i u s ' s biuret base) Dileucylglycylglycine
The hydrolysis of these substances by acids would, no doubt,
likewise reveal considerable differences between the velocities.
But, as K a s 1 1 e and Loevenhart's experiments with
esters show, the order is not always the same for hydrolysability
by enzymes and by other catalysts. The lack of parallelism
between the two cases may be due to enzymes and acids being
combined to different extents by different esters.
The enzymes exhibit the strictest specificity towards optical
antipodes.
After E . Fischer had shown how new optically active
products are obtained by purely chemical syntheses (Chem.
Ber., 1894, 27, 3230), the fundamental difference assumed by
Pasteur between natural and artificial syntheses fell to the
ground. Four years later Fischer arrived at the conclusion
that the specificity of enzymes towards optical antipodes is con-
ditioned by the stereochemical structure of the enzymes (H.,
* 1898,26,60).
1 Peptides marked * are the racemic compounds.
SPECIFICITY OF ENZYME ACTION
277
He himself, partly in conjunction with his collaborators,
obtained in the hydrolysis of the methylglucosides the most
striking examples of the influence of configuration on the attack-
ability of a substrate.
Both a- and @-methyl-d-glucosides are acted on by enzymes,
but a- and $-methyl-/-glucosides remain unchanged. While,
however, a-methyl-d-glucoside is hydrolysed only by yeast-
enzymes, emulsin attacks (3-methyl-d-glucoside alone.
In general, it appears that a-glucosides are decomposed by
maltase and ^-glucosides by almond-emulsin.
OCHs
CH 3
HO-C-H
r.
H-C
K-C OH
CH 2 OH
a-methylglucoside.
H-C
H-C-OH
I
CH 2 OH
/3-methylglucoside.
Further cases in which enzymes hydrolyse stereoisomeric
compounds with very unequal velocities are given by the inves-
tigations of Fischer and of Abderhalden on poly-
peptides. Some of their results are as follows:
Hydrolysed.
d-Alanyl-d-alanine
d-Alanyl-Z-leucine
Z-Leucyl-Meucine
/-Leucyl-c?-glutamic acid
Not hydrolysed.
d-Alanyl-Z-alanine
Z-Alanyl-rf-alanine
Z-Leucylglycine
Z-Leucyl-d-leucine
d-Leucyl-Weucine
Since the view was advanced that enzymes act as optically
active catalysts, numerous cases of enzymic, asymmetric syntheses
and decompositions have been observed.
Asymmetric Syntheses. If a symmetrical mole-
cule gives rise to an asymmetric one, the dextro- and laevo-
278 GENERAL CHEMISTRY OF THE ENZYMES
modifications are formed in equal quantities, so that an inactive,
racemic preparation is obtained. It is, however, otherwise if a
molecule which is already asymmetric is employed for further
syntheses. If in one of the two optical antipodes, I and II,
say, in I, one of the substituents 6 is replaced by another
radicle r, two new forms, i and @i, may arise. Since in these
two forms the new group r is at different distances from the
remaining constituents of the molecule, it is evident that the
molecules i and gi will be formed with unequal velocities.
Just as two diastereomeric, 1 asymmetric products i and @i
are formed from configuration I, so also II, which is the mirror-
image of I, yields two corresponding diastereomerides, a 2 and @ 2 ,
these being mirror-images to ai and gi. The forms I and II
thus give the two pairs of optical isomerides, ai+a2 and Pi + fe,
in different amounts.
As an example of an asymmetric synthesis analogous to
those occurring in the living organism, the following case, which
was investigated by Marckwald, may be taken :
Methylethylmalonic acid was converted into an acid salt:
C0 2 H C0 2 M CO 2 M
CH 3 -C-C 2 H 5 a CH 3 -C-C 2 H 5 & C 2 H 5 -C-CH 3
C0 2 H C0 2 H C0 2 H
The two forms of the acid salt, a and g, . as optical antipodes'
possess similar properties, except as regards the sense of their
rotation. This is the case, however, only if M itself is optically
inactive. But if M itself be an optically active radicle, as, for
instance, if the acid brucine salt of the acid were formed, the
1 Stereoisomeric compounds related as an object to its image in a mirror,
are termed optical or enantiomorphous isomerides.
On the other hand, stereoisomeric compounds which are not mirror imageSj
one of the other, are named diastereoisomerides or dias-
tereomerides.
SPECIFICITY OF ENZYME ACTION 279
two forms a and @ will no longer be enantiomorphs but diastereo-
merides and hence will exhibit different physico-chemical be-
haviour. If the mixture of a- and $-forms is heated so as to
remove the free carboxyl groups, the dextro- and laevo-salts
must be formed in unequal amounts. The free valeric acids
obtained by removal of the brucine residue, form an optically
active mixture. According to Fischer's expression, from
one active molecule (brucine), " another is born."
A series of interesting syntheses was also carried out by
McKenzie (Journ. Chem. Soc., 1904, 85, 1249) who, by
reduction of Z-menthyl benzoylformate with aluminium-amalgam,
obtained a mixture of Z-menthyl d-mandelate with a slight excess
of Z-menthyl Z-mandelate.
Shortly afterwards, by the reduction of Z-menthyl pyruvate,
he succeeded in preparing laevo-lactic acid (Journ. Chem. Soc.,
1905, 87, 1373). With the help of Grignard's reaction,
other asymmetric syntheses, such as that of laevo-atrolactinic
acid from menthyl benzoylformate and magnesium menthyl
iodide, were effected (Journ. Chem. Soc., 1906, 89, 365). Mc-
Kenzie and Wren prepared the optically active tartaric
acids by oxidation of d- and /-bornyl and menthyl fumarates
(Journ. Chem. Soc., 1907, 91, 1215).
Of the investigations in this direction those of D a k i n
deserve special mention. After W. Marckwald and
A. McKenzie had succeeded in showing that the velocities
of esterification of two opposed optically active acids by one
and the same optically active alcohol were not equal (Chem.
Ber., 1899, 32, 2130; 1901, 34, 469), Da kin found (Journ. of
Physiol., 1903, 30, 253) that, when partially hydrolysed by
lipase, inactive menthyl mandelate yields a strongly dextro-
rotatory mandelic acid, while the remaining ester is correspond-
ingly laevo-rotatory; the dextro-component of the ester is hence
hydrolysed more rapidly than the laevo-component. 1 Further
experiments by this investigator have led to a number of inter-
esting conclusions (Journ. of Physiol., 1905, 32, 199). It must
be remembered that two optical antipodes, in combining with
one and the same asymmetric substance, do so with unequal
velocities and that, on the other hand, the products of such
1 The hydrolysis of racemic esters has found practical application also
in the preparation of optically active amino-acids (Warburg).
280 GENERAL CHEMISTRY OF THE ENZYMES
reactions decompose at different rates. This was the case with
D a k i n ' s asymmetric ester-hydrolysis by lipase; the latter
must therefore be an optically active substance which enters
into combination with the ester it hydrolyses. Experiment
showed further that, in the fractional hydrolysis of a series of
structurally allied racemic esters, the components which are the
more rapidly attacked always possess similar configurations but
not necessarily rotations of the same sign.
Asymmetric hydrolysis by means of lipase is also effected if
an asymmetric carbon atom is present, not in the acid- but in
the alcohol-residue of the ester. D a k i n therefore drew the
conclusion that combination between enzyme and ester takes
place, not exclusively at the acid-group but probably with the
molecule of the ester as a whole.
Finally, a number of earlier (S c h u 1 z e and Bosshard,
H., 1886, 10, 134) or isolated observations, and also the more
recent ones of A. McKenzie and A . Harden (Journ.
Chem. Soc., 1903, 83, 424) show that the specificity of the action
of micro-organisms on optical antipodes is not complete. The
enantiomorph less preferred as nutriment is also consumed by
micro-organisms, although considerably more slowly and imper-
fectly, and even in cell-free (active) enzyme solutions, in certain
cases at least, neither of the two forms seems to remain unat-
tacked. Here also, quantitative measurements of the relative
attackability of the antipodes promise valuable results.
While D a k i n ' s experiments dealt entirely with asymmetric
hydrolyses, Rosenthaler has recently described (Biochem.
Z., 1908, 14, 238) a true asymmetric synthesis, namely, the forma-
tion of d-benzaldehydecyanohydrin from benzaldehyde and
hydrocyanic acid under the influence of emulsin. Of his exper-
iments the following may be described :
To 5 grms. of emulsin, macerated with 20 c.c. of water, was added
0-675 grm. of hydrocyanic acid; after an hour, 20 grms. of benzaldehyde
were slowly added, the liquid being kept thoroughly shaken meanwhile.
The liquid was then agitated in a shaking machine for an hour, after
which the nitrile was isolated and hydrolysed, and the mandelic acid
extracted from the aqueous solution by means of ether. The residue
from the ether, after crystallisation from benzene, showed a specific
rotation of []/>= -153-78, which is in good agreement with the value
SPECIFICITY OF EJNZYME ACTIOJN
for mandelic acid. The cyanohydrin formed was free from the laevo-
form, as was shown by hydrolysis to mandelic acid.
As Rosenthaler found, in the emulsin there is a sub-
stance which conditions the asymmetry of the synthesis and
another constituent which accelerates the addition of hydro-
cyanic acid to aldehyde or ketone. The latter of these substances
proves to be a compound of magnesium, calcium or potassium.
The explanation of this phenomenon is probably to be sought
in Franzen's recent investigation (Chem. Ber., 1909, 42,
3293), which showed that aldehydes and ketones, with calcium,
barium, strontium or magnesium cyanide, lead to the formation
of the salt of the corresponding nitrile. This reaction proceeds
as follows:
/0-Ca-Ov
2C 6 H 5 C<f +Ca(CN) 2 = C 6 H 5 Ctt( >CH - C 6 H 5 .
X H \CN CW
Of great interest is the fact that Rosenthaler (Biochem.
Z., 1910, 26, 1 and 28, 408) has succeeded in preparing from
emulsin, besides the nitrile-synthesising enzyme (a-emulsin)
which we shall term n i t r i 1 e s e , an enzyme which exerts
solely a hydrolytic action and was named by him B-emulsin.
By protracted heating at 40-45 the B-emulsin is inactivated
completely and the hydrolytic enzyme partially, whilst the
nitrilese remains active.
Suitable treatment with acid and subsequent neutralisation
with alkali also destroys the amygdalin-resolving action of emulsin,
while the synthetic action (of the nitrilese) is to some extent
retained.
The nitrates obtained after precipitating with copper sul-
phate, saturating with magnesium sulphate or half saturating
with ammonium sulphate, contain no nitrilese but still hydrolyse
amygdalin.
In the decomposition of amygdalin, three enzymes must
hence take part (cf. p. 23): an amygdalase, a g-glucosidase
and a n i t r i 1 a s e which resolves the mandelonitrile; the two
first are hydrolysing enzymes, whilst the last has a purely decom-
posing action.
Rosenthaler has attempted to separate his -emulsin
further into these three constituents in the following manner:
282 GENERAL CHEMISTEY OF THE ENZYMES
1. The filtrates obtained after precipitation with copper sul-
phate and half saturating with ammonium sulphate contained
all the three enzymes.
2. The filtrate obtained after saturating with magnesium
sulphate contained hydrolysing enzyme, but no nitrilase.
No other means could be discovered of separating the hydror
lysing enzyme from that which decomposes the nitrile.
No satisfactory theoretical treatment of the co-existence
and co-operation of a synthetic and a decomposing enzyme has,
in the author's opinion, yet been advanced. Mention must,
however, not be omitted of the theory developed by F a j a n s
(Dissertation, Heidelberg, 1910; Zeitschr. f. physikal. Chem.,
1910, 73, 25).
An interesting case, closely related to the above, has been
described by B r e d i g and F a j a n s (Chem. Ber., 1908, 41,
752). The two optically active camphocarboxylic acids, which
readily decompose into camphor and carbon dioxide when heated :
CioHi 5 CO 2 H = CioHi G 0+C0 2 ,
do so with different velocities when they are dissolved either in
pure nicotine or in a solvent containing nicotine. The follow-
ing results were obtained:
Velocity of liberation of carbon dioxide in nicotine at 70.
Per 1 grm. dextro-acid.
fed
Dissolved in 3 c.c. nicotine . 0-00493
" 5 0-00493
" 10 " . 0-00479
Mean.. . 0-00488
Per 1 grm. laevo-acid.
Dissolved in 5 c.c. nicotine . 0-00436
" 5 " . 0-00444
10 " . 0-00421
Mean.. . 0-00434
Hence in nicotine as solvent, the d-acid decomposes about
13% more rapidly than the Z-acid. Salt-formation evidently
takes place between the active acid and the active base, the
diastereomeric bodies thus formed differing in their chemical
behaviour.
In the case studied by B r e d i g , the carbon dioxide liberated
is evolved, so that the nicotine previously combined with the
camphocarboxylic acids becomes free after the decomposition
and can form salt with fresh quantities of acid.
SPECIFICITY OF ENZYME ACTION 283
The difference between the experiments of Marckwald
and those of B r e d i g consists in the employment by the latter
of a weaker base. The reaction studied by B r e d i g hence
assumes the character of a catalytic process.
Nevertheless, both the results obtained by Marckwald
and those of B r e d i g support the view, first expressed by
Fischer, that the enzymes are optically active catalysts.
Their mode of action may apparently be expressed as follows:
By combination with the racemic substrate, the optically
active enzymes give rise to diastereomeric substances, which
decompose with different velocities and hence result in the forma-
tion of optically active material.
E . Fischer made an interesting experiment to ascertain
if two oppositely active acids, d- and Z-camphoric acids, hydro-
lyse cane-sugar with different velocities, but the result was neg-
ative. The probability of the assumption that catalysing acids,
like enzymes, combine with the substrate, suggests the extension
of this experiment and the making of others in which a substrate
consisting of two enantiomorphs shall be decomposed by an
optically active catalyst. The author has been occupied with
such experiments for several years. It is evident that all facts
are of value which furnish further knoweldge of the union between
enzyme and substrate.
By the representation of the key fitting the lock, that is, by
the hypothesis that the enzymes are optically active catalysts,
the enzymes are brought into close relation with other catalysts.
The development of this hypothesis is undoubtedly one of the
most important aims of the chemistry of the enzymes.
CONCLUSION
WHAT then can be given as the results of the investigation
of the enzymes?
As regards the chemical nature of the enzymes, the result
of our survey is but negative, inasmuch as the analyses and chem-
ical reactions of various enzyme preparations furnish no evidence
in support of the statement often found in the literature 1
that enzymes are protein substances; further there is nothing
to indicate that all enzymes belong to a single class of substances.
1 See, for instance, V e r n o n , Ergeb. der Physiol., 1910, 9, 227.
284 GENERAL CHEMISTRY OF THE ENZYMES
On the other hand, no fact is known which definitely disproves
the protein character of any of the hydrolytic enzymes, since the
results indicating the failure, of enzyme solutions to give protein
reactions do not give the concentrations of the solutions and have
not been sufficiently controlled by means of similarly dilute solu-
tions of undoubted proteins.
All that has yet been stated concerning the chemical con-
stitution is mere supposition. Better than from the purely
chemical investigations, we could, from the physico-chemical
measurements of thermo-sensibility, i.e., from the inactivation
constants, attempt to derive certain relations with the proteins,
the denaturation of these by heat bearing a close resemblance
to that of the enzymes. But perhaps the saponins exhibit still
more marked analogies to those remarkable colloidal poisons,
the physico-chemical behaviour of which is still insufficiently
investigated.
The view that, for the development of their activity towards
the substrate, certain enzymes require the presence of other
substances co-enzyme, acid, etc. which are classed together as
activators, has resulted in a thorough qualitative investigation
of the chemistry of enzyme-action, and the consideration of
these activators is of the utmost importance to the chemico-
dynamic study of the enzymes.
The many deviations of the best-known enzymes, e.g.,
invertase, from the simple relations required by the law of mass
action, had led to a formal treatment of enzymic reactions, but
the results of this correspond, by no means, with the amount
of labour expended. Only in the most recent times has the nec-
essary revision of the earlier experiments been commenced; never-
theless, it cannot be regarded as premature to assert that the
reactions induced by enzymes the enzymic hydrolyses being
here especially referred to follow the laws which hold generally
for catalytic reactions in solution and are deducible theoretically
from the law of mass-action. Correspondence of the time-law
with that for unimolecular reactions, and proportionality between
concentration of the enzyme and velocity of reaction were fre-
quently observed. Where these relations are not obeyed, the
disturbing influence exerted by the products of the reaction
either is known with certainty or may be assumed with a high
degree of probability. In individual cases, the ultimate cause
SPECIFICITY OF ENZYME ACTION 285
of this disturbance is still uncertain; sometimes it must be the
enzymes themselves, but in many instances the activators,
which are combined, the latter case appearing to be the more
common. In any case, we have seen that a simple explanation
is forthcoming for S c h u t z ' s rule; relations similar to that of
S c h ii t z can also occur with inorganic catalysts, one of the
best-known, apparent peculiarities of enzymic reactions thus
falling to the ground. For, that the numerous " laws " such as
7 1 a+x
etc., possess neither real significance nor validity may be regarded
as an established and pleasing fact.
The enzymes are, therefore, catalysts. Do they, like the
inorganic catalysts of the best-known reactions, for instance,
hydrolysis of esters, leave the equilibrium of the reaction
unchanged? Perhaps, under some circumstances and more fre-
quently than now appears to be the case, they do so, but it can
be stated with certainty that this does not always happen, and
the conception of a catalyst must hence be made more compre-
hensive than experience of non-enzymic reactions requiies. 1
But this leads to no disagreement with the principles of chemical
dynamics or with the fundamental laws of thermodynamics, it
being only necessary to assume that a considerable proportion
of the enzyme can combine with the substrate or with the pro-
ducts of the reaction. The equilibrium must evidently depend on
the concentration of these new molecules, enzyme-substrate or
enzyme-reaction product, and any circumstance altering this
concentration alters also the equilibrium or the stationary con-
dition of the reaction.
The configuration of the substrate, the spatial arrangement
of its atoms, is, as was seen in the preceding chapter, of deter-
mining importance for the occurrence of an enzymic reaction.
Emil Fischer has given us the theory for these facts, the
underlying assumption being that an enzyme is an optically
active catalyst. This forms, with the two components of a racemic
mixture, " active molecules " which are not enantiomorphous
but diastereomeric products, differing in their chemical properties
1 Cf. Taylor, Journ. of Biol. Chem., 1910, 8, 503.
286 GENERAL CHEMISTRY OF THE ENZYMES
and hence leading to the decomposition of the components with
unequal velocities. Thus, what formerly appeared to char-
acterise the catalysts of living matter presents itself, in the light
of this theory, as a consequence of our views of the configuration
of molecules.
But what is the nature of the combination between the
enzyme and the substance succumbing to chemical attack,
and how does the living organism maintain the equilibrium
between enzyme and substrate so important to its existence?
Here we meet the great riddle of the formation of enzymes
and anti-enzymes in the organism which opens out a region of
investigation of immeasurable breadth.
APPENDIX
PRACTICAL METHODS
IN what follows, a short description is given of those methods
of investigating enzyme-preparations and of following enzymic
decompositions which have been or might be generally applied
either in medicinal practice or in industrial work.
Scientific investigation of enzymic reactions has often been
effected with the acid of physico-chemical methods, but a detailed
account of these would occupy too much space here, so that
reference must be made to the special literature of the subject.
Particular mention may be made of :
O s t w a 1 d and Luther : Manual of Physico-chemical
Measurements.
W. A . R o t h : Exercises in Physical Chemistry, London,
1909.
Hamburger : Osmotischer Druck und lonenlehre in den
medizinischen Wissenschaften, Wiesbaden, 1902-1904.
Also shorter references by:
H. Friedenthal, L. Michaelis, etc., in Abder-
halden's Handbuch der biochemischen Arbeitsmethoden, Berlin,
1910-1912.
A . K a n i t z , in Oppenheimer's Handbuch der Biochemie
des Menschen und der Tiere, Band I, 1908.
As regards the methods of obtaining enzymes, these have
been given in the first chapter of this book, where also the
preparation of the separate enzymes in a pure state has been
described.
It is only necessary here to call attention to the fact that
enzymes which exert their actions within the walls of a cell are
often either not at all or only very incompletely extractable, so
287
288 GENERAL CHEMISTRY OF THE ENZYMES
long as the cell- wall is living and uninjured; the dead cell-wall
allows of much readier passage to the enzyme, which can thus
be obtained in several ways.
1. The enzymic material is dried as rapidly, and at as low a
temperature, as possible. By this treatment, the cell-walls are
rendered, in st)me cases, more permeable and in others, more
easily ruptured; these effects are enhanced if the dehydrated
cells are heated at about 50-70 [cf. E. Fischer's
method of preparing invertase (Chem. Ber., 1894, 27, 2985);
and Wiechowski and Wiener's method (Hofm. Beitr.,
1907, 9, 232) for preparing from the kidneys the enzyme which
oxidises uric acid].
2. The finely-divided material, e.g., yeast, after being freed
mechanically from water, is introduced into absolute alcohol
or anhydrous acetone. Here also, the dehydration of the mate-
rial by the organic solvent should be as rapid as possible.
3. The cell-walls may be destroyed by autolysis (see O ' Sul-
livan and Tompson's method, p. 26).
4. The cells, while still living, are ruptured mechanically.
The method so successfully employed by Buchner (see p.
56) is well known. Rowland (Journ. of PhysioL, 1901,
27, 53) gave a somewhat different method, in which a mixture
of the cells with sand is made to assume a vigorous rotatory
motion; the action resembles that of a sand-blast.
Bacteria, soft organs, etc., can be hardened by cooling in
liquid air and are then readily broken up.
The analytical methods employed may now be
mentioned.
L i p a s e s . Pancreas-lipase is very suitably tested by
means of an aqueous emulsion of egg-yolk, as in the work of
V o 1 h a r d and others. The quantity of fat hydrolysed in time
t is measured and the total amount hydrolysable then cal-
culated.
To this end, the egg-yolk emulsion containing the lipase is
extracted with ether: (I) An aliquot part (50 c.c.) of the ethereal
extract is titrated after addition of 50 c.c. of alcohol and then
hydrolysed with 10 c.c. of normal sodium hydroxide solution,
the salts of the fatty acids being decomposed after 24 hours by
means of 10 c.c. of normal sulphuric acid. (II) The fatty acids
APPENDIX 289
obtained by hydrolysis are estimated by titration and the per-
centage x of fatty acid split off by the enzyme calculated by the
formula, I : I+II = z : 100.
In shaking the fat-emulsion with ether, more rapid separation
is effected if 2-10 c.c. of alcohol are added to the ether.
In this connection see Stade, Hofm. Beitr., 1902, 3, 291,
and E n g e 1 , Hofm. Beitr., 1905, 7, 78.
Esterases of lower esters. Ethyl butyrate is
best employed as substrate. The course of the reaction is fol-
lowed by direct titration or by observation of the change of the
electrical conductivity.
Vegetable lipases. Ricinus seeds are skinned, freed
from oil by pressing and treating the pressed cake with ether,
and finely ground. The seed-juice formed is separated from the
inactive constituents of the seed in a centrifuge. This juice is
allowed to stand for 24 hours, during which time the enzymic
emulsion, in which the acid (lactic) necessary for activation is
formed, collects at the surface and can be removed. One hun-
dred grms. of oil and 0-2 grm. of manganous sulphate are stirred
up with this emulsion (5-10 grms.) and the mixture left. Here
also the lipolysis can be followed by titration.
A m y 1 a s e s . For the estimation of the diastatic power of
malt for brewery purposes, L i n t n e r (Zeitschr. f. prakt.
Chem., 1886, 34, 386) gave the following method, which, in prac-
tised hands, gives good results.
Separate volumes; of 0-1, 0-2, 0-3, .... 1-0 c.c. of malt
extract [25 grms. of the ground malt +500 c.c. of water, allowed
to stand at 21 (70 F.) for 3 hours and then filtered bright]
are added to a series of 10 test-tubes, each containing 10 c.c. of
2% soluble starch solution, the contents of each tube being well
mixed. After exactly 1 hour's rest at 21, 5 c.c. of Fehling's
solution are mixed with the liquid in each tube and the tubes
then immersed in a boiling water-bath for exactly 10 minutes,
after which the precipitate is allowed to settle. If the Fehling's
solution in the tube containing 0-1 c.c. of malt extract is just
completely reduced, the diastatic power of the malt is taken as
100; if that in the one containing 0-2 c.c. of the extract, the
diastatic power is 50, and so on. A more exact result may be
290 GENERAL CHEMISTRY OF THE ENZYMES
obtained, if necessary, by taking 0-1, 0-15, 0-2, 0-25, etc., c.c.
of malt extract for the series of tubes.
The cold-water malt extract itself contains a small amount
of sugars which reduce Fehling's solution; the extent of this
reduction may be determined by direct experiment, but for all
ordinary purposes it is sufficiently accurate to deduct 1*5 from
the value obtained for the diastatic power in the manner described
above.
The following modification of the above method has been
recently devised by Ling and is widely used : 3 (or 1 0, 2 0,
or 4-0, according to the expected diastatic power) c.c. of the
malt extract (prepared as already described) are added to 100
c.c. of 2% soluble starch solution in a 200-c.c. flask, the mixture
being kept at 21 (70 F.) for 1 hour. At the end of this time,
20 c.c. of N/10-sodium hydroxide solution are added and the
liquid made up to 200 c.c. with water. After mixing, this solu-
tion is introduced into a burette and gradually run into 5 c.c.
of Fehling's solution diluted with a little water and kept boiling;
this is continued until the solution just loses its blue colour or
fails to give a brown coloration with a drop of ferrous thiocyanate
solution on a white tile. If, say, 25 c.c. of the liquid (100 c.c.
of which corresponds with 1 grm. of soluble starch and 1-5 c.c.
of malt extract) are required tc reduce 5 c.c. of Fehling's solution,
the diastatic power of the malt will be
1000 =26-7.
25X1-5
This method gives excellent results, in exact agreement with
those given by L i n t n e r 's method.
With preparations of diastase, L i n t n e r dissolves 2-0 5
grm. (according to the activity) in 50 c.c. of water and adds 0-1,
0-2, . . .1-0 c.c. of this solution to a series of 10 test-tubes, each
charged with 10 c.c. of 2% starch solution. The subsequent
procedure is exactly similar to that employed in the case of malts.
A soluble starch which can be readily prepared with constant
properties, is obtained by allowing potato starch to remain
under 7 5% hydrochloric acid solution for 7 days at the ordinary
temperature, then removing the acid completely by washing
with cold water, and drying the starch in the air. This method
APPENDIX 291
yields a product dissolving readily in hot water to a clear solution
(cf. G.C.Jones, Journ. Inst. of Brewing, 1908, 14, 13).
The determination of the velocity of hydrolysis by oxidation
of the liquid with F e h 1 i n g ' s solution (cf . Wroblewski,
H., 1898, 24, 173) is, indeed, the most reliable of the methods
yet developed, although it is still capable of improvement.
A gravimetric method, which seems to give good results,
has recently been proposed by Sherman, Kendall and
Clark (Journ. Amer. Chem. Soc., 1910, 32, 1073), who have
also compared the older methods.
A number of other methods are based on the colorations
produced by iodine in starch and dextrin solutions. Of the
earlier methods, those of D e t m a r (H., 1882, 7, 1) and
Roberts (Proc. Roy. Soc., 1881, 32, 145) may be referred to,
whilst the following method, given by Wohlgemuth
(Biochem. Z., 1908, 9, 1), deserves special mention.
To each of a series of test-tubes containing different quanti-
ties of the enzyme solution to be tested are added 5 c.c. of 1%
starch solution, each tube being placed immediately in a wire
basket standing in ice-water, so as to prevent the slightest enzyme
action. When all the tubes are ready, the wire basket contain-
ing them is transferred to a water-bath at 40; by this means,
the enzyme action in each tube begins at the same moment.
After 30 or 60 minutes, the basket is placed again for a short time
in ice-water, so that all the actions are interrupted at the same
instant. The strength of the enzyme solution is then deter-
mined as follows:
All the tubes are filled with water up to within about the
thickness of the finger from the top, and to each is added a drop
of decinormal iodine solution, the liquid being then mixed.
Different colorations dark blue, bluish-violet, reddish-yellow
and yellow are thus obtained. The tubes showing a yellow or
reddish-yellow colour contain disregarding further degradation
of the starch to maltose or isomaltose and dextrose only achro-
odextrin or erythrodextrin, the bluish-violet ones contain a mixture
of erythrodextrin and starch, whilst those with a dark-blue
colour contain mainly unaltered starch. As the lower limit of
the activity (limes) Wohlgemuth takes the first tube in
which the blue colour cannot be detected, i.e., the one showing
292 GENERAL CHEMISTRY OF THE ENZYMES
a violet colour. In the preceding tube, the whole of the starch
is broken down to the dextrin stage at least; from this, calcula-
tion is made of the number of c.c. of 1% starch solution degraded
to dextrin by 1-0 c.c. of enzyme in the time employed for the
experiment.
Suppose that the tube which has become just colourless
contained 0*02 c.c. of saliva; this amount was then able to trans-
form 5 c.c. of 1% starch solution into dextrin in 30 minutes,
so that 1 c.c of saliva corresponds with 250 c.c. of 1% starch
solution. To indicate the diastatic power of 1 c.c. of the enzyme
solution, Wohlgemuth contracts the word diastase to D
and suggests that the temperature and duration be given for
each experiment. The result of the above determination would
then be stated thus: D 3Q , = 250. To calculate the diastatic
power from an experiment in which tube No. 5 was taken as the
lower limit, while tube No. 4, containing 0-0125 c.c. of saliva,
was coloured red (see the scale of colour given, 1 o c . c i t .) ,
we must proceed as follows:
In 30 minutes, 0-0125 c.c! degrades 5 c.c. 1% starch solution.
In 30 minutes, 1-0000 c.c. degrades 400 c.c. 1% starch solution,
Other colorimetric iodine methods are given by J u n g k
and by Johnson (Journ. Amer. Chem. Soc., 1908, 30, 798).
A criticism of these methods will be found in Sherman,
Kendall and C 1 a r k 's paper (1 o c. cit.).
G 1 i n s k i and Walther (Pawlow, Arb. d. Ver-
dauungsdrlisen, Wiesbaden, 1898) have applied M e 1 1 ' s method
to the estimation of diastase. Narrow glass tubes, open at both
ends, are filled with starch-paste and immersed in the enzyme
solution, the length of the dissolved cylinder of starch being meas-
ured after a certain lapse of time. The velocity of this reaction
is evidently influenced considerably by the diffusion of the enzyme
to the surface of the starch and by the rate at which the hydrolytic
products are removed from this surface; movement of the liquid
or of the tubes in the liquid has, therefore, considerable effect.
On the other hand, this method of procedure measures the liquefac-
tion of the starch-paste which is not a direct measure of the sac-
charifying action of the amylase. The method is therefore
applicable only in special cases.
APPENDIX 293
According to E d . M u 1 1 e r (Zentralbl. f. inn. Med., 1908),
the use of plates of starch-paste presents certain advantages.
Enzymes of the Disaccharides and G 1 u-
c o s i d e s . In these cases, the change of the optical rotation
affords a simple and accurate method of following the reaction.
As was pointed out on p. 159, it is essential to destroy the muta-
rotation of glucose; this is best effected by the addition of soda
immediately before reading the rotation.
Another method consists in determining the reducing power
of the solution. This is carried out with Fehling's solution in
one of a number of ways, of which that of Bertrand is one
of the most accurate and convenient.
Bertrand (Bull. Soc. Chim., 1906, 35, 1285) boils the sugar
solution to be tested with Fehling's solution of definite com-
position for three minutes, the time being reckoned from the instant
when the first bubbles form. The precipitated cuprous oxide is filtered
on an asbestos filter and washed with hot water. The cuprous oxide
remaining in the E r 1 e n m e y-e r boiling flask and also that collected
on the filter are dissolved in a solution of ferric sulphate in sulphuric
acid, the following reaction occurring:
Cu 2 +Fe 2 (S0 4 ) 3 +H 2 S0 4 = 2CuS0 4 +H 2 +2FeS0 4 .
The ferrous salt is titrated with permanganate solution, standardised
by means of ammonium oxalate. Bertrand has prepared tables
for the most important reducing sugars, so that the amount of sugar
can readily be obtained from that of the cuprous oxide formed.
The solutions employed have the following compositions:
F~e hling's solution. Iron solution.
Copper sulphate 40 grms. Ferric sulphate 50 grms.
Rochelle salt 200 Sulphuric acid 200
Sodium hydroxide 150 " Water to 1 litre
Water to 1 litre
Permanganate solution
5 grms. potassium permanganate per litre.
The iron solution should not reduce the permanganate. If this
does occur, the permanganate solution is gradually added to the iron
solution until the latter assumes a slight pink colour; it is then ready
for use.
294 GENERAL CHEMISTRY OF THE ENZYMES
When a sugar solution is to be titrated, 20 c.c. of it are intro-
duced into an Erlenmeyer flask of 125-150 c.c. capacity. The
amount of sugar in this volume of the solution is preferably
0-010-0-090 grm. and should not exceed 0-100 grm.
To the sugar solution are added 20 c.c. of the copper sulphate
solution and 20 c.c. of the alkaline tartrate solution which
are best kept separate the liquid being then boiled for just 3
minutes after the first appearance of bubbles.
For separating the cuprous oxide, use is made of a Gooch
crucible packed with asbestos and fitted as usual to a pump-
flask. The precipitate is washed with hot water and as little as
possible of it collected on the filter. When the washing is complete,
the main quantity of cuprous oxide in the Erlenmeyer flask is
dissolved in a known volume of the ferric sulphate solution;
the oxide changes from bright red to blue-black and finally
yields a clear, pale-green solution. This is then poured through
the filter to dissolve the remaining cuprous oxide, more ferric
sulphate solution being added if necessary. When all the oxide
is dissolved, the Erlenmeyer flask and the filter are washed with
water and the combined liquids titrated in the pump-flask with
the permanganate solution. The change in colour from green
to pink is extremely sharp.
The equation given above shows that 2 atoms of copper
correspond with 2 mols. of ferrous sulphate and hence with
2 atoms of iron to be oxidised by the permanganate. The
iron-titre of the permanganate has thus only to be multiplied
by the ratio,
63-6 : 55-9-1-1377,
in order to obtain the amount of copper, the corresponding
quantity of the sugar being then given by the tables.
The permanganate solution is standardised as follows: a
weighed quantity of about 0-25 grm. of ammonium oxalate is
dissolved in a beaker in 50-100 c.c. of water and 1-2 c.c. of pure
sulphuric acid. The liquid is heated to 60-80 and the perman-
ganate solution run in from a burette until a pink colour is obtained.
One molecule of ammonium oxalate, (NH4) 20264 -HH^O
(mol. wt., 142-1) corresponds with 2 Fe and hence with 2 Cu.
rO > vx O
Multiplication of the weight of oxalate by ~ 142 -1 ' *' 6 '' ^
APPENDIX 295
0-8951, gives the quantity of copper corresponding with the
volume of permanganate solution required to produce the pink
coloration. One litre of the permanganate solution will cor-
respond with about 10 grms. of copper.
The tables to be used with this method are given at the end
of this section (pp. 306-311).
I. B a n g (Biochem. Z., 1906, 2, 271) has described a new
method for the estimation of reducing sugars which may be applied
to the study of enzymic processes. It depends on the fact that,
in presence of potassium thiocyanate, cuprous oxide separates
as white, insoluble copper thiocyanate. The description of the
method is readily accessible and will not be given in detail here.
E m u 1 s i n . In discussing the possible methods for measur-
ing the decomposition of amygdalin, A u 1 d points out that the
estimation of the sugar liberated is affected by a number of
considerations. Here also, the influence of mutarotation must
be removed. He employs, therefore, in the investigation referred
to on p. 173, D u n s t a n and Henry's titrimetric method
(Proc. Roy. Soc., 1903, 72, 287) of estimating the free hydro-
cyanic acid by means of standard iodine solution.
The reaction proceeds according to the equation:
HCN+I 2 = CNI+HI.
Excess of sodium bicarbonate is employed to combine with the
hydriodic acid formed.
Proteolytic Enzymes
On account of the great importance of measurements of
digestive action to pure enzymology and also to practical medicine,
the number of communications dealing with methods employed
in these measurements is very large.
1 . An optical method was employed byE.Schiitz
(compare p. 175). He removed the undigested protein from
the peptic albumin solutions and estimated the quantity of
peptone formed from the optical rotation of the residual liquid.
S c h ii t z and H u p p e r t also used this polarimetric
method (see p. 179) whilst Abderhalden and K o e 1 k e r
296 GENERAL CHEMISTRY OF THE ENZYMES
have recently studied the action of tryptic enzymes on optically
active polypeptides by direct measurement of the change of
rotation of the solution.
Ib. Obermayer and Pick (Hofm. Beitr., 1905, 7, 331)
attempted to apply the alteration of the refractive index to the study
of enzymic reactions. But this magnitude changes only in the case
of tryptic digestion.
Griitzner (Pfliig. Arch., 1874, 8, 452; 1905, 106, 463)
has devised a colorimetric method. Fibrin, which has been
softened by immersion in 0-1% hydrochloric acid containing
carmine, is distributed as uniformly as possible into test-tubes
of equal diameters, each containing 15 c.c. of 0-1% HC1, the
pepsin solution to be tested being then added. The fibrin stained
with carmine is dissolved and thus reddens the liquid; the inten-
sity of the red colour indicates approximately the extent of the
digestion. The digested liquid is compared with a number of
tubes containing carmine solutions of definite dilutions, which
are so chosen that the pepsin-content is proportional to the num-
bers of the colour scale. This method has been modified by
Roaf (Bio-Chemical Journ., 1908, 3, 188).
2. Measurement of the electrical conductivity was first
employed for the study of peptic digestion by Sjoqvist
(cf. p. 176). This method was subsequently largely used and was
applied in investigations on tryptic digestion by Henri and
by B a y 1 i s s (Journ. of PhysioL, 1907, 36, 221) and on the
hydrolysis of dipeptides by erepsin (E u 1 e r, see p. 188); in the
last experiments, a similar quantity of alkali solution was added
to each solution in order to increase the variation of the
conductivity.
3. Valid objections have been raised against the suggestion
made by S p r i g g s (cf. p. 181) to determine the progress of
proteolysis by measuring the viscosity of .the protein solutions,
and this method cannot be recommended.
4. A purely chemical method of general applicability and
great accuracy was proposed several years ago by Sorensen
(Biochem. Z., 1908, 7, 45).
By this method, the content of protein or its decomposition
products in a solution is determined from the number of free
carboxyl-groups. The latter can be estimated by titration if
APPENDIX 297 '
the free amino-groups of the protein are first combined. This
is readily effected by addition of excess of formaldehyde, which
unites with the amino-groups, giving methylene-compounds.
The increase of carboxyl-groups represents the extent of pro-
teolysis, which can hence be expressed by the number of c.c.
of N/5-barium hydroxide solution employed in the titration.
On the assumption that each carboxyl-group formed during
proteolysis corresponds with one amino-group, the amount of
proteolysis can also be stated as milligrams of nitrogen, this
being obtained by multiplying the number of c.c. of the N/5-
barytaby 2-8.
The titration is best carried out in presence of thymolphthalein
as indicator, the solutions used being as follows:
(a) 0-5 grm. of thymolphthalein (G r ii b 1 e r ' s) dissolved
in 1 litre of 93% alcohol.
(6) 50 c.c. of commercial formaldehyde solution are mixed
with 25 c.c. of absolute alcohol and 5 c.c. of the thymolphthalein
solution, N/5-baryta being then added until a faint green or
blue colour results; this solution should be prepared fresh for
each series of experiments.
As a control solution, 20 c.c. of boiled water are used. To
this are added 15 c.c. of the formaldehyde solution (b) and about
5 c.c. of the baryta solution, the liquid being then titrated back
with N/5-HC1 until it assumes a faint blue opalescence. An
addition is then made of two drops of baryta, which should change
the colour to a distinct blue, and finally of two further drops,
which should produce a vivid blue colour.
It is this last colour which is obtained in titrating the protein
solution, 20 c.c. of which is mixed with 15 c.c. of the formalde-
hyde solution (6) and a slight excess of baryta solution; it is
then titrated back with HC1 until the colour is fainter than that
of the control solution, the baryta solution being finally added
in drops until the deep blue of the control is obtained.
For the description of the titration with phenolphthalein,
the original paper must be consulted.
The methods of V o 1 h a r d (Munch. Med. Wochens., 1903,
No. 49) and L 6 h 1 e i n (Hofm. Beitr., 1905, 7, 120) are based
on methods given by T h o m a s and Weber, and by M e u -
nier (1901). In both, casein is employed; Thomas and
Weber dissolve 100 grms. of casein in 1900 c.c. of water with
298 GENERAL CHEMISTRY OF THE ENZYMES
the aid of 3-2 grms. of sodium hydroxide ( = 80 c.c. normal
NaOH) or 5-04 grms. of hydrochloric acid ( = 138 c.c. normal
HC1). The alkaline and acid solutions serve for the estimation
of trypsin and pepsin respectively. After the digestion, the
liquid is acidified, if necessary, with sulphuric acid and salted
out with 20% sodium sulphate solution. After filtration, the
precipitate is washed on the filter with hot water until the last
trace of sulphuric acid is removed; the filter and precipitate are
dried and weighed and the weight of undigested protein compared
with that obtained in a blank experiment without pepsin or
trypsin. The amount of protein dissolved gives a measure of
the digestive power of the gastric juice examined.
In Meunier's method, the gastric juice (14 c.c.) is
mixed with pure hydrochloric acid (0-4 c.c.) and casein (1 grm.).
After the casein has settled, 2 c.c. of the clear liquid are removed
and the content of free hydrochloric acid estimated. The
remainder of the liquid with the undissolved casein is kept for
24 hours in a water-bath at 40, the hydrochloric acid being again
estimated in 2 c.c. of the filtrate. Since hydrochloric acid com-
bines with protein during peptic digestion, the diminution in the
amount of the free acid expresses the extent of the action.
V o 1 h a r d employs a modification of the gravimetric
method given by Thomas and Weber. As we have seen,
the latter method is based on the observation that pure, unal-
tered casein, dissolved in the hydrochloric acid of the digest, is
completely precipitated by sodium sulphate. Hence, if different
quantities of the enzyme are allowed to act on similar amounts
of casein solution for equal intervals of time at 40, the precipitate
produced by addition of sodium sulphate will be the smaller,
the less the proportion of casein remaining undigested, i.e., the
larger the proportion peptonised by the enzyme; the larger the
residue, the smaller the quantity of enzyme. Thomas and
Weber collect the precipitate on a tared, pleated filter-paper,
wash with distilled water and dry and weigh. The difference in
weight between the residues from experiments in which pepsin
has, and has not been employed, serves as a measure of the peptic
action.
V o 1 h a r d avoids the inconvenience of this weighing by
titration of the filtrate. He proceeds on the assumption that
peptonisation of the casein solution is accompanied by increase
APPENDIX 299
of the acidity of the filtrate, the peptone hydrochlorides being
non-precipitable by sodium sulphate and hence passing through
the filter. His experiments showed that, when equal quantities
of the same acid casein solution without pepsin were used, the
acidity of the filtrate was always constant and much smaller
than corresponded with the true acidity of the original solution.
This depends on the fact that, under similar experimental con-
ditions, the casein precipitate always contains the same amount
of hydrochloric acid, only the free acid passing into the filtrate.
It is therefore justifiable to refer the excess of acidity over this
constant value to the peptone hydrochlorides in the filtrate,
and hence to regard the increase of acidity as a measure of the
extent of digestion.
The undigested residues are in inverse proportion to the
acidities of the filtrates.
As example may be given the following results of V o 1 h a r d
taken from L 6 h 1 e i n ' s paper (1 o c . c i t .) :
One hundred c.c. of casein solution, previously heated with 150 c.c.
of water, were digested with O'l, 0-4, or 0-9 c.c. of gastric juice (acidity
59 : 87) for one hour. Each solution was then made up to 300 c.c.
in a graduated cylinder and precipitated with 100 c.c. of 20% sodium
sulphate solution. Titration of 200 c.c. of the filtrate from the solution
which contained no gastric juice in presence of phenolphthalein, gave
the acidity as 19-15.
Two hundred c.c. from the other experiments gave
Increase of
acidity.
1. 0-lc.c. gastric juice, 22-25-19- 15 -acid of the juice (0-043)=3'06
2. 0-4c.c. ,25-5-19-15- (0-17) =6-18
3. 0-9c.c. ,28-5^19-15- " (0-387)=8-96
The casein precipitates were collected on weighed filters, washed,
completely dried, washed again and finally dried until constant in
weight.
Weight of precipitate from original solution, A =4- 104 grms.
1 3-607 "
2 3-053 "
3 2-585 "
The amount digested is hence, b v 1 (0-1 c.c.) AI =0-497 grm.
' 2 (0-4 c.c.) A -2 = 1-051 grms.
" 3 (0-9 c.c.) A -3 = 1-519 ''
300 GENERAL CHEMISTRY OF THE ENZYMES
The proportionality between the degree of acidity of the nitrate
and the digested amount determined by weighing, is shown by the
quotients:
Of the investigations in which this method has been widely
used, that ofS.Kuttner (H., 1907, 52, 63) may be mentioned.
In the practical application of Volhard's method, the
formulation of the results proposed by V o 1 h a r d himself is
to be recommended. The pepsin-unit is taken to be that quan-
tity of enzyme which renders the nitrate from the whole of the
casein used more acid by 1 c.c. of decinormal acid.
The digestion which would be produced by 1 c.c. of gastric juice in
1 hour is found from the experimental numbers by dividing the increase
of acidity by the product of the time, t, and number of c.c. of juice
employed, /. This number is to be multiplied by 2 or by 4, according
as 200 or 100 c.c. of the nitrate are titrated. The values thus obtained
for the increase of acidity follow S c h ii t z ' s rule, the pepsin-unit
being given by the formula,
_v*_
'"f-f
Example: Suppose the acidity of 200 c.c. of the original solution,
after precipitation and nitration, is 18*0, i.e., 36-0 per 400 c.c., and
that of the juice, 20 c.c. per 100 c.c. of juice. Then, if in the digestion
of 100 c.c. of casein solution made up to 300 c.c. with 3 c.c. of gastric
juice for 3 hours, 200 c.c. of the nitrate obtained after adding 100 c.c.
of sodium sulphate solution show an acidity of
32 -7( =65-4 per 400 c.c.),
the calculation is as follows:
less 36-0, for the original solution,
and less 0-6, for the gastric juice
v=28-8
OQ C
o Xo
= 3 2, and x = 10 24 pepsin-units.
Two simple methods, apparently well suited to the clinical
estimation of pepsin, are due to J a c o b y and F u 1 d .
According to Jacoby's method (Biochem. Z., 1906, 1,
APPENDIX 301
58), 0-5 grm. of ricin is dissolved in 5% sodium chloride solution
and filtered. An opalescent solution is obtained which becomes
turbid on addition of decinormal hydrochloric acid. Equal
volumes of this solution are mixed with diminishing quantities
of differently diluted gastric juice and then made up with dis-
tilled water or boiled gastric juice to a constant volume. After
3 hours in a thermostat, the liquids are examined to ascertain
the smallest quantity of gastric juice able to clear the solution,
i.e., to digest the protein present completely.
If, after dilution of the juice a hundredfold, 1 c.c. is just
sufficient for this purpose, the number of pepsin-units in the
original gastric juice is taken as 100 (normal gastric juice con-
tains 100-200 pepsin -units).
F u 1 d ' s method (F u 1 d and L e v i s o n , Biochem. Z.,
1907, 6, 473; see also Zeitschr. klin. Med., 1907, 64, 376) is as
follows: A clear boiled solution (1 : 1000) of crystalline edestin
in N/300-hydrochloric acid is prepared, the edestin being thus
converted into the so-called edestan.
The gastric juice to be examined is now diluted in the pro-
portion 1 : 20 and a series of dry test-tubes charged with diminish-
ing amounts of this diluted juice by means of a 1 c.c. pipette
reading to 0-01 c.c. These tubes should have a diameter of
not more than about 1 c.m., so that mixing may be avoided on
subsequent addition of ammonia.
The selected amount, say 2 c.c., of edestin solution is then
rapidly added to each tube and after a lapse of 30 minutes ammonia
solution is poured carefully into each tube, starting with the one
containing most pepsin. The tubes are then observed in incident
light against a black background, the one containing the smallest
amount of pepsin and showing no ring being noted.
The number of c.c. of pepsin solution or gastric juice con-
tained in this tube is divided by the product of its dilution and
the number of c.c. of edestin solution digested. If, therefore,
0-25 c.c. of the 1 : 20 concentration of the gastric juice is suf-
ficient to prevent the formation of the ring in 2 c.c. of edestin
solution, the number required is 0-25:20X2 = 1:160. The
gastric juice is then termed a 1 : 160 pepsin or is said to contain
160 pepsin-units.
The methods of J a c o b y and F u 1 d have been repeatedly
tested and found to be of general utility.
302 GENERAL CHEMISTRY OF THE ENZYMES
Witte (Berl. klin. Wochens.j 1907, 44, 1338) suggests in
Jacoby's method a slight but not unimportant modification :
before use, the gastric juice is exactly neutralised; the results
are thus rendered more exact.
Also Reicher (Wien. klin. Wochens., 1907, 20, 1508)
gives these methods the preference over all the older quantitative
methods. He, too, found the influence of acidity to be very
considerable and agreed with W i 1 1 e ' s proposal to neutralise
the gastric juice. S o 1 m s (Zeitschr. klin. Med., 1907, 64, 159)
likewise obtained favourable results with Jacoby's method.
E i n h o r n (Berl. klin. Wochens., 1908, 45, 1567) suggests
the simplification of Jacoby's method by the use of an appa-
ratus of special construction. This apparatus, which is a glass
vacuum-vessel, contains water at 50-60 and a stand holding
graduated test-tubes charged with the digestion mixture. The
time of a test may thus be shortened to 30 minutes.
Of the researches in which F u 1 d and L e v i s o n ' s method
has been successfully employed, those of Wo 1 f f and von
Tomaszewski (Berl. klin. Wochens., 1908), 45, 1051) deserve
special mention.
In M e 1 1 ' s method, hens'-egg albumin is drawn up into
a glass tube 1-2 m.m. in diameter and coagulated in the tube at
95, lengths of about 2 c.m. of the tube, cut sharply off, being
then immersed in the peptic liquid. The length of the digested
cylinder of the albumin is measured after 10 hours; it should
not exceed about 6 m.m. The amount of pepsin is proportional
to the square of this length.
According toNierenstein and S c h i f f (Archiv. f. Verd.
Krank., 1902, 8, 559), the gastric juices to be compared should
be brought to equal degrees of acidity.
These methods, which are open to the objections indicated
on p. 292, resemble that of F e r m i founded on the solution of
layers of solidified gelatine.
H a 1 1 o r i (Arch, internat. de Pharm. et de Therap., 1908,
18, 255), points out that, in general, gelatine is digested much
more rapidly than coagulated albumin and that two different
enzymes may be in question. Such a statement is, however,
hypothetical and the criticism of Fermi's method based thereon
insufficiently supported.
APPENDIX 303
Estimation of Trypsin and Erepsin
Most of the methods given above for the estimation of pepsin
may be used, with suitable modification, to follow tryptic diges-
tion. This is the case with the optical methods, the conductivity
method (cf. Henri, and B a y 1 i s s , p. 186), etc.
The application of V o 1 h a r d' s method (cf . p. 297) to
the estimation of trypsin is described by L 6 h 1 e i n (1 o c .
c i t .) ; the only difference is that the hydrochloric acid is added
to the casein after the digestion, whilst in the investigation
of pepsin it is added before digestion.
A similar method was employed by R. Goldschmidt
(Deut. med. Wochens., 1909, 35, 522).
J a c o b y ' s ricin method also serves for the estimation of
trypsin (Biochem. Z., 1908, 10, 229). Two c.c. of a solution of
1 grm. of Merck's ricin in 100 c.c. of 1-5% sodium chloride
solution are placed in each of a series of tubes together with
0,0-1, 0-2,0-3, 0-5, 0-7, 1-0 c.c.
of a 1% solution of Griibler's trypsin. Water is added to
bring the volume to 3 c.c. in each tube, to which 0-5 c.c. of 1%
soda solution is then added. The tube without trypsin remains
persistently turbid whilst the others gradually clear, that with
0-1 c.c. of trypsin becoming quite bright after 6 hours in an
incubator.
C h y m o s i n . The activity of a solution of this enzyme
is estimated by determining in what dilution it just coagulates
a certain quantity of milk in 30 minutes at40(K.Glaessner,
Hofm. Beitr., 1901, 1, 1, 24; Hammarsten, H., 1896, 22,
103).
Since the milk used for the estimation of rennet varies very
considerably as regards its chymosin-content, Blum and
Fuld (Berl. klin. Wochens., 1905, 42, 107; Biochem. Z., 1907,
4, 62) propose to replace the milk by a preparation of milk-
powder, which is prepared commercially and is of constant
composition. Three grms. of the milk-powder are mixed with
9 times the weight of water in the following manner : the weighed
(or measured, after pressing down and smoothing in a measure)
quantity of the powder is added in small portions to, and stirred
304 GENERAL CHEMISTRY OF THE ENZYMES
with, sufficient distilled water to form a semi-solid paste, to
which the remainder of the water is then added. On stirring,
almost the whole of the powder goes into solution without heating,
and this solution, which can be prepared in a couple of minutes,
can be used immediately if the sediment is rejected. It will,
on the other hand, keep for 3 days in an ice-chest. An addition
of calcium salt which was recommended by these authors in their
first communication, is unnecessary.
Twenty tubes are now charged with the following amounts:
(1) of the undiluted gastric juice,
0-10, 0-15, 0-21, 0-32, 0-46, 0-68, 1-0 c.c.;
(2) of the dilution 1 : 10,
0-10, 0-15, 0-21, 0-32, 0-46, 0-68 c.c.;
(3) of the dilution 1 : 100,
0-10, 0-15, 0-21, 0-32, 0-46, 0-68 c.c.
The last tube, contained 1-5 c.c. of the boiled gastric juice,
serves as a control.
The solutions are then made up to 10 c.c. with the milk solu-
tion, so that they contain the gastric juice in dilutions varying
from 1 : 10 to 1 : 10,000 (a preliminary test being thus unneces-
sary); the tubes are then placed in a large water-bath at 17-5.
At the end of 2 hours, a drop of 20% calcium chloride
solution is added to each of the tubes, which are then transferred
to a water-bath at 40 for 5 minutes. The ratio, gastric juice :
milk solution in the clotted liquid containing the least amount
of the juice gives directly the rennetic value of the gastric juice
and also its enzyme-content in general. A more accurate es-
timation can afterwards be made, either immediately or, if an ice-
chest is available, on the following day.
Z y m a s e . For technical purposes and, indeed, whenever
great accuracy is not desired, the fermenting power of pressed
yeast-juice or permanent yeast is determined by placing in a
small Erlenmeyer flask (100 c.c.) furnished with a M e i s s 1
valve, 20 c.c. of the pressed juice, 8 grms. of cane-sugar (or 2 grms.
of permanent yeast, 10 grms. of water and 4 grms. of cane-sugar),
and a little toluene, the loss in weight being determined after 1,
2, 3 or 4 days at 22. The evolution of carbon dioxide amounts
to about 1-2 grms. (E. and H. Buchner and M. H a h n ,
Die Zymasegarung, 1903, p. 80).
APPENDIX 305
For more accurate estimations, the carbon dioxide is expelled
from the solution by a gentle stream of air, or the evolution of
gas is allowed to take place under diminished pressure, the
amount of carbon dioxide liberated being then determined
volumetrically.
An excellent volumetric method has been devised by
S 1 a t o r (Journ. Chem. Soc., 1906, 89, 128).
Oxydase and peroxydase. The numerous reac-
tions which have been employed for the detection and estimation
of the oxydases have been referred to on p. 59. Since, as was
previously mentioned, no general method for the quantitative
determination of the oxydases exists, the methods given for the
study of special oxydases will not be described here. Reference
may be made to the brief outlines given on pp. 220-223.
C a t a 1 a s e. Since the determination of the catalase-
content of the blood and other liquids of the body is now one of
the more common tests of physiological chemistry, the most
important methods may be shortly mentioned.
With aqueous solutions of purified catalases, the undecom-
posed hydrogen peroxide is usually determined by titration with
potassium permanganate. The most suitable concentrations
of the peroxide are N/20 N/50; the solutions are acidified
with sulphuric acid and titrated with centinormal permanganate.
In many cases this method is, as B r e d i g found in his researches
on colloidal metals, preferable to measurement of the volume
of oxygen evolved.
Where the fluid of an organ is investigated directly, volume-
and pressure-methods may possess decided advantages. A
volume-method was employed by L. von Liebermann
(Pflug. Arch., 1904, 104, 176) and more recently also by S a n -
tesson, while W. L 6 b (Biochem. Z., 1908, 13, 339) has
described an arrangement, which apparently allows of rapid and
accurate measurement of catalase-content. In the same com-
munication, Lob describes a pressure-method, which also serves
well in certain cases.
306
GENERAL CHEMISTRY OF THE ENZYMES
TABLES FOR THE ESTIMATION OF SUGARS BY BERTRAND'S
METHOD
Glucose
4-07X50-l c.c.
Fourth crystallisation: [a]/>
1-960 grm.X2d.m.
+52.'
Sugar in
m grins.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
10
20-4
41
79-3
71
131-4
11
22-4
42
si -i
72
133-1
12
24-3
43
82-9
73
134-7
13
26-3
44
84-7
74
136-3
14
28-3
45
86-4
75
137-9
15
30-2
46
88-2
76
139-6
16
32-2
47
90-0
77
141-2
17
34-2
48
91-8
78
142-8
18
36-2
49
93-6
79
144-5
19
38-1
50
95-4
80
146-1
20
40-1
51
97-1
81
147-7
21
42-0
52
98-9
82
149-3
22
43-9
53
100-6
83
150-9
23
45-8
54
102-3
84
152-5
24
47-7
55
104-1
85
154-0
25
49-6
56
105-8
86
155-6
26
51-5
57
107-6
87
157-2
27
53-4
58
109-3
88
158-8
28
55-3
59
111-1
89
160-4
29
57-2
60
112-8
90
162-0
30
59-1
61
114-5
91
163-6
31
60-9
62
116-2
92
165-2
32
62-8
63
117-9
93
166-7
33
64-6
64
119-6
94
168-3
34
66-5
65
121-3
95
169-9
35
68-3
66
123-0
96
171-5
36
70-1
67
124-7
97
173-1
37
72-0
68
126-4
98
174-6
38
73-8
69
128-1
99
176-2
39
75-7
70
129-8
100
177-8'
40
77-5
APPENDIX
307
Invert-sugar
A 0-5% solution was prepared by hydrolysing 4-750 grms. of cane-sugar
with 50 c.c. of 2% hydrochloric acid. The solution was heated for 10-15
minutes, cooled, neutralised, and diluted to a litre.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
10
20-6
41
79-5
71
130-8
11
22-6
42
81-2
72
132-4
12
24-6
43
83-0
73
134-0
13
26-5
44
84-8
74
135-6
14
28-5
45
86-5
75
137-2
15
30-5
46
88-3
76
138-9
16
32-5
47
90-1
77
140-5
17
34-5
48
91-9
78
142-1
18
36-4
49
93-6
79
143-7
19
38-4
50
95-4
80
145-3
20
40-4
51
97-1
81
146-9
21
42-3
52
98-8
82
148-5
22
44-2
53
100-6
83
150-0
23
46-1
54
102-3
84
151-6
24
48-0
55
104-0
85
153-2
25
49-8
56
105-7
86
154-8
26
51-7
57
107-4
87
156-4
27
53-6
58
109-2
88
157-9
28
55-5
59
110-9
89
159-5
29
.57-4
60
112-6
90
161-1
30
59-3
61
114-3
91
162-6
31
81-1
62
115-9
92
164-2
32
63-0
63
117-6
93
165-7
33
64-8
64
119-2
94
167-3
34
66-7
65
120-9
95
168-8
35
68-5
66
122-6
96
170-3
36
70-3
67
124-2
97
171-9
37
72-2
68
125-9
98
173-4
38
74-0
69
127-5
99
175-0
39
75-9
70
129-2
100
176-5
40
77-7
308
GENERAL CHEMISTRY OF THE ENZYMES
Galactose
16-03X25c.c.
Fifth crystallisation: [a]z> =
2 5 grms. X 2 d.m.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
10
19-3
41
75-6
71
126-6
11
21-2
42
77-4
72
128-3
12
23-0
43
79-1
73
130-0
13
24-9
44
80-8
74
131-5
14
26-7
45
82-5
75
133-1
15
28-6
46
84-3
76
134-8
16
30-5
47
86-0
77
136-4
17
32-3
48
87-7
78
138-0
18
34-2
49
89-5
79
139-7
19
36-0
50
91-2
80
141-3
20
37-9
51
92-9
81
142-9
21
39-7
52
94-6
82
144-6
22
41-6
53
96-3
83
146-2
23
43-4
54
98-0
84
147-8
24
. 45-2
55
99-7
85
149-4
25
47-0
56
101-5
86
151-1
26
48-9
57
103-2
87
152-7
27
50-7
58
104-9
88
154-3
28
52-5
59
106-6
89
156-0
29
54-4
60
108-3
90
157-6
30
56-2
61
110-0
91
159-2
31
58-0
62
111-6
92
160-8
32
59-7
63
113-3
93
162-4
33
61-5
64
115-0
94
164-0
34
63-3
65
116-6
95
165-6
35
65-0
66
118-3
96
167-2
36
66-8 '
67
120-0
97
168-8
37
68-6
68
121-7
98
170-4
38
70-4
69
123-3
99
172-0
39
72-1
70
125-0
100
173-6
40
73-9
APPENDIX
309
Maltose
Third crystallisation: [<Z]D :
26-10X 25c.c.
2-5 grms. X 2 d.m.
The above rotation refers to the hydrate,
lowing tables to the anhydrous sugar.
but the fol-
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
10
11-2
40
44-1
70
76-5
11
12-3
41
45-2
71
77-6
12
13-4
42
46-3
72
78-6
13
14-5
43
47-4
73
79-7
14
15-6
44
48-5
74
80-8
15
16-7
45
49-5
75
81-8
16
17-8
46
50-6
76
82-9
17
18-9
47
51-7
77
84-0
18
20-0
48
52-8
78
85-1
19
21-1
49
53-9
79
86-1
20
22-2
50
55-0
80
87-2
21
23-3
51
56-1
81
88-3
22
24-4
52
57-1
82
89-4
23
25-5
53
58-2
83
90-4
24
26-6
54
59-3
84
91-5
25
27-7
55
60-3
85
92-6
26
28-9
56
61-4
86
93-7
27
30-0
57
62-5
87
94-8
28
31-1
58
63-5
88
95-8
29
32-2
59
64-6
89
96-9
30
33-3
60
65-7
90
98-0
31
34-4
61
66-8
91
99-0
32
35-5
62
67-9
92
100-1
33
36-5
63
68-9
93
101-1
34
37-6
64
70-0
94
102-2
35
38-7
65
71-1
95
103-2
36
39-8
66
72-2
96
104-2
37
40-9
67
73-3
97
105-3
38
41-9
68
74-3
98
106-3
39
43-0
69
75-4
99
107-4
100
108-4
310
GENERAL CHEMISTRY OF THE ENZYMES
Fifth crystallisation: [<Z]D
Lactose
13
2-5 grms.XS d.m'
The above rotation refers to the hydrate, Ci 2 H 2 2Oii+H 2 O, but the fol-
lowing tables to the anhydrous sugar.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cuin
mgrms.
10
14-4
41
56-7
71
95-4
11
15-8
42
58-0
72
96-6
12
17-2
43
59-3
73
97-9
13
18-6
44
60-6
74
99-1
14
20-0
45
61-9
75
100-4
15
21-4
46
63-3
76
101-7
16
22-8
,47
64-6
77
102-9
17
24-2
48
65-9
78
104-2
18
25-6
49
67-2
79
105-4
19
27-0
50
68-5
80
106-7
20
28-4
51
69-8
81
107-9
21
29-8
52
71-1
82
109-2
22
31-1
53
72-4
83
110-4
23 '
32-5
54
73-7
84
111-7
24
33-9
55
74-9
85
112-9
25
35-2
56
76-2
86
114-1
26
36-6
57
77-5
87
115-4
27
38-0
58
78-8
88
116-6
28
39-4
59
80-1
89
117-9
29
40-7
60
81-4
90
119-1
30
42-1
61
82-7
91
120-3
31
43-4
62
83-9
92
121-6
32
44-8
63
85-2
93
122-8
33
46-1
64
86-5
94
124-0
34
47-4
65
87-7
95
125-2
35
48-7
66
89-0
96
126 5
36
50-1
67
90-3
97
127-7
37
51-4
68
91-6
98
128-9
38
52-7
69
92-8
99
130-2
39
54-1
70
94-1
100
131-4
40
55-4
APPENDIX
311
Third crystallisation: [a]z>
Mannose
3-48X25c.c.
1-25 grm.X 5d.m
= +13-92(=21),
Sugar in
mgrms.
Cu in
mgrms.
Sugar in
mgrms.
Cu in
mgrms.
10
20-7
60
113-3
20
40-5
70
130-2
30
59-5
80
146-9
40
78-0
90
163-3
50
95-9
100
179-4
INDEX OF AUTHORS
Abderhalden, 9, 33, 38, 39, 40, 41,
43, 64, 103, 107, 116, 119, 142, 143,
190, 192, 193, 195, 196, 228, 236,
276, 277, 287, 293
Abelous, 69, 265
Aberson, 209, 210, 241
Achalme, 269, 271
Acree, 264
Agulhon, 116
Albert, 57
Armstrong, E. F., 18, 21, 22, 23, 53,
141, 165, 166, 168, 170, 173, 174,
261, 262
Armstrong, H. E., 11, 22, 23, 97,
128, 150, 161, 166, 170, 174, 275
Arrhenius, 75, 76, 131, 133, 134, 135,
136, 147, 151, 178, 183, 200, 202,
232, 236, 237, 267, 272
Arthus, 11, 116
Ascoli, 14, 15, 38, 268, 270
Asher, 117, 120
Aso, 59
Auld, 22, 24, 30, 101, 140, 172, 173,
174, 239, 262, 295
Bach, 61, 63, 64, 65, 66, 67, 68, 69,
108, 128, 217, 219, 220, 224, 225,
226, 228, 246
Baker, 14
Bang, 36, 45, 105, 109, 201, 202, 295
Barendrecht, 165
Barker, 74
Bashford, 272
Battelli, 63, 64, 67, 70, 272
Baur, 10
Bayliss, 36, 37, 84, 91, 103, 104, 106,
128, 142, 186, 241, 262, 266, 270,
296, 303
Beam, 123, 238
Bechhold, 84
Behrend, 11
Beijerinck, 14, 19, 28, 30
Beitzke, 266, 268
Benjamin, 48
Berg, 29, 95
Bergell, 36, 249, 268, 269.
Bergmann, von, 271
Berkeley, 194
Bernard, Claude, 14
Bertarelli, 267, 268
Bertrand, 21, 33, 61, 62, 64, 66, 105,
107, 108, 221, 275, 293
Bezzola, 270
Bial, 15
Bickel, 249, 250
Biedermann, 13
Biernacki, 237
Bierry, 24, 28, 83, 87, 91, 100, 109,
171, 275
Blake, 85
Bliss, 35
Bloch, 41
Blum, 269, 303
Blunt, 246
Bodenstein, 128, 129, 131, 152, 159,
252, 256, 263
Bokorny, 171
Boldyreff, 10, 93
Bolin, 62, 66, 79, 83, 221
Bonfanti, 14, 15
Bordet, 116, 272
Borissow, 176
Bosshard, 280
Bourquelot, 5, 13, 18, 19, 21, 22, 23,
24, 28, 30, 32, 59, 98
Boysen-Jensen, 52
Braun, 11, 268
Breandat, 30
Bredig, 67, 69, 122, 282, 283, 305
Brieger, 270
Brown, A., 128, 162
Brown, H. T., 13 ; 14, 15, 19, 128, 155,
157
Briicke, 35
Brugsch, 10
Bruno, 92
Bruschi, 48
BuchnerE., 3, 5, 41, 51, 52, 53, 57,
58, 60, 81, 83, 84, 94, 105, 116, 117,
118, 119, 211, 288, 304
314
INDEX OF AUTHORS
Buchner, H., 57, 304
Buckmaster, 91
Buglia, 93
Burian, 44, 59, 119, 230
Butkewitsch, 14, 16, 43, 100
Caemmerer, 107, 116, 119
Caldwell, 22, 23, 39
Campbell, 17
Camus, 11, 120, 240, 269
Carlson, 15
Cathcart, 270
Chanoz, 12
Charrin, 270
Chigin, 196
Chittenden, 39, 98, 109, 120
Chocensky, 56
Chodat, 48, 63, 64, 65, 66, 67, 143,
224, 225, 240, 241
Chodschajew, 83
Chrzascz, 14
Clark. 25, 291, 292
Claus, 55
Coehn, 85
Coggi, 269
Cohnheim, J., 16
Cohnheim, O., 38, 55, 91, 92
Cole, 97, 98, 110, 112
Connstein, 10, 11, 149
Courtauld, 22, 23
Cramer, 123, 238
Cremer, 264
Czapek, 13
Czyhlarz, von, 59, 66
Dakin, 12, 42, 279, 280
Dam, van, 105
Danilewski, 92, 113, 265
Davidsohn, 160, 161
Dean, 18
Delbruck, 11
Deleano, 66, 84
Delezenne, 91, 93, 106, 244, 270
Detmer, 158, 291
Devaux, 32
Dietz, 10, 152, 256, 263
Dietze, 102
Doblin, 270
Donath, 92, 93
Donnan, 74
Dony-Henault, 61, 62, 220
Downes, 246
Dox, 31
Doyon, 11, 12
Dreyer, 247
Drjewezki, von, 101
Duchacek, 54, 57
Duclaux, 5, 116, 117, 158
Dunstan, 30, 295
Durham, 108
Duuren, van, 61, 220
Edelstein, 249
Edmunds, 46
Effront, 13, 17, 97, 109, 110, 116, 158,
239
Ehrenreich, 82
Ehrlich, 44, 223
Einhorn, 302
Elvove, 12, 69, 146
Embden, 55
Emmerling, 39, 245, 261, 264
Engel, 10, 147, 289
Engler, 137
Erben, 37
Eriksson, 122, 123
Ernest, 56
Euler, 13, 25, 26, 27, 41, 62, 66, 67.
68, 79, 83, 88, 100, 102, 104, 105,
107, 112, 113, 116, 118, 121, 137,
142, 148, 165, 188, 193, 206, 209,
214, 216, 218, 221, 231, 232, 236,
241, 259, 283, 296
Faitelowitz, 67, 106, 116, 119
Fa jans, 282
Falloise, 10
Falta, 250
Farr, 119
Faubel, 186
Fermi, 24, 25, 83, 117, 245, 269, 271,
Fernbach, 83, 108, 114, 264
Finsen, 245
Fischer, E., 18, 19, 21, 22, 25, 29
30, 31, 36, 53, 79, 115, 118, 119,
168, 261, 276, 277, 279, 283, 285,
288
Fischer, W., 24, 283
Foa, 98
Ford, 93, 97
Foster, 15
Frankel, 17
Franzen, 52, 281
Freederickz, 64
Freudenreich, 118
Freundlich, 73, 76, 88
Freidenthal, 35, 273, 287
Fromme, 10
Furth, von, 59, 66, 93, 228
Fuhrmann, 11, 31
Fuld, 48, 49, 119, 201, 205, 241, 272,
300, 301, 302, 303
Gatin, 70
Gaunt, 60
Gengou, 116, 272
Gerard, 11, 23, 24
INDEX OF AUTHOKS
315
Gerber, 48, 49, 104, 109, 111, 202,
241, 244
Geret, 40, 41, 119
Gerhartz, 249
Gessard, 272
Gewin, 47
Giaja, 24, 28, 275
Gigon, 108, 142, 143
Gizelt, 120
Gjaldbak, 265
Glaessner, 270, 303
Glendinning, 128, 140, 155, 157, 264
Gley, 19, 270
Glimm, 238
Glinski, 292
Glover, 161, 166, 170
Gockel, 120
Godlewski, 55
Golden, 31
Goldschmidt, H., 144
Goldschmidt, R , 303
Gonnermann, 24, 64
Gottlieb, 70
Grafe, 228
Green, Reynolds, 11, 13, 16, 18, 39,
49, 96, 247
Gries, 95
Grignard, 279
Grigoriew, 210
Gross, 180
Grass, 82
Griitzner, 96, 117, 296
Gudzent, 250
Guggenheim, 64, 228, 236
Guignard, 23, 29, 244
Gulewitsch, 36
Guthrie, 93
Hagglund, 75
Haehn, 54
Hafner, 26
Hahn, 40, 41, 57, 95, 119, 250, 269,
304
Hairs, 30
Hall, 92
Halliburton, 15
Hamburg, 17
Hamburger, 14, 19, 30, 91, 287
Hammarsten, 36, 45, 46, 47, 48, 49,
50, 83, 97, 104, 200, 205, 272, 303
Handovsky, 85, 87
Hanriot, 11, 128, 240, 263
Hansen, 19, 201
Hanssen, 247
Harden, 3, 32, 53, 54, 56, 94, 109,
206, 211, 213, 214, 280
Hardy, 89
Hart ,'31
Hattori, 302
Hedin, 37, 77, 78, 81, 82, 101, 122,
128, 187
Heffter, 69
Hekma, 30, 91
Henneberg, 115
Henri, 87, 91, 128, 129, 130, 132, 140,
141, 156, 158, 159, 163, 165, 167,
175, 184, 249, 252, 253, 296, 303
Henriques, 265
Henry, 24, 30, 262, 295
Herissey, 13, 21, 22, 23, 24, 28, 30,
32, 118, 168
Herlitzka, 35
Heron, 19
Hertel, 245
Herzog, 79, 137, 167, 172, 196, 210,
240, 241, 265, 269
Higuchi, 49
Hildebrandt, 101, 267, 268
Hill, Croft, 4, 20, 83, 168, 257, 261,
262
Hinkins, 264
Hober, 88, 122
Hoff, van't, 138, 231, 251, 253, 254,
257, 261
Hoffmann, 96, 117
Hofmeister, 87, 89
Holderer, 21, 82
Homfray, Miss, 75
Hongardy, 91
Horton, 22, 23, 174
Hoyer, 11, 94, 97, 108, 111, 149
Huber, 116
Hubert, 83
Hudson, 18, 19, 21, 26, 27, 90, 98,
99, 100, 114, 127, 159, 160, 161,
163, 165, 171, 237, 238, 262
Huerre, 243
Huiskamp, 50
Huppert, 179, 293
Issaew, 25, 216, 217
Italic, van, 215
Iwanoff, 42, 214
Izar, 109
Jacobson, 105
Jacoby, 35, 36, 83, 84, 272, 300, 301,
302, 303
Jalander, 152
Jamada, 246, 248
Jastrowitz, 136
Jerusalem, 228
Jochmann, 37, 269
Jodlbauer, 246, 247, 248
Johansson, 209
Johnston, 146
Jones, 42, 44
Jones, G. C., 291
Jonescu. 244
316
INDEX OF AUTHORS
Jorissen, 30
Johnson, 292
Jungk, 292
Kalaboukoff, 9, 93
Kalanthar, 21
Kanitz, 10, 102, 107, 148, 149, 287
Kantorpwicz, 269
Karamitsas, 248
Kasarnowski, 79
Kastle, 12, 25, 59, 67, 69, 116, 117,
118, 122, 128, 146, 147, 215, 224,
225, 239, 241, 244, 263, 275, 276
Kaufmann, 116, 118, 119
Kayser, 108
Kendall, 156, 264, 291, 292
Kiesel, 43
Kikkoji, 42, 43, 101
Kirchoff, 15
Kjeldahl, 117, 158, 241, 242
Klatte, 81, 84
Klempin, 157
Knauthe, 13
Kobert, 24
Koelker, 103, 190, 192, 193, 295
Kottlitz, 205
Kohl, 246, 264
Kossel, 42
Kostytschew, 55, 56
Kotake, 70
Krafft, 79
Krawkow, 16
Krober, 118, 168, 240
Kriiger, 25, 110
Kudo, 102, 110
Kiihne, 139, 142
Kuttner, 300
Kullberg, 25, 26, 79, 104, 121, 165,
214, 236, 243
Kurajeff, 265
Kussmaul, 268
Kutscher, 39, 42
Laborde, 30
Lalou, 175
Landsteiner, 270
Langley, 94, 98
Lanzenberg, 108
Laqueur, 93, 120
Larguier des Bancels, 184
Larin, 95
Lawrow, 265
Lea, 43
Lebedew, von, 57, 214
Lesser, 66, 67, 215
Leube, 43
Leuchs, 15
Levaditi, 270
Levene, 42
Levison, 301, 302
Levites, 110
Lewkowitsch, 10
Liebermann, von, 67, 215, 305
Lindberger, 102
Lindet, 63
Lindner, 19, 240
Ling, 14, 290
Lintner, 16, 97, 100, 110, 118, 168,
288, 290
Lippmann, von, 5
Lobassow, 196
Lockemann, 37, 110, 246, 249
Loeb, Jacques, 107, 115, 184
Loeb, L., 50
Lob, W., 305
Lohlein, 186, 297, 299, 303
Lorcher, 104
Loevenhart, 12, 63, 67, 92, 93, 116,
117, 118, 122, 128, 146, 147, 215,
224, 225, 239, 241, 263, 275, 276
Loew, 16, 67, 107, 215, 217
Loewenherz, 275
Loewenthal, 249
Lohmann, 39
London, 195, 198, 200
Luckhardt, 15
Lundeqvist, 137
Luther, 137, 287
McCollum, 31
MacGillawry, 13
McKenzie, 279, 280
Maclean, 56
Macleod, 10
Madsen, 203, 245, 272
Magnus, 12, 92, 93, 273
Malfitano, 106
Malleyre, 33, 107
Mangin, 32
Maquenne, 14, 155, 264
Marchand, 108
Marckwald, 278, 279, 283
Martin, 205, 237
Martinand, 62, 66
Martini, 101
Maxwell, 71
Mayer, 9, 249
Mays, 37
Medigreceanu, 42
Medwedew, 128, 219
Meisenheimer, 3, 5, 51, 52, 58, 60, 94,
211
Meltzer, 236
Mendel, 15
Mett, 222
Meunier, 297, 298
Meyer, Kurt, 180, 271
Michaelis, 36, 77, 78, 82, 84, 85, 86,
87, 160, 161, 190, 267, 287
Millner, 73
INDEX OF AUTHORS
317
Minami, 250
Miquel, 240
Moitessier, 66
Moll, 43, 271
Montesano, 24, 25
Moore, 139
Moraczewski, von, 95
Morawitz, 50, 91, 107
Morel 11
Morgenroth, 271, 272
Moritz, 13, 140, 264
Morris, 13, 14, 15
Mouton, 244
Miiller, Ed., 293
Muller-Thurgau, 158, 240
Muntz, 118
Nagayama, 38
Nasse, 15, 119
Neilson, 110
Nencki, 34, 35, 47, 92
Neppi, 38
Neuberg, 10, 23, 65, 249, 266, 268
Nicloux, 11, 151, 236
Niebel 19, 25
Nierenstein, 302
Norris, 53
Novy, 35
Niirnberg, 265
Obermayer, 296
Ohlsen, 214
Okuneff, 265
Oppenheimer, C., 287
Oppenheimer, S., 69
Ormerod, 97, 150, 275
Osaka, 137
Osborne, 17, 25, 26, 109
Oshima, 26
Ostwald, Wilh., 72, 129, 234, 287
Ostwald, Wo., 72, 246, 287
O'Sullivan, 26, 98, 158, 159, 161, 162,
165, 237, 241, 242, 262, 288
Overton, 264
Pagenstecher, 10
Paine, 21, 100, 114, 171, 237, 238
Palladin, 55, 56
Pantanelli, 264
Parnas, 70
Parrozzani, 8
Pasteur, 276
Patten, 114
Pauli, 85, 87, 88, 89
Pawlow, 34, 36, 37, 47, 91, 92, 106,
111, 113, 157, 176, 276, 292
Payen, 15
Peirce, 154
Pekelharing, 34, 35, 47, 49, 50, 86
Pernossi, 83, 117, 245
Persoz, 15
Pewsner, 198
Pfleiderer, 94, 96, 117
Philoche, 157, 167
Pick, 15, 296
Pinkussohn, 107, 116, 118, 119
Plimmer, 30
Pohl, 59
Pollak, 38, 102, 123, 184
Pomeranz, 257
Porter, 123
Posternak, 88
Pottevin, 10, 14, 22, 107, 263
Pozerski, 244
Pozzi-Escot, 68
Preti, 110
Price, 231
Pringsheim, 43
Pugliese, 15, 118, 269
Quincke, 80
Rachford, 92, 93
Rapp, 105
Raudnitz, 67
Reichel, 94, 118, 120, 201
Reicher, 302
Reinhard, 63
Reiss, 67
Resenscheck, 84
Rey-Pailharde, de, 68
Ribaut, 265
Richter, 249
Rinckleben, 57
Ringer, 86
Roaf, 296
Roberts, 291
Robertson, 25, 104, 266
Roden, 272
Rohmann, 20, 59
Roger, 15, 109
Rogozinski, 66
Rona, 78, 84, 97
Rosenberg, 108
Rosenblatt, 64
Rosenfeld, 63, 120, 265
Rosenheim, 10, 92, 93
Rosenthaler, 5, 22, 23, 45, 173, 264
280, 281
Rostocki, 33
Roth, 287
Rouge, 11, 48, 148
Rowland, 101, 288
Rozenband, 105
Sachs, 101, 269
Saiki, 15, 268
Sailer, 119
Salaskin, 265
318
INDEX OF AUTHORS
Salazar, 100, 171
Salkowski, 25, 26, 41
Samojloff, 176
Santesson, 117, 305
Sarthou, 63
Sawitsch, 47
Saw j alow, 265
Schade, 52
Schaeffer, 36, 83, 87, 110
Schaer, 59
Schapirow, 92
Schardinger, 68, 69
Schellenberg, 13
Schiff, 302
Schilow, 137
Schittenhelm, 41, 43, 44
Schlesinger, 18, 118
Schmidt, Alex., 49
Schmidt, C. L. A., 104
Schmidt, G. C., 75, 76, 78
Schmidt-Nielsen, Signe, 36, 47, 236,
247
Schmidt-Nielsen, Sigval, 236, 247,
248, 249
Schmiedeberg, 12, 59
Schneegans, 27
Schondorff, 120
Schorstein, 13
Schreiner, 69
Schrumpf, 35
Schiitz, E., 132, 133, 175, 179, 295
Schiitz, J., 93, 110, 179, 285
Schiitze, 268, 269
Schutzenberger, 28
Schulze, 280
Schumoff-Siemanowski, 9
Schumoff-Siemanowski, Mme., 34
Schunck, 29
Schwarz, 123, 250
Schwarzschild, 36, 37
Schwiening, 101
Scurti, 8
Segelke, 201
Senter, 67, 105, 106, 117, 119, 215,
216, 217, 236, 241
Shaklee, 236
Shaw-Mackenzie, 93
Shedd, 59
Sherman, 18, 156, 264, 291, 292
Shibata, 43
Shiga, 43
Shigeji, 49
Shore, 19
Sieber, 9, 34, 35, 47
Sieber, Mme., 55
Siedentopf, 80
Sigmund, 11, 28, 117, 149
Simnitzki, 270
Sjoqvist, 95, 96, 176, 178, 296
Slator, 51, 53, 210, 240, 243, 274, 305
Slowtzoff, 63, 220
Sorensen, 27, 90, 96, 98, 99, 100, 160,
161, 165, 184, 219, 266, 296
Solms, 302
Sommer, 69
Souder, 93
Soxhlet, 201
Spatzier, 29
Spiro, 48, 94, 118, 119, 120, 201, 203,
272
Spitzer, 59, 63
Spohr, 231
Spriggs, 181, 296
Stade, 10, 147
Stangassinger, 70
Starling, 91, 106, 270
Steche, 216
Stenitzer, von, 269
Steppuhn, 52
Stern, 64, 70, 272
Stevens, 63
Stiles, 114
Stoecklin, de, 59, 62, 65, 66, 223
Stoklasa, 56
Stoll, 12
Stone, 16
Storch, 201
Strada, 83
Sullivan, 69
Sundberg, 35
Suzuki, 31
Takaishi, 31
Takamine, 16
Takemura, 40
Tammann, 139, 158, 171, 173, 233,
240, 241, 242, 255, 258
Tanret, Ch., 29, 262
Tanret, G., 29
Tappeiner, von, 248
Taylor, 47, 104, 154, 156, 157, 161,
194, 241, 256, 257, 264, 266, 285
Tebb, Miss, 14, 19
Terroine, 9, 36, 93, 167
Terry, 110
Teruuchi, 39
Thierfelder, 53
Thies, 110, 246, 249
Thomas, 297, 298
Tichomirow, 36
Titoff, 75
Tomaszewski, von, 302
Tompson, 98, 158, 159, 161, 162,
165, 237, 241, 242, 262, 288
Trebing, 270
Trillat, 62
Trommsdorff, 69
Tscherniak, 65
Tschirch, 63
INDEX OF AUTHORS
319
Twitchel, 113
Twort, 24
Ugglas, af, 27, 100, 112, 121, 165,
241, 242, 243
Umber, 10
Vandevelde, 83, 109, 116, 117, 271,
273
Vernon, 37, 38, 46, 103, 115, 183, 223,
237, 241, 283
Victorow, 120
Vines, 39, 40, 41, 100, 194
Vinson, 25
Visser, 242, 258
Volhard, 10, 83, 97, 147, 288, 297,
298, 299, 300, 303
Vulquin, 101
Waele, de, 273
Waentig, 216
Walbum, 272
Walker, 92
Walther, 176, 292
Warburg, 279
Wartenberg, 11, 149
W T eber, 297, 298
Weevers, 28, 228
Weinland, 31, 269, 271
Weis, 39, 41, 100, 181, 246
Welter, 264
Wheldale, Miss, 228
White, Miss, 244
Whitney, 85
Wichern, 110, 246, 249
Wiechowski, 60, 288
Wiener, 60, 288
Wijsman, 14, 15
Wilcock, 249
Wilhelmy, 127
Willstatter, 12, 108
Windisch, 41
Witte, 302
Wittich, von, 15, 16
Wohl, 51, 238
Wohlgemuth, 93, 94, 110, 249,291,292
Wolff, J., 59, 66, 109, 223, 264
Wolff, W., 302
Wren, 279
Wright, 16
Wroblewski, 16, 17, 26, 96, 291
Yoshida, 61
Yoshimoto, 101
Yoshimura, 31
Young, 3, 32, 53, 54, 94, 109, 206,
211, 213, 214
Zahorski, 64
Zaleski, 42, 63
Zeller, 247, 248
Zellner, 148, 246
Zemplen, 21
Zinsser, 10
Zsigmondy, 80
Zunz, 91, 106, 107, 271
INDEX OF SUBJECTS
Acids; activation by, 94
Activators, 90 et seq.
Adenase, 7, 44
Adenine, 7,^44
Adsorption, 75 et seq.
Adsorption media; acid or basic, 84
; neutral, 81
Aesculase, 28
Alcoholic fermentation, 51 et seq.
Alcoholoxydase of acetic acid bac-
teria, 7, 60
Aldehydases, 7, 70, 219
Alkaloids, 119
Amicrons, 80
Amidases, 43
Amygdalase, 23, 173
Amygdalin, 22, 23, 171-175
Amylase, 6, 13, 155
; 'activation by acids and salts, 97,
158, 289
; dynamics, 155
; occurrence, 14
; preparation, 16
Amylopectinase, 6, 14, 155
Anti-enzymes, 267
Arbutase, 28
Arginase, 42
Arginine, 42
Arsenic, 109, 116
Asymmetric syntheses, 277
Autocatalysis, 111, 128
Autolytic enzymes, 101
Bases; activation by, 94
Betulase, 27
Blood; clotting of, 49, 50, 205
Borax, 29
Boric acid, 116
Bromelin, 39
" Buffers," 106, 115
Butyrases, 6, 11
Carbamases, 6, 33
Carbonases, 56
Caroubinase, 13
Casein, 45, 200
Catalase, 7, 67, 87, 116, 117, 305
; activation by acids and alkalis,
105
; dynamics, 215
; occurrence, 67
; preparation, 67
Catalysis, 127
Cellase, 21
Cellulase, 13
Charcoal as adsorbent, 81
Choral, 29, 118
Chloroform, 118
Chlorophyllase, 6, 12
Chymosin, 45, 303
; activation by acids, 104
; dynamics, 200
; occurrence, 45
; preparation, 46
Classification of enzymes, 6
Coagulating enzymes, 45 et seq.
Co-enzymes, 12, 53, 90
Colloids, 78
; inorganic, 118
Coupled reactions, 137
Creatine, 70
Cresols, 118
Cynarase, 48
Cytase, 6, 13
Desamidases, 7, 43
Diastase, 6, 13, 97, 118, 120, 289 et
seq.
; preparation, 16, 17
Digestion; dynamics of, 195
Dihydroxyacetone, 52
Dynamics; chemical, of reactions, 124
'Elaterase, 29
Electric transference of enzymes, 85
Emulsin, 6, 22, 87, 261, 266, 295
; activation by acids, 101
; dynamics, 171
; occurrence, 23
; preparation, 24
321
322
INDEX OF SUBJECTS
Endotryptase of yeast, 40
Enterokinase, 91
Enzymic equilibria, 252
Erepsin, 6, 38, 116, 118, 303
activation by alkali, 102
dynamics, 188
occurrence, 38
preparation, 38
Erythrozyme, 29
Esterases, 6, 9 et seq., 289
dynamics, 146
preparation, 10, 12
Fermentation; mechanism of, 51
Fermentation enzymes, 50
Fibrin ferment : see thrombin
Fibrinogen, 49
Formaldehyde, 118
Galacto-lactase, 22
Galacto-zymase, 53
Gastric juice, 34, 97
Gaultherase, 27
Gease, 28
Gels, 89
Gluco-lactase, 22, 23, 170
a-Glucosidase, 6, 19
/3-Glucosidase, 6, 19, 21, 171
Glucp-zymase, 53
Glutinase, 38, 102, 184
Glyceraldehyde, 52
Glycerol, 118
Glycolytic enzymes, 55
Guanase, 7, 44
Guanine, 7, 44
Hexosephosphatase, 32, 54
Hexosephosphatese, 104, 214
Histozyme, 12
Hydrocyanic acid, 119
Hydrogen peroxide, 67, 117
Hydrogen sulphide, 117
Hypoxan thine, 44, 60
Indigo enzyme, 30
Inhibiting agents, 115
Internal pressure, 71
Intestinal juice, 101
Inulinase, 6, 98
occurrence, 18
Invertase, 6, 24, 87, 117, 284
activation by acids, 98
destruction by acids and alkalis,
237
dynamics, 158
occurrence, 25
preparation, 25
Isomaltose, 14
Isomerising enzyme, 70
Kaolin as adsorbent, 84
Kinases, 90, 91
Laccases, 61, 62, 105
; artificial, 62
Lactacidase, 53
Lactase, 6, 30
; activation by acids, 100
; dynamics, 168
; occurrence, 30
; preparation, 31
Lactic acid bacteria; zymase of, 7, 58
Light; influence of, on enzymes, 245
Linamarase, 30
Lipases, 6, 9, 10, 116, 117, 118, 119,
263, 264, 275, 288
; dynamics, 146
; occurrence, 10-11
Lotase, 30
Maltase, 6, 19-20
; dynamics, 166
; occurrence, 19
; preparation, 19
Mandelonitrile glucoside, 23
Manno-zymase, 53
Maximum temperatures, 243
Melibiase, 31
Mercuric chloride, 116
Mercuric cyanide, 116
Mesothorium; influence of, on en-
zymes, 250
Methylglyoxal, 52
Microns, 80
Myrosin, 6, 29, 118
; occurrence, 29
Neutral salts; activation by, 106
Nitrilase, 45
Nitrilese, 5
Nomenclature of enzymes, 5
Nuclease, 6, 41
Nucleinases, 42
Nucleosidases, 42
Nucleotidases, 42
Oenoxydase, 63
Olease* 63
Optimum temperatures, 243
Ornithine, 42
Oxydase of acetic bacteria, 60
Oxydases, 58, 118, 219, 305
; detection, 59
Oxynitrilase, 45
Ozone, 117
Pancreatic juice, 107, 276
Papain or papayotin, 39, 40, 101
Paracasein, 45, 200
Parachymosin, 45, 105
INDEX OF SUBJECTS
323
Paralysors, 115
Pectase, 6, 32
Pectinase, 7, 32, 100
Pepsin, 6, 33, 46, 47, 83, 116, 117, 118
175 et seq., 265, 295 et seq.
; activation by acids, 94 st seq.
; dynamics, 175
; occurrence, 34
; preparation, 34
Pepsinogen, 34
Peptases, 40
Permanent yeast, 57
Peroxydases, 7, 65, 105, 223, 305
; dynamics, 223
; preparation, 65
Phaseolunatase, 30, 172
Phenol, 118
Phenolases, 7
Philothion, 68
Phosphates, 109
Phosphatese, 5, 7, 104
Phytase, 6, 31
Plastein formation, 265
Poisons, 115 et seq.
Potassium cyanide, 119
Preparation of enzymes, 7 et seq.
Press yeast juice, 53, 54, 56
; dynamics, 206
; preparation, 56
Prochymosin, 46
0-Proteases, 40
Protective agents, 115
Proteinases, 6, 33, 38, 295
Proteolytic enzymes of plants, 38,
181 et seq.
Ptyalin, 15, 97
; preparation, 16
Purification of enzymes, 7 et seq.
Radiation; influence of, on enzymes,
245
Radium; influence of, on enzymes,
249
Reductases or reducing enzymes, 68
Rennet: see chymosin
Reversible reactions, 137
Revertase, 264
Rhamnase, 6, 29
Rontgen rays; influence of, on en-
zymes, 249
Salicase, 28
Salicylic acid, 29, 119
Salts, activation by, 106
; inhibition by, 116
Schinoxydase, 63
Schiitz's rule, 132
Seminase, 13
Sodium chloride, 109
Sodium fluoride, 116
Specificity of enzyme actions, 274
Spermase, 63
Statics; chemical, in enzyme reac-
tions, 251
Steapsin: see lipase
Stimulin, 92
Submicrons, 80
Sucrase: see invertase
Surface energy, 72
Surface tension, 72
Syntheses by enzymes, 261
Taka-diastase, 16
Tannase, 6
Tannin, 29
Temperature; influence of, on en-
zyme reactions, 231
Temperature coefficients of enzyme
reactions, 239
Thrombin, 7, 49, 107, 116, 205
Thymol, 58, 118
Toluene, 58, 118
Transference; electric, 85
Trehalase, 20
Trypsin, 6, 36, 43, 84, 86, 102, 116,
117, 118, 119, 123, 183, 184, 186,
194, 266, 303
; dynamics, 184
; occurrence, 37
; preparation, 37
Tyrosinase, 64, 120, 228
Urea, 43
Urease, 7, 43 ,
Uric acid, 60
; enzyme oxidising, 41, 60
Xanthine, 44, 60
Yeast; permanent, 57
Zymase, 7, 54 et seq., 94, 105, 109,
116, 118, 120, 304
; co-enzyme, 211
; dynamics, 206
; occurrence, 54
; preparation, 56, 57'
Zymin, 58
Zymogen, 40, 49
; activation of, 96
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Association of State and National Food and Dairy Departments, Hartford
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Whipple's Microscopy of Drinking-water 8vo, 3 50
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CIVIL ENGINEERING.
BRIDGES AND ROOFS. HYDRAULICS. MATERIALS OF ENGINEER.
ING. RAILWAY ENGINEERING.
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Comstock's Field Astronomy for Engineers 8vo, 2 50
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Crandall's Text-book on Geodesy and Least Squares 8vo, 3 00
Davis's Elevation and Stadia Tables 8vo, 1 00
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Folwell's Sewerage. (Designing and Maintenance.) 8vo, 3 00
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Johnson's (L. J.) Statics by Algebraic and Graphic Methods 8vo, 2 00
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Venable's Garbage Crematories in America 8vo, 2 00
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Wait's Engineering and Architectural Jurisprudence 8vo, 6 00
Sheep, 6 50
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Warren's Stereo tomy Problems in Stone-cutting 8vo, 2 50
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Webb's Problems in the Use and Adjustment of Engineering Instruments.
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BRIDGES AND ROOFS.
Boiler's Practical Treatise on the Construction of Iron Highway Bridges.. 8vo, 2 00
* Thames River Bridge Oblong paper, 5 00
Burr and Falk's Design and Construction of Metallic Bridges 8vo, 5 00
Influence Lines for Bridge and Roof Computations 8vo, 3 00
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Heller's Stresses in Structures and the Accompanying Deformations.. . .8vo, 3 00
Howe's Design of Simple Roof-trusses in Wood and Steel 8vo, 2 00
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Ricker's Design and Construction of Roofs. (In Press.)
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Waddell's De Pontibus, Pocket-book for Bridge Engineers 16mo, mor. 2 00
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HYDRAULICS.
Barnes's Ice Formation 8vo, 3 00
Bazin's Experiments upon the Contraction of the Liquid Vein Issuing from
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Church's Diagrams of Mean Velocity of Water in Open Channels.
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Hydraulic Motors 8vo, 2 00
Mechanics of Fluids (Being Part IV of Mechanics of Engineering) . .8vo, 3 00
Coffin's Graphical Solution of Hydraulic Problems , 16mo, mor. 2 50
Flather's Dynamometers, and the Measurement of Power 12mo, 3 00
Folwell's Water-supply Engineering 8vo, 4 00
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Ganguillet and Kutter's General Formula for the Uniform Flow of Water in
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Hazen's Clean Water and How to Get It Large 12mo, 1 50
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Wilson's Irrigation Engineering 8vo, 4 0:)
Wood's Turbines. . . . . 8vo, 2 50
MATERIALS OF ENGINEERING.
Baker's Roads and Pavements 8vo, 5 00
Treatise on Masonry Construction 8vo, 5 00
Black's United States Public Works Oblong 4to, 5 00
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Bleininger's Manufacture of Hydraulic Cement. (In Preparation.)
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Burr's Elasticity and Resistance of the Materials of Engineering Svo, 7 50
Byrne's Highway Construction Svo, 5 00
Inspection of the Materials and Workmanship Employed in Construction.
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Church's Mechanics of Engineering Svo, 6 00
Mechanics of Solids (Being Parts I, II, III of Mechanics of Engineer-
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Du Bois's Mechanics of Engineering.
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* Eckel's Building Stones and Clays Svo, 3 00
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Fowler's Ordinary Foundations ; Svo, 3 50
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Holley's Analysis of Paint and Varnish Products. (In Press.)
* Lead and Zinc Pigments Large 12mo, 3 00
S
* Hubbard's Dust Preventives and Road Binders Svo, $3 00
Johnson's (C. M.) Rapid Methods for the Chemical Analysis of Special Steels,
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Johnson's (J. B.) Materials of Construction Large Svo, 6 00
Keep's Cast Iron Svo, 2 50
Lanza's Applied Mechanics Svo, 7 50
Lowe's Paints for Steel Structures 12mo, 1 00
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* Martin's Text Book on Mechanics. Vol. I. Statics 12mo, 1 25
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Metcalf's Steel. A Manual for Steel-users 12mo, 2 00
Morrison's Highway Engineering .. Svo, 2 50
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Patton's Practical Treatise on Foundations Svo, 5 00
Rice's Concrete Block Manufacture .Svo, 2 00
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Richey's Building Foreman's Pocket Book and Ready Reference. 16mo, mor. 5 00
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Spalding's Hydraulic Cement 12mo, 2 00
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Thurston's Materials of Engineering. In Three Parts Svo, 8 00
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Tillson's Street Pavements and Paving Materials . .Svo, 4 00
Turneaure and Maurer's Principles of Reinforced Concrete Construction.
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Waterbury's Cement Laboratory Manual 12mo, 1 00
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Wood's (De V.) Treatise on the Resistance of Materials, and an Appendix on
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Wood's (M. P.) Rustless Coatings: Corrosion and Electrolysis of Iron and
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RAILWAY ENGINEERING.
Andrews's Handbook for Street Railway Engineers 3X5 inches, mor. 1 25
Berg's Buildings and Structures of American Railroads 4to, 5 00
Brooks's Handbook of Street Railroad Location 16mo, mor. 1 50
* Burt's Railway Station Service I2mo, 2 00
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Ives and Hilts's Problems in Surveying, Railroad Surveying an^I Geodesy
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Molitor and Beard's Manual for Resident Engineers 16mo, 1 00
Nagle's Field Manual for Railroad Engineers 16mo, mor. 3 00
* Orrock's Railroad Structures and Estimates 8vo, 3 00
Philbrick's Field Manual for Engineers 16mo, mor. 3 00
Raymond's Railroad Field Geometry 16mo, mor. 2 00
Elements of Railroad Engineering 8vo, 3 50
Railroad Engineer's Field Book. (In Preparation.)
Roberts' Track Formulae and Tables 16mo. mor. 3 00
Searles's Field Engineering IGmo, mor. 3 00
Railroad Spiral 16mo, mor. 1 50
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Webb's Economics of Railroad Construction Large 12mo, 2 50
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12
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14
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Carnot's Reflections on the Motive Power of Heat. (Thurston.) 12mo, 1 50
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Peabody's Manual of the Steam-engine Indicator 12mo, 1 50
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MECHANICS PURE AND APPLIED.
Church's Mechanics of Engineering 8vo, 6 00
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Stones for Building and Decoration 8vo, 5 00
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MINING.
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