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CKJt^^^ <fq<^ ,\^
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FROM TMfc BKQ^
DANIEL TRF
4
Rumford Prof'
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■
Ti\i» book should be returne
■ the Library on or before the last
stamped below.
A fin© of five cents a day is inci]
by retaining it beyoad the spe*
time.
Please return promptly*
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C^U^^^ <t^^ ,\^
f^arfaarti College Eibrars
FROM THB BSqyBST OF
DANIEL TREADWELL
Rumford Professor and Lecturer on the Application
of Science to the Useful Arts
1834-1845
SCIENCE CENTER LIBRARY
a
INTRACELLULAR ENZYMES
INTRACELLULAR
ENZYMES
A COURSE OF LECTURES GIVEN IN THE
-PHYSIOLOGICAL LABORATORY
UNIVERSITY OF LONDON - ^t^^w-.
BY H. M. VERNON, M.A., M.D.
FXLLOW OF MAGDALEN COLLBOE, AND LBCTURBB ON PHY8IOL0OY AT IXETSB
AND queen's COLLSOBS, OXFORD
LONDON
JOHN MURRAY, ALBEMARLE STREET, W.
1908
^U^^ff, /^
PREFACE
The subject of these lectures might at first sight be regarded
as too small and unimportant to warrant their reproduction
in book form, but I hope that such an opinion may be
dispelled by a study of the lectures themselves. The progress
of research renders it more and more evident that the
cellular protoplasm of all living organisms is made up very
largely of ferments or enzymes, and that many or most of its
properties are dependent upon their activities. The literature
dealing with these intracellular enzymes is scattered and some-
what fragmentary, and comparatively little of it has as yet found
its way into text-books. This is partly because of its recent origin,
for reference to the authorities cited at the foot of these pages
will show that almost the whole of the research work described
has been carried out during the course of the last decade. If
such rapid rate of progress be continued in the future, the sub-
ject of intracellular enzymes bids fair to become, if it has not
already become, one of the most important branches of bio-
chemistry, for it alone seems to offer a clue to the solution of
the most fundamental of all biological problems, the nature and
constitution of protoplasm.
The matter in this book closely follows that of the spoken
lectures, with some amplification of detail. I take this oppor-
tunity of thanking Dr A. D. Waller for his kindness in invit-
ing me to give the course of lectures in the Physiological
Laboratory of the University of London, for I should scarcely
have had the energy to collect and publish the material without
the stimulus of such an invitation. Also, I am indebted to
Dr W. M. Bayliss for his kindness in looking through the MS.,
and offering valuable criticism. H. M. V.
SepUtnder 1908.
CONTENTS
LECTURE I
PROTEOLYTIC ENDOENZVMES
FAOl
Dependence of chemical activities of living tissues on endoenzymes.
Liberation of endoenzymes on death of cells : gradually, if tissues
be kept intact ; immediately, if minced up. Methods of extract-
ing endoenzymes. Classification of proteolytic endoenzymes.
Proteases, endoerepsins, arginase, urease. Action of enzymes on
polypeptides. Amide nitrogen in relation to enzymes and acids . i
LECTURE II
PROTEOLYTIC ENDOENZYMES (continued)
Action of pepsin, trypsin, and proteases on nucleoproteins. Presence
of nuclease in all tissues. Irregular distribution of adenase,
guanase, and xantho-oxidase, Uricolytic enzyme and its action.
Relation between endoenzymes and functional capacity of tissues,
as shown by effects of development, disease, food, and hibernation.
Presence of proteolytic endoenzymes in all organisms, and their
reaction to acids and alkalis. Plant enzymes, both peptic and
ereptic ••....., 26
LECTURE III
FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
Lipolytic endoenzymes in animal tissues. Action upon esters, and
upon natural fats. Vegetable lipolytic enzymes, and their
relation to acids and alkalis. Glycogen content of tissues of
adult and embryonic animals, in relation to intracellular amylase.
Maltase, ihvertase, and lactase in animal tissues. Lactase and
adaptation* Vegetable diastatic and sucroclastic enzymes.
Glucoside-splitting enzymes . . • . • 53
X CONTENTS
LECTURE IV
ZYMASE AND OTHER GLYCOLYTIC ENZYMES
PAG 8
Zymase of yeast* Its action on various sugars. Cause of its
instability. Effect of filtration. Action of antiseptics. Influence
of phosphates. Zymase and lactacidase enzymes. Action of
inorganic catalysts on glucose. Glycolytic power of mixed
pancreas and muscle juice. Alcohol in animal tissues.
. Anaerobic respiration in living and dead plants. Formation of
acids in aseptic and antiseptic autolyses . . . .81
LECTURE V
OXIDISING ENZYMES
/ Oxygenases or aldehydases and their various activities. Peroxidases
and their estimation. Doubtful enzymic nature of oxidases.
Tyrosinase^, laccase, and alcohol-oxidase. Catalases: their
estimation, mode of action, and relation to functional capacity.
Inotganic ferments. Respiration in dead animal and plant
tissues^ before and after disintegration, and its relation to
respiratory entyines. Intramolecular oxygen. Respiratory pro-
cesses in biogens . . . . -IIS
LECTURE VI
i THE CONSTITUTION AND MODE OF ACTION OF ENZYMES
Preparation of pure pepsin. Its protein -like nature. Influence of
proteins on stability of enzymes. Precipitability of enzymes.
Relation of rennin to pepsin, trypsin, and other proteolytic
enzymes. Adsorption of enzymes and of dyes. Slight difiUsi-
bility of enzymes. Optical activity of enzymes. Chemical
combination of enzyme with substrate. Velocity of enzyme
action, and its deviations from law of mass action • • . 145
CONTENTS xi
LECTURE VII
REVERSIBLE ENZYME ACTION
^ Stereoisomeric sugars and their corresponding enzymes. Stereo-
isomeric polypeptides and proteolytic enzymes. Retardation
exerted by products of action. Interaction of organic acids,
alcohols, esters, and water. Reversible action of sucroclastic
enzymes. Synthesis of maltose, revertose, isomaltose, isolactose,
cane-sugar, emulsin. Synthesis of ethyl butyrate, glycerin tri-
acetate, methyl oleate, mono-olein and triolein by lipolytic
enzymes. Action of organic and inorganic catalysts compared.
Synthetic action of proteolytic enzymes. Formation of plastein.
Energy relations of reacting systems. Transformation of
radiant energy of sun into chemical energy by catalytic agents.
Synthesis in plants and animals . . . . .169
LECTURE VIII
ENDOENZYMES AND PROTOPLASM
Comparison of living and dead tissues. Disintegration effected by
chloroform and by lactic acid saline. Autodigestion of intact
and of minced tissues. Comparison of enzymes with agglutinins
and lysins. Zymoids. Antiferments. Constitution of biogens.
Action of antiseptics on living organisms and on enzymes.
Influence of temperature on enzymes, on metabolism of
organisms, on heart beat, and on rate of propagation of nervous
impulse. Optimum and maximum temperatures . . • 197
Index of Authors ....... 229
Index of Subjects ....... 235
4^W
INTRACELLULAR ENZYMES
LECTURE I
PROTEOLYTIC ENDOENZYMES
Dependence of chemical activities of living tissues on endoenzymes.
Liberation of endoenzymes on death of cells : gradually, if tissues be
kept intact ; immediately, if minced up. Methods of extracting
endoenzymes. Classification of proteolytic endoenzymes. Proteases,
endoerepsins, arginase, urease. Action of enzymes on polypeptides.
Amide nitrogen in relation to enzymes and acids.
Of the numerous subjects included under the head of Physi-
ological Chemistry, or Bio-Chemistry, few have attracted
so much attention within recent years as that of Enzymes.
And great as has been the increase in our knowledge of the
nature and mode of action of these substances, the further we
advance the wider becomes the field of research opening out
before us. This is especially true in respect of the group of
enzymes known as Intracellular or Endo-enzymes. These
enzymes differ from the exo-enzymes, such as are found in many
of the secretions of living organisms, by reason of the fact that
they are bound up in the protoplasm of the cells, and, so long as
these cells retain their vitality, can only exert their activity
intracellularly. On death of the cells, the protoplasm dis-
integrates, and many of the constituent enzyme groupings
gradually split off and pass into solution. It is inferred, though
strict proof of the inference is wanting, that any zymolysing
powers possessed by such solutions were, in all probability,
possessed by the protoplasm before disintegration. And as a
living tissue would scarcely elaborate and store up within itself
enzymes which were useless to it, it is supposed that any enzyme
which can be extracted from a tissue after death — apart from
A
2 PROTEOLYTIC ENDOENZYMES
such enzymes as may be secreted externally during life — was of
functional importance during the life of the tissue. A thorough
study of all the zymolysing powers possessed by the dis-
integration products of various typical tissues, vegetable as well
as animal, is therefore of paramount importance, for the know-
ledge so attained may lead us far towards the explanation of the
properties of living matter. It is possible that it may show us
that many or most of the katabolic processes of living tissues,
and perhaps the anabolic processes as well, are due to nothing
more than the ceaseless activity of a vast variety of endo-
enzymes, bound up together in the biogens, and exerting their
powers as they are needed. Thus Duclaux suggested that the
life of bacteria is nothing more than the sum total of the
activities of the enzymes contained within them, and Hofmeister ^
supposed that the multifarious activities of the liver cell, such as
the synthesis and hydrolysis of glycogen, the formation of
bilirubin and bile acids, of ethereal sulphates and urea, merely
represent the action of the different enzymes contained within
it This hypothesis of cellular metabolism is not at present by
any means completely established on a sound experimental
basis, but it is at least a working hypothesis, and one which can
only stimulate research, not retard it Hence, even if it ulti-
mately prove erroneous, it needs no further justification. It is
frbm the point of view of the probable validity of this hypothesis
that the experimental data collected together in these lectures
are described.
To pass from theory to fact, it is first necessary to mention
briefly the methods used for extracting endoenzymes from the
tissues. These enzymes are fixed by some definite chemical
bond in the cellular protoplasm, and even after death they are,
as a rule, only set free very gradually, provided the tissue be
left intact This is well shown by perfusing an excised organ
{e,g,, a mammalian kidney) for some days with an antiseptic
medium, such as 2 per cent NaF solution. Even after six
days' continuous perfusion, the amount of endoenzymes washed
out is so small that it can scarcely be estimated ; but a sudden
change in the conditions of perfusion, such as the substitution of
^ Hofmcister, Die chemische Orgardsation der Zelle^ Brunswick, 1901.
AUTOLYSIS OP TISSUES 3
chloroform saline for the sodium fluoride, causes an immediate
and very rapid setting free of the endoenzymes.*
Another and simpler method of measuring the autolysis of
intact tissues is to keep the organ under investigation in a
moist chamber for the required time, and then wash out the
autolytic products with saline solution. The results obtained
in three experiments with rabbits' kidneys are given in the
table. A kidney kept for three days at 14** C, yielded 12 units
of erepsin during the first four hours' perfusion, and 82 more
during the next six days, whilst at the end of this time it still
retained 18 units bound up in the tissues. Two other kidneys,
kept for seven and eight days respectively before perfusion,
yielded three to five times as much erepsin during the first
CiOBditloii of
Kidney.
Number
ofDaTi
Perftiaed.
Units of Biepsin washed out during
BNpitB
remaining
inKidnef
at end of
PsrfHsion.
Otolhoar.
lto4]ioiin.
4 hout onwards.
Kept 3 days at 14''
» 7 .. 12*
Fresh .
»f • • •
i» • • •
6
7
4
I
5
6
43
a8
:l
•5
6
23
10
•7
•5
83
61
63
188
18
15
I
I
6
four hours' perfusion, but in spite of this, anid of a subsequent
four to seven days' perfusion, they still retained a fair amount
of the enzyme bound up in their tissues. The amount so bound
up after a long-continued perfusion is very variable, and it will
be seen that the three kidneys which were perfused immediately
after excision, and which yielded very little enzyme during the
first four hours' perfusion, retained very little after five to seven
days' perfiision under various abnormal conditions as of putre-
faction and of chloroform treatment
When organs and tissues are disintegrated by mechanical
means, the endoenzymes break free from their anchorage and
pass into solution very much more rapidly. If a tissue be finely
minced or chopped with a knife, and an extracting medium be
added, a large portion of the endoenzymes pass into solution at
once, but it is a matter of days or weeks before filtered samples
* Vernon, Z^;V./. ai/^em, pAystoL,6jp, 399, 1907 ; see also, Lcct. viii.,p. 199.
4 PROTEOLYTIC ENDOENZYMES
of the extract show their maximum zymolysing power. For
instance, I found ^ that extracts of most animal tissues, made
by adding two parts of glycerin to one part of chopped tissue,
attained their maximum ereptic activity in about three weeks.
If, on the other hand, the extracting medium consisted of a
mixture of three parts of glycerin and two parts of water, the
maximum activity was attained in four or five days.^
If the state of division of the tissue particles be fine enough,
it IS probable that most of the endoenzymes are set free
immediately, and pass into solution. Hence, if sufficient
material is available, the method first adopted by Buchner^
for obtaining endoenzymes should be used. This method
consists in mixing the tissue — or micro-organisms — ^with an
equal weight of quartz-sand, and grinding up the mixture till
the cells are thoroughly broken up. If the mass becomes too
liquid, some of the moisture-absorbing siliceous earth, kieselguhr^
should be added in addition. The mass of disintegrated tissue
and sand is wrapped up in hydraulic chain-cloth, and by means
of a hydraulic press subjected to considerable pressure. The
juice of the broken-up tissue cells is thereby squeezed out, and
this juice probably contains the major part of the soluble
endoenzymes. But it has been shown by Dauwe* that
kieselguhr is capable of taking up enzymes, whilst Hedin ^ finds
that it has a specific absorption power. For instance, if mixed
with a solution of spleen enzymes, it may absorb and remove
from solution more than half of the o-protease (an enzyme
digesting in alkaline solution), but leave the /8-protease (which
digests in acid solution) almost untouched. In order to obtain
the maximum yield of endoenzymes, it would probably be best,
therefore, to avoid using kieselguhr altogether, and keep to sand
alone. Also, in the case of soft tissues, such as most animal
tissues, the preliminary grinding with sand is unnecessary, as
the cells are sufficiently broken up by the impact of the sharp
sand grains upon them in the hydraulic press. In the case of
micro-organisms, the mechanical disintegration is best effected
* Vernon, /<?«r«. Physiol,^ 32, p. 34, 1904. ^Jbid.^ 33, p. 93, 1905.
* E. Buchner, Ber,y 30, p. 117, 1897.
* Dauwe, Hofmeister^s Beitr.^ 6, p. 427, 1906.
* Hedin, Biochem. Joum,^ 2, p. 112, 1907.
ENDOENZYME PREPARATIONS 5
by placing the mixture of organisms and sand in a metal
cylinder surrounded by a jacket through which cold brine is
circulated. A steel axis provided with a series of horizontal
vanes is rotated rapidly (up to 5000 revolutions per minute)
in the mixture, and the violent intercoUision of the sand particles
and micro-organisms results in a very complete disintegration.^
The alternative method adopted by Macfadyen and Rowland,^
and one suitable for animal tissues as well as micro-organisms,
is probably the best of all if expense be no object. It consists
in triturating the organisms at the temperature of liquid air
(—180° to — 190"* C). At this low temperature, the cells become
so brittle that no sand need be added to assist disintegration.
Also, all chemical decomposition processes are for the time
being cpmpletely prevented.
It is to be borne in mind that the methods just described can
only afford information concerning the soluble and fairly stable
endoenzymes of the tissues. As will be pointed out in subse-
quent lectures, there is very little doubt that insoluble
endoenzymes exist in addition, and probably other endoenzymes
which are so unstable as to lose their activity directly they break
free from the tissues.
Another convenient method for obtaining intracellular
enzyme preparations is that employed by Wiechowski.* The
organ is chopped up very finely, and the tissue pulp, mixed with
toluol, dried in thin layers on glass plates in a warm room.
The dried tissue is then ground up thoroughly, washed with
toluol, and is ultimately obtained in such a fine state of division
that an aqueous suspension of it is completely filterable through
filter paper, and on standing only very slowly forms a deposit.
Wiechowski claims that this organ powder contains the tissue
proteins and ferments in an uninjured condition.
Proteolytic Endoenzytnes, — We now pass on to a description of
the more important endoenzymes hitherto identified. First and
foremost come the proteolytic endoenzymes. All living matter
is continually breaking down its protein constituents and
excreting protein disintegration products. Hence, if any or all
* Cf, Macfadyen, Rowland, and Morris, Proc, Roy, Soc,^ 67, p. 250, 1900.
* Macfadyen and Rowland, Centralb,f. Bakt.y 30, p. 753, 1901.
^ Wiechowski, Hofmeister^s Beitr.y 9, p. 232, 1907,
6
PROTEOLYTIC ENDOENZYMES
of the processes of degradation occurring during life are the work
of endoenzymes, we should expect to be able to demonstrate the
existence of such enzymes in the tissues after death. In the
study of endoenzymes, we should most appropriately examine
those of the lowest and simplest of living organisms first, and
pass thence to those of the higher animals ; but in the present
state of our knowledge this is not advisable. Almost all the
work hitherto done upon proteolytic endoenzymes concerns
mammalian tissues, and so this will be described first.
The existence of proteolytic endoenzymes was first estab-
lished in 1890, when Salkowski* showed that liver and muscle,
if minced and kept in chloroform water, underwent what he
termed ** autodigestion." The data given in the table show the
result of the autodigestion of dog's liver. The gland was
chopped up, and half of it was placed in ten times its volume of
chloroform water, and kept for sixty-eight hours at blood heat.
The digest was then filtered, boiled to remove coagulable protein,
again filtered, and analysed. By comparison with the control
Soluble Constituents from 1000 gn».
of Liver.
Control
Bxperimeiil.
Chief
Experiment.
Organic Substances
Nitrogen (calculated as Protein) .
Phosphoric Add
Purin Bases
Gms.
33-7
19.7
1.36
I-IO
Gms.
460
39-0
1.96
I •22
experiment, in which the other half of the chopped-up liver was
first sterilised by a current of steam for an hour and a half,
and was then incubated in chloroform water, we see that the
autodigestion caused a large amount of organic substances to
pass into solution. Probably these consisted mostly of protein
decomposition products, for the digestion liquid of the chief
experiment gave no distinct biuret test, but yielded a consider-
able quantity of leucin and some tyrosin. The liquid of the
control experiment, on the other hand, gave a biuret test, and
yielded no leucin. Judged by the increase of phosphoric acid
and purin bases, there must have been some autodigestion of the
1 Salkowski, Zeitf. klin, Med., 17, p. ^^ (suppL), 1890.
CLASSIFICATION OF ENDOENZYMES 7
nucleoprotein constituents of the tissues as well as of the
proteins. The autodigestion, or, to adopt Jacoby's term, the
autolysis, was undoubtedly the work of endoenzymes and not of
bacteria, as complete sterility was maintained throughout the
experiment.
The increase of purin bases found by Salkowski had been
noticed by Salomon ^ nine years previously. He found that
the amount of purins in muscle and liver kept at room tempera-
ture was doubled during the first twenty-four hours after death.
This purin formation he attributed to the action of a ferment
set free at the time of death of the tissue cells. He thus fore-
shadowed the more complete and extensive researches of
Salkowski. However, these researches were lacking in one
particular, and this was supplied by Schwiening^ two years
later, Schwiening showed that filtration of the liver extracts
did not prevent autolysis : in other words, that the autolysis was
the work of soluble endoenzymes, and was not determined by
insoluble cell constituents. Schwiening also showed that alkalis
paralysed the autolysis, whilst acids did not.
In all probability, the autodigestion occurring in tissue
extracts and juices is the work not of one or two, but of many
endoenzymes. But little attempt has been made to isolate
these several ferments, but their existence is clearly indicated
by the differential actions of extracts of various organs
upon different proteins and protein decomposition products.
They are best classified according as they have power to
hydrolyse :
(a) Native proteins (as fibrin, albumins, globulins).
(b) Proteoses, peptones, and polypeptides.
(c) Individual amino acids (as glycocoU, hippuric acid,
arginin).
(d) Urea.
Endoenzymes of the first class are somewhat poorly
developed, and the most active press juice obtainable from
mammalian tissues is weak in comparison with gastric juice
or activated pancreatic juice, or with extracts of gastric mucous
membrane or pancreas. At least this is the case if the action
1 Salomon, Arvk,/, {Anat u,) PhysioLy 1881, p. 361.
2 Schwiening, VtrcAow's ArcJk^ 136, p. 444, 1894,
8
PROTEOLYTIC ENDOENZYMES
of the press juice upon protein from external sources be tested.
Boiled fibrin and coagulated egg white are very slowly attacked,
whilst unboiled fibrin passes into solution only after some hours.
As a rule, therefore, the action of the endoenzymes upon the
native proteins already present in the press juice is investigated,
and the rate of conversion of these proteins into non-coagulable
proteoses, peptones, and amino acids determined. Any or all
of the stages of digestion can be investigated by making
Kjeldahl determinations of the total nitrogen in the filtrate from
samples of the press juice which have been precipitated by
suitable reagents, and comparing them with the nitrogen in
the unprecipitated juice. Hot trichloracetic acid precipitates
native proteins, but not proteoses, peptones, or amino acids.
Saturated zinc sulphate, and 7 per cent, tannic acid pre-
cipitate proteoses, but not peptones or amino acids, whilst a
mixture of 40 per cent, phosphotungstic acid with 10 per cent,
sulphuric acid precipitates proteoses, peptones, and di-amino
acids such as hexone bases and cystin, but not mono-amino
acids.
Working with such precipitants, Hedin and Rowland^
demonstrated the existence of a fairly active /8-protease enzyme
in the expressed juice of a number of organs. They kept the
Nitrogen at or beyond the Peptone Stage.
Before
Digestion.
After
16 hours.
After
22 hours.
After
40 hoars.
Spleen Juice alone . . . ,
+ .25% Acetic Acid
+ .i%HCl . . .
+ -37%Na2C03 . .
Boiled Juice + .25% Acetic Acid .
7.2
f-4
17-4
26.7
25^
9.6
7.6
17-6
27-8
27.8
9.4
19-8
30*0
30*2
10-8
juice at body temperature in the presence of toluol, and at fixed
times precipitated samples of it with 7 per cent, tannic acid, and
estimated the nitrogen in the filtrate. Spleen juice and kidney
juice proved to be the most active of all, and some of the data
obtained with ox spleen juice are given in the table. The total
nitrogen in 5 c.c. of the juice corresponded to 35-1 c.c of -A^io
^ Hedin and Rowland, ZeiLf.physioL Chem.^ 32, pp. 341 and 531, 1901.
AUTOLYSIS
9
acid, and we see that 7-2 parts of this nitrogen was at or beyond
the peptone stage before digestion began. After forty hours,
19-8 parts of it had got to this stage, but when acetic acid or
hydrochloric acid was added to the juice, the hydrolysis was so
much accelerated that 30'0 parts (or 8$ per cent, of the whole)
reached this stage. The /8-protease is, in fact, an acid-acting
ferment, and the moderate digestion occurring in the unacidified
juice is dependent on this juice having a considerable natural
acidity (corresponding in the present instance to NJ4,o NaOH).
Slight over-neutralisation of the juice with sodium carbonate
almost stopped the autolysis. Such as did occur was probably
due to another endoenzyme, called by Hedin a-protease, which
digests in alkaline solution.
Jtdee of Skeletal Muscle of
Horse.
(Total N=:66-8.)
Nitrogen at or beyond Peptone Stage.
Before
Digestion.
After
2 days.
After
5 days.
After
1 month.
Juice alone ....
„ +.25% Acetic Add.
„ +CaC08
17-8
31-3
36.5
33-8
37-1
51.4
45-0
53-4
The press juice of lymphatic glands was found to be almost
as active as that of the spleen and kidneys, whilst that of the
liver was distinctly less active. That of heart muscle was less
active still, and weakest of all was the juice of skeletal muscle.
The best results obtained with this juice are given in the table.
Juice of Heart of Ox.
(Total N=86-9.)
Nitrogen at or beyond Peptone Stage.
Before
Digestion.
After
16 hours.
After
8 days.
After
16 days.
Juice alone ....
„ +.25% Acetic Acid.
„ +CaC03
9-6
10-5
I3-0
10-3
13-2
17.4
22.6
13-9
and they show that the protease acted better in neutral solution
(neutrality being effected by addition of excess of calcium
carbonate) than in acid solution. That of heart muscle acted
B
10 PROTEOLYTIC ENDOENZYMES
best in acid solution, but neutralisation with CaCOg or MgO
did not diminish the relative rate of autolysis to so great an
extent as in the case of spleen, kidney, and liver juice. We
must therefore conclude, either that tlie proteases of one
organ differ in kind from those of another, or that the relative
amounts of acid-acting and alkali-acting proteases vary con-
siderably.
The influence of acids and alkalis upon tissue autolysis has
been studied by Wiener,^ Baer and Loeb,^ Preti,^ and Arinkin,*
and their observations confirm the observations of Hedin and
Rowland. Arinkin investigated the influence of various acids
upon liver autolysis, and he found the optimum acidity to be
•056 per cent, of HCl, '075 per cent, of H2SO4, -lo per cent,
of H3PO4, and -277 per cent of lactic acid. This optimum
acidity concerns only the hydrolysis of the protein constituents
of the tissues, as the nuclein autolysis of the tissues is diminished
by the acidification.
A feeble alkali-acting protease, or a-protease, has been
isolated by Hedin ^ from the residue left by digesting minced
spleen with •! per cent, acetic acid. This residue is extracted
with S per cent. NaCl, and the saline solution dialysed for one
or two days. The very scanty precipitate thrown down contains
an enzyme which is fairly active when dissolved in -25 per cent.
NagCOg. The slight action which it showed when in acid
solution was probably due to the presence of some /8-protease
as impurity The feeble action of the a-protease is perhaps
dependent in part on the presence of an anti-body, for Hedin ®
made the curious discovery that if fresh spleen pulp were kept
for twenty-four hours in presence of ^2 per cent, acetic acid, and
were then made alkaline and allowed to act upon a suitable
protein such as 2*5 per cent, casein solution, it digested it more
rapidly than if it had not previously been treated with acid.
For this and other reasons he concluded that the acid treat-
^ Wiener, Centralb.f, Physiol,^ 19, p. 349, 1905.
2 Baer and Loeb, ArcKf. exp. Patk,y 53, p. i, 1905 ; Bsieryidtd,, 56, p. 68.
3 Preti, Zeit, f, physioL Chetn,^ 52, p. 485, 1907.
* Arinkin, ibid,^ 53, p. 192, 1907.
^ Hedin, /<?«r«. PhysioL^ 30, p. 155, 1904.
• Hedin, Hatnmarstetis Festschrift^ Upsala, 1906.
AUTOLYTIC PRODUCTS 11
ment had destroyed an anti-ferment present in the spleen
pulp.
Of the two endoenzymes, the a-protease shows considerable
resemblance to a weak tryptic ferment, but the /8-protease is
evidently quite different from both trypsin and pepsin. Like
pepsin, it acts in an acid medium: like trypsin, it hydrolyses
native proteins to proteoses, peptones, and amino acids. The
formation of proteoses in the autolysis of tissues was first
demonstrated by Biondi^ for calfs liver. However, Biondi
failed to find either peptones or tryptophan among the auto-
lytic products, and so concluded that the course of hydrolysis
is different from that effected by trypsin. Jacoby^ failed to
find either proteoses or peptones in liver autolyses, but in
addition to leucin and tyrosin he proved the presence of
tryptophan, and likewise of glycocoll, hippuric acid, and urea.
In ox spleen juice which had been incubated for two to four
months in presence of toluol, Leathes* found tryptophan and
traces of proteoses, and he isolated leucin, tyrosin, amino-
valerianic and aspartic acids, arginin, histidin, and lysin. From
a filtered aqueous extract of minced kidney, which had been
allowed to digest itself at 36** in presence of -2 per cent, acetic
acid, Dakin* isolated all of the products obtained by Leathes
except arginin and aspartic acid, and in addition found alanin,
o-pyrrolidin carboxylic acid, phenyl-alanin, cystin, and hypo-
xanthin. Both Leathes and he conclude that the products
of action are the same as those formed by trypsin in alkaline
media, or those formed by the hydrolytic action of mineral
acids, but their results agree with Biondi's conclusion that the
course of hydrolysis is different from that effected by trypsin,
for neither of them found any peptone. Umber ^ likewise
failed to find peptone in the ascitic fluid of two patients
examined by him, though he found proteoses, leucin, tyrosin^
and traces of hexone bases. We may assume, therefore, not
that no peptone is formed at all by the hydrolysis of the
* Biondi, Virckow's Arch.y 144, p. 373, 1896.
* Jacoby, Zeit f. physiol, Chem.^ 30, p. 149.
' Leathes, /<7iyn!r. PhysioL^ 28, .p. 360, 1902.
^ Dakin, ibid,^ 30, p. 84, 1904.
^ Umber, Munch^ Med, Wach.^ 1902, No. 28,
12 PROTEOLYTIC ENDOENZYMES
proteoses, but that when formed it is immediately split up
further into amino acids. Endoenzymes in this respect differ
considerably from trypsin, which, though it quickly splits up
some of the peptones formed by the hydrolysis of proteoses,
has little if any action on others of them (^.^., the so-called
anti-peptone of Kuhne). The powerful peptone-splitting action
of endoenzymes is probably to be attributed, not to the a- and
/8-proteases mentioned above, but to the erepsin-like enzymes
described in the next section.
The action of Hedin's a-protease was examined more in
detail by Cathcart,^ who digested coagulated blood serum with
it for seven and a half months ^t 37° in presence of -25 per
cent. NagCOg and toluol. The digest showed a faint biuret
reaction, and a well-marked tryptophan reaction, and was
found to contain leucin, amino-valerianic acid, alanin, phenyl-
alanin, tyrosin, a-pyrrolidin-carboxylic acid, lysin, histidin,
and arginin. It differed from the acid digest examined by
Leathes in containing little or no aspartic acid, but a large
amount of glutamic acid, and in the fact that the arginin was
optically inactive, instead of being of the usual dextro-rotatory
form.
Erepsin. — In 1901 Cohnheim ^ found that if the press juice
of intestinal mucous membrane were allowed to act upon
proteoses and peptones, these bodies disappeared, just as
Hofmeister^ found that they disappeared from the intestinal
mucous membrane of an animal after death. This disappear-
ance was attributed by Hofmeister to the synthetic action of
the still living intestinal cells, but Cohnheim found that in his
mixtures of press juice and peptones there was never any
increase in the total protein content On the contrary, the
peptones were broken down, and converted into crystalline
decomposition products. This hydrolysis was effected by the
enzyme erepsin, a body quite distinct from trypsin, in that it
has no action upon ns^tive proteins but only upon their
decomposition products. According to Cohnheim, the proteins
* Cathcart, Joum. PhysioLy 32, p. 299, 1905.
* Cohnheim, Zeit /. physioL Ckem.^ 33, p. 451, 1901 ; and 35, p. 134,
1902.
3 Hofnieisteri ildd,y 6, p. 69 ; and Arch.f. exp, Patk.y 19, p. 8, 1885.
EREPSIN 13
of horses' plasma, of ascitic fluid, of muscle, and vitellin and
globtn are not acted upon by erepsin even during several weeks.
Primary proteoses are not appreciably attacked in thirty-six
hours, though they undergo gradual hydrolysis in the course of
days. Deutero-proteose B (prepared by Pick's method) is
split up in nineteen hours to the stage at which it no longer
gives the biuret test, and peptone (prepared by the prolonged
peptic digestion of muscle) in as little as two hours. The
protamine clupein sulphate is quickly hydrolysed, and, con-
trary to expectation, casein is likewise decomposed sbtnewhat
readily.
Erepsin has been shown by Kutscher and Seemann ^ to be
present in succus entericus, and doubtless it is of importance in
assisting the pancreatic trypsin to break up the proteoses and
peptones in the gut But Cohnheim thinks that in all proba-
bility its more important seat of action is within the cells of the
intestinal mucous membrane, not outside them. Whether the
intra- and extra-cellular erepsins have identically the same
action upon proteoses and peptones has not been determined,
but there is no reason to suppose that the two enzymes are
different bodies. They both act best in fairly alkaline solution,
and have little or no digestive power in an acid one, and both
give similar decomposition products, so far as is known. It
is suggested by Abderhalden and Teruuchi^ that the term
"erepsin," being comparable to ** trypsin" and " pepsin," ought
to be confined to the proteolytic enzyme of the succus entericus,
and not applied to the corresponding endoenzyme. If this
suggestion be adopted, the endoenzyme could be spoken of
as " endoerepsin," or "ereptase."
In the light of our present knowledge, it might be predicted
that most. intracellular enzymes found in one tissue or organ of
the body would, }n all probability, be found in greater or less
degree in other tissues. This is true of erepsin, as in 1903' I
showed that the pancreas contains an ereptic enzyme which is
quite distinct from trypsin, and in 1904* that erepsin is probably
* Kutscher and Seemann, Zeitf.physiol, Ckem.y 35, p. 432, 1902.
^ Abderhalden and Teruuchi, ibid,^ 49, p. i, 1906.
^ Vernon, /<wr;i. Physiol,^ 30, p. 330, 1903,
* IHd,, 32, p. 33, 1904.
14
PROTEOLYTIC ENDOENZYMES
present in all animal tissues. In order to obtain comparable
results^ the relative amounts of enzyme in the tissue extracts
were determined quantitatively by a colorimetric method
dependent on the biuret test I found that the time required
to split up any given percentage of a standard solution
of Witte's peptone to the non-biuret-test-giving stage varied
inversely as the quantity of enzyme present. For instance, in
one experiment 8, 4, 2, and i parts of enzyme split up 20 per
cent, of the peptone in -7, i«4, 2«8, and 66 hours respectively.
The same amounts of enzyme split up 30 per cent, in vg, 3-4,
68, and IS«7 hours respectively, and 40 per cent in 4*2, 7'4, 13-8,
and 33-8 hours. In the majority of cases the time required by a
tissue extract to split up 20 per cent of a 2-5 per cent, peptone
solution was determined, when acting at 38"* C. in presence of
• I per cent NagCOg and toluol. The relative amounts of
erepsin found to be present in the various tissues of the cat were
the following : ^ —
Tiiisae.
Breptio Value.
Tissue.
Ereptic Value.
Duodenal muc. memb. .
27.7
Submaxillaiy gland
Thyroid gland .
5-3
Jejunal „
18.2
4-3
Ileal
14.4
Suprarenal gland ,
2-5
Large intest „
5.8
Cardiac muscle
1-6
Gastric „
3-9
Brain .
1-2
Kidney . . .
14-3
Ovary . . .
l-O
Spleen.
7-6
Skeletal muscle
.8
Lung . , , ,
6.9
Blood . .
•3
Pancreas
6.4
Serum .
•I
Liyer . . . •
5-0
It will be seen that the duodenal mucous membrane is
richest of all in erepsin, and that there is a steady diminution in
enzyme on passing down the gut. Of the other tissues, the
kidney comes easily first, being twice as rich in erepsin as any
other organ. Blood and serum contain only a very small
amount of the ferment.
The dependence of the hydrolytic power of erepsin upon an
alkaline medium is shown by the following data, obtained with
cat's kidney extract : —
Vernon, t^'d. ; and/wr« PAystol,^ 33, p. 81, 1905.
EREPSIN
16
Bztnct acting in presence of
Relative Digeetiye Power.
Water only
•I per cent. Na^CO,
.2 „ Na,CO, . . .
K)5 " Acetic Acid .
•I ,, Acetic Add .
3-7
13-3
22*2
28*2
•4
•17
It will be seen that the rate of hydrolysis is greater and
greater the more alkaline the solution. However, this is true
only if the time of hydrolysis of 20 per cent, of the peptone be
taken as a measure of digestive power. The strong alkali
destroys the enzyme, so that 40 per cent, of the peptone was
more quickly hydrolysed in presence of •! per cent. NagCOsthan
in -4 per cent. Na^COg.
The question arises as to whether the ereptic enzyme in the
various tissues is in all cases the same body. The evidence,
though not conclusive, points distinctly to the existence of
different enzymes in different tissues. I found that various
partially hydrolysed peptones were further split up at very
different relative rates by the different extracts. Again, the
different extracts were differently affected by the alkalinity
and acidity of the medium in which they were acting. Those of
cat's- tissues, for instance, were in some cases found to be 60 or
70 times more active in presence of • i per cent. Na^COg than
in *i per cent, acetic acid, whilst in others they were only 12
times more active. The erepsin in pigeon's tissues was much less
retarded by acid, for it was only three to five times less active
in -I per cent, acetic acid than in -i per cent. NagCOg. It
seems probable, therefore, that the tissues not only contain
somewhat different erepsins, but different relative amounts of
acid-acting ferment and of alkali-acting ferment But it is to be
remembered that the tissue extracts undoubtedly contain pro-
teins and other substances which may retard or accelerate the
action of the enzymes, and until these enzymes can be prepared
free from such impurities, it is best not to speak dogmati-
cally.
The differences between endoenzymes and trypsin is well
shown by their action upon polypeptides. Abderhalden, in
16
PROTEOLYTIC ENDOENZYMES
conjunction with Emil Fischer/ Teruuchi,^ Rona,^ and Hunter,*
found that the press juice of liver, kidney, and muscle, hydrolyses
certain polypeptides such as dl-leucyl-glycin, glycyl-dl-alanin
and glycyl-glycin which trypsin has no action upon. Kidney
juice was the most active; liver juice somewhat less so, and
muscle juice very much less so. The first two of the three
polypeptides mentioned are racemic bodies, and in each case
only one of th^ two stereoisomers was attacked by the endo-
enzymes. The active aniino acid liberated was that which is
present in native proteins, viz., 1-leucin in the one case and
Polypeptide.
Ox or Dog's
Liver Juice.
Dog's Kidney
Juice.
Ox of Dog's
Muscle Juice.
Trypsin.
Dl-leucyl-glvcyl-glydn .
Dl-alanyl-g^(^l-giydn .
GlycyM-tyrosm
Dl-leucyl-glydn
Glycyl-ai4Uimn .
Glycyl-fflydn . .
LevLcyl-ltaan .
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
d-alanin in the other. Other peptides, such as leucyl-leucin, resist
the attack of endoenzymes as well as of trypsin, whilst the first
three peptides recorded in the table are split up by both endo-
enzymes and trypsin. They are not attacked by pepsin, however,
so by means of these synthetic polypeptides we are able to
differentiate sharply between the three proteolytic enzymes. If
the proteases, endoerepsins and other proteolytic endoenzymes
could be separated from one another, it would probably be found
that they too exerted a differential action upon certain of the
polypeptides : but at present we are ignorant as to the precise
endoenzyme responsible for the peptide hydrolyses. No acid or
alkali was added to the digests, but in that the tissue juices have a
considerable natural acidity, it might be supposed that the /9-pro-
tease enzyme was chiefly responsible. However, Abderhalden
and Teruuchi found that fresh succus entericus, which has a con-
* Fischer and Abderhalden, ZeiL f, physioL Chem^ 46, p. 52, 1905.
^ Abderhalden and Teruuchi, ibid.^ 47, p. 466 ; and 49, p. i, 1906.
^ Abderhalden and Rona, ibid,^ 49, p. 31.
* Abderhalden and Hunter, ibid.^ 48, p. 537.
TRYPSIN AND ENDOENZYMfiS
17
siderable natural alkalinity, could split up glycyl^glycin just tike
the tissue juices, and also we have seen that these juices can
hydrolyse peptones slowly in faintly acid solution, so it is
possible that the peptide hydrolysis is due to endoerepsin, or to
this enzyme and j3-protease acting concurrently.
Other evidence differentiating trypsin arid endoen2ymes is
yielded by quantitative studies of the course of protein
hydrolysis. Abderhalden, working in conjunction with Rein-
bold^ and Vogtlin,* found that certain of the amino acid
constituents of proteins, such as tyrosin and tryptophan, were
quickly split off from the protein complex by the action of activated
pancreatic juice, and could be recovered quantitatively. Other
constituents, such as glutamic acid, aspartic acid, leucin, valin,
and alanin, were only gradually and often not completely
liberated, whilst others again, such as prolin and phenyl-alanin,
were scarcely liberated at all. As already mentioned, Dakin
isolated both of these latter bodies from an acid kidney autolysis,
and Cathcart isolated them from a digest of blood serum with
a-protease in alkaline solution. Hence the tissue endoenzymes
carry the protein hydrolysis to a further stage than trypsin.
But it is not yet proved that they can carry it to absolute
completion in the same way that inorganic catalysts can do.
Abderhalden and Prym ^ hydrolysed the proteins of liver pulp
by boiling with hydrochloric acid, and from the mixture they
isolated, by the ester method, 42*5 to 4S«8 gms. of mono-amino
acids for each loo gms. of liver protein taken. Some of the liver
pulp was incubated in presence of water and toluol for ten to fifty
days, and the following amounts of mono-amino acids (calculated
for 100 gms. of liver protein) were isolated from the autolysis : —
DiufationofAvtolyste.
10 dftys.
20 days.
80 days.
40 days.
50 days.
Mono-amino adds .
Gms.
1-85
Gms.
5-5
Gms.
lO'I
Gms.
20.2
Gms.
29.1
Assuming that the ester method yields roughly quantitative
^ Abderhalden and Reinbold, Zeit /. physioL Chem,^ 44, p. 284, 1905 ;
and 46, p. 159, 1905.
* Abderhalden and VSgtlin, iHd.^ 53, p. 315, 1907.
3 Abderhalden and Prym, ibid,^ 53, p. 320, 1907.
C
18 PROTEOLYTIC ENDOENZYMES
results, we see that even after fifty days* autolysis only about
two-thirds of the total mono-amino acids were liberated. Still
the autolysis seemed to be progressing at a steady rate at the
time it was stopped, and it is very probable that if only it had
been continued for another twenty or thirty days, it would have
attained the completeness of the acid hydrolysis.
An interesting and important fact noted by Abderhalden
and Prym in their autolyses was the early disappearance of the
biuret test It was distinct for the first day or two, but had
disappeared on the fourth day. At this time extremely little
of the protein had been hydrolysed to the mono-amino acid
stage, and hence presumably the protein decomposition products
consisted chiefly of various unknown dipeptide amino acid
combinations which were too simple in constitution to yield
the biuret test. Thus the work of Schiff ^ has shown that in order
to give this test a substance must contain two — CO.NH — group-
ings, joined indirectly or directly. So tripeptides give it, but
dipeptides — unless they are amides — do not.
Arginase and similar Endoenzymes, — The third class of
proteolytic endoenzymes is at present even less clearly defined
than the two classes so far described. It includes enzymes
which act upon individual amino acids, and split them up into
still smaller molecules. The best known member of this class
is arginase. This enzyme was discovered by Kossel and Dakin ^
in 1904, and it has the special property of hydrolysing arginin
to urea and ornithin (diamino-valerianic acid). Kossel and
Dakin point out that this hydrolysis diflers from those effected
by other " imidolytic " ferments such as trypsin and erepsin, for
they attack the — CO — NH — C — groupings of proteins and
polypeptides, and split them into — COOH and NHg — C —
groupings. Arginase, on the other hand, splits off urea
according to the equation:
NH NHg
-C-NH-CH2-CH2-CH2-CH-COOH + H2O
' '. ' ' NH,
^NHg-CG-NHg + NHg-CHa-CHg-CHa-CH-COGH
* Schiff, Ber,^ 29, p. 298, 1896 ; Ann, Chem, Pharm,^ 299, p. 236, 1897.
' Kossel and Dakin, Zeitf.physioL Chem,y 41, p. 321, 1904.
NH2
ARGINASE 19
Arginase was found to be present in largest quantity in the
liver. For instance, 25 gms. of minced liver, placed in an
incubator with 1000 c.c. of water and toluol, completely
hydrolysed 5-0 gms. of arginin in six hours. In another
experiment, 25 gms. of liver hydrolysed 2-7 gms. out of 3*2 gms.
of arginin in ten minutes. No other organ proved anything
like so active. The best of them was the kidney, and it was
found that 25 gms. of minced calfs kidney, when allowed to
act upon 2-2 gms. of arginin for three days, decomposed 1-14 gms.
of it. Slightly less active were thymus and lymph glands.
Intestinal mucous membrane was considerably less active, and
muscle still less. Blood contained only traces of arginase, and
spleen, suprarenal gland, and pancreatic juice apparently none
whatever. These conclusions are in agreement with the
observations of various investigators upon the products of tissue
autolysis. Thus Kutcher and Seemann^ found no arginin in
autolyses of the thymus and of intestinal mucous membrane,
and Dakin * found none in those of the kidney. On the other
hand, Leathes ' obtained a good yield of arginin from a spleen
autolysis which had been digesting two to four months.
Dakin * concludes that arginase is a specific enzyme adapted
for the exclusive hydrolysis of dextro-rotatory arginin, or of
substances containing the dextro-arginin grouping. He finds
that it has no action upon guanidin, creatin, or creatinin, or on
protamines or other proteins : but it acts upon the arginin
complex present in certain protones, and liberates urea therefrom.
The definite localisation of arginase in particular organs
proves that the enzyme is quite distinct from proteases and
erepsins, for these ferments are probably present in every tissue
of the body. There is reason to think that other proteolytic
enzymes exist which are similarly confined to particular organs,
and to particular purposes, but in their case the evidence is not
very complete or convincing. Lang^ made a number of
* Kutscherand Seemann, Zeit.f, physioU Chem»^ 34, p. 114, 1901 ; and 35,
p. 432, 1902.
. * Dakin, /<wf7i. PhysioLy 30, p. 84, 1903.
3 Leathes, ibid,^ 28, p. 360, 1902.
* Dakin, /(7f#f7i. Biol, Chem,^ 3, p. 43$, 1907.
* Lang, Hofmeister^s Beitr,^ 5, p. 321, 1904.
20 PROTEOLYTIC ENDOENZYMES
determinations of the power possessed by various tissues to
split off ammonia from certain amino acids and other nitro-
genous bodies. The method consisted in mixing up the minced
tissue thoroughly with -9 per cent. NaCI, toluol and the amino
acid, and allowing it to incubate for two to thirty-two days.
It was then acidified, and 5 to 8 per cent, tannin solution added.
The filtrate was distilled with magnesia in a vacuum at 40** to
45*" (Nencki and Zaleski's method),^ and the ammonia which
came off collected and estimated. Lang found that from glycin
minced intestinal mucous membrane and pancreas separated a
considerable amount of ammonia; kidney, suprarenal gland,
testis, and liver separated a moderate amount ; and spleen and
lymph glands none at all. From glucosamin, kidney and
suprarenal gland separated the most ammonia ; liver, intestine,
testis, and spleen a moderate amount ; muscle very little ; and
pancreas none at all Other experiments were made with
tyrosin, leucin, and cystin, and every one of these substances
was apparently hydrolysed to some extent by one or other of
the tissues experimented with. Unfortunately the results,
though of considerable interest, cannot be accepted unreservedly.
Many of them are based upon a single experiment only, and
are sometimes self-contradictory. For instance, liver digested
with glucosamin for five days yielded 62 mg. of NH3, as
against 45 mg« from liver digesting without glucosamin.
After digesting nine days, however, liver plus glucosamin gave
only 53 mg. of NH3, but liver alone, 92 mg. Again, in
some cases liver and kidney tissue to which glycin had been
added yielded less NH3 than without any addition, and indeed
Jacoby^ has stated that minced liver is quite incapable of
hydrolysing glycin. Still there can be no doubt that the tissues
contain enzymes which have the power of liberating ammonia
from some or other amino acids and amides, and that certain
structures, such as the liver, are much more active than others
such as muscle.
Previous to Lang, the ammonia-liberating power of liver
endoenzymes was studied in considerable detail by Jacoby.^
He found that if ground-up liver tissue were kept at 38^ with an
1 Nencki and Zaleski, Zeit f. pkysiol, Ch$m,y 33, p. 193, 1901.
* Jacoby, ibid,y 30, p. 149, 1900. ^ y^y;
AMIDE NITROGEN 21
equal volume of water and toluol, the amount of ammonia set
free on boiling with magnesia, or the " amide " nitrogen, steadily
increased. For instance, in one experiment it was -0013 gm.
after one day's autolysis, -0035 gm. after two to five days',
•0047 gm. after eleven to fifteen days', and -0067 gm. after twenty
days' autolysis. In another experiment, he found that after
fourteen days' digestion the nitrogen still present in the form of
protein bad fallen from 94 per cent, of the whole down to 27
per cent, whilst the amide nitrogen had increased from M per
cent to 5-6 per cent In another experiment, after eighteen days*
digestion, it bad increased from •4 per cent to 8*4 per cent. Other
observers have obtained similar results with other tissues.
Dakin^ allowed kidney juice to digest itself under antiseptic
conditions, and he found that the ammonia increased slowly and
steadily for about two months.
The only amide known to be present in the protein
molecule from which the amide nitrogen could be derived is
urea, and this can account for only a small fraction of the
amide nitrogen obtainable. But in the light of Lang's
experiments, we may assume that part of it is derived from
certain of the amino acids, though we cannot definitely say
which of them.
Hausmann,* Giimbel,* Osborne and Harris,* and others, have
shown that if proteins be dissociated by boiling with HCl, they
yield a definite proportion of their nitrogen in the form of
ammonia which can be driven off by subsequent distillation
with magnesia. Casein yields 10 to 13 per cent of its total
nitrogen in this way, edestin 12 per cent, serum albumin 7 per
cent, but gelatin only i-6 per cent Hirschler,^ Stadelmann,*
and others, showed that trypsin has the power of liberating
ammonia from the protein molecule, whilst Zunz,^ and
Dzierzgowski and Salaskin,® found that pepsin likewise has this
^ Dakin, Jaum. Pkysio^ 30, p. 84, 1904.
* Hausmann, Zeitf,phyHoL Ch$m.^ 27, p. 95, 1899 ; and 29, p. 136, 1900.
' Giimbel, Hofmeister's Beitr,^ 5, p. 297, 1904.
* Osbonie and Harris, /<7«f7i. Amer, Ckem, Soc.y 25, p. 323, 1903.
* Hirschler, Zeit f, physiol, Chem,^ 10, p. 302, 1886.
* Stadelmann, Zeitf, BioL^ 24, p. 261, 1888.
^ Zunz, Zeit f, physioL Chem,^ 28, p. 151, 1899.
^ Dzierzgowski and Salaskin, Centralb,/, Physiol,^ 15, p. 249, 1902.
22
PROTEOLYTIC ENDOENZYMES
power. The latter investigators found that peptic digestion
of egg albumin for eighteen days split off 2-6 per cent,
of the total nitrogen in the form of ammonia, whilst peptic
digestion of casein for ten days split off 3-5 per cent. Zunz
found that peptic digestion of serum albumin for fifteen days
split off 2' I per cent, and of casein for fifteen days, 3-8 per cent
These amounts are only a half to a third those observed by
Jacoby for liver autolysis, and by Hausmann and others for acid
hydrolysis.
The amino acids and amides hydrolysed by endoenzymes
are probably to some extent different from those hydrolysed by
acids, for Jacoby found that if liver juice were first allowed to
digest itself, and were then boiled with HCl, it yielded consider-
ably more ammonia than in the absence of an initial autolysis.
The data obtained in one experiment were : —
Without
Autolysln.
After
Autolysis.
Amide Nitrogen, directly separable .
„ separable by adds .
Total Amide Nitrogen
Per cent.
2.6
8.7
Per cent.
9.4
6-3
II-3
15-7
In another experiment the amide nitrogen amounted to 9-6 per
cent without autolysis, and 15.5 per cent with it
The Formation and Hydrolysis of Urea by Enzymes, — Charles
Richet^ in 1893 found that dog's liver, freshly removed from the
body and kept at 39°, showed an increase in its content of urea.
Later on^ he found that an aqueous filtered extract of liver
tissue, digesting in presence of NaF, likewise possessed this
urea forming power, and even that the precipitate thrown down
by the addition of alcohol to the extract possessed it Gottlieb *
observed urea formation in liver pulp kept at 40° under aseptic
conditions, whilst Schwarz* found that a digest of liver pulp
showed an increase in its nitrogenous ether-alcohol extractives.
^ Richet, Comptes Rendus^ 118, p. 1125, 1893.
* Richet, C. R, Sac. BioL^ 1894, pp. 368 and 525.
3 Gottlieb, Munch. Med. Wochenschr.^ 1895.
* Schwarz, Arch./, exp. Path,^ 41, p. 60, 1898.
UREA FORMATION 23
These extractives were not ammonia, and so were presumably
urea.
There can be no doubt, therefore, as to the formation of urea
in liver pulp and liver extracts, but the amount produced is
small, and, as Kossel and Dalcin suggest, it may be formed by
the arginase hydrolysing the arginin formed by autolysis.
There can be no doubt that some of it arises in this way, but
probably not all. O. Loewi ^ found that the liver enzymes could
convert glycin and leucin into a nitrogenous body which, though
not actually urea, behaved like urea in respect of its solubility in
alcohol, its easily separable nitrogen, and in other ways. Not
only could liver extracts effect this change, but also an aqueous
extract of the precipitate thrown down by 97 per cent, alcohol.
The enzyme had no influence upon alanin or ammonium salts,
however. Previous to Loewi, Chassevant and Richet * observed
that liver extracts could convert alkaline urates into urea, but
Spitzer^ failed to observe any urea formation when liver pulp
and extracts were allowed to act either upon leucin, urates, or
ammonium salts. The origin of urea from any other source
but arginin is therefore unproved, and the subject requires re-
investigation.
The hydrolysis of urea by enzyme action is less open to
question than its formation. Jacoby* found that if liver juice
were allowed to act upon urea in presence of toluol for thirty-six
hours, more than twice the amount of ammonia was set free on
subsequent boiling with magnesia, as in the control experiment
in which no urea was added. Lang ^ found that liver pulp split
off a small quantity of ammonia from urea, whilst minced
pancreas split off a larger amount
The existence of a urea-splitting enzyme in fermenting urine
was demonstrated by Musculus* as long ago as 1874. This
urease^ as he cgiUed it, is formed by the activity of the Micrococcus
ureae^ and it seems to be entirely an endoenzyme, for Sheridan
^ O. Loewi, Zeit f. physiol. Chem,^ 25, p. 511, 1898.
^ Chassevant and Richet, Comptes Rendus Sac, BioL^ 1897, p. 793.
3 Spitzcr, Pfluger's Arch,,, 70, p. 60, 1898.
* Jacoby, Zeitf,pkysioL Chem,y 30, p. 149, 1900.
* Lang, HofmeisUr^s Beitr,^ 5, p. 321, 1904.
^ Musculus, Compies Rendus^ 78, p. 132, 1874 ; $2, p. 334, 1876.
24 PROTEOLYTIC ENDOENZYMES
Lea ^ found that fermenting urine, when freed from the Micrb-
coccus by adequate filtration, has no power of splitting up ur6a.
Again, Leube ^ did not find any soluble ferment in filtrates from
pure cultures of the organism. Musculus precipitated alkaline
pathological urine with alcohol, and found that an aqueous
extract of the dried precipitate was very active. Lea precipi-
tated fermenting urine in the same way, and found that the dried
alcohol precipitate, or an aqueous extract of it, when mixed with
2 per cent, urea solution and kept at 38"*, turned it alkaline and
liberated ammonia in a few minutes. The destruction of the
micro-organisms by the alcohol precipitation apparently sets
free the urease, and renders it capable of extraction. However,
Beijerinck * was unable to extract it from dead micrococci, and
he considers that it is firmly bound up in the cells. Moll * found
that even small quantities of antiseptics, such as toluol and
chloroform, render it inactive, but that sodium fluoride is not so
harmful.
This urea-splitting enzyme is not by any means confined to
the Micrococcus ureae. Miquel ^ demonstrated its presence in a
number of other Micrococci and Bacilli, and in a Sarcina* He
cultivated no less than 14 different species of micro-organisms in
peptone solutions containing -2 to '3 per cent, of ammonium
carbonate, and he found that after some days these solutions
contained a good deal of soluble enzyme which had been
excreted by the micro-organisms. But he gives no details of
his experiments, and in the face of the contrary assertions of the
other investigators, we cannot accept his statements as proven.
The urease of micro-organisms acts best in an alkaline
medium. The urease of the liver and other tissues mentioned
above was allowed to act at the natural acidity of the tissue
juices. Hence it is probably a different enzyme. But until it
is to some extent isolated from other endoenzymes and tissue
constituents, and obtained in a state of greater concentration, we
cannot decide the point definitely.
' l^ts^Joum. Physiol^ 6, p. 136, 1885.
•-« Leube, Virchov^s Arch,^ 100, p. 540, 1885.
3 Beijerinck, Centralb.f. Bakt (2) 7, p. 33, 1901.
* Moll, Hoftneister's Beitr., 2, p. 244, 1900.
6 Miquel, CompUs Rendus, iii, p. 397, 1890-
SUMMARY 25
Summary, — We have seen that the proteolytic endoenzymes
of mammalian tissues, so far as they are known, form a graduated
series, each succeeding member of which acts more especially
upon the digestion products produced by a preceding member.
In addition to the acid- and alkali-acting proteases and
erepsins, and to arginase, there are probably other distinct
enzymes concerned in the hydrolysis of individual amino acids,
but at present we have no exact knowledge of them. It is
possible that urease is identical with the enzyme or enzymes
which split off ammonia from the individual amino acids, and the
fact that the Micrococcus ureae has been, found by Van Tieghem ^
to decompose hippuric acid into glycin and benzoic acid, lends
support to this view. But it seems more probable, judging from
what is known as to the close correlation between individual
sugars and sucroclastic enzymes, * and of the capacity of the
tissues to elaborate innumerable protein molecules, such as
antitoxins, antiferments, lysins, agglutinins, precipitins, etc., each
of a different configuration and with a special function, that these
tissues are built up of a very large number of different endoen-
zymes, each specially adapted for some particular and closely
defined purpose.
^ Van Tieghem, Comptes Rendus^ 58, p. 533, 1864.
2 See Lect. VII.^p. 170.
LECTURE II
PROTEOLYTIC ENBOENZYMES— continued
Action of pepsin, trypsin, and proteases on nucleoproteins. Presence of
nuclease in all tissues. Irregular distribution of adenase, guanase, and
xantho-oxidase. Uricolytic enzyme and its action. Relation between
endoenzymes and functional capacity of tissues, as shown by effects of
development, disease, food, and hibernation. Presence of proteolytic
endoenzymes in ail organisms, and their reaction to acids and alkalis.
Plant enzymes, both peptic and ereptic.
The nucleoproteins, like the proteins, seem to need a whole
group of endoenzymes peculiar to themselves to bring about
the later stages of their decomposition. The earlier stages are
readily induced by the enzymes of the alimentary canal.
Pepsin and hydrochloric acid quickly split up nucleoproteins
into a protein fraction, which undergoes further hydrolysis into
proteoses and peptones, and leave an insoluble precipitate of
nuclein. Milroy^ and Umber ^ have shown that some of this
nuclein is split up further to the nucleic acid stage, but probably
most of this hydrolysis is effected in the ordinary course of
digestion by the pancreatic trypsin. Apparently trypsin has no
further action upon nucleic acid, and the succeeding stage of
hydrolysis is effected by the erepsin of the intestinal juice ; but
our information upon this point is very vague. The action
of intracellular enzymes upon nucleoproteins is much more
complete. No special observations on the earlier stages of
hydrolysis have been made, but there is no doubt that the tissue
proteases, probably those acting in presence of alkalis, as well as
* Milroy, Zeit f, physioL Chem.^ 22, p. 307, 1896.
2 Umber, Zeit.f, klin. Med.^ 43, 1901.
NUCLEASE 27
those acting with acids, readily split up nucleoproteins to the
nucleic acid stage. The further hydrolysis is effected by special
nuclease, guanase, adenase, and other enzymes.
The existence of intracellular nucleoprotein-hydrolysing
enzymes was first pointed out by Salomon/ who in 1878 showed
that muscle, pancreas, and liver contain an enzyme which
can liberate xanthin bases from the tissues. These xanthin
bases were presumably derived from the nucleins present.
Salkowski and his pupils showed that the enzyme responsible
for this hydrolysis was present in filtered aqueous extracts of
glands and of yeast cells. The enzyme is distinct from trypsin,
in that its action is retarded by alkalis. Also Iwanoff* found
that the " nuclease," as he termed it, which is present in Fungi
such as Aspergillus niger and Penicillium glatuum^ rapidly split
up nucleic acid into phosphoric acid and xanthin bases, but was
incapable of liquefying gelatin. Jones ' showed that the nucleo-
protein of thymus, freed from all other glandular constituents,
retained an enzyme which was capable of hydrolysing it to
phosphoric acid and xanthin bases, and that this hydrolysis was
quickly stopped by alkalis. Again, Sachs * tested the action of
nuclease lipon the sodium salt of the a-nucleic acid obtained
from the thymus gland. A 4 per cent, solution of this body forms
a firm jelly at room temperature, and nucleases liquefy this jelly
and convert it first into the soluble /9-nucleate, and then liberate
phosphoric acid and xanthin bases; but trypsin is without
action upon it. Recently prepared pancreatic extracts and
pancreas press juice, though possessing little or no tryptic power
owing to the trypsin being in the zymogen form, were able to
split up the nucleate, as they contained some nuclease. If the
extracts were kept, however, the trypsin was liberated and soon
destroyed the nuclease, just as the writer^ found that the trypsin
of pancreatic extracts gradually destroys the endoerepsin
present.
Nuclease has been found by Jones, Partridge, and Winternitz^
* Salomon, Zeit f, phystoL Chem,^ 2, p. 65, 1878.
* Iwanoff, ibid,^ 39, p. 31, 1903. s Jones, iHcL^ 41, p. loi, 1904.
* Sachs, ibid,^ 46, p. 337, 1905.
* Vernon, /47«r«. PhystoL^ 30, p. 341, 1903.
* Jones and Partridge, Zeit /. physioL Chem,^ 42, p. 343, 1904 ; Jones
and Winternitz, ibid^ 44, p. i, 1905.
28 PROTEOLYTIC ENDOENZYMES
in many other organs, such as the spleen, suprarenal gland, and
liver, so it is probably present in all glands. In that all tissues
contain nucleoproteins, one would expect nuclease to be univer-
sally present. But Sachs, though he found it in the pancreas,
thymus, and kidney, failed to trace it in muscle. Doubtless
muscle contains much less of the enzyme than glandular tissues,
as it is so much poorer in nucleoproteins, but there can be little
doubt that it would be found if the methods of detection were
sufficiently delicate.
It seems probable that nuclease is an enzyme sui generis^
and not to be confounded with any of the proteolytic endo-
enzymes mentioned in the previous lecture. It is distinct from
/8-protease, for Arinkin^ found that the addition of -3 per cent,
or less of various mineral and organic acids, though it accelerated
the autolysis of the protein constituents of liver pulp, retarded
the formation of purin bases. Lactic acid retarded the nucleic
acid hydrolysis least of all, and in one experiment in which -06
per cent, of the acid was added, it accelerated it
The non-identity of nuclease and erepsin seems to be proved
by the fact that nuclease acts best in a neutral medium
or at the natural acidity of tissue extracts,^ whilst erepsin acts
best in alkaline solution. Nakayama^ found that ground-up
extracts of intestinal mucous membrane, when acting at the
natural acidity of the extract, decomposed sodium a-nucleate,
and he attributed this action to erepsin; but there can be no
doubt that such an extract contained nuclease, and the whole
of the observed action may have been effected by this enzyme.
Before discussing the further changes undergone by the
purin bases liberated from nucleic acid, it will be well to point
out briefly the chemical relationships of these bodies to one
another and to uric acid. As we see from the structural
formulae, adenin is an amino-hypoxanthin, and it can react with
water to form ammonia and hypoxanthin. Similarly, guanin
is amino-xanthin, which can react to form ammonia and
xanthin. Xanthin contains one oxygen atom less than uric
acid. Uric acid, when acted on by a mild oxidising agent
such as potassium permanganate, is converted into allantoin
^ Arinkin, Zett f. phystol, Chem.^ 53, p. 192, 1907.
2 Kikkoji, ibid.^ 51, p. 201, 1907. ^ Nakayama. ibid,^ 41, p. 348, 1904.
PURINS
29
and carbon dioxide, whilst a stronger oxidising agent, such
as cold nitric acid, converts it into alloxan and urea. By other
N=C— NH,
CH
I
NH.
N— C N<^
Adenin.
CH
NH-CO
CH C— NH.
II I >CH
N— C N^
Hypoxanthin.
NH— CO
NH,— C C-
NH
11 II
N— C-
N>
NH— CO
cU-
A
NH
\,
-Ln>
;CH
Ouftuin.
Xanthin.
NH-CO
CO C— NH^
I II >co
NH— C— NH/
Uric Acid.
.NH— CH— NH.
CO I CO
-NH— CO NH,
AllAntoin.
/
treatment this alloxan can be split up into carbon dioxide,
oxalic acid and another molecule of urea.^
The nature and number of the molecules of purin bases
present in a molecule of nucleic acid is a matter of considerable
doubt. Bang ^ prepared a nucleic acid from the pancreas which
contained only guanin, and the structural formula he gives for
the acid contains four guanin molecules. He represents its
hydrolysis by the following equation : —
C44H66N20P4O34 + loHp
Nadeio Acid.
= 4C5H5NP + aCjHioOs + sCsHgO, + 4H,P0,
Ouanin. Pentose. Glycerin. Phosphoric
Acid.
As a rule, the nucleic acids prepared from various tissues
yield adenin as well as guanin, but there is no good proof
that the preparations are not mixtures of two nucleic acids,
^ C/, article by Macleod in Hill's Recent Advances in BiO'Ckemtstry^ p.
388, 1906.
2 Bang, ZeiLf.pkysioL Chem,^ 31, p. 411, 1900.
30 PROTEOLYTIC ENDOENZYMES
one containing only guanin, and the other only adenin. Some
analyses of nucleic acids show the presence of xanthin and
hypoxanthin, as well as of adenin and guanin. For instance,
Inoko ^ found that the nucleic acids prepared from the spermatic
fluid of the salmon, from the pancreas, and from yeast, in each
case yielded all four purin bases. Steudel ^ observed the same
thing in the case of thymus nucleic acid, but he subsequently
concluded that the xanthin and hypoxanthin isolated from the
decomposition products are formed secondarily during the
disintegration of the nucleic acid. This was effected by
boiling with hydriodic acid and phosphoric acid; but when
a less violent hydrolytic agent such as cold nitric acid was
substituted, no xanthin and hypoxanthin whatever were formed.
Steudel ^ thinks that the decomposition of thymus nucleic acid
may be represented by the following equation : —
C48H57Ni50aoP4 + 8H20 + 02
Nneleio Acid.
= QH,N,0 + C^H^Nj + CjHeNp, + QH^N.O + 4C,Hi A + 4HPO,
Qoanin. Adenin. Thymin. Gytosin. Hexose. Metophos-
phoiic Acidi
The- formula given for the nucleic acid differs somewhat from
that derived by Miescher, Schmiedeberg, and Kostytschew from
their analyses, but Steudel shows that it stands in fair agree-
ment with their quantitative results. Also, from the hydrolytic
products of a known weight of the acid, Steudel isolated approxi-
mately the quantities of guanin, adenin, and thymin required by
this equation ; but the cytosin obtained was too low in amount,
as some of it underwent a secondary oxidation to uracil. No
pentose was isolated by him, though we saw that the pancreas
nucleic acid examined by Bang contained the whole of its
carbohydrate in this form. Burian* gives a tentative formula
* Inoko, Zeit f, physioL Chem.^ i8, p. 540, 1893; see also, G. Mann's
Chemistry iff the Proteids^ London, 1906, p. 44a
2 Steudel, Zeit, f. physioL Chem.^ 42, p. 165, 1904.
^ Ibid,^ 53, p. 14 ; also, Centralb, /. Physiol,^ 21, p. 472, 1907.
^ Burian, quoted from Loeb^ University of Calififmia Publications in
Physiology, 3, p. 62, 1907.
ADENASE AND GUANASE 31
for nucleic acid with two hexose groups and two pentose
groups : —
Tfaymin— Hexose — Ov yO — Hexose — Cytosin
Pentose— O—N p<^ \ p q P \ ^ P<^—0— Pentose
Adenin^ X \ ^ \ ^Guanin
OlH
/
HO Oft HO
These several formulae show how greatly the nucleic acids of
various tissues differ from one another in constitution ; but we
may provisionally conclude that none of them contain xanthin or
hypoxanthin in their molecules.
Adenase and Guanase, — The action of intracellular enzymes
upon the purin bases was first studied by Horbaczewski ^ in
1 89 1. Minced spleen, kept with nine parts of water at 50° for
eight hours, was found by him to contain a large amount of
xanthin and hypoxanthin, whereas fresh spleen contained none.
If the spleen mixture were oxygenated by shaking with air or by
adding hydrogen peroxide or blood to it, it was found to contain
uric acid instead of hypoxanthin and xanthin. Horbaczewski's
experiments were carried out in the absence of antiseptics, so a
certain amount of putrefaction ensued. The reaction was not
dependent on putrefaction, however, as Spitzer^ obtained the
sanie result in the presence of chloroform or thymol. Also,
Spitzer found that if xanthin and hypoxanthin were added to
extract of spleen or liver which was kept at 50° with a con-
tinuous stream of air bubbling through, they were directly con-
verted into uric acid. In one experiment, at least 90 per cent,
of the xanthin and hypoxanthin added was converted. On the
other hand, extracts of kidney, pancreas, and thymus, acting
under similar conditions, yielded no uric acid whatever. Spitzer
found that liver and spleen extracts likewise possessed the power
of converting adenin and guanin into uric acid, though the conver-
sion was not effected so readily as that of xanthin and hypo-
xanthin. That is to say, the enzymes which split off the amino
grouping from the guanin and adenin, and so converted them
* Horbaczewski, Sitzungsben d. Wiener Akad, d, Wtss. Mathemnaturw,
Ciasse, 100, 1891.
^ Spitzer, Pfliiger^s Arch,, 76, p. 192, 1899.
32
PROTEOLYTIC ENDOENZYMES
into xan thin and hypoxanthin respectively, were not so active as
the oxidising enzyme which converted these bodies into uric
acid. Schittenhelm ^ found that the enzymes concerned in this
conversion could be salted out from extracts of spleen, liver, and
lung by means of ammonium sulphate. The largest amounts of
oxidase were thrown down at two-thirds saturation, and the
precipitate so obtained, after removal of the ammonium sulphate
by dialysis, gave an active solution which converted adenin and
guanin almost quantitatively into uric acid. At least this was
the case if air were bubbled through the solution. Ah experi-
ment made with guanin showed that in absence of air only the
xanthin stage was reached.
Arguing from comparisons of the activities possessed by
various tissue extracts, Jones, Austrian, and Winternitz^ conclude
that in the conversion of the amino purins into uric acid three
distinct enzymes are concerned. A guanase converts guanin
into xanthin ; an adenase converts adenin into hypoxanthin,
whilst a xantho-oxidase oxidises these two oxypurins into uric
acid. The hypoxanthin presumably passes through an inter-
mediate xanthin stage. According to their observations, these
three enzymes are distributed in various tissue extracts in the
following manner: —
Tissue.
Guanftse.
Adenase.
Xantho-oxidase.
Liver of Ox
„ Rabbit .
» Dog .
Spleen of Dog .
" Pig .
Pancreas of Pig .
Dog
+
+
+
+
+
+
trace
+
+
+
+
+
+
+
+
According to the data of this table the distribution of the
three enzymes is very irregular and capricious. The liver of one
animal contains all three enzymes, that of another only two,
and that of another only one. The same thing is true of the
' Schittenhelm, Zeitf.physiol. Chem.^ 42, p. 251, 1904 ; 43, p. 228, 1904.
2 Jones and Austrian, Zeit, f. fhysioL Ckem,^ 48, p. no, 1906; Jones
and Wintemitz, Zeit f, physioL Chem.y 44, p. i, 1905.
ADENASE AND GUANASE 38
other organs, only we find, for instance, that dog's spleen
contains all three enzymes, and pig's spleen only one of them,
whilst dog's liver contains one, and pig's liver two. Schittenhelm
and Schmidt ^ do not altogether accept this scheme of ferment
distribution. Contrary to Jones and Austrian, they found that
there was no lack of adenase in rabbit's liver. In fact, Schitten-
helm ^ denies the necessity of assuming the independent
existence of adenase and guanase enzymes, for he finds that any
tissue extract which can desaminate the one amino purin can
likewise attack the other. Still, he admits that the rate of action
upon the two purins is very different in different cases, and that
pig's spleen, for instance, acts upon adenin much more rapidly
and completely than it does upon guanin, whilst dog's liver
acts much more rapidly upon guanin than upon adenin. Jones,
whilst admitting that some of the tissue extracts in which he
found adenase or guanase to be lacking might contain traces of
the enzyme, and so might exhibit some activity if only permitted
to act for a long enough time, insists upon the existence of the
two distinct enzymes. The evidence— admitted by Schitten-
helm — as to the very different rates of action of different tissue
extracts upon the two amino purins seems sufficient to uphold
Jones's view, and, moreover, in some cases Jones failed to find
any trace of enzyme, however long the tissue extract was per-
mitted to act.
As regards the distribution of xantho-oxidase, Schittenhelm
is in agreement with Jones. He found, for instance, that this
enzyme is present in large amount in ox spleen, but is wanting
in pig's spleen. Burian^ made a preparation of the oxidase
from minced ox liver which contained only traces of nucleo-
piroteins and purin bodies, and on keeping it at 37° with xanthin
and hypoxanthin in presence of oxygen, these substances were
rapidly oxidised to uric acid, without the oxidase apparently
undergoing any diminution of activity.
Accepting the experimental results given in the above table
as substantially correct, we are by no means clear as to their
interpretation. The simple and straightforward interpretation,
* Schittenhelm and Schmidt, Zeit f, physioL Chem,^ 50, p. 30, 1906.
2 Schittenhelm, ilnd,^ 45, p. 152 ; 46, p. 354, 1905.
5 Burian, ibid,^ 43, p. 497, 1904.
34 PROTEOLYTIC ENDOENZYMES
viz., that these results accurately indicate which enzymes were
present in the tissues before their death and disintegration, and
which were not, cannot be accepted with any confidence. It is,
on the face of it, highly improbable that the distribution of the
three enzymes in the various organs of one and the same animal
should be so irregular, and still more so that the corresponding
organs of another animal should show quite a different distribu-
tion. As will be pointed out more fully in a subsequent portion
of this lecture, these intracellular enzymes must be of functional
importance, and so they must be studied and re-studied in
connection with their probable functions. They are presumably
concerned in the conversion of the purin bodies derived from
their own tissues — and in some cases those derived from external
sources — into uric acid, in prder that the uric acid, wholly or in
part, may pass into the blood and be excreted from the system.
Supposing, therefore, the nucleins of any given organ be found
to contain either guanin or adenin, but not both of these purins,
then one would naturally expect that an extract of that organ
would, as a rule, contain only the corresponding enzyme. Con-
versely, if an organ extract be found to contain only adenase, or
guanase, then one would expect that the nucleins of that organ
would contain only adenin or guanin. Supposing that this
relationship between organ enzyme and composition of organ
nuclein is not established, as seems more than likely to be the
case from the few data at present available, then one is driven to
the conclusion that the simple and straightforward interpretation
of experimental results above referred to is invalid. There are
several possible ways in which apparent contradictions of this
kind can arise. In the first place, the mechanical treatment of
breaking up and extracting a tissue may be so violent as entirely
to destroy some of the more unstable endoenzymes bound up in
the tissues. Again, the action of some of the enzymes in an
extract may be entirely neutralised by the presence of anti-bodies.
Again, other enzymes may be present which do not interfere
with the action of the enzyme under examination, but which
convert the product formed by this latter enzyme, the amount of
which is taken as a measure of its activity, into some other
unknown substance.
As far as I am aware, a comparison between the nucleins
URICOLYTIC ENZYME
35
and the enzymes present in a tissue has been made in only one
case. Schittenhelm ^ found that fresh pig's spleen contains
adenin and guanin, and, as already mentioned, he likewise found
that it contains both adenase and guanase. His result is
almost certainly correct, therefore, but the fact that Jones failed
to find any trace of guanase is a point requiring re-investigation.
Until other comparisons have been made between the composition
of the nucleic acids and the related enzymes of tissues, the
question of their correspondence or otherwise must remain in
abeyance.
Uricolytic Enzyme. — In addition to uric acid forming enzymes,
most tissues contain also a uric acid destroying enzyme. The
disintegrative action of animal tissues upon uric acid was
noted by Stokvis as long ago as 1859. In recent years the
subject has been studied by Chassevant and Richet,* Ascoli,*
Jacoby,* Wiener,^ Schittenhelm,® Burian,^ Austin,® Almagia,® and
by Wiechowski and Wiener.^^ Some of the results obtained by
these observers are collected in tabular form, and it will be seen
Tissue.
Ox.
Horse.
Pig.
Dog.
Babbit.
Liver . .
Kidney .
Spleen
Muscle
Bone marrow
Intestine .
Lung .
+
+ -
+
+ (?)
+
+
+
+
+
that most of the tissues examined were found to contain the
enzyme. As in the case of the uric acid forming enzymes,
however, the corresponding organs of different animals do not
^ Schittenhelm, Zeit f, physioL Ckem.^ 46, p. 354, 1905.
*-* Chassevant and Richet, C R, Soc. BtoL^ 1897, p. 743.
3 Ascoli, PflUger^s ArcMv^ 72, p. 340^ 1897.
* Jacoby, Virchau/s Arck.^ 157, p. 235, 1899.
* Wiener, Arck,f, exp, Path,^ 42, p. 375, 1899.
* Sphittenhelm, Zeitf.pkysioL Ckem,^ 45, pp. 121 and 161, 1905.
^ Burian, ibid.^ 43, pp. 487 and 532, 1904.
^ Austin, y47i/r;i. MeiL Research^ I5) P' 2P9y and 16, p. 71.
^ Almagia, Hofmeister^s Beiir,^ 7, p. 459, 1906.
^^ Wiechowski and Wiener, ibid,^ 9, p. 247, 1907.
ie PROTEOLYTIC ENDOENZYMES
always agree. The kidney of the ox and horse contained the
enzyme, but that of the dog and rabbit apparently did not. A
result such as this must be accepted with reserve. In the case
of ox spleen, Austin found the enzyme, whilst Schittenhelm
was unable to detect it.
The uricolytic and uricbgenic enzymes have nothing in
common with one another, as they do not run parallel in various
tissues. The uricolytic enzyme is a soluble body, which can be
thrown out of solution by means of uranium acetate, and then
isolated from the precipitate by digestion with -2 per cent,
sodium carbonate. Wiechowski and Wiener consider it to be
an oxidase, as it acts best in presence of plenty of air. It is
a moderately active body, for they found that 4 gms. of dried and
powdered dog's liver, when kept with -05 per cent NagCOg
solution and sodium urate at 40°, decomposed -51 gm. of uric
acid in four hours. Or again, the 100 gms. of dry powder
obtained from the kidneys of an ox were able to split up about
70 gms. of uric acid in twenty-four hours.
The fate of the uric acid is not known with certainty.
Chassevant and Richet thought that minced liver changed it
into urea. Subkow,^ after acting upon uric acid with dog's liver
under anaerobic conditions, isolated urea. Wiener thought that
under aerobic conditions some of it was converted into glycin.
Jacoby found that dog's liver converted it into allantoin.
CipoUina * concluded that the spleen, and perhaps also the liver
and muscles, could oxidise it to oxalic acid. Almagia found
that It was converted, first into allantoin, and then, into glyoxylic
acid. However, none of these observers offered a complete
proof of the formation, from the uric acid, of the various products
isolated by them. But Wiechowski ^ found that if sodium urate
solution were shaken up for four to eight hours at 40® with
purified emulsions of organs such as the liver and kidney, the
whole of the urate was destroyed, and was in some cases oxidised
quantitatively to allantoin. Hence there could have been no
formation of urea or glycin, though possibly under anaerobic
conditions such as Subkow used, the changes may have been
^ Subkow, Diss,^ Moscow^ 1903, cited hy Jahrb,f, Tierchem,^ 33, p. 873.
2 CipoUina, BerL klin. Wochensckr,, 1901, p. 544.
3 Wiechowski, Hofmeistet's Beitr.^ 9, p. 295, 1907.
ENZYMES AND FUNCTIONAL CAPACITY S7
different Or perhaps the allantoin formed first may have
undergone a subsequent conversion into urea and other
substances.
If purin bases be added to a suitable tissue extract, it follows
that several changes will occur at one and the same time. The
purins, if in the form of adenin and guanin, will gradually be con-
verted into hypoxanthin and xanthin. These oxypurins, directly
they are formed, will in turn be oxidised to uric acid. This uric
acid will then undergo oxidation into allantoin, and this allantoin
very probably will undeigo further decomposition into urea, glycin,
or glyoxylic acid. The course pf these changes, so far as they
concern the uric acid, has been examined in detail by Burian.^
Under the particular conditions of experiment, in which a
purified ox liver extract was allowed to digest a well oxygenated
solution containing xanthin, the uric acid was found to increase
to a maximum during the first three hours' digestion, and then
to diminish gradually.
Intracellular Enzymes and Functional Capacity. — We have
seen that so far as our meagre information avails us, the intra-
cellular enzymes concerned in nucleoprotein metabolism do not
correspond at all closely with the character of the work that one
imagines they are called upon to perform. No quantitative
determinations of the relative amounts of the enzymes in the
various tissues have been made, though Almagia found that in
uric acid destroying power the liver was the most active, and
then in order came the kidney, lymph gland, muscle, bone
marrow, spleen, and thyroid. The nucleoprotein content of
these tissues has a very different order, or seems to bear no
relationship to enzyme activity ; but obviously the whole subject
needs investigating in much greater detail before a definite
opinion be adopted. In the case of certain other proteolytic
enzymes we are a little better informed, though as yet only the
fringe of the subject has been touched upon. The enzyme
erepsin has been investigated by the writer ^ in some detail, as
it is of universal distribution, and it seems to occur in fairly
constant proportion in the tissues of very different classes of
animals. From the mean results given in the table, we see that
^ Burian, Zett f, physioL Chem.y 43, p. 497, 1904.
' V tmotiy Joum. PhysioL^ 33, p. 81, 1905.
38
PROTEOLYTIC ENDOENZYMES
in the carnivorous hedgehog, the omnivorous cat and man, and
in the herbivorous rabbit and guineapig, the extreme values of
a tissue such as the liver vary only from 1-9 to 3-6. The kidney
Animal.
Hedge.
hog.
Gat.
Man.
Babbit.
Guinea,
pig.
Number of Observations
2
8 to 10
2
8
7
Kidney
Liver
Cardiac muscle ....
Skeletal muscle ....
Brain
7.9
1.9
.48
•25
•23
1 1.6
3-6
IK)
5-2
2.7
(•18)
(•27)
10-9
2.3
'•la
•49
8.8
3-4
1-8
.66
.68
contains two to four times more erepsin than this, and the other
tissues less than half as much ; but having regard to the small
number of observations made, and the great differences in the
conditions of life of the animals examined, one is led to conclude
that* given similar conditions, the average ereptic value of a
tissue may be roughly constant through a wide range of the
mammalian kingdom. There can be little doubt, therefore, that
this enzyme is closely bound up with certain proteolytic activities
of the tissues. Perhaps its amount is directly proportional to the
protein metabolism, but of this we know nothing at present
Though the (tuerage amount of erepsin in a tissue is nearly
constant, yet it varies very greatly with the functional activity of
the tissue. This is strikingly indicated by the data in the table,
Age of Guineapig.
Foetal.
Oday.
8 days.
81 days and
oveT.
Weight .
37gms.
57gms.
81 gms.
585 gms.
Kidney .
Liver.
Cardiac muscle .
Skeletal muscle .
Brain.
1.8
1.6
.48
.56
•45
6.8
2.6
.67
•79
.26
8k)
3-3
•70
8.9
3^3
1-6
•65
•73
which show the effect of growth upon ereptic power. In the
foetal tissues the erepsin is at a minimum, and it rapidly
increases as the animal develops, till it reaches its full value — in the
ENZYMES IN EMBRYOS 39
case of the guineapig — after about eight days of postnatal
existence. Similar results were obtained with the rabbit and
the cat, though in the case of the cat especially, the tissues did
not reach their full ereptic power so soon after birth. This is
probably connected with the fact that the kitten is much slower
in attaining maturity than the young guineapig or rabbit. Very
small fcetal rabbits, weighing only 4 gms. were found to have
only about a tenth as much erepsin in their tissues as the fcetal
guineapig above recorded, so presumably in a still earlier stage
of development the embryo is almost enzyme-free. Battelli and
Stem ^ obtained similar results to this in respect of the hydrogen
peroxide decomposing ferment catalase. They found that the
liver and kidney of the guineapig showed a rapid increase of
enzyme during embryonic development, and the first few days
after birth, but that subsequent to the seventh day there was
little or no further increase. At this period the liver contained
five times more ferment than that of the new-born guineapig,
whilst the kidney contained twice as much. The other tissues
examined, viz., the spleen, lung, muscle, and brain, showed only
a slight increase of enzyme with growth.
Analogous results have been obtained ' by Jones and
Austrian 2 in respect of the nuclein ferments. They found
that in the earlier stages of foetal development, the liver of pigs
contained no nuclein ferments whatever. Embryos less than
150 mm. in length had no appreciable quantity of adenase, but
those of 165 to 200 mm. had a distinct amount. Xantho-
oxidase did not appear till a later period, perhaps after birth,
whilst guanase was invariably wanting, both before and after
birth, Mendel and Mitchell* made independent observations
with pigs' embryos averaging 50, 75, and 100 mm. in length.
They found adenase in the liver of even the smallest embryos,
but as far as one can judge from their incomplete data, it
distinctly increased in amount with growth. As in Jones and
Austrian's experiments, no xantho-oxidase or guanase were
found in the liver, but an extract of the other viscera contained
guanase.
^ Battelli and Stem, Archiv, d, Fisiologia^ 2, p. 471, 1905.
^ Jones and Austrian^ /^ti^/ti. BioL Chem,^ 3, p. 227, 1907.
' Mendel and Mitchell, Amer.Joum. Physiol,^ 21, p. 69, 1908,
40 PROTEOLYTIC ENDOENZYMES
Other observations, upon the fat- and carbohydrate-splitting
enzymes of embryos, are recorded in the next lecture. These
enzymes increase with the growth of the embryo in the same
way as the proteolytic enzymes.
In apparent contradiction to these conclusions is the work of
Schlesinger,^ who found that in new-bom rabbits the autolysis
of ground-up liver tissue was maximal, whilst in eight-day
rabbits it was considerably greater than in older animals.
Again, Mendel and Leavenworth ^ investigated the autolysis of
pig's liver, and they found that in the presence of '02 per cent,
acetic acid the minced liver of embryos 50 to 280 mm. in length
showed slightly more autolysis than that of adult pigs; but
when no acid was added, the embryo liver hydrolysed only a
third or fourth as much of its proteins to the non-coagulable
stage as the adult liver. In considering these results, one must
remember that the tissues of young animals are much more
fragile and easily digestible than those of older ones, and so
would more readily undei^o autolysis in spite of a deficiency of
enzyme. The only valid method of comparing the enzyme
activity of tissues from animals of various ages is to separate
the enzymes as far as possible from the tissue proteins (^^., by
making glycerin extracts) and test their action on fibrin,
caseinogen, or some other foreign protein.
The explanation of all these results seems clear. Embryonic
animals receive most of their food ready formed, in an easily
assimilable condition, and do not need to elaborate it for
themselves. Even after birth the new-born animals are lacking
in vigour for some days, and they are feeding on the easily
digestible maternal milk; so their tissues, though more active
than in the embryonic state, are not functioning so conlpletely
as later on, when they have to perform the same duties as those
of the full-grown animals. In growing animals, therefore, the
ereptic power of the tissues is closely related to their functional
activity and functional capacity.
The ereptic power of the tissues is moderately aflfected by
diet. I found that cats fed for eleven to twenty-nine days on
a meat and fat diet had on an average 1-3 times more erepsin
» Schlesinger, Hofmeistet's Beitr., 4, p. 87, 1904.
2 Mendel and Leavenworth, Amer.Joum, Physiol^ 21, p. 69, 1908,
HIBERNATING ANIMALS
41
in their tissues than cats fed on bread and milk diet, whilst cats
kept on a mixed diet had most enzyme of all. The response
of the intracellular enzymes to changes of nutrition, just as that
of the extracellular ones,^ is only a slow and gradual one, and
is not marked as a rule. Probably in cases of starvation they
would be more striking, but I made no direct observations to
test the point. However, I compared the tissues of hibernating
hedgehogs with those of non-hibernating, and a comparison of
the mean results given in the table shows that most of the
Tissue.
Non-hibemating
Hibernating
Hedgehogs.
Ratio.
Kidney ....
Pancreas ....
Spleen ....
Liver ....
Cardiac muscle
Skeletal muscle
Brain ....
7.9
3-0
5-2
1-9
.48
•25
•23
3-8
3-0
.76
•30
•24
•23
2*1 : 1
10
7-4
10
10
tissues of the active animals were considerably richer in erepsin
than those of the passive ones. The spleen is especially
noticeable, as it contained seven times more ferment in the
one case than in the other. The differences of ereptic power
are not immediately connected with the differences of tempera-
ture of the hibernating and the non-hibernating animals, for
one of the hibernating hedgehogs was examined and found to
have a temperature of 9*2°, but it was not killed till the following
day. By that time its temperature had shot up to 35°, yet this
animal had practically the same amounts of erepsin in its tissues
as another animal which was killed when its temperature was
7-5°. Probably, therefore, the diminution in the ereptic power
of the tissues only gradually ensues in the hibernating hedge-
hogs after the onset of the winter sleep, whilst the increase
which must occur when the animal becomes active again in the
spring is likewise a gradual one.
The effect of disease on ereptic power may be very great.
As we see from the data in the table, guineapigs which had
wasted away gradually for some weeks before death, and were
^ Cf. Vassiliew, Arck, d, ScL BioLy 2, p. 219, 1893.
42
PROTEOLYTIC ENDOENZYMES
reduced to less than half the normal weight, contained less than
half as much erepsin in their tissues as healthy animals, though
a guineapig which died three days after a Staphylococcus injec-
tion, and which consequently had had no time to waste away,
showed no defect of the enzyme. Again, in the case of man,
Tissue.
Normal Guineapigs
(7a7 grms.).
Wasted Ouineapigs
(805 grms.).
Died 8 days after
injection (677 grms.).
Kidney .
Liver
Cardiac muscle
Skeletal muscle
Brain
8-9
3-3
•73
37
1-4
•72
•59
•31
7-8
3^6
2-8
•72
•41
I found that healthy kidneys had an average ereptic value of
5*4. Slightly diseased kidneys showing some cloudy swelling
or fatty degeneration, had a value of 3-4. Kidneys showing
interstitial nephritis had one of 2-8, and a kidney with
very advanced nephritis one of only '86. Here again ereptic
power was very closely correlated with functional activit}'^ and
capacity.
What is true of erepsin is probably true of the other proteo-
lytic endoenzymes of the tissues, though there is but little exact
information upon the subject. In so far as one can deduce
comparable data, the relative amounts of /S-protease enzyme
found by Hedin and Rowland^ in the press juice from the
tissues of the ox, correspond moderately well with the relative
amounts of erepsin in the tissues of the cat. The figures given
Tissue.
Ereptic Valaes of
Gat's Tissues.
Per cent. Protein
hydrolysed in Acid
Solution (/3-protease).
Spleen ^ .
Pancreas
Liver .
Cardiac muscle
Skeletal muscle
Submaxillary gland
Blood .
Serum .
7.6
6.4
1-6
•8
5-3
•3
•I
89
about 90
50
about 45
slight
very slight
very slight
nil
^ Hedin and Rowland, Zeit, f. physioL Chem.y 32, pp. 341 and 531, 1901.
ENZYMES AND FUNCTIONAL CAPACITY 43
in the table for )8-protease represent the percentage of protein
hydrolysed to or beyond the peptone stage when digesting in
the presence of '25 per cent, of acetic acid. The only real lack
of correspondence is shown by the submaxillary gland, and of
course a gland such as this might vary considerably in different
animals.
The variation of proteolytic endoenzymes with functional
activity is shown by the observations of Hildebrandt,^ who
concluded that the proteolytic ferments in the cow's mammary
gland at the height of its activity and secretory powers are
greater in' amount than in non-secreting or feebly secreting
glands. Also the mammary glands of seven women who had
died after childbirth were found by him to contain much more
active proteolytic enzymes than those of non-pregnant women.
The nitrogen hydrolysed to or beyond the albumose stage by
autolysis was about four times greater in the former case than
in the latter. Again, in correspondence with the diminished
functional activity produced by wasting disease, Schlesinger*
found that the autolytic power of minced liver tissue varied
with the degree of atrophy at the time of death. In children
who weighed only 35 per cent, of their normal amount, the
autolysis was only half as great as in those who weighed 80
per cent, of the normal. Brugsch and Schittenhelm ' conclude
that in gouty patients the whole series of ferments connected
with purin metabolism is in a condition of enfeebled activity.
They found that such patients, when fed with nucleic acid,
were much slower than normal people in metabolising it and
excreting the uric acid formed.
Special Endoenzymes, — In addition to the numerous pro-
teolytic endoenzymes which are common to all tissues, and
which are concerned in the hydrolysis of proteins and their
normal decomposition products along well-known lines, it is
probable that certain organs and tissues possess other enzymes
more or less peculiar to themselves, or at any rate not of
universal distribution. These enzymes act upon nitrogenous
bodies which are formed by what may be termed the abnormal
* Hildebrandt, Hofmeister's Beitr.^ 5, p. 463, 1904.
' Schlesinger, loc, cit
3 Brugsch and Schittenhelm, Zeitf. exp. Path, u. Therap,^ 4, p. 438.
44 PROTEOLYTIC ENDOENZYMES
cleavage of the protein molecule, or in other ways. Two of the
most interesting of such nitrogenous bodies are creatin and
hippuric acid. As far as is known, creatin is found in appreciable
quantity only in muscle, and to a less extent, in nervous tissue.
Hence, if the hypothesis of correlation between endoenzyme and
functional capacity be valid, one would expect to find creatin-
forming and creatin-destroying enzymes in these two tissues,
but not in the other tissues of the body. The evidence is very
incomplete, but so far as it goes, it must be admitted that it
does not give much support to the hypothesis. Gottlieb and
Stangassinger ^ found that many tissues such as muscle, liver,
kidney, spleen, and suprarenal gland contain an enzyme which
gradually converts creatin into creatinin, and other enzymes
which destroy both creatin and creatinin. The press juice of
muscle, and to a less extent that of the kidney, was found to
contain in addition a creatin-forming enzyme. Hence it follows
that the amounts of creatin and creatinin present in press juice
undergoing autolysis gradually rise or fall, • according to the
relative rates at which the four enzymes are exerting their
activities. For instance, in one experiment the press juice of
muscle (to which some creatin had been added) showed an
increase of its total crestin plus creatinin from *^g per cent, to a
maximum '63 per cent, after forty hours* autolysis, and then a
gradual diminution down to '33 per cent, after ninety-one days.
No evidence of the existence of a creatin-forming enzyme in
extracts of the tissues was obtained, and observations upon
press juice do not seem to have extended beyond the two tissues
mentioned. Further investigation upon the press juice of other
tissues might show that they contained no creatin-forming
enzyme, or very much less than is present in muscle, and
hence might conform to the hypothesis of correlation between
endoenzyme and functional activity. The widely distributed
creatin- and creatinin-destroying enzymes are probably not
specific bodies of limited activity, but may be identical with the
enzyme or enzymes described in the last lecture, which hydrolyse
glycin, leucin, tyrosin, and other amino acids. They are distinct
from arginase, for Dakin^ states that this enzyme is without
' Gottlieb and Stangassinger, ZeiLf,physioL Chem,, 52, p. i, 1907.
5 Dakin, Joum, Biol. Ckem,^ 3, p. 435, 1907.
HIPPURIC ACID 45
action upon creatin and creatinin. But at present it is best to
suspend judgment upon the whole question of these enzymes,
for Mellanby^ has recently repeated some of Gottlieb and
Stangassinger's experiments, and he has been unable to confirm
them in any single particular. He found that if creatin and
creatinin were kept with extracts of liver or muscle for two or
three days at 37° in presence of toluol, they underwent no change
whatever, whilst the creatin content of muscle which was allowed
to undergo autolysis aseptically or antiseptically, was likewise
unchanged. The conversion of creatin to creatinin observed by
Gottlieb and Stangassinger in some of their experiments is
attributed by Mellanby to the fact that they evaporated to
dryness on the water-bath, a process known to lead to a
conversion of creatin to creatinin.
The reaction of hippuric acid to endoenzymes has been
known since 1881, when Schmiedeberg ^ described a "histozym "
which could decompose it This ferment was found by him in
moderate quantity in pig's blood, and in the liver of the dog
and kidney of the pig, whilst the kidney of the dog and lung of
the pig contained a small amount of it. Nencki and Blank*
found that pancreatic extracts decomposed hippuric acid, but
Gulewitsch * and Schwarzschild ^ showed that pepsin and trypsin
do not attack it, whilst Cohnheim ® found that it likewise resisted
the action of erepsin.
The hippuric acid-splitting power of liver juice has been
demonstrated by Jacoby,^ and that of minced kidney by
Berninzone® and by Wingler.® No adequate comparative
observations upon the hippuric acid-splitting power of the tissues
appear to have been made, but probably it would be found that
organs other than the liver and kidney, if they contain the
enzyme at all, have it in but small quantity.
* Mellanby, /wr«. Physiol^ 36, p. 447, 1908.
2 Schmiedeberg, Arch, f. exp. Path,^ 14, p. 379, 1881.
' Nencki and Blank, ibid,^ 20, p. 367, i886.
* Gulewitsch, Zeitf.physioL Chem,y 27, p. 540, 1899.
^ Schwarzschild, Hofnuistet^s Beiir,^ 4, p. 155, 1904.
^ Cohnheim, Zeit f. phystol. Chem,^ 52, p. 526, 1907.
7 Jacoby, ibid.^ 30, p. 149, 190a
^ Beminzone, Atti, d, Soc. ligusL d. Set, naty xi^ 190a
^ Wingler, Inaug, Diss, Abstr,^ in yi2Xf^ Jahresb,^ p, 92, 19C50.
46 PROTEOLYTIC ENDOENZYMES
Proteolytic Endoenzymes in Lower Animals, — Most of the
evidence concerning endoenzymes adduced thus far concerns
mammalian tissues, but what is true of one living tissue is
probably true to a greater or less extent of all, and so far as has
been investigated the tissues of every living organism, vegetable
no less than animal, contain proteolytic endoenzymes.
In the protozoa it is self-evident that all digestion must be of
an intracellular nature, but it is often comparable to the extra-
cellular digestion of higher animals, in that a watery vacuole
is formed around the particle of ingested food, and enzymes
are secreted by the protoplasm of the organism into this vacuole.
It was noted by Engelmann ^ that granules of blue litmus, if
ingested by Amoebae and Paramoecia, were changed to a red
colour in a few minutes. Brandt ^ tested the intracellular
staining reactions of amoebae by means of haematoxylin, and
likewise found that the watery vacuoles contained acid.
Metschnikoflf * observed that the plasmodia of different species
of Mycetozoa secreted an acid liquid round ingested litmus
grains. Miss Greenwood and Miss Saunders * made numerous
observations upon the Infusorian, Carchesium Polypinum^ and
the Plasmodia of certain Mycetozoa, and they found that the
ingestion of solid matter, whatever its nature, stimulates the sur-
rounding cell substance to secrete acid fluid. A definite watery
vacuole need not necessarily be formed, for in the case of the
Mycetozoa the ingested mass is as a rule merely permeated with
acid. The vacuole, after a time, gradually loses its free acid, and
finally the ingested litmus shows a neutral or alkaline reaction.
These results are of importance in their bearing upon the
endoenzymes of higher animals. We saw in the previous
lecture that the most powerful proteolytic enzyme of the
tissues acts in an acid medium, not an alkaline one. Hence, it
is possible that intracellular digestion occurs in higher animals,
just as in the Protozoa, by the secretion of an acid liquid and of
the appropriate acid-acting enzyme around the particle of pro-
tein matter which the cell desires to break down. It seems
* EngelmaDn, Hermantfs Handhuch d, Physiol,^ i, p. 349, 1878.
> Brandt, Biol. Centralb,^ 1, p. 202, 1881.
8 Metschnikoff, Ann. de PInst. PasUur, 1889, p. 25.
* Greenwood and Saunders, y<7«r«, Physioh^ 16, p. 441, 1884.
ENZYMES IN LOWER ANIMALS 47
improbable, however, that this is the normal mechanism of
degradation of protein matter which is always going on in all
tissues, and which forms a large part of the protein metabolism
of a normal animal, and the whole of it in a starving one. It is
much more probable that the intracellular enzymes exert their
activities when still attached to and forming part of the structure
of the living protoplasm of the cell. A local acid environment
might still be induced, however, whereby they could act upon
neighbouring particles of protein matter under the most favour-
able conditions; but such surmises are useless in the present
state of knowledge.
The fact that the watery vacuoles were noticed to assume ulti-
mately a neutral or alkaline reaction, is likewise a significant one,
for it implies that the endoerepsin, which is chiefly an alkali-
acting enzyme, would find itself under the most suitable condi*
tions for carrying on and completing the digestion of the food
substance which had been broken down in its earlier stages by
the acid-acting j8-protease.
It is stated by Krukenberg^ that glycerin extracts of the
Mycetozoa plasmodia contain a very active "peptic" enzyme,
/.^., an enzyme which digests in an acid medium, but not in an
alkaline one. It is sufficiently powerful to digest boiled fibrin.
Krukenbergdid not determine whether it could split up peptones
to the amino acid stage, but probably it would be found to
resemble /8-protease rather than pepsin.
In the sponges and many of the Coelentera^ digestion is
mainly intracellular, as in the Protozoa. Krukenberg* found
that glycerin extracts of the parenchyma of many different
species of sponges could digest raw fibrin in an alkaline medium,
as well as in an acid one. Frdd^ricq * found a tryptic ferment in
Actinia, whilst Krukenberg^ found that they contained an intra-
* Krukenberg, Untersuch, d, Physiol. Inst Heidelberg^ 2, p. 273, 1878.
* Cf. von Furth, Vergleichende Ckemiscke Physiologie der niederen Tiere^
Jena, 1903, pp. 153 and 161. The whole subject of digestion in inverte-
brate animals, both intracellular and extracellular, is dealt with fully in this
book.
3 Krukenberg, Vergl, Studien^ i Reibe, i Abt., 1880, p. 64 ; Untersuck. d,
PhysioL Inst Heidelberg^ 2, p. 339, 1882.
* Fr^d^ricq, Bull. Acad. Roy. de Belgique^ 47, 1878.
^ Krukenberg, Untersuck. d. Physiol. Inst. Heidelberg^ 2, p. 338, 1882.
48 PROTEOLYTIC ENDOENZYMES
cellular acid-acting ferment MesniP found that an aqueous
extract of ground-up filaments of the mesenteries of Actinia
contained a proteolytic enzyme which acted both in acid and
in alkaline solution. It acted best at 36°, and both tyrosin
and tryptophan could be detected among the products of
digestion.
Like Protozoa and Mycetozoa, the cells of sponges and
Actinia can secrete acid intracellularly. Loisel ^ observed that
litmus grains ingested by the parenchyma cells of living sponges
showed an acid reaction, whilst Chapeaux* observed an intra-
cellular secretion of acid in Actinia.
Most of the other observations upon the enzymes of inverte-
brate animals were made with extracts of the alimentary canal,
and of the glands pouring secretions into it, and so concern
exoenzymes rather than endoenzymes. Still, judging from
the observations above described, there can be no doubt that
the tissues of the lower animals contain both acid- and alkali-
acting proteases of a similar nature to those found in higher
animals. They likewise contain enzymes of an erepsin-like
nature, />., enzymes which hydrolyse proteoses and peptones,
but which have little or no action upon native proteins. Thus
I found* that the tissues of the frog, for instance, contained
as a rule about a third as much erepsin as the correspond-
ing tissues of mammals, whilst those of the eel contained a fifth
to a tenth as much. The tissues of the lobster were about
equally poor in ferment, whilst those of the fresh water mussel
Anodon contained much less still. To test the character of the
erepsin, comparative digests were carried out in acid as well as
in alkaline media. In the presence of • i per cent, of acetic acid,
I found that extracts of cats' tissues, on an average, digested
Witte's peptone forty-two times more slowly than in presence of
• I per cent. NagCOg — /.^., the erepsin is almost entirely an alkali-
acting one. Extracts of the tissues of the frog, eel, lobster, and
Anodon, on the other hand, digested on an average only five
times more slowly in acid solution than in alkaline, and with
^ Mesnil, Ann, de, PInst Pasteur, 15, p. 352, 1901.
2 ho\st\^ Journ. Anat Physiol,, 34, p. 187, 1897.
^ Chapeaux, Arch, de ZooL Exp, (3), i, p. 139, 1893.
* Vernon, Joum. Physiol,, 32, p. 33, 1904.
VEGETABLE ENDOENZYMES 49
three extracts the digestion rate was only 1-4 to 2-0 times slower.
Evidently, therefore, the erepsin is relatively much less affected
by acidity and alkalinity than that of mammalian tissues.
Proteolytic Endoenzymes in Plants, — In plant tissues, so far
as they have been investigated, the proteolytic enzymes act
better in an acid medium than in an alkaline one. Vines ^ made
a very extensive series of observations upon the proteolytic
enzymes of plants of many different Natural Orders, and he
finds that a peptolysing or erepsin-like enzyme is present in
every plant, and as a rule in every part of the plant ; leaves,
stems, roots, bulbs, tubers, fruits, and seeds. The enzyme digests
Witte's peptone best at the natural acidity of the plant juice,
but it can also act, though less vigorously, through a fairly wide
range of acid and alkaline reaction. For instance, an aqueous
extract of pulped mushroom digested best at its natural acidity,
and when neutralised with calcium carbonate : less well in
presence of i«2S per cent. NagCOg or in -i per cent. HCl, and
feebly in •2 per cent. HCl. Again, an aqueous extract of
pounded barley which had been allowed to germinate for
eleven days, digested best at its natural acidity, less well in
presence of -5 per cent. NagCOg, and feebly in presence of *2 per
cent. HCl. On the other hand, a glycerin extract of Dahlia root
digested best in presence of -5 per cent, citric acid, less well at
natural acidity, and feebly in presence of -5 per cent. NagCOg.
We see, therefore, that the endoerepsins of vegetable tissues,
though very different from those of the lower animals examined,
differ from them less widely than from those of the higher
animals. Hence it seems possible that the enzymes of the
lowest members of the animal kingdom will be found to differ
still less, and may show comparatively little preference for an
alkaline medium as against an acid one.
Though endoerepsins are probably of universal occurrence in
plants, it seems probable that enzymes capable of digesting the
higher proteins are of more restricted distribution, or at any
rate they are frequently present in such small amount that it is
not possible to test for them. Such enzymes, sometimes known
1 Vines, Annals of Botany^ 15, p. 563, 1901 ; 16, p. i, 1902; 17, p. 237,
1903 I 18, p. 289, 1904 ; 19, p. 171, 1905 ; 20, p. 113, 1906.
G
50 PROTEOLYTIC ENDOENZYMES
as vegetable trypsins,^ have been recognised and worked upon
for years. They were first described by Gorup-Besanez ^ in ger-
minating seeds in 1874, though he regarded them as rather of a
peptic than a tryptic nature. The enzymes which have been
examined in most detail are bromelin, from the fruit of the pine-
apple: papain, from the fruit of the papaw {Car tea papaya) \
cradein, from the latex of the fig, and the endotrypsin of yeast.
This last is a somewhat active body, but as a rule the vegetable
enzymes are very weak compared with those of animal origin.
The term " vegetable trypsin " was adopted because these plant
enzymes, like animal trypsin and in contradistinction to pepsin,
split up proteins into leucin, tyrosin, tryptophan, and other
decomposition products. But this was before the existence of
more than one class of proteolytic enzymes was recognised, and
in the light of modern knowledge Vines ^ regards the protein-
digesting or peptonising enzyme of plants as more comparable
to pepsin than trypsin, and attributes their peptone-splitting
power to an entirely distinct vegetable erepsin. He has suc-
ceeded* in separating the peptase and ereptase enzymes of
hemp seed {Cannabis sativa). He extracted the crushed seeds
with 10 per cent. NaCl solution, and faintly acidified the extract
with acetic acid. The j5recipitate thrown down was washed with
acid saline, and when dissolved in water gave a solution which
digested fibrin, but had no action upon Witters peptone. On
the other hand the filtrate from the precipitate contained
ereptase, but no peptase.
Professor Vines informs me that be has recently effected a
separation of the peptonising and peptoly tic enzymes of papain.
He first extracted papain powder with twenty-five times its
weight of 2 per cent NaCl, whereby most of the protein and
erepsin, and much of the peptonising ferment, were removed.
The residue was then washed with 20 parts of water, whereby
the remainder of the protein and erepsin still present was got
rid of. A further extraction of the residue with 2 per cent.
* Cf. Reynolds Green, The Soluble Ferments and Fermentation^
Cambridge, 2nd ed., chap, xiii., 1901.
* Gorup-Besanez, Sitzben d, pkys» med, Soc. zu. Erlangen^ 1874, p. 75.
^ Vines, loc, city 20, p. 121, 1906.
* Vines, Ann, Bot^ 22, p. 103, 1908.
VEGETABLE ENDOENZYMES 61
NaCl now gave a solution which could digest fibrin in twenty-
fouf hours, but which was unable to split up Witte's peptone
into tryptophan and other decomposition products. That is to
say, it contained a peptonising ferment, but not a peptolytic
one. The peptolytic enzyme was most active in acid media,
whilst the peptonising enzyme digested well in a neutral or
slightly alkaline medium, or in the presence of '5 per cent, of
an, organic acid such as citric acid, but was inhibited in its
action by -05 to •! per cent. HCl.
The function of proteolytic endoenzymes in plants is similar
to that in animals. They are concerned in the protein meta-
bolism of the tissues, and in rendering the supplies of insoluble
and indiffusible nitrogenous food material which may be stored
up in certain parts of the plant, available for the whole organism
whenever they are needed. For this purpose they must be
dissolved, and to effect this solution proteolytic endoenzymes
are requisite. To quote Professor Vines,^ " their importance is
strikingly illustrated in a germinating seed, where the reserve
materials, whether deposited in the cotyledons or in the
endosperm, have to be made available for the nutrition of the
growing embryo." We accordingly find that as the reserve
stores of protein are called upon in increasing degree, so the
endoenzymes which render them effective are correspondingly
elaborated. Reynolds Green ^ observed that the resting seed
of the lupin {Lupinus hirsutus) did not yield any active enzyme
to glycerin, but the ground cotyledons of seeds which had been
allowed to germinate for four days, yielded an extract containing
a fibrin-digesting enzyme. This enzyme worked best in
presence of '2 per cent. HCl, an acidity which corresponds
roughly to the natural acidity of the germinating seeds. The
enzyme was inactive in presence of weak alkalis, and '5 per cent.
NagCOg destroyed it entirely. Neumeister ^ made observations
upon seedlings of the poppy, barley, wheat, maize, and rape.
No enzyme was present in the early stages of germination, but
it developed with growth of the plants, and reached a maximum
when they had attained a length of 1 5 to 20 cm. The enzyme
> Vines, Proc, Linn, Soc, 1903, p. 16.
2 Green, PAil. Trans. Ray. Soc., 178 B., p. 39, 1887.
3 Neumeister, Z«V. Bt'ol,, 30, p. 447, 1894.
52 PROTEOLYTIC ENDOENZYMES
digested only in acid liquids. An organic acid was necessary,
oxalic acid being the best. Mineral acids such as HCl destroyed
it. Vines ^ found that ungerminated seeds of the pea, broad
bean, scarlet runner, white haricot bean, blue lupin, and maize,
contain an ereptase, and also an enzyme which acts slowly
on the reserve proteins of the seeds, but scarcely at all upon
fibrin. The germinated seeds, on the other hand, contained
in addition a weak fibrin-digesting enzyme. And lastly
Bruschi^ made observations upon the seeds of the castor oil
plant, and found that the endosperm of ungerminated seeds was
unable to undergo autolysis: but directly germination began,
autolytic power developed.
* Vines, Ann, Bot^ 20, p. 113, 1906.
^ Bnischi, Rendic, d, R, 4ccad, d, Lincei (5a), xv., 9, p. 563.
LECTURE III
FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
Lipolytic endoenzymes in animal tissues. Action upon esters, and upon
natural fats. Vegetable lipolytic enzymes, and their relation to acids
and alkalis. Glycogen content of tissues of adult and embryonic
animals in relation to intracellular amylase. Maltase, invertase, and
lactase in animal tissues. Lactase and adaptation. Vegetable dia-
static and sucroclastic enzymes. Glucoside-splitting enzymes.
Lipolytic Endoenzymes, — Our knowledge of intracellular fat-
splitting enzymes in animal tissues is in a somewhat frag-
mentary state. It is only within the last year or two that
direct proof has been afforded of the existence of enzymes
capable of hydrolysing the glycerides of the higher fatty acids.
Previous to this, all observations had been made, not upon
naturally occurring fats, but upon artificially prepared esters.
That this is by no means the same thing has been emphasised
by Connstein,^ who showed that the lipase of the castor-oil
plant acts best upon natural fats, and hardly attacks other
ethereal salts at all.
The existence of lipolytic enzymes in animal tissues other
than the pancreas was first demonstrated by Nencki and LUdy ^
in 1887. The action of various tissue extracts upon the ester
salol (phenyl salicylate) was tested, both in faintly acid and
faintly alkaline solution. As can be seen from the data in the
table, about two to four times more salol was split up in an
alkaline medium than in an acid one. Argfuing from these
* Connstein, " Ergebnisse der Physiol.," Biochemie^ 3, p. 194, 1904.
2 Nencki and Liidy, Therapeut Monatshefte^ 1887, p. 417, quoted from
Connstein, ibid.
54 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
Tissue.
Acid
Alkaline
Solation.
Solution.
Per cent.
Per cent.
Pancreas ....
16.3
24.9
Liver ....
6-4
24.8
Intestinal mucosa
60
25.0
Stomach ....
5-1
II'I
Muscle ....
5-6
24.2
results, Nencki concluded that all tissues possess fat-splitting
properties.
The next observations were made by Hanriot,^ who allowed
extracts of the minced tissues, previously neutralised with sodium
carbonate, to act upon monobutyrin. The formation of butyric
acid and glycerin was proved by the acid reaction which
developed. The lipolytic enzyme was present in considerable
quantity in the liver, and in small amount in the spleen and
suprarenal gland, whilst from muscle, testis, and thyroid gland
it was absent. The blood serum was very rich in it, for one
drop of eel's serum (which contained five to seventeen times
more enzyme than the serum of other animals) split up •!/ gm.
of monobutyrin. The enzyme could split up the ethyl esters
of formic, acetic, propionic, and isobutyric acids, and Hanriot
also found that in three days it could completely hydrolyse
the neutral fat present in the blood. Arthus^ confirmed
Hanriot with regard to the action of blood upon monobutyrin,
but denied that it has any action upon olein, palmitin, and
stearin.
The action of various tissue extracts on ethyl butyrate was
studied in detail by Kastle and Loevenhart.^ These observers
ground up the tissue with sand, extracted it with five or ten
times Its volume of water, and allowed the extract to act upon
ethyl butyrate. The free acid was estimated by titration with
Njio KOH. When mixtures of 4 c.c. of water, i c.c. of extract,
•26 C.C. of ethyl butyrate, and -i c.c. of toluol were kept for forty
^ Hanriot, C. R, Soc. Biol,^ 48, p. 925, 1896 ; Comptes Rendus, 123, p. 753,
and 124, p. 778, 1897 ; Arch, de PhysioL {5), 10, p. 797, 1898.
2 Arthus, Joum, de PhysioL^ 4, pp. 56 and 455, 1902.
2 Kastle and Loevenhart, Amer. Chem, Joum.^ 24, p. 491, 1900.
ACTION ON ESTERS
55
minutes at 40°, the following amounts of butyric acid were
liberated :
Per cent.
Per ceut.
Liver of pig
„ sheep .
„ duck . . .
„ ox . . .
9-5
4.8
2.7
...
Liver of chicken
Pancreas of pig .
Kidney of pig .
Submaxillary gland of pig .
1.95
3-5
1.8
1-3
We see that liver extracts were the most active. That of
the pig hydrolysed three times more ester than the correspond-
ing pancreatic extract. Extract of the mucosa scraped from the
duodenum of the pig's intestine hydrolysed 4- 1 per cent, of the
butyrate in thirty minutes, whilst extract of gastric mucous
membrane had a smaller action. Comparative experiments
made with the ethereal salts of the four lowest members of
the fatty acid series showed that in fifteen minutes a pancreatic
extract split up 175 per cent, of ethyl formate: 175 percent,
of ethyl acetate : 287 per cent, of ethyl propionate, and 4-37
per cent, of ethyl butyrate. It might be supposed, therefore,
that the higher fatty acid compounds would be split up even
more readily, but Kastle and Loevenhart found that they were
acted on very much more slowly than ethyl butyrate.
The lipolytic enzyme appears to cling to a large extent to
the solid particles of tissue cells. For instance, a liver extract
which has been strained through cloth, and so contained such
particles, split up 6-3 per cent, of the ethyl butyrate, but after it
had been repeatedly filtered through filter paper, it split up only
2-8 per cent.
The lipolytic action of liver press juice (pig) upon the methyl,
ethyl, amyl, and bfenzyl esters of optically inactive mandelic acid,
QHg.CHOH.COOH, has been studied by Dakin.i The action
was rather feeble, but in every case Dakin found that the rate of
hydrolysis of the dextro-rotatory component was greater than
that of the laevo-rotatory component, so that if the hydrolysis
were incomplete, an excess of free dextro-rotatory acid was
liberated, and a residue of laevo-rotatory ester left.
The first adequate evidence of the existence of endocellular
1 Dakm^/oum, PhysioL^ 30, p. 253, 1904 ; 32, p. 199, 1905.
56 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
Upases capable of hydrolysing natural fats was obtained by
Umber and Brugsch.^ In their experiments, the whole of the
body of the animal was washed out by injecting saline into the
jugular vein. The various organs were rubbed up with kieselguhr,
and the juice pressed out. This juice was allowed to act upon
an emulsion of yolk of egg, in presence of -25 to -5 per cent.
NagCOg. Volumes of 2 c.c. of the organ juice were put with
5 C.C. of emulsion for fifteen to twenty-two hours at 37°, and, as
can be seen from the data in the table, from 1 8-6 to 43-9 per cent.
Fasting Dog.
Digesting Dog.
Pancreas juice
Liver juice ....
Spleen juice ....
Intestinal mucosa juice .
Serum
Corpuscles ....
Per cent.
26.7
370
37-4
} ■" {
Per cent.
43-9
19-5
40.4
1 9*0
20>2
19-0
of the fat was thereby split up. In the case of the fasting dog, the
liver and intestinal mucosa juices were considerably more active
than the pancreas juice, but in the dog killed whilst digesting a
meat and fat meal, the pancreas juice was the most active. In
that the fat digestion of the intestine is chiefly dependent upon
the pancreas, it seems remarkable that its juice should not always
possess much greater lipolytic activity than that of any other
organ. It must be borne in mind, however, that we know
nothing about the condition of pancreatic steapsin before
secretion. It may exist in zymogen form, and for the most
part continue so to exist in the expressed juice of the gland, just
as the trypsin does unless specially activated. The considerably
greater lipolytic power of the pancreas juice of the actively
digesting dog may have been due to the presence of a certain
amount of secreted steapsin, in addition to the normal intracellular
lipase.
Umber and Brugsch also made observations upon the
lipolytic powers of mixtures of their tissue juices. The most
striking results were obtained by acting upon S c.c. of emulsion
1 Umber and Brugsch, AtxhJ, exp. Path., 55, p. 164, 1906.
LIPASE IN EMBRYOS 57
with 2 c.c. of pancreas juice plus 2 c.c. of liver juice of the fasting
dog, when 718 per cent of the fat was hydrolysed ; whilst with
2 C.C. of pancreas juice //«^ 2 c.c. of spleen juice of the digesting
dc^ no less than 82' i per cent, of the fat was hydrolysed. This
seems to show the existence of activating bodies in one or other
of the organ juices, but unfortunately none of the experiments
described were repeated, and so it is not permissible for us to
draw far-reaching conclusions from them.
It is probable that the lipolytic power of the tissues increases
during embryonic development in the same way as their
proteolytic power. Wohlgemuth^ found that if egg yolk were
shaken with water and toluol, and allowed to undergo autolysis
for four to ten weeks at 38°, it contained free glycerin, phosphoric
acid, and cholin. This was presumably formed by the action of a
lipolytic enzyme on the lecithin of the yolk : but a positive result
was obtained only in five out of eight experiments, hence the
amount of enzyme present is probably very small in all cases.
Buxton and Shaffer ^ found a trace of lipase in very small
embryos of the pig, rabbit, and sheep, and they state that the
amount of enzyme increases with the age of the embryos.
Mendel and Leavenworth^ found that aqueous extracts of the
ground-up liver and intestine of pig's embryos in every case had
some hydrolytic action upon ethyl butyrate, but they were not
nearly so active as the extracts of the corresponding tissues of
adult pigs. For instance, extracts of the liver of embyros 50
to 75 mm. in length hydrolysed 5-4 per cent, of the ester
on an average: those of the liver of embryos 150 to 215 mm.
in length, 7-8 per cent., and those of the liver of adult pigs,
28-6 per cent.
Upon the lower animals scarcely any observations of lipolytic
enzymes have been made. Cotte* describes a fat-splitting
ferment in sponges, and Mesnil ^ found one in aqueous extracts
of the ground-up mesentery filaments of Actinia, but no other
^ Wohlgemuth, Zett f, physiol, Chem,y 44, p. 540, 1905.
^ Buxton and Shaffer, /<7i^f7i. Med» Research^ I3) P* 549> 1905. Quoted
by Mendel and Leavenworth, Amer, Joum, physioL^ 21, p. 95, 1908,
^ Mendel and Leavenworth, ibid,
* Cotte, C. y?. Soc. BioL^ 53, p. 95, 1901.
* Mesnil, Ann, de PInst Pasteur^ 15, p. 352, 1 901.
H
58 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
animals seem to have been examined, though doubtless many or
all of them contain such an enzyme.
Lipolytic Endoenzymes in Plants. — Upon plants a consider-
able number of observations have been made, especially within
recent years. As long ago as 1876 Schiitzenberger ^ showed
that when an oil-containing seed is pounded in water, an
emulsion is obtained which is found after a time to contain
free glycerin and fatty acid. He attributed this hydrolysis to
the action of an enzyme. The enzyme itself was discovered by
Reynolds Green ^ in 1889 in the germinating seeds of Ricinus,
the castor-oil plant. The endosperms of seeds which had
germinated for five days were ground up with 5 per cent,
sodium chloride solution containing -2 per cent, of potassium
cyanide, and after standing for twenty-four hours the liquid was
filtered. It remained slightly opalescent. Some of the extract
was incubated with castor-oil emulsion, and after about half an
hour it began to develop acidity owing to the formation of free
fatty acid. When allowed to digest for a week in a dialysing
tube suspended in distilled water, the reaction of the mixture
became more and more acid, whilst the liberated glycerin
dialysed out. After concentration of the dialysate, it was
detected by means of the acrolein test. This vegetable lipase
was found to act best in neutral solution. It also acted well in
presence of dilute alkalis, but '066 per cent, of HCl reduced its
activity to a fourth, and '13 per cent. HCl stopped it almost
entirely. Green found that there was no lipase in the resting
seeds of Ricinus, but that ground seeds, if kept at 35° for a few
hours in the presence of dilute acetic acid, gradually developed
their lipolytic power. A saline extract of the resting seeds,
faintly acidulated, underwent a similar change, so presumably
the acid liberated the enzyme from a zymogen.
The existence of lipase was subsequently demonstrated by
Sigmund' in both the resting and germinating seeds of rape,
hemp, flax, maize, and the opium poppy. The seeds, crushed
^ Schiitzenberger, quoted from R. Green's Soluble Ferments and
Fermentation^ 2nd ed., Cambridge, 1901, p. 242.
2 R. Green, Proc, Roy, Soc,, 48, p. 370, 1890.
' Sigmund, Monats, / Chem. Wien^ 1 1, p. 272, 1890 ; Sitzungsber d. k,
Akad, d, Wiss, in Wien, 99^ p. 407, 1890, and 100, p. 328, 1891.
VEGETABLE LIPASES 59
in water, yielded an emulsion which gradually increased in
acidity on standing. The lipase also possessed the power of
hydrolysing olive oil. Sigmund found that the enzyme could
be precipitated from aqueous extracts of bruised mustard and
almond seeds by alcohol, and the precipitate, washed with
alcohol and dried at 40", yielded an active solution.
A lipase was prepared by Gerard ^ from the mould
Penicillium^ and by Camus ^ from Aspergillus niger, Biffen*
worked with the mycelium of a fungus which sometimes attacks
coconuts during germination. He cultivated it on sterilised
coconut milk, and then ground it up with kieselguhr. On
filtering under pressure through several thicknesses of filter
paper, he obtained an opalescent fluid which not only decom-
posed monobutyrin, but also coconut oil. The enzyme could be
precipitated with alcohol, and the precipitate dried and dissolved
up again without loss of activity.
Lipolytic ferments both of animal and vegetable origin have
always been found to offer especial difficulties to the investi-
gator, and hence the literature of the subject teems with
contradictory statements. As regards the lipase of Ricinus
seeds, for instance, Connstein, Hoyer, and Wartenberg * found
that the activity is most marked in a strongly acid medium,
and does not show itself at all in an alkaline medium as Green
stated. Taylor^ obtained an active preparation in the form of
a powder from pounded Ricinus seeds which had been extracted
with ether, and this preparation hydrolysed the ester triacetin
best in a feebly acid medium. H. E. Armstrong® also found
that Ricinus lipase could hydrolyse only in presence of acid, and
that practically any acid is effective. For instance, i gm. of
fat-free castor-oil seed, kept with 5 c.c. of olive oil and 10 c.c.
of 3/100 N sulphuric acid for eighteen hours at 38°, liberated
4- 1 gms. of oleic acid. Aspartic and glutamic acids were also
very efficient
^ Gerard, Comptes Rendus^ 124, p. 370, 1897.
2 Camus, C. R. Sec, BioL^ 49, pp. 192 and 230, 1897.
3 Biffen, Ann, Bot^ 13, p. 336, 1899.
* Connstein, Hoyer, and Wartenberg, Ber,^ 35^ p. 3988, 1902.
° Taylor, /<7«r«. BioL Chem,^ 2, p. 87, 1907.
^ H. E. Armstrong, Proc, Roy, Soc,^ B. 76, p. 606, 1905.
eO FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
We saw above that Green obtained a filtered extract of
Ricinus lipase, only slightly opalescent, which had the power of
hydrolysing castor oil. Astrid and Euler ^ likewise found that
the filtered juice which had been expressed from rape seed
(germinated for sixteen days), hydrolysed ethyl butyrate, but
about five times more slowly than the press cake. The enzyme
acted best at the natural acidity of the juice. Armstrong,
however, was quite unable to obtain an active filtered extract,
either of the freshly ground material, or after the extraction of
fatty matter or addition of acid. Also Hoyer ^ denies that the
lipolytic ferment is soluble in water. Nicloux ^ goes so far as to
say that the fat-splitting body of Ricinus seeds is not an enzyme
at all. By mechanical means he was able to separate the cyto-
plasm of pounded Ricinus seeds from all the other cellular
elements, and this cytoplasm had a considerable lipolytic power.
It acted on fats in the same way as an enzyme, and followed the
laws of enyme action. Nevertheless the active substance, which
appeared to be attached to the cytoplasm, is not a true enzyme,
according to Nicloux, in that it is destroyed by water as soon as
it is no longer protected by fats. He calls it lipaseldine.
From this mass of conflicting evidence it is impossible for the
present to pick out the true and the false. We can only await
the results of further investigation.
Carbohydrate-splitting Endoenzymes, — We have seen in the
two preceding lectures that the tissues contain a series of proteo-
lytic endoenzymes, which effect the gradual degradation of
native proteins and nucleoproteins through numerous inter-
mediate stages till they finally split them up into simple bodies
such as ammonia, urea, and other products. Similarly, many of
the tissues contain a series of carbohydrate-splitting enzymes,
which hydrolyse complex polysaccharides like glycogen through
intermediate stages of dextrins and maltose to dextrose, and
finally, perhaps, split the dextrose still further, with the forma-
tion of alcohol and lactic acid, and ultimately of carbon dioxide
and water.
1 Astrid and Euler, Zeit. f. physiol. Chem., 51, p. 244, 1907.
2 Hoyer, i3i</., 50, p. 414, 1907.
» Nicloux, Proc. Roy, Soc.^ B. 77^ p. 454, 1906, where further literature
is given.
GLYCOGEN DISTRIBUTION
61
Distribution of Glycogen, — Before discussing the glycogen-
hydrolysing enzyme, it is desirable to enquire briefly into the
distribution of glycogen in the tissues. As is well known, the
liver is generally the richest of all in glycogen, and it sometimes
contains as much as all the other body tissues put together.
The amount present is extremely variable, the liver of a dog
having been found by Schondorff^ to contain as much as 187
per cent, of it. The muscles come next in their glycogen content,
as much as 3*7 per cent, being found by Schondorff in the
muscles of the dog. The percentage varies considerably in
different muscles as is shown by the following data, which were
obtained by Aldehoff^ for a twenty-five year old horse which
had been starved nine days before being killed.
Tisstte.
Per cent,
of Glycogen.
Liver ..... r *
.42
•82
2-44
1*29
1.71
1-47
Heart
I'Glutaeus maximus .
KfiierlPQ 1 Latissimus dorsi
L Biceps brachii .
The richness of the muscles of this particular animal in
glycogen is remarkable, and much above the average of that
found in well-fed animals.
Practically all the other tissues of the body contain glycogen.
It has been detected in the kidneys, salivary glands, lungs, testes,
ovaries, gastric mucous membrane, involuntary muscle, brain,
connective tissues and epithelial tissues, not only of vertebrate
animals, but also in the corresponding tissues of invertebrate
animals.^ The amounts of glycogen present are generally much
smaller than those found in liver and muscle. For instance,
Handel* found '03 per cent, in the spinal cord of the ox, and
•10 per cent, in cartilage, -03 per cent in tendon, and '008 per
^ Schondorff, Pfluget^s Arch,^ 99, p. 191.
2 Aldehoff, Zeitf. Biol^ 25, p. 147.
3 Cf, Pfliiger, Pfluget^s Arch,^ 96, p. 159, 1903.
* Handel, ibid,^ 92, p. 104.
62 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
cent in bone of the dog. Cramer ^ found -07 to '10 per cent,
in the human placenta, -lo to -19 per cent, in the lungs of new-
born children, -008 to •018 per cent, in the human brain,. '85
per cent, in the intestine, and 'OS to -07 per cent, in the skin.
Upon the glycogen content of embryonic tissues erroneous
statements have gained wide currency, for their supposed
richness in the carbohydrate is unsupported by the results of
recent analysis. Microscopical examination is, of course, value-
less for quantitative purposes, though it is useful in showing that
glycogen is absent from some embryonic organs as the spleen,
connective tissues, bones, and nervous tissues, but present in
striped muscle and cartilage.^ Adamoff^ has made some exact
quantitative determinations by Pfliiger's method, and he finds
that newly hatched chicks contain only traces of glycogen in
their bodies. New-born rabbits contain -17 to -86 per cent of
glycogen, or less than well-nourished adult animals, whilst the
human liver at a late foetal period contains '46 to i-68 per cent
Bernard* found no glycogen at all in the liver in early
embryonic life, though it appeared towards the middle of
intra-uterine development Pfliiger^ found glycogen in the
liver of all the embryos he examined. It was very variable in
amount, and sometimes was present only in traces: but the
muscles always contained a considerable store. Lochhead and
Cramer® found that the liver of foetal rabbits contained very
little glycogen up to the twenty-fifth day of gestation, and then
it rose above that of the rest of the foetal tissues. At the same
period the glycogen in the maternal placenta, which had
previously been considerable, showed a rapid and progressive
diminution till the end of gestation. Mendel and Leavenworth ^
estimated the glycogen in pig's embryos, and they found -25 per
cent, of it in the total body tissues of a 50 mm. embryo, -5 per
cent, in a 137 mm. embryo, and a maximum of -69 per cent in the
1 Cramer, ZeiLf. BioL, 24, p. 75> 1887.
2 Lubarsch, Arch. f. path. Anat, 1906, 183, p. 192.
3 Adamoff, Zeitf, BioL, 46, p. 281, 1905.
* Bernard, /?«r«. de la PhysioL^ 2, p. 326, 1859.
6 Pfliiger, Pfiuget^s Arch., 95, p. 19, 1903 ; 102, p. 305, 1904.
• Lochhead and Cramcty /oum. Phystol., 35, p. xi., 1906 ; Proc. Roy. Soc,^
B. 80, p. 263, 1908.
^ Mendel and Leavenworth, Amer, Joum, Physiol.^ 20, p. 117, i907«
AMYLASES 63
largest embryo examined — one of 212 mm. The liver and brain
substance of 85 to 230 mm. embryos contained no glycogen at all,
but the combined muscular and skeletal structures contained -47
per cent, in the smallest embryos, and i- 10 per cent, in the biggest.
Amylases. — Corresponding to this almost universal presence
of glycogen, we should expect to find a glycogen-hydrolysing
enzyme, which was richest in the liver and muscles. The
existence of a diastatic enzyme in the liver was first
demonstrated by von Wittich^ and by Claude Bernard,^ but
subsequent to them, several observers failed to obtain satisfactory
evidence of its existence. However, Pavy^ has demonstrated
it by a convenient and quite unexceptionable method. He
removed the liver from rabbits directly after death, minced it
up finely, pounded it in a mortar, and stirred the pulp with a
large volume of absolute alcohol. After standing with the
alcohol for as long as six months, he washed it with ether, dried
and powdered it. Two grammes of this dry powder were thrown
into boiling water to destroy the ferment, whilst another 2 gms.
were kept with 20 c.c. of i per cent. NaCl solution in an
incubator for four hours at 46°. The boiled control was found
by titration to contain '46 per cent, of reducing sugar (on the
weight of liver taken), whilst the incubated liver yielded 4-27
per cent. This great increase of sugar must have been due to
the action of a diastatic enzyme on the glycogen present in the
liver substance. Pavy found that the enzyme was not destroyed
even on boiling the liver powder with absolute alcohol.
Though the liver undoubtedly contains a fairly active
enzyme, it is doubtful if it is a soluble body to the same extent
as the proteolytic endoenzymes described in the previous
lectures. It seems to cling somewhat firmly to the liver tissue,
and can only be extracted therefrom in small quantities. Miss
Eves* extracted powdered alcohol-coagulated sheep's liver for
forty-eight hours with twice its weight of 10 per cent, sodium
chloride solution, and added 2 c.c. of the filtered solution to
10 c.c. of '5 per cent, starch paste, and another 2 c.c. to 10 c.c. of
* von Wittich, PflUget's Arch,^ 7, p. 28, 1873.
2 Bernard, Comptes Rendus^ 85, p. 519, 1877.
3 V?ivy ^Joum, Physiol., 22, p. 391, 1898.
* Eves, ibid.^ 5, p. 342, 1884.
64 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
•5 per cent, glycogen solution. After twenty minutes' digestion
at 38°, no reduction could be obtained with Fehling's solution,
but there was a copious one after an hour. The reducing power
of the solutions increased in succeeding hours, but there was still
some unaltered starch and glycogen left after twelve hours*
incubation. The enzyme could be precipitated from the saline
solution by absolute alcohol, and when re-dissolved six days
later furnished a weak diastatic solution. Again, Miss Tebb^
found that the enzyme could be extracted from dried liver
powder by means of 5 per cent, sodium sulphate solution. The
salt was removed from this solution by dialysis, and on
incubating it with 4 per cent, of glycogen at 37° for twenty-one
hours, dextrose was formed in some quantity. Such positive
evidence as this clearly outweighs the negative results obtained
by Noel Paton ^ and other observers.
In that liver tissue after death and disintegration undoubtedly
contains an amylolytic enzyme, there can be practically no
doubt that such an enzyme exists during life, bound up in the
protoplasm of the cells, and exerting its activity whenever it is
required. In the light of recent investigation the half-forgotten
controversy as to the causation of the post-mortal formation of
sugar in the liver is readily set at rest. Noel Paton maintained
that in the excised liver there is an early rapid amylolysis
preceding, and probably accompanying, the disintegrative
changes in the cells, such change being effected by the vital
processes of the organ ; and that subsequently there is a much
slower amylolysis which continues after the disintegration of
the liver cells and lasts for many hours, this change being due
to the development of an enzyme formed by the disintegration
of the cells. Pavy,* on the other hand, maintained that the
conversion of glycogen into sugar is entirely the work of an
enzyme, such enzyme not being present in the cells during
life, but only formed on their death from some pre-existent
zymogen. If Pavy's hypothesis be accepted for the diastatic
enzyme of the liver, then it must be accepted for any and every
^ Ttihhy /oum. PhysioL^ 22, p. 423, 1898.
^ Paton, ibid,^ 22, p. 121, 1897.
^ Pavy, The Physiology of the Carbohydrates^ An Epicriticism, 1895,
p. 102.
GLYCOGEN IN MUSCLE 65
intracellular enzyme, whether proteolytic, liipolytic, or amylolytic.
Such an hypothesis, though it cannot for the present be definitely
disproved, is sufficiently improbable to carry its own condemna-
tion.
Observations upon the glycogen-hydrolysing enzyme of
muscle are almost as numerous as those upon the liver, and
just as contradictory. This is largely because of the unsatis-
factory methods used by most of the earlier observers for the
estimation of glycogen. Almost all the observations concern
the rate of disappearance of glycogen from muscles after death.
Takdcz^ concluded, from experiments on rabbits, that this
disappearance is very rapid, as he found no trace of glycogen
thirty minutes after death. Praussnitz ^ found that from the
muscles of hens 25 to 59 per cent, of the glycogen disappeared
in thirty to sixty minutes. A. Cramer^ found that muscle
separated from the body and kept at 40° showed a considerable
diminution of glycogen in four hours, and Seegen * obtained a
similar result. On the other hand, Boruttau ^ found that only -2 to
IM per cent, of the glycogen disappeared from skeletal muscle
in twenty-four to thirty-eight hours, whilst 24 to 100 per cent,
disappeared from cardiac muscle in the same time. E. Kiilz®
likewise found only a slow disappearance of glycogen from
muscle after death, and Bohm ^ even stated that there was no
disappearance at all in six to twenty-four hours, provided that
putrefaction was prevented.
These observations, taken as a whole, undoubtedly proye the
post-mortal disappearance of glycogen from muscle, but they
do not definitely show how far this disappearance is the work
of an enzyme, and how far dependent on the vital activities of
the. still living muscle. However, Nasse^ has demonstrated
the existence of an amylolytic enzyme in muscle jtiice, and
1 Takdcz, Zeit f, physiol. Chem,, 2, p. 372, 1878.
« Praussnitz, Z«/./. Biol., 26, p. 377, 1890.
^ Cramer, i^V/., 24, p. 66, 1888.
* Seegen, Centralb.f. med. Wiss., 1887, No. 20 and 21,
* Boruttau, Zeit, f, physiol Chem,, 18, p. 513.
« Kulz, PflUget^s Arch,, 24, p. i, 1881.
^ B6hm, ibid,, 23, p. 44> 1880.
8 Nasse, Zur, Anat, u, Physiol, d, qutrgestrtiften Muskelsubstanz,
Leipzig, 1882.
I
66 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
Halliburton ^ found that a watery extract of the dried alcoholic
precipitate of muscle juice would change glycogen into a reduc-
ing sugar. It had a similar but slower action upon starch, and
at a temperature of 40*" no sugar was discoverable till the
enzyme had acted for five or six hours. Recently Kisch ^ has
made an exhaustive research upon the enzyme. He chopped
up the muscles of the back and the lower extremities of rabbits
a few minutes after death, and mixed 100 gm. of muscle with
100 C.C. of saline, -5 gm. of glycogen, and toluol, and one to
four and a half hours later estimated the glycogen still present
by the exact method of Pfluger. In one hour at room temper-
ature, 8 to 68 per cent, of the glycogen disappeared, whilst in
parallel experiments in which a current of air was drawn through
the tissue pulp, 18 to 75 per cent, disappeared. If a small
quantity of oxalate blood were added, there was likewise an
increased hydrolysis of glycogen, especially if air were drawn
through in addition. The rate of glycolysis was not appreciably
influenced by the addition of 20 per cent of decifiormal sulphuric
acid or caustic soda. Temperature had a great effect, the
amount of glycogen hydrolysed by 100 gm. of muscle in one
hour being on an average -07 gm. at is"", 'iS gm. at 22°, and
•40 gm. at 36^
Though the rates of amylolysis were very different in different
animals, yet the muscles of each individual gave fairly similar
rates with the exception of cardiac muscle. This was much
more active than skeletal muscle. For instance, in the case of
two rabbits the cardiac muscle hydrolysed 71 and 72 per cent,
respectively, and the skeletal muscle 36 and 27 per cent,
respectively.
As might be expected from the observations upon the
relation of ereptic value to functional capacity described in the
last lecture, the enzyme content of a muscle was found to be
practically uninfluenced by brief changes in its condition. For
instance, the sciatic nerve on one side of a dog was cut, and the
remaining muscles were thrown into convulsions for fifty minutes
to three hours by means of strychnin injections. Though some
of the glycogen had disappeared from the stimulated muscles as
» Halliburton, /wm. Physiol., 8, p. 182, 1887.
2 Kisch, Hofmeistet^s Beitr,, 8, p. 210, 1906.
AMYLASES 67
the result of contraction, they contained no more enzyme than
the resting muscles. Again, the muscles of a rabbit which had
undergone violent contractions as the result of strychnin
injection, were found to contain as much enzyme when tested
two hours post mortem as when tested directly after death, and
nearly as much when tested five hours post mortem.
Upon tissues other than liver and muscle extremely few
observations have been made. Foster ^ observed that extracts of
kidney and bladder wall, and also pleural, peritoneal, and peri-
cardial fluids, had a very slight amylolytic action upon starch, whilst
extracts of lymphatic glands were inert, von Wittich ^ found
small quantities of diastatic ferment in the kidney, brain, and
gastric mucous membrane. These observations were made
forty years ago, and apparently have not since been repeated
and extended, except in one instance. Pick* made a single
comparison of the digestive powers of minced liver and kidney
tissue, and found that in three hours lOO gm. of liver
hydrolysed '6g gm. of glycogen, whilst lOO gm. of kidney
hydrolysed 2*39 gm. Hence the kidney tissue is apparently
much richer than the liver in the amylolytic enzyme, just as it is
richer in )8-prctease and erepsin. But it is impossible to draw
conclusions from a single observation, and until a complete
series of comparisons has been made of the amylolytic power
of the various tissues, we cannot say definitely whether, and to
what extent, the enzyme activity corresponds with the power of
the cellular protoplasm to store up and utilise glycogen. At
least this is the case as regards the tissues of full-grown animals.
Upon embryos a series of observations has recently been made
which strongly supports the hypothesis of correlation. Mendel
and Saiki * minced up the liver and the muscles of pig*s embryos
of various sizes, allowed the pulp to stand some days under
alcohol, and then dried and powdered it. They kept *S S^-
samples of these powders with 40 cc. of i per cent, glycogen
solution and toluol for forty-eight hours at 24**, and determined
the amount of sugar formed by gravimetric analysis. The data in
^ Foster, Joum, Anat and PhystoLy i, p. 107, 1867.
2 von Wittich, Pfliiget^s Archiv,, 3, p. 340, 1870.
' Pick, Hofineistet's Bettr,^ 3, p. 174, 1903.
* Mendel and Saiki, Amer.Joum, Physiol,^ 21, p. 64, 1908.
fe8 FAt- ANt) CARBOHYDRATE-SPUTTING ENDOENZYMES
the table show the weights of CuO obtained for each lOO gm.
of fresh tissue taken. We see that the reducing power of the
hver digests increased very greatly with the growth of the
embryos, till in the largest embryos it reached that of the full-
grown organ. Muscle had a considerably greater initial
amylolytic power, but this increased relatively less rapidly with
Avenge Length of
Embryo.
GuO from
Liver digest.
CaO from
Muscle digest.
mm.
gm.
gm.
33
•79
1.40
8i
.62
1.56
125
1.12
1.58
188
1.46
174
225
2-99
3-42
275
5-90
4-93
Adult pig
3-82
growth than that of the liver. These results agree well with the
previously recorded observations to the effect that the glycogen
content of the pig embryo liver is small at first, and increases
considerably with growth, whilst that of the muscles is always
large, and increases less markedly with growth.
Probably the time and rate of increase of the amylolytic
power in the liver and other tissues varies in different animals,
for Pugliese ^ found that the blood and liver of new-bom dogs
and cats contained very little diastatic enzyme, or occasionally
none at all, but that it quickly increased in amount with growth
of the animal, especially in the case of the liver. Again,
Stauber ^ found that an ox embryo 1 5 cm. in length contained no
diastatic ferment in the pancreas, parotid and thymus. On the
other hand, the thymus of embryos 23 cm. in length possessed
strong diastatic properties, though the brain, lungs, stomach,
liver, spleen, kidneys, and muscle were practically free from the
ferment. After birth the ferment gradually disappeared from
the thymus.
Since the time of Majendie and Claude Bernard it has been
^ Pugliese, Arch, d, Farfnacologia e Terap,^ 12, p. i.
2 Stauber, -Py&f^rr'j Arck.^ 114, p. 619.
AMYLASES 69
known that blood serum can change starch into sugar. Pick^
found that lOO c.c. of blood digested -31 gm. of glycogen in
three hours, as against the -69 gm. digested by 100 gm. of liver,
hence the small amylolytic activity of the tissues other than
liver, muscle, and kidney may be due entirely to the blood
enzyme. Bial ^ found that the enzyme could be extracted from
the serum of blood and the lymph, but not from blood corpuscles.
He stated that the enzyme converted starch into glucose, and
so differed from the diastatic enzyme of the salivary gland.
Rohmann,' and later Hamburger,* arguing from the fact that
saliva, pancreatic juice, intestina] juice, and blood serum converted
starch into maltose and maltose into dextrose at very different
relative rates, concluded that they all contain different propor-
tions of two distinct enzymes, viz. a diastase or amylase which
converts starch into dextrin and maltose, and a glucase or
maltase, which converts these products into glucose. Ascoli
and Bonfanti ^ go still further, and think that the blood serum
contains several amylases. They find that serum has different
rates of action upon different starches, and that it acts more
quickly upon a mixture of two starches than upon either starch
individually. For instance, human serum, when acting upon
2 per cent potato starch paste, yielded -095 per cent, of reducing
sugar in a given time ; when acting upon 2 per cent, rice starch,
•076 per cent, of sugar, but when acting upon a paste containing
I per cent, of potato starch and i per cent, of rice starch, it gave
•no per cent, of sugar. In the light of recent knowledge upon
the multiplicity of toxins, precipitins, lysins and other bodies in
the blood, it seems very probable that Ascoli and Bonfanti are
correct in their contention, though many more observations are
required before it can be accepted as proven.
But there can at least be no doubt as to the existence of two
distinct classes of carbohydrate-splitting enzymes in the blood
and tissues, viz., enzymes which hydrolyse polysaccharides like
starch, glycogen, and dextrins into maltose or some other
^ Pick, Hofmeistet^s Beitr,^ 3, p. 174, 1903.
2 Bial, Pfliiget's Arch.y 52, p. 137, 1892 ; and 53, p. 156, 1893.
3 Rohtnann, Ber,^ 27, p. 3251, 1894.
* Hamburger, Pfiuge?s Arch,^ 60, p. 543, 1895.
^ Ascoli and Bonfanti, Zeitf,physioL Chem,^ 43, p. 156, 1904.
70 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
disaccharide, and enzymes which hydrolyse maltose and other
disaccharides into monosaccharides such as glucose. The
existence of these two classes of enzymes was suggested by the
investigations of Brown and Heron ^ in 1880. Brown and
Heron found that aqueous extracts of the minced intestine of
the pig had but little action upon starch or cane-sugar, but that
if the well washed intestine were dried rapidly in a current of
air at 35°, fine shreds of it, added to the starch or sugar under
examination, exerted a powerful hydrolytic action. It seemed,
in fact, that the endoenzymes clung somewhat firmly to the
tissue, and only passed into solution in small quantity.
Maltose. — To test the activity of the dried intestine, Brown
and Heron kept 5 gm. of it with 100 c.c. of 3 per cent, soluble
starch at 40" for three and a half hours, and then for another
sixteen hours at room temperature. Analysis showed that about
half of the starch had been converted into sugar, but somewhat
unexpectedly, this sugar was found in four experiments out of
five to consist entirely of dextrose. In the single experiment in
which maltose was present at all, it formed less than a fourth of
the total sugar. Further experiment showed that the whole of
the starch must have passed through the maltose stage, but
that the enzymes of the intestine had a more energetic action
upon maltose than upon starch or dextrins, and so directly the
starch had been converted into maltose, this maltose was seized
upon and broken down further into dextrose. For instance, the
shredded dried intestine, if allowed to act upon 3*1 per cent,
maltose solution, converted it entirely into dextrose when
digested for sixteen hours at 40°. Pancreatic extract, in
contradistinction to dried intestine, converted starch rapidly to
the maltose stage, and then very slowly converted some of this
maltose into dextrose. But malt extract, according to Brown
and Heron, had no further action upon the maltose. Following
Rohmann's interpretation, we therefore conclude that malt
extract contains no trace of maltase enzyme ; pancreatic extract
contains a small quantity of it, and intestinal extract a large
quantity.
Arguing from the presence of maltase in pancreatic extracts,
it has been generally assumed that the juice secreted by the
^ Brown and Heron, Proc, Roy. Soc^ 30, p. 393, 1880.
MALTASE
71
pancreas likewise contains a small amount of maltase, but as far
as I am aware, this has never been proved, and it is more
probable that the maltase in extracts consists entirely of a
soluble endoenzyme, such as is present in most if not all of the
other body tissues, and is not secreted into the pancreatic juice
as an exoenzyme.
The proof of the general distribution of a maltose-splitting
enzyme in the tissues we owe to Miss Tebb.^ Working on
similar lines to Brown and Heron, Miss Tebb dried and shredded
various tissues of the pig, and added 5 gm. of each to 100 c.c.
of 2'7 per cent, maltose. After twenty hours* digestion at 38°,
the following percentages of the maltose were found to have
undergone conversion into dextrose : —
Percent.
Per cent.
Tissue.
ofMaltoM
Tlnae.
of Maltose
hydiolysed.
hydrolysed.
Mucous membrane of
Kidney ...
40
small intestine .
76
Gastric muc memb. .
31
Spleen.
57
Pancreas .
24
Lymphatic gland .
48
Salivary gland .
17-4
LiveTT . . .
44
Skeletal muscle .
16.7
Intestinal mucous membrane gave the best result of all,
whilst the pancreas and salivary glands contained less of the
enzyme than any tissue but muscle. A result such as this shows
very clearly how completely independent of one another are the
powers possessed by a gland of elaborating a diastatic enzyme
for secretion externally, and of storing up another kind of carbo-
hydrate-splitting enzyme within its cell substance. The figures
also seem to imply that the richness of a tissue in intracellular
maltase bears but little relationship to its richness in intracellular
amylase, for muscle, which seems to be rich in glycogen-
hydrolysing enzyme, is very poor in maltase.
In contrast to Brown and Heron, Miss Tebb found that the
maltase readily passed into solution when fresh intestinal mucous
membrane was minced and kept in chloroform water. Even the
dried intestine, when extracted with 5 per cent, sodium sulphate,
yielded an active solution. Extracts of dried lymphatic gland,
^ Tebb, /i?«f7r. Physiol.^ 15, p. 421, 1894.
72 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
pancreas, and liver were likewise moderately active. Probably
Brown and Heron did not mince the tissue sufficiently, and they
certainly did not allow sufficient time for adequate extraction.
This requires several days for aqueous extracts, and three weeks
or more for glycerin extracts, whereas they allowed only ten to
fifteen hours. There can be no doubt as to the solubility of the
enzyme, once it has broken free from its anchorage in the tissues,
for Miss Tebb found that serum was far richer in it than any of
the organs investigated. Serum diluted with three volumes of
water converted the whole of the 27 per cent, of maltose added
to it into dextrose in twenty-three hours at 38°. Assuming
that serum contains 8 per cent, of solids, it follows that only
2 gm. of solid serum were used for each 100 c.c. of maltose,
as against 5 gm. of solids in the case of the tissues above
mentioned. This richness of serum in maltase introduces a
disturbing factor into the above recorded maltase values of the
tissues, for they were obtained with organs from which the blood
and lymph had not been removed, and so an unknown fraction
of their apparent maltose-splitting power is due to retained
serum. A repetition of the observations with previously
perfused organs is therefore desirable.
Upon the maltase content of embryonic tissues no quantita-
tive observations have been made. Bierry ^ found the enzyme
in the intestines of embryonic sheep and cattle, whilst Mendel
and Mitchell ^ found that it was present in the intestines of all
pigs* embryos over 50 mm. in length. A single observation on
the kidneys of a 120 mm. embryo showed no maltase, whilst one
on the liver of a 200 mm. embryo showed a trace of it.
Invertase. — Though there is some doubt as to the existence
of more than one amylolytic enzyme in the tissues, there are
certainly at least three distinct enzymes which act upon di-
saccharides, viz. maltase, invertase, and lactase. Invertase, which
has the power of hydrolysing cane-sugar to glucose and fructose,
was isolated by Berthelot* from yeast in i860. Subsequently
Claude Bernard showed that an infusion of intestinal mucous
membrane likewise contained the enzyme. He demonstrated its
1 Bierry, C. R. Soc. Bioi,, 52, p. 1080.
2 Mendel and Mitchell, Amer,/oum. Physiol^ 20, p. 81, 1907.
3 Berthelot, Cpmptes Rendus^ 50, p. 980, i860.
INVERTASE 73
presence in the intestine of dogs, rabbits, birds, and frogs.i
Other observers found it in the intestinal tract of man, the horse,
ox, and cat It is also present in succus entericus, but Rohmann *
found that extracts of the mucous membrane are much richer in
enzyme than the secretion, so probably it is chiefly intracellular
in its action, and splits up the cane-sugar during its passage
through the intestinal wall. Rohmann,^ and also Miura,* found
that the upper part of the small intestine contains more
invertase than the lower part, and Miura found that the enzyme
is also present in small amount in the tissues of the colon,
stomach, and pancreas. However, Harris and Gow* failed to
find it in the pancreas, and Widdicombe® found none in
lymphatic glands. Hence it is not an enzyme of widespread
distribution like maltase, but is practically limited to the mucous
membrane of the alimentary canal.
The inverting ferment of the small intestine is much less
active than the maltase. Brown and Heron found that in two
parallel experiments 27 and 24 per cent, respectively of the cane-
sugar was split up, but 74 and 58 per cent, respectively of the
maltose. Widdicombe observed an interesting relationship of
the enzyme to the reaction of the medium in which it was
digesting. He found that in -3 to -5 per cent. HCl the enzyme
of intestinal mucous membrane was inactive, though the inverting
action was merely suspended under the influence of the acid, not
destroyed. Gastric mucous membrane, on the other hand, and
likewise gastric juice, inverted cane-sugar readily in an acid
medium, but not in an alkaline one.
Lactase. — The distribution of lactase in animal tissues is even
more limited than that of invertase, for it is confined entirely to
the intestine, and in some cases to the intestine of young animals
only. It was discovered by Rohmann and Lappe^ in the
mucous membrane of the small intestine of dogs and calves.
^ Bernard, cf, R. Green, Soluble Ferments and Fermentation^ p. 115,
1901.
2 Rohmann, Internal. PhysioL Kongr.^ Turin^ 1901.
3 Rohmann, Pfliiger^s Arch,^ 41, p. 411, 1887.
* Miura, Zeit, /. BioL^ 32, p. 266, 1895.
° Harris and Oovr^ Joum. Physiol.^ 13, p. 469, 1892.
® Widdicombe, ibid,^ 28, p. 175, 1902.
^ Rohmann and Lappe, Ber,^ 28, p. 2506, 1895.
74 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
Aqueous extracts slowly split up lactose into glucose and
galactose. The enzyme could be precipitated from the extracts
by alcohol, and a solution of the precipitate possessed the power
of hydrolysing lactose. In all probability lactase is purely an
endoenzyme, as PregP found that the intestinal juice of the
lamb, collected from a Thiry-Vella fistula, contained none of it.
Fischer and NiebeP found that intestinal extracts made from
young animals were frequently more active than those from old
ones. Portier^ found lactase in the intestine of old rabbits, but
not in that of pigs and birds. Weinland * examined the intestine
of young and old animals, and found that lactase was always
present in young animals, and also in old dogs, pigs, and horses,
but not in the intestine of old oxen, sheep, rabbits, and fowls.
As a rule the amount of lactase present is small, and its
detection by no means easy, so the results obtained cannot be
accepted implicitly. However, Aders Plimmer*'* has recently
repeated these observations, and using an accurate gravimetric
method for estimating the reducing sugar formed, has in the
main confirmed them. He ground up the mucous membrane
with sand, and treated it for six to twenty-four hours with toluol
water. The extract was then filtered through lint, and so still
contained cells and cell fragments. Its action was only slow at
best. For instance, 150 C.c. of extract of cat's intestine, kept
with 150 c.c. of 5 per cent, lactose solution and toluol at 37°,
hydrolysed 13 per cent, of the lactose in twenty-six hours,
23-4 per cent, in seventy-two hours, and 465 per cent, in
170 hours. Plimmer found that the omnivorous cat and
pig have lactase in their intestine during the whole of their
lives. The herbivorous guineapig has it only when it is
young, but in that the adult rabbit has plenty of lactase, there
can be no hard and fast distinction between the two classes of
animals. Probably, also, one is not justified in drawing con-
clusions from a limited number of observations, for Orbdn,* just
1 Pregl, Pfluget's Arch,, 61, p. 359, 1895.
2 Fischer and Niebel, Sitzber, Akad. Wiss, Berlin^ p. 73, 1896.
3 Porticr, C R. Soc. BioLy 52, p. 423, 1900 ; 53, p. 810, 1901.
* Weinland, Zeitf, Biol.^ 38, p. 16, 1899.
^ Plimmer, /<7«/r«. Physiol,^ 35, p. 20, 1906.
• Orbdn, Malfs Jahresb,^ 1899, p. 384.
LACTASE 75
like Weinland, was unable to find lactase in full-grown rabbits.
Plimmer found that neither the frog nor the fowl had lactase in
their intestines, so probably the enzyme is confined to mammalia.
Guineapigs one to three days old were found to be rich in
lastase, whilst animals five or more weeks old had practically
none. Hence the ferment must have dwindled down within
these weeks, more or less synchronously with the change of diet
from milk to vegetable food. The interesting and important
question arises as to whether this is a case of direct adaptation.
Plimmer endeavoured to solve it by feeding adult guineapigs
upon a milk and lactose diet for five to eleven weeks, but in no
case did any appreciable amount of lactase appear in their
intestines. One might almost expect such a result as this, for
once the tissues have given up the elaboration of any particular
enzyme, the mechanism for such elaboration probably disappears,
and cannot be acquired again at any rate in a short time.
Quite otherwise is the result one would expect if young animals,
still possessing lactase-forming powers, were kept permanently
on a milk diet. Plimmer records no experiments of this kind,
but he says that adult rats and rabbits, when fed on milk and
lactose, show no increase in the normal lactase-content of their
intestines. However, his experimental data do not altogether
bear out this contention. For instance, two rabbits, kept three
and fifteen weeks respectively without milk, gave ii'2 and 55' i
per cent, of hydrolysis of the standard lactose solution, whilst
two other rabbits, kept for similar periods on a milk and lactose
diet, gave 23-4 and 62'3 per cent of hydrolysis. Again, Sisto^
obtained results which accord with expectation, for he found that
though lactase is present only in small quantities in the intestine
of adult mammals, or is not present at all, it appears on continued
feeding with milk or milk sugar.
As regards the variation of lactase with growth, Plimmer
found that rat embryos had none of the enzyme in their
alimentary canal forty-eight hours before birth, whereas it was
present twelve hours before birth. On the other hand, Mendel
and Mitchell ^ found it in all pigs' embryos more than 50 mm.
in length. Probably the lactase attains a maximum within a
^ Sisto, Arch, d. FisioLy 4, p. 116.
2 Mendel and Mitchell, Amer,Joum, Physiol^ 20, p. 81, 1907.
76 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
very few days after birth, and then dwindles down again.
Comparative observations upon invertase do not seem to have
been made, but Cohnheim^ states that it is present in the
intestine of foetal and new-born cats and dogs. In fact it might
be the only demonstrable ferment in the foetal animals. On the
other hand Mendel and Mitchell found that it was uniformly
lacking from the intestines of pigs' embryos.
Intracellular diastatic enzymes are probably as widely
distributed in the lower animals as in the higher. Miss
Greenwood 2 found that Rhizopods do not attack starch, but
Meissner found that Infusoria, if deprived of protein food, will
take it up and dissolve it. De Bary states that starch grains
ingested by the plasmodium of Mycetozoa are strongly corroded
in the course of some days, whilst Lister concludes that certain
of the Mycetozoa have little or no action upon raw starch, but
speedily digest swollen starch. Hartog and Dixon found that
the large protozoon Pelomyxa palustris contained a diastatic
enzyme which quickly converted starch into erythrodextrin, but
only slowly transformed this body into sugar. In sponges
Krukenberg found that diastatic ferments capable of converting
starch into sugar are widely distributed, whilst Mesnil found a
weak ferment in aqueous extracts of the pulped mesentery
filaments of Actinia.
Amylases in Plants, — In plants, diastatic and sucroclastic
enzymes are much more widely distributed than in animals,
and by reason of their great activity and of their economic
importance they have been the subject of a much more thorough
study. It is unnecessary to describe them in detail, however, as
they have been dealt with fully elsewhere.^ It will be sufficient
to refer to them chiefly in relation to the intracellular diastatic
and sugar-splitting enzymes of animal tissues.
The diastase of germinating barley, discovered by Kirchoff
in 1 8 14, is the first of the soluble ferments known. Payen and
* Cohnheim, NagePs Handbuch d, Physiol,^ 2, p. 599, 1907.
2 Greenwood. See v. Fiirth's Vergleichende Chem, physioL d, niederen
Tierey for this and other references, and for fuller details.
3 See especially Reynolds Green's Soluble Ferments and Fermentation,
Cambridge, 1901, chaps, ii. and iv.-x., from which this brief summary is
mainly drawn.
VEGETABLE DIASTASES 77
Persoz found the same ferment in germinating wheat, oats,
maize, and rice, and in 1874 Gorup-Besanez found it in other
varieties of germinating seeds. Kosmann and Krauch demon-
strated it in the leaves and shoots of the higher plants, and in
various algae, lichens, mosses, and fungi. Baranetzky found it
in buds and in potato tubers, and in the light of what was known
of its distribution, he suggested that it is present in all vegetable
cells. Subsequent investigations have tended to confirm this
hypothesis, and they have established the additional fact of
the existence of at least two different vegetable diastases, viz.
the so-called diastase of translocation, and diastase of secretion.
The first is the more widely distributed, as it occurs in the seed
during the development of the embryo, as well as being present
in the vegetative organs. It is most readily prepared from
barley, and it differs from secretion diastase in that it dissolves
starch grains without corrosion : acts very slowly on starch paste,
but quickly on soluble starch : works best at 45° to 50°, and is
much more active at a low temperature than secretion diastase.
Secretion diastase, on the other hand, is especially connected
with the process of germination, and is most readily prepared
from malt extract. It corrodes starch grains and disintegrates
them before solution: acts rapidly upon starch paste: works
best at 50° to 55°, and can be heated to 70° without destruction.
Arguing from the fact that vegetable proteins differ from animal
proteins, and that enzymes are probably bodies related to
proteins, it follows that vegetable diastases cannot be chemically
identical with animal diastase. The course of action of the two
enzymes is likewise very different,^ but the final product of
activity is in both cases maltose. Probably no isomaltose,
dextrose, or other sugar is- formed at the same time, and it
seems likely that glycogen is also converted by diastase into
maltose only.
In addition to diastase, vegetable tissues contain at least
two other amylolytic enzymes, which seem to have no analogues
in the animal kingdom. Certain of the Compositae, such as the
genera Dahlia and Helianthus, possess tubers or fleshy roots in
which stores of the carbohydrate inulin are situated, whilst the
bulbous Liliaceae and related plants contain stores of it in their
^ Cf. Wtrnoti^Joum. Physiol.^ 28, p. 156, 1902 ; see also Lect. VI., p. 159.
78 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
leaves and elsewhere. This inulin is usually in solution in the
sap of cells. In the process of growth of the plants it is
transformed into fructose by the action of an enzyme inulase
(Reynolds Green).^ Again, the walls of many vegetable cells
consist partly of cellulose, pectose, and related substances, the
hydrolysis of which is effected by the enzyme cytase,
Sucroclastic Enzymes, — In sucroclastic enzymes, as in amylo-
lytic ones, plants are likewise richer than animals, for in addition
to maltase, invertase, and lactase, they contain at least three
others, viz. trehcUase^ raffinase^ and melizitase, Maltase was
first shown to exist in the vegetable kingdom by Bourquelot,
who ground up the mycelia of the moulds Aspergillus niger and
PeniciUium glaucum with sand, extracted with water, precipitated
the maltase in the aqueous extract with alcohol, and on redissolv-
ing this precipitate in water obtained an active preparation.
Bourquelot also detected maltase in yeast, whilst Cuisinier
found it in barley malt, and Geduld in maize.
Invertase is more widely distributed in plants than maltase,
for Berthelot discovered it in yeast (i860) ; B^champ found it in
moulds, and in the petals of several flowers ; Brown and Morris
found it in the air-dried leaves of Tropoeolum, and Kosmann in
the buds and leaves of young trees. It is also present in the
rootlets of germinated barley. Probably cane-sugar and
invertase play an important part in the nutrition of actively
growing vegetable protoplasm.
In plant tissues, as in those of animals, lactase seems to be
of very restricted distribution. Beyerinck found it in extracts
of the Kephir organism, whilst Fischer found it in certain
yeasts.
Of the remaining three sucroclastic enzymes, trehalose is
found in certain moulds such as Aspergillus niger and PeniciUium
glaucum^ where its function is to hydrolyse the disaccharide
trehalose into glucose. Raffinase is found in the root of the
beet, in germinating barley and wheat, and elsewhere. It
acts upon the hexatriose body raffinose, CigHggOie, and splits
it up into glucose, fructose, and galactose. Melizitase is found
in the manna exuded from the leaves and branches of the
leguminous plant, Alhagi maurorum^ and elsewhere, and it
* R. Green, Ann. Bot,^ i, p. 223, 1888.
GLUCOSIDE^SPLITTING ENZYMES 79
hydrolyses the melizitose therein contained to glucose. Melizitose
is a hexatriose like raffinose.
In some plants still another class of sugar-splitting enzymes
is represented, viz. the glucoside-splitting enzymes. These
enzymes have the property of breaking down various complex
bodies with the formation of a sugar, generally glucose, and
other products. The best known of them, emulsin^ has the
power of decomposing the glucoside amygdalin into benzoic
aldehyde, hydrocyanic acid, and glucose. It will also effect
the hydrolysis of other glucosides such as salicin and phlorizin.
Emulsin is found in the young stems and leaves of the
Cherry- Laurel, in certain Lichens, and in many fungi, as well as
in the seeds of sweet and bitter almonds. Another enzyme,
myrosifiy is widely distributed throughout the Cruciferae, and in
several allied Natural Orders, and moreover it is present in the
roots, stems, leaves, flowers, and seeds of many of these plants.
It splits up the glucoside sinigrin into allyl sulphocyanate,
potassium hydrogen sulphate, and glucose.
It is unnecessary to refer to the other glucoside-splitting
enzymes which have been described in various plant tissues. Of
greater interest to us is the occurrence of similar ferments in
animal tissues. Kolliker and Miiller^ showed that pancreatic
juice is capable of decomposing amygdalin, but they did not
isolate the specific enzyme. Recently Gonnermann ^ has made
a number of observations upon the hydrolytic power of various
animal tissues upon glucosides and alkaloids. He found that
fresh minced liver of the ox and hare were able to decompose
the glucosides amygdalin, arbutin, and sapotoxin, but that the
liver of the dog, horse, and fish had no action on amygdalin,
a feeble one on sapotoxin, and a well marked one on arbutin.
Trypsin, or presumably a pancreatic extract, was said to split
up amygdalin, but not the other two glucosides, whilst pepsin
acted on none of them. The glucoside sinigrin was not attacked
by any of the tissues or enzymes investigated. This capacity
of animal tissues to split up glucosides seems to imply that a
specific enzyme is not required for the process, but that one of
the enzymes normally present, perhaps maltase, is able to effect
^ Kolliker and Miiller, see R. Green's Soluble Ferments^ p. 154, 1901.
2 Gonnermann, Pfliiget's Arch,^ II3> P- 168, 1906.
80 FAT- AND CARBOHYDRATE-SPLITTING ENDOENZYMES
it. But even then it is difficult to account for the results
obtained by Gonnermann, as one would expect that amygdalin,
for instance, if attacked by the liver tissue of one animal, would
be similarly decomposed by that of other animals. Before any
definite conclusion can be adopted, therefore, a good deal more
investigation is necessary.
LECTURE IV
ZYMASE AND OTHER GLYCOLYTIC ENZYMES
Zymase of yeast. Its action on various sugars. Cause of its instability.
Effect of filtration. Action of antiseptics. Influence of phosphates.
Zymase and lactacidase enzymes. Action of inorganic catalysts on
glucose. Glycolytic power of mixed pancreas and muscle juice.
Alcohol in animal tissues. Anaerobic respiration in living and dead
plants. Formation of acids in aseptic and antiseptic autolyses.
In the last lecture we were able to trace the endoenzymes which
can convert the carbohydrates of the tissues and food into
disaccharides, and subsequently into the simpler monosac-
charides. But of the further changes which these monosac-
charides undergo we know very little with certainty. Until
recent years we had no conception of what happened to them,
other than that they were oxidised in the tissues to carbonic
acid and water. But in the light of recent investigation it seems
possible, even probable, that they undergo the same kind of
changes as those which have long been known to occur in
alcoholic and lactic acid fermentations.
Alcoholic fermentation^ was first proved to be the work of
a definite organism by Cagniard de Latour and by Schwann in
1837. Both these observers thought that the yeast cells formed
alcohol and carbon dioxide by their vital processes, and though
this view was combated by Liebig, it was supported by the
remarkable researches of Pasteur. The recognition of substances
which though not composed of living organisms were derived
^ For a detailed account of the history of alcoholic fermentation,
see Reynolds Green, Soluble Ferments and Fermentation^ Cambridge,
2nd ed., 1901.
82 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
from such organisms, and which were able to induce somewhat
similar changes to those effected by yeast, led to a separation
of fermentations into two classes, viz., those produced by
organised ferments such as yeast and putrefactive bacteria, and
those produced by unorganised ferments. Many attempts have
been made in the past by Naegeli, Sachs, and other investigators,
to show that these ferments are radically different, but with
increase of knowledge the supposed differences and distinctions
were found to dwindle down more and more, till the discovery
of zymase by Buchner^ in 1897 indicated that they could no
longer be supported. The further they are analysed the more
have the activities of the micro-organisms of fermentation been
shown to depend upon intra- or extra-cellular enzymes, and
hence it would seem to be only a question of time before they
will all of them be referred to this source.
The method used by Eduard Buchner for isolating an
enzyme which could decompose sugar into alcohol and carbon
dioxide is as follows. Washed yeast is subjected to a pressure
of fifty atmospheres in an hydraulic press, whereby 70 per cent, of
the water contained in it is squeezed out, and a fine dry white
powder left. This powder is mixed with quartz sand and the
siliceous earth kieselguhr in the proportions of 10 of yeast, 10 of
sand, and 2 or 3 of kieselguhr, and the mixture ground up in a
porcelain mortar by means of a very heavy (8 kg.) iron pestle.
The yeast cells are broken up by the sharp sand grains, and in
it few minutes the dry powder becomes converted into a grey-
brown plastic mass, which sticks together in the form of balls.
The mass is wrapped up in a strong "press-cloth," and
subjected to a gradually increasing pressure by means of a
hydraulic press. One kilogram of yeast, subjected to 300
atmospheres pressure, yields as much as 450 to 500 c.c. of juice.
In some cases the press cake was taken out of the press and
ground up with 100 c.c. of water and again subjected to pressure,
when 100 to 150 c.c. more juice was obtained.
Macfadyen, Morris, and Rowland,^ who have repeated many
^ Most of the experimental details recorded in the next few pages are
taken from Die Zymasegdhrung^ by E. Buchner, H, Buchner, and M.
Hahn : Munich and Berlin, 1903, pp. 1-416.
* Macfadyen, Morris, and Rowland, Proc, Roy. Soc,^ 67, p. 250, I9cx>*
ZYMASE 83
of Buchner's experiments in this country, adopted a more
thorough method of disintegrating the yeast cells. They mixed
the yeast with silver sand and placed it in a vessel in which a
many-toothed spindle was rapidly rotated by mechanical power.
The sand grains and yeast cells were driven violently against
one another, and a microscopical examination at the end of the
process showed that every cell was ruptured. Brine at a
temperature of — 5° was circulated round the vessel, and this kept
the disintegrating mass at about 15°.
The press juice, after filtration, was obtained as a yellowish,
slightly opalescent liquid. It had a specific gravity of 1-031 to
1-057, 2iwd contained 8-6 to 13-9 per cent, of solids, of which 1-3
to 1-9 per cent, was ash, and the remainder mostly protein. On
boiling, this coagulated and yielded a solid white mass. In
addition to zymase, the juice contained the enzymes invertase,
maltase, endotryptase, rennin, a glycogen-hydrolysing enzyme,
and a reducing enzyme which could liberate sulphuretted
hydrogen from sodium thiosulphate and from sulphur, and
decolorise methylene blue. If sugar were added to this juice,
carbon dioxide began to bubble off in a few minutes, and a
steady stream was evolved for hours or days, according to the
temperature. At 35° the outflow was very rapid, but almost
ceased after a day, whilst at 6° its initial rate of evolution was
six times slower, but it continued with undiminished vigour for
ten days or more. At 22* a mixture of 20 c.c. of juice with
8 gm. of cane-sugar, gave it off at the following rate : —
1st day . ... . i-oo gm.
2nd „ . . . . -36 „
3rd „ . . . . KH „
4th , -oi „
Together with this carbon dioxide, alcohol is formed in
almost equal quantity, in accordance with the equation :
CeHigOg = 2C02 + 2C2HeO.
This requires the formation of twenty-three parts by weight of
alcohol for each twenty-two parts of COg. One of the most
active juices obtained yielded 12-2 gm. of COg and 14-4 gm.
of alcohol per 100 c.c. of juice; another, 12-2 gm. of COg and
84
ZYMASE AND OTHER GLYCOLYTIC ENZYMES
12-4 gm. of alcohol ; another, 8-9 gm. of CO2 and 8-9 gm. of
alcohol.
Numerous experiments were made by Buchner and Rapp ^
upon the fermentability of various sugars, and from the data
given in the table we see that glucose and fructose gave the
20 C.C OF Juice +13 per cent, of Sugar, kept 40 hours at I5^
YIELDED :— .
In pxeaenee of
•2 C.C. of Toluol.
Another Sample
of Juice,
without Antiseptic.
Glucose
Fructose
Galactose
Saccharose
Maltose
Lactose
Glycogen
gm.
•70
.70
•12
.66
.69
02
.29
gm.
.72
•73
•13
•72
.72
•03
*23
best yield of CO2. Cane-sugar and maltose were only slightly
inferior, in spite of the fact that they had to be converted by the
invertase and maltase enzymes of the juice into the monosac-
charide form before they could be acted on by the zymase.
On the other hand, galactose was attacked but slightly by the
zymase, and lactose practically not at all. Glycogen was acted
on somewhat slowly, presumably because the glycogen-splitting
enzyme of the juice is only a weak one. Potato starch was even
less readily attacked, as a mixture of 20 c.c. of juice (a different
sample from the above) with i gm. of starch and -2 cc. of toluol
yielded only -lo gm. of COg in sixty-four hours. Four grammes
of soluble starch, acted on under similar conditions, gave -13 to
•28 gm. of COg, whilst dextrin gave -57 to 75 gm. of COg.
Buchner found that the greatest yield of CO2 is obtained by
using concentrated sugar solutions containing 30 to 40 per cent,
of sugar. (See table on page 85.) The sugar protects the juice
against auto-destruction, and so the evolution of CO2 continues
longer with greater concentrations than with smaller ones;
but the initial rate of evolution is less. For instance, in
the first six hours of the experiment, the 10 per cent, sugar
^ Buchner and Rapp, Ber,^ 31, pp. 1084 and 1090, 1898.
ZYMASE
85
mixture gave out '17 gm. of COg, but the 40 per cent, mixture
only -105 gm. Macfadyen, Morris, and Rowland found that
their juice (which was made from top fermentation yeast, not
bottom yeast like Buchner's) gave the best yield of COg with
5 to ID per cent, cane-sugar. Also it gave a considerable
20 c.c. Juice + Cane-sugar + -2 cc. Toluol, kept 96 hours
AT 22** TO 25^ YIELDED :—
Sugar.
CO,,
gm.
Per cent.
10
.56
15
.64
20
•73
2S
•79
• 30
.81
40
.82
yield when no sugar whatever was added, so presumably it
was richer in glycogen than Buchner's juice, which under such
conditions evolved scarcely any CO2.
The properties of zymase have been described thus far
without any comment upon their resemblance to or difference
from those of enzymes in general on the one hand, and of living
yeast cells on the other. In fact, they have been tacitly assumed
to be those of an enzyme, though such an assumption is not
justifiable without further explanation. This is especially the
case in regard to the extreme instability of zymase. This and
other curious properties for a time led some investigators to
believe that zymase consisted of fragments of still living
protoplasm, which, for the short time they remained alive,
were able to exert the functions normally confined within the
living cell. In the light of more complete knowledge, however,
this hypothesis has been almost entirely discarded, and for
reasons which are given in detail below, we may now regard the
enzymic nature of zymase as undoubtedly established.
The property of extreme instability which seemed more
especially to differentiate zymase from other enzymes is
dependent in large measure upon the powerful proteolytic
enzyme always present in yeast juice. This enzyme, called
endotryptase, has been subjected to a detailed study by Hahn
86
ZYMASE AND OTHER GLYCOLYTIC ENZYMES
and Geret.^ It is similar to the j8-protease of animal tissues,
in that it acts best in feebly acid solutions — the optimum
being -2 per cent. HCl — whilst its activity is paralysed by
alkalis. It digests native proteins somewhat slowly, as fibrin
flakes require about twenty-four hours, for solution by yeast
juice, but it rapidly converts albumoses and peptones into amino
acids. The coagulable protein present in yeast juice is speedily
digested by it to the non-coagulable stage, as the following data
show : —
Ooagulable
Protein.
N.ln
CoBgulam.
N.ln
Filtrate.
Fresh juice
After I day at room temperature
„ 6 days „ „ . .
Percent.
4-38
1-43
.14
Per cent.
.64
.19
•02
Per cent.
.42
.85
103
Hahn and Geret also estimated the phosphates in the filtrate
from juice which had been precipitated by HgCl^+HCl, and
they observed a very rapid passage of organically bound
phosphorus into the soluble inorganic state. Thus of the total
•528 per cent, of PgOg present they found that : —
P2O5 in fresh filtrate = .024 per cent
PqOs after 12 hours = '360 „
P2O5 after 5 days = .416 „
In another experiment the filtrate from the fresh juice contained
•020 per cent, of PgOg, and after seventy minutes' autodigestion
at 39°, no less than -384 per cent. After nine days it was -506
per cent, the total PgOg present being -600 per cent
Similar evidence of the rapid autodigestion of yeast juice
has been obtained by Macfadyen, Morris, and Rowland, by
Petruschewsky^ and others. The endotryptase, when digesting
the coagulable protein, doubtless digests the zymase at the same
time, so if it were possible to separate the two enzymes it might
be found that zymase by itself is no more unstable than other
enzymes. No attempts at such separation appear to have been
^ Hahn and Geret, Zeit. /. BioLy 40, p. 117 ; also. Die Zymasegdhrung^
p. 29 et seq.
2 Petruschewsky, Zeit f, physioL Chetn,^ 50, p. 251, 1907.
ENDOTRYPTASE 87
made, though there is no reason why a partial separation
should not be effected by fractional precipitation with alcohol
or ammonium sulphate solution. However, it is improbable
that the endotryptase would be completely removed in this way.
Perhaps a better method of neutralising its digestive action
upon the zymase would be by means of an antiferment.
The destructive action of the endotryptase upon the zymase
can be diminished or increased in a number of different ways.
The protective action of concentrated cane-sugar solutions has
already been commented on. Glycerin also exerts some
influence, and small quantities of alcohol retard the action of
endotryptase more than that of zymase. Probably proteins act
best of all, in that a good deal of the endotryptase becomes
bound to the added protein, and exerts its digestive powers
upon it, whilst the zymase is correspondingly spared. A few
data proving this point will be quoted below in another con-
nection.
It is much easier to hasten the rate of digestion of the
zymase, and so its rate of destruction, than to retard it. The
effect of adding another proteolytic enzyme is well shown by the
following data : —
20 cc. Juice + 8 gm. Cane-Sugar + 2 c.c Toluol, in 96 hours
at 22% gave ...... '95 gm. COg.
20 cc. Juice + 8 gm. Cane-Sugar + 2 cc Toluol + '4 gm.
Trypsin, in 96 hours at 22**, gave . . . •! I „
20 cc J nice + 8 gm. Cane-Sugar + 2 cc Toluol + -4 gm.
Pancreatin, in 96 hours at 22°, gave . . • 05 m
20 cc Juice + 8 gm. Cane-Sugar + 2 c«c Toluol + -4 gm.
Diastase, in 96 hours at 22**, gave . . • '83 „
Trypsin and pancreatin reduced the CO2 output to a tenth or
twentieth its normal amount, whilst a non-proteolytic enzyme
like diastase depressed it but slightly.
The readiest method of increasing the rate of destruction of
the zymase is to dilute the press juice. For instance, the
dilution of a sample of juice with an equal volume of water
reduced its yield of COg from -46 gm. to -33 gm. Probably such
small dilution acts chiefly by rendering the conditions of action
of the endotryptase upon the zymase more favourable. When
the dilution was made with a solution of cane-sugar (9 to 29
88 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
per cent.) instead of with water, there was practically no
diminution of COg output. This is presumably due to the sugar
forming a loose combination with the zymase, and so protecting
it from the endotryptase. However, the activity of the zymase
is very greatly reduced by considerable dilution, as the following
data show : —
20 C.C. Juice + 5 or lo gm. Cane-Sugar + Th3anol, in 96
hours at 22**, gave ..... ^7 gm. CO2
20 cc Juice + 5 or 10 gin. Cane-Sugar + 500 cc. Saline, in
96 hours at 22°, gave ..... 032 „
20 CO. Juice + 500 cc. of 10 per 'cent. Glycerin containing
I per cent Cane-Sugar, in 96 hours at 22**, gave . oS „
20 cc. Juice + 500 cc of 10 per cent Egg White containing
I per cent Cane-Sugar, gave . • . . •28 ,,
The saline used contained KgHPO^, MgSO^, CaSO^, NaCl,
and I per cent, of cane-sugar. It will be seen that 10 per cent
glycerin solution had a slight protective effect upon the zymase,
whilst 10 per cent, egg white had a more considerable one. In
another experiment a mixture of juice, sugar, and thymol with
500 cc. of solution containing 10 per cent, of egg white and
2 per cent, of cane-sugar, yielded no less than -88 gm. of COg, as
against the i-20 gm. yielded by the control. The protective
influence of proteins referred to above is strikingly shown by
these two experiments.
The influence of dilution varies greatly with different samples
of press juice, for Wroblewski ^ found that the evolution of CO2
practically stopped on tenfold dilution. Macfadyen, Morris, and
Rowland ^ observed a much greater influence still, for their juice
sometimes ceased to evolve COg on dilution with twice its
volume of water. Saline solution (-75 per cent. NaCl) acted
even more unfavourably, as the addition of an equal volume of
it to the juice almost stopped the CO2 output. Presumably the
juice contained a more active endotryptase than Buchner*s, for
its initial content of zymase — as judged by the COg formation of
the undiluted juice — was often as great.
The influence of filtration upon the activity of yeast juice has
been the subject of much experiment and discussion, as the
* Wroblewski, CentralLf, Physiol.^ 13, p. 284, 1899.
2 Loc. ciU
FILTRATION OF ZYMASE 89
results are somewhat contradictory. Buchner found that the
juice could be filtered through a Berkefeld kieselguhr filter
without losing much of its activity, e,g,y a reduction of COg-
forming power from 2-o8 gm. to 1-62 gm. The pores of the
filter are suflSciently small to stop the passage of yeast cells, but
not of bacteria, hence it seemed possible that small fragments of
living protoplasm might be forced through. Filtration through
a Chamber land biscuit porcelain filter, which is said to stop the
passage of the smallest bacteria, reduced the activity of the juice
in some cases to vanishing point. But Buchner obtained more
favourable results by first of all filtering the juice through a
kieselguhr filter, whereby the larger particles of cell membrane,
protoplasm, and intact yeast cells were removed, and then forcing
it through the porcelain filter. The pores of this filter were not
so rapidly clogged up as before, and the first portions of juice
coming through had about half the activity of the unfiltered
juice: but the subsequent portions contained less and less
zymase, as the following data show: —
In 88 hours at 16", 20 c.c Juice (unfiltered) + 8 gm. Sugar
gave ....... 'Ssgrn. CO2.
First 20 C.C Juice (filtered) + 8 gm. Sugar, in 88 hours
at 16", gave . . • . . • '30 „
Next 20 cc. Juice (filtered) + 8 gm. Sugar, in 88 hours
at 16**, gave . . . . . . '12 „
Next 20 cc Juice (filtered) + 8 gm. Sugar, in 88 hours
at 16**, gave . . . . . . -08 „
Next 20 cc Juice (filtered) + 8 gm. Sugar, in 88 hours
at 16", gave . . . . . . -08 „
This considerable and increasing reduction in the activity of
the filtered juice is easily explained in the light of C. J. Martin's ^
observations on gelatin filters. Martin found that if the pores of
a Pasteur-Chamberland candle filter were filled with a 10 per cent,
gelatin solution, they are rendered impermeable to colloids
such as proteins and starch, but readily permit the passage of
crystalloids. Filtration of serum or egg white diluted with an
equal bulk of salt solution gave a clear colourless solution
absolutely free from protein. Harden and Young ^ found that
yeast juice similarly gives an enzyme-free — so presumably
^ C. J. Martin, /<?«r«. PhysioL^ 20, p. 364, 1896.
2 Harden and Young, Proc, Roy, Soc,^ yj B, p. 405, 1906.
M
90 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
protein-free — filtrate. In Buchner's experiments the pores of
the filter must have got gradually clogged up with protein, till
they finally became almost as impermeable as those of the
gelatin filter.
Zymase, though so unstable in solution, is almost if not
quite as stable as other enzymes when dried. Buchner evapo-
rated the juice in vacutio at 20° to 25°, and in about half an hour
it reached a syrupy condition. He then spread it in a thin layer
on glass plates, and dried it in vacuuo or in air at 35°. After
twenty-four hours he powdered it and dried it still more thoroughly
by keeping it over sulphuric acid in vacuuo. The dry powder
was almost completely soluble in water, and had lost scarcely
any of its initial activity. For instance, a sample of fresh juice,
mixed with sugar, yielded 1-28 gm. of CO2, whilst an equal
amount of it dried and dissolved up again to the same volume,
gave I'll gm. of CO2. The following data show how permanent
is the stability of dried zymase : —
3 gm. Dried Juice (fresh) + 18 c.c. HgO + S gm. Cane-Sugar
+ Toluol, in 168 hours at 17", gave
3 gm. Dried Juice (kept i month) +18 cc H2O + 8 gm.
Cane-Sugar + Toluol, in 168 hours at 17°, gave
3 gm. Dried Juice (kept 2 months) +18 cc H2O + 8 gm,
Cane-Sugar + Toluol, in 168 hours at 17°, gave
3 gm. Dried Juice (kept 5 months) +18 cc H2O + 8 gm.
Cane-Sugar + Toluol, in 168 hours at 17°, gave
3 gm. Dried Juice (kept 7 months) +18 cc HgO + S gm.
Cane-Sugar + Toluol, in 168 hours at 17°, gave
3 gm. Dried Juice (kept 9 J months) +18 cc H2O + 8 gm.
Cane-Sugar + Toluol, in 168 hours at 17', gave
3 gm. Dried Juice (kept 12 months) +18 cc H2O + 8 gm.
Cane-Sugar + Toluol, in 168 hours at 17°, gave
I.91 ^Oi, COo.
2.00
2-07
2.19
176
2-03
1.87
The thoroughly dried juice could be heated to 85° for eight
hours without any substantial loss of activity, and even when
kept for six hours at 97° it did not lose its activity entirely.
Zymase can be dried in quite another manner, viz., by
precipitation with absolute alcohol, or a mixture of alcohol and
ether. The precipitate must be collected on a filter, and the
retained alcohol quickly removed by washing with ether. It is
then dried in vacuuo over sulphuric acid, and the dried powder
so obtained, if rubbed up in a mortar with cane sugar and the
volume of water necessary to bring it up to the original volume
ACTION OF ANTISEPTICS 91
of juice, is found to possess nearly its initial activity. In two
experiments, for instance, it yielded -53 and 1-28 gm. of COg
respectively, as against -54 and 1-33 gm. of CO2 in the corre-
sponding experiments with fresh juice.
The effects of antiseptics upon the activity of zymase and of
living yeast cells have been studied by several investigators, and
the results of such study are • instructive, as they demonstrate
very clearly that unorganised ferments and living organisms
differ from one another only in degree in their reaction to
antiseptics. Both are affected to some extent, and zymase,
being more unstable in most respects than other enzymes, is as
a rule affected more than they are by antiseptics; but even
living organisms, though much more sensitive than zymase, can
retain vitality in the presence of low concentrations of anti-
septics.1
Buchner and Rapp ^ divide antiseptics into two classes, viz.,
those which enter into chemical combination with the proteins
of the yeast juice, and those which do not enter into combination.
In the first class fall corrosive sublimate and probably ammonium
fluoride, both of which produce a precipitate when added to
yeast juice. Potassium metarsenite may perhaps be included
as well, for it produces a precipitate when in tolerable concentra-
tion. These bodies exert a harmful influence in proportion to
the concentration they bear to the total amount of protein
present in the juice, or of protoplasm in the living organism.
In the second class of antiseptics fall concentrated glycerin and
sugar, toluol, thymol, and chloroform. These are substances
which do not combine with proteins, and which are supposed
to exert a harmful influence in proportion to their absolute
concentration, apart from their amount and the amount of
I gm. Yeast + 50 c.c 8 per cent. Cane-Sugar "^ ,^^ ^^ . ^^ ,
u. r ««, rwri r =■• ''52 gm. COg in 22 hours.
+ '5 gm. UrlUi3 . . . .J
10 gm. Yeast + 50 cc. 8 per cent. Cane-Sugar 1 -,- «. nr>i • , u
+ .5gm.CHCl, . . . *.|=-5i5gm.CO,mlhour.
I gm. Yeast+ 50 cc lo per cent. Cane-Sugar | ^ ^.^ .^ ^^^^^
+ '5 gm. Toluol . . . . J
10 gm. Yeast + 50 cc 10; per cent. Cane-Sugar 'i _
+ .5 gm. Toluol . . . ,)= 709 gm. 00^11,24 hours.
' C/. Lecture VIII., p. 215, ' Buchner and Rapp, Ber., 32, p. 127, 1899.
92
ZYMASE AND OTHER GLYCOLYTIC ENZYMES
protein present in the juice or yeast cells. However, Abeles^
has obtained results which seem to disprove this hypothesis, for
the data adduced show that with the larger quantity of yeast
CO2 was evolved at a relatively much faster rate than with the
smaller quantity, when this was acting in the presence of the
same amount of chloroform or toluol. No explanation of this
result was arrived at, but probably the larger quantity of yeast
did not become saturated with the antiseptic so quickly as the
smaller quantity.
Of the first class of antiseptics, ammonium fluoride proved
itself especially harmful to the activity of zymase, as the
addition of -55 per cent of it to the juice paralysed the enzyme
completely. Bokorny * found that living yeast cells were much
more sensitive still, as -i to -5 per cent of it killed them. The
action of arsenious acid, dissolved in K2CO3 and so partly
converted into KAsOj, was somewhat irregular, as is indicated
by the following data : —
CO2 evolved In gnmmes.
Mmui.
20 cc Juice + 8 gm. Sugar
alone
20 C.C Juice + 8 gm. Sugar
alone + 2 per cent As^Oj .
•90
•43
•S7
•49
.48
•63
•42
.69
•42
•37
.42
•38
gm.
•54
•50
These were obtained with different samples of juice, which were
allowed to act upon cane-sugar for forty hours at about 17°.
Upon diluted juice potassium metarsenite had a much more
harmful influence, but this influence could be to a large extent
neutralised by the addition of protein. The proteins of yeast
juice, previously inactivated by heating for ten minutes to 55°,
gave the best result of all, as the following data show : —
In 64 hours at 16", 20 cc Juice + 16 gm. Sugar + 2 i>cr cent
A82O3 + 20CC Water gave ....
In 64 hours at 16°, 20 cc Juice + 16 gm. Sugar + 2 per cent
AsjOj + 20 cc Egg White gave
In 64 hours at l6^ 20 cc Juice + 16 gm. Sugar + 2 per cent
A83O3 + 20 cc Blood Serum gave
In 64 hours at i6', 20 cc Juice + 16 gm. Sugar + 2 per cent
AsgOgH- 20 cc Inactivated Yeast Juice gave
.i4gnL
COg.
•18
11
•5S
»»
.86
»»
1 Abclcs, Ber.y 31, p. 2261, 1898. * Bokorny, PflUgef^s Arck.^ m, p. 37i»
ACTION OF ANTISEPTICS
93
Analogous results to these have been obtained by Abeles with
living yeast cells. A mixture of i gm. of yeast with 50 c.c. of
nutritive solution containing 8 per cent of cane-sugar and i gm.
of sodium metarsenite evolved only 002 gm. of COg in three
hours at 30°. On the other hand, 20 gm. of yeast mixed with
20 C.C. of water, 10 gm. of sugar and i gm. of sodium metar-
senite evolved no less than 3-62 gm. of COg in twenty-six hours.
In this instance probably the protoplasm of a portion of the
yeast cells combined with the arsenite, and the remaining cells
formed CO2, whilst in the case of yeast juice the proteins, both
those already present and those added artificially, similarly
combined with much of the arsenite, and protected the zymase.
Of the second class of antiseptics chloroform and toluol were
found to exert comparatively little influence, but thymol was
much more harmful, as is shown by these two sets of fairly
concordant data :
CO2 evolved.
gm.
gm.
20 C.C Juice + 8 gra.
Sugar + no Antiseptic, in 92 hours gave
1.24
1*84
»»
„ +.2 C.C. Toluol „ „ .
l-OO
1.87
i»
i» T" ^ ^^ i» n f» • •
IK)4
I '69
tt
+ •2 gm. Thymol „ „ .
•74
1-18
M
„ +1 gm. „ „ „
.62
.71
In remarkable contrast to the effect of toluol upon zymase,
is its action upon living yeast cells. Buchner found that i gm.
of living yeast cells, added to 1 5 c.c. of water -f- 5 c.c. of beer
wort -f- 4 gm. of cane-sugar -f- '2 c.c. of toluol, evolved only
•03 to -05 gm. of CO2 when kept for ninety-six hours at 22°.
In control experiments without toluol, the same quantity of yeast
gave 2- 10 to 2-13 gm. COg : ?>., forty to seventy times as much.
It is to be remembered, however, that in the absence of
toluol the yeast cells would be continually reproducing themselves
and generating fresh zymase, whilst in the presence of toluol
there would be no mechanism for increasing the store of zymase
originally present. This store would be somewhat greater than
that obtained in the juice of the ground-up cells, for doubtless a
good deal of the enzyme is retained in the press cake. Arguing
from the known chemical composition of yeast and from the fact
94 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
that a kilogram of ground yeast cells yields 500 c.c. of juice, we
may assume that the total amount of juice actually present in
them is about 700 c.c. One gramme of yeast would therefore con-
tain 7 C.C. of juice. Now Buchner found that 100 c.c. of juice
gave off from 3-5 to 12-2 gm. of COg under the most favourable
circumstances, so the juice in i gm. of yeast would give off '024
to -085 gm of CO2, or about the amount actually obtained from
1 gm. of yeast kept with sugar, beer wort, and toluol. Hence
the only legitimate deduction that can be made from these com-
parative observations is that toluol stops all reproductive activity
and regeneration of zymase in living yeast cells. They cannot
be taken to show that toluol paralyses the formation of CO2 by
the intracellular zymase of an intact but dead yeast cell any
more than it paralyses the action of the zymase in press juice.
A number of interesting observations have been made by
Albert^ and by Buchner which seem to show that the powers
of actual reproduction and of formation of intracellular zymase
by yeast cells are independent of one another, and that the one
power may be destroyed without the other. Albert found that
if yeast were put in a mixture of absolute alcohol and ether
(250 gm. yeast, 3 1. alcohol and i 1. ether) for five minutes, were
then washed with ether and dried in air at 20° to 45°, the
yeast cells were rendered sterile. But if suspended in cane-
sugar solution in presence of toluol they evolved ten or even
twenty times more COg than is evolved by the juice pressed
from an equal weight of ground-up yeast cells. For instance,
2 gm. of this sterile yeast +4 gni. sugar +10 cc. water +2
gm. toluol gave *92 to 1-05 gm. COgin ninety-six hours at 22°.
Again, Albert, Buchner, and Rapp ^ found that yeast could be
rendered sterile by keeping it in acetone for ten minutes, and
then washing with ether and drying. Another method con-
sisted in heating the carefully dried yeast for six hours to 100°,
In each case the yeast, when placed in sterile beer wort, failed
to show any signs of reproduction, but still retained this con-
siderable fermentative power. The retention of this power
is difficult to reconcile with the above-mentioned fact that
living yeast cells, when kept under similar conditions in presence
1 Albert, Ber., 33, p. 3775, 1900.
2 Albert, Buchner, and Rapp.^^r., 35, p. 2376, 1902.
ACTION OF PHOSPHATES 95
of toluol, yielded no more COg than the press juice which could
be extracted from them ; for this shows that the toluol par-
alyses the zymase-forming power of the cells, as well as the
reproductive power. Presumably these two functions are so
intimately bound up together in the living cell that if the one
is paralysed the other is likewise, but in the sterilised cell the
destruction of the one leaves the other independent. Of course
it is possible though not probable that the fermentative power of
sterilised yeast is due entirely to zymase present from the outset
in the sterilised cells, and that there is no new formation of
zymase whatever. If this is the case, then the much smaller
activity of press juice implies that the major part of its
zymase is destroyed during the violent mechanical methods of
extraction.
Zymase, in addition to its extreme instability, differs from
other enzymes in that its activity seems to be absolutely depen-
dent on the presence of phosphates. Wroblewski ^ noticed that
disodium phosphate exerted a favourable influence on zymase
activity, the addition of 1-25 per cent, of it giving the best result.
Buchner found that any concentration up to 4 per cent, of it acted
equally well, and that its addition increased the output of CO2 by
over 30 per cent. Harden and Young ^ obtained still more
striking effects by the addition of the phosphates of the juice
itself. They found that if boiled and filtered juice were added
to fresh juice, together with glucose and toluol, its COg output
might be doubled or trebled. On the other hand, if the phos-
phates already present in the juice were removed, it entirely lost
its fermenting power. A fairly complete separation of the
colloidal and crystalloidal constituents of the juice was effected
by filtration through a Martin gelatin filter. A saline filtrate
containing no enzyme was obtained thereby, whilst a brown
viscid mass was left on the filter. This residue was dissolved
in water and made up to the volume of the juice filtered, but it
yielded little or no COg when kept with glucose. On addition
of the saline filtrate, however, it recovered a good deal of its
initial activity. Buchner and Antoni ^ repeated this experiment
* Wroblewski, /£>»r«.//rai/. Chem, (2), 64, p. 11, 1901.
2 Harden and Young, Proc. Roy. Soc,^ B. T]^ p. 405, 1906.
3 Buchner and Antoni, Zeit f,physioL Chem., 46, p. 136, 1905.
96 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
in another form. They separated the salts from the juice by
dialysis, and found that the residue in the dialysis tube had Irttle
or no fermenting power until some of the dialysed salts were
added. Thus : —
20 cc Dialysed Juice + lo cc. Water + 8 gm. Cane-Sugar gave 02 gm. COj-
„ „ + 10 cc Evaporated Dialysate gave . '48 „
„ >, + 20 cc Boiled Juice gave . . '59 „
The increased evolution of COg produced by the addition of
phosphates to yeast juice plus sugar was found by Harden and
Young to be within certain limits strictly proportional to the
phosphates added. Each atom of phosphorus added as
phosphate (a solution of NagHPO^ and NaH2P04 saturated
with CO2) caused the evolution of a molecule of COg. For
instance, in four experiments the amounts of COg calculated
on this basis were -055, -086, -112, and -197 gm. respectively,
whilst the amounts actually observed were -054,' -090, -106, and
•196 gm. respectively. The mode of action of the phosphates is
unknown. It might be thought that the COg was formed in the
usual way by the zymase, but that it remained in loose combina-
tion with certain constituents of the juice, presumably the protein
constituents, and was only liberated therefrom by an equivalent
amount of phosphate taking its place. However, recent investi-
tion indicates that the reaction is not so simple as this, for Harden
and Young ^ found that ^he addition of soluble inorganic phos-
phates to a solution of the inactive yeast juice residue was quite
unable to provoke fermentation. Apparently the fermentation
depends on the presence of some crystalloidal thermostable
" co-enzyme " in the yeast juice as well as on the phosphates.
Again, Buchner and Klappe ^ found that if fresh yeast juice were
kept for three or four days with sugar and toluol, until its COg
output had ceased, it was still able to give a very considerable
further output of COg if boiled juice were added gradually. In
one experiment 20 cc. of the fresh juice gave out "73 gm. of COg
altogether. Then volumes of 20 cc. of boiled juice, together
with sugar and toluol, were added on seven successive occasions
1 Harden and Young, Proc. Roy, Soc.y B. 78, p. 369, 1906 ; 80, p. 263,
1908.
2 Buchner and Klappe, Biochem, Zeit^ 8, p. 520, 1908.
LACTACIDASE 97
at two- to five-day intervals, and they provoked a further
discharge of -32, 17, 42, 29, -19, -07, and -05 gm. of COj
respectively, or 1-5 gm. of COg in all. It follows, therefore,
that some of the zymase must have remained active for as long
as twenty-seven days. The addition of boiled juice to yeast
juice which had been kept standing without any sugar for three
days failed to provoke any CO2 output, and also this inactive
juice, when boiled, was found to have lost its activating power
upon juice kept with sugar. As the result of some not very
convincing experiments, Buchner and Klappe conclude that
the co-enzyme in boiled juice may be an organic ester of
phosphoric acid, which in kept (sugarless) juice is split up and
rendered inactive by the action of a lipase.
The molecular changes involved in the conversion of sugar
into alcohol and CO2 are not known, but recent research seems
to point with some probability to there being at least two stages
in the process. Buchner and Meisenheimer ^ suggest that each
molecule of glucose is first converted into two molecules of lactic
acid, and that this lactic acid is subsequently broken down into
alcohol and COj. The experimental support for this hypothesis
is at present somewhat slender. It depends chiefly on the fact,
first noted by Meisenheimer,* that yeast juice forms quite
appreciable quantities of lactic acid. Buchner and Meisenheimer
estimated the percentage of the acid in a number of samples of
juice, and they found that whilst fresh juke contained •01 to ^14
per cent, of the acid, juice which had undergone autolysis for
four to six days contained -09 to -40 per cent of acid. The
addition of sugar to the juice made very little diflference, so
probably the lactic acid was formed at the expense of intra-
cellular glycc^en. On repeating these observations during the
summer months of two successive years, Buchner and
Meisenheimer found that the small qusmtities of lactic acid
originally present in the juice disappeared on autolysis, and that
if some lactic acid were added to the juice it was likewise
destroyed. It seemed probable, therefore, that the juice con*
tained variable proportions of two distinct enzymes, a " zymase,"
* Buchner and Meisenheimer, Ber.^ 37, p. 417, 1904; 38, p. 620,
1905.
2 Meisenheimer, Zeit f. pkysiql Chem^^ 37, p. 526, 1903-
N
98 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
which has the power of converting sugar into lactic acid
according to the equation
CftHigOg = 2C8Hg03,
and a 'Hactacidase " enzyme, which splits up the lactic acid
into CgHgO+COg. It is supposed that in the winter months the
lactacidase was lacking in amount, whilst in the summer months
it was in excess. In that living yeast cells form scarcely a trace
of lactic acid, it would seem that in their case there is always
plenty of lactacidase available.
Attempts have been made to isolate a lactic acid-forming
enzyme from various bacteria. Herzog^ found that if lactic
acid bacteria were killed by ipmersion in acetone, they were
still able to split up milk sugar and form a body which he
identified, not altogether satisfactorily, as lactic acid. Buchner
and Meisenheimer 2 used a preparation of Bacillus Delbrilcki
which had been placed for ten to fifteen minutes in acetone
and washed with ether. They found that it readily formed
lactic acid when placed in cane-sugar or maltose in presence of
toluol. Ten grammes of the preparation yielded '75 to i«26 gm.
of lactic acid. A control experiment with bacteria previously
heated to 91"* gave no lactic acid at all Somewhat unexpectedly
the lactic acid formed was found to be the optically inactive
variety, whilst the living bacillus produces the laevo-rotatory
acid. Juice expressed from the ground-up bacteria showed no
lactic acid-forming power, though the press cake, even after
treatment with acetone, still retained its activity. This seems
to show that the enzyme is insoluble, or more probably, as
Buchner and Meisenheimer think, that the active constituents
of the bacterial cells are not pressed out in the juice. It seems
very unlikely, however, that none of the enzyme should be
driven out by the forcible mechanical means adopted, so one
is perforce driven to question the validity of the whole evidence.
It is true that these " Acetondauerpraparate " of yeast cells and
of bacteria, if placed in a sterile nutrient medium, show no signs
of reproductive power, but must one on that account look upon
them as dead? Reproduction in all organisms calls for the
1 Herzog, Zeit f, physioL Chem,^ 37, p. 381, 1903.
^ Buchner and Meisenheimer, LUbigs Ann,y 349, p. 125.
ACTION OF ALKALIS ON SUGAR S9
highest degree of vitality, and if this vitality be reduced to a
low ebb by treatment with acetone and ether, it may be
insufficient to bring about growth and reproduction, though
still adequate for the normal metabolic processes of the cell.
On the other hand, we must remember that these acetone
preparations act perfectly well in the presence of toluol.
"Dauerhefe," for instance, evolves ten times as much COg as
living yeast kept under similar conditions. Again, it is possible
that the lactic acid-forming enzyme is so unstable that it is
destroyed by the act of forcible rupture from its protoplasmic
basis in the cell
The action of inorganic catalysts such as caustic alkalis
upon sugar seems to throw light upon the processes of decom-
position by enzymes. As long ago as 1871 Hoppe-Seyler ^
and Schiitzenberger showed that caustic alkalis split up sugar
with the formation of a considerable amount of lactic acid.
Nencki and Sieber^ found that if a 10 per cent, glucose
solution were incubated with 20 per cent of KOH for twenty-
four hours, it was almost completely decomposed, and as much
as 50 per cent, of it was converted into lactic acid. More dilute
alkali effected a similar decomposition but at a slower rate, and
even -3 per cent. KOH split it up in ten days at 37°. Duclaux*
found that if glucose were kept in sunlight in presence of weak
alkalis such as baryta or lime water, some 50 per cent, of it was
converted into lactic acid, but if the weak alkali were replaced
by a strong one such as KOH, then alcohol and COg were
formed. He suggested that lactic acid was formed in every
case, but that only a strong alkali was able to break it down
further. Duclaux likewise found that if an aqueous solution of
calcium lactate were kept in sunlight in presence of air, it formed
alcohol, calcium carbonate, and calcium acetate. Hanriot^
stated that if calcium lactate were heated with excess of
calcium hydrate, considerable quantities of ethyl alcohol and
acetone were produced* Buchner and Meisenheimer ^ confirm this
^ Hoppe-Seyler, Ber.y 4, p. 396, 1871.
^ Nencki and Sx^hex^ Joum, f. prakt, Qkem.y 24, p. 502, 1881.
3 Duclaux, Ann, de VInsK Nat Agronomique^ 10, 1886. Ann, de PInst.
Pasteur^ 7, p. 751, 1893 ; 10, p. 168, 1896.
* Hanriot, Bull, Sac, Chim,^ 43, p. 417, 1885 ; 45, p. 80, 1886.
* Buchner and Meisenheimer, BcK^ 38, p. 620, 1905.
loo ZYIAASE AND OtttfiTa GLYCOLYTIC EN7YMES
statement, but they find that a good deal of isopropyl alcohol is
formed as well. They also confirm the experiments of Duclaux,
and find that even at room temperature and in darkness caustic
potash slowly converts glucose into lactic acid and other products.
Of the intermediate stages between glucose and lactic acid
we know nothing for certain, but probably the glucose first
breaks up into two molecules of glyceric aldehyde, CHgOH •-
CHOH — CHO. Nefi has shown that in the conversion of
glucose into lactic acid by the action of caustic alkali, pyruvic
aldehyde, CH3— CO — CHO, is formed as an intermediate product,
and Buchner and Meisenheimer suppose that this substance like*-
wise represents a stage in the conversion of glyceric aldehyde into
lactic acid by zymase. Such an assumption makes the molecular
changes more complex than if the glyceric aldehyde be supposed
to pass directly into lactic acid, so it should not be adopted unless
supported by stronger evidence than is at present available. In
the conversion of lactic acid into alcohol and COg, it is probable
that acetic aldehyde and formic acid are first produced :
CHg-CH.OH-COOH = CH3 - CHO + H . COOH.
The formic acid then breaks up into COg and hydrogen, and
this hydrogen reduces the aldehyde to alcohol. Thus lactic
acid splits up into aldehyde and formic acid if heated with
dilute sulphuric acid to 130°, or if electrolysed,^
In the autolysis of yeast juice acetic acid is formed as well
as lactic acid. Buchner and Meisenheimer found -03 to '28
per cent, of this acid in the juice after four to six days autolysis.
They attribute its formation to an alcohol-oxidase enzyme.^ It
seems probable that yeast juice contains other enzymes in
addition to those recorded. Pasteur found that in the fermenta-
tion of sugar by living yeast, there were always certain amounts
of glycerin and succinic acid formed. These amounts varied
under different conditions, and the slower the fermentation the
greater they were, but as a rule the glycerin formed 2-5 to 3*6
per cent on the weight of sugar fermented, and the succinic acid
•5 to 7 per cent. Buchner found that these two substances are
likewise produced in the fermentation of yeast juice, though in
* Ne^ AnnaUn^ 335, p. 247, 1904.
* Erienmeyer, Zeitf. Chtm.^ 1868, p. 343. ^See Lecture V., p. 126.
ZYMASE IN ANIMAL TISSUES 101
smaller proportion. Thus 1250 cc. of juice gave -5 gm. of
glycerin and -3 gm. of succinic acid. Buchner and Meisen-
heimer ^ subsequently found that no succinic acid is formed as a
rule, but that the glycerin amounted to from 5-4 to i6«S per
cent, on the sugar fermented.
The isolation of a glycolytic enzyme from yeast suggested
that the glycolytic powers possessed by all living tissues might
depend, wholly or in part, on a similar enzyme, whilst the
alcohol formed by the action of this zymase was supposed to be
split up by another enzyme into COg and water. In support of
this hypothesis^ BlumenthaP found that the juice expressed
from the pancreas has a strong glycolytic action upon glucose.
Carbon dioxide was formed thereby, but Blumenthal could not
demonstrate the formation of alcohol. Umber* could not
confirm the formation of COg, but Herzog* obtained doubtful
evidence of it. In 1903 Stoklasa,^ working in conjunction with
Jelinek, Czerny, and Vitek, stated that he had been able to
isolate an active zymase, not only from the roots and seeds of
plants, but also from several different animal tissues. He
demonstrated the existence of this animal zymase by several
methods. The simplest method consisted in dipping pieces of
the tissue (heart, liver, lungs, and muscle) in -5 per cent, corrosive
sublimate solution for fifteen to thirty minutes to render them
aseptic, and then placing them in a sterile 5 per cent glucose
solution at a temperature of 37°. The vessel containing the
glucose was filled with hydrogen, so the tissue enzyme was
acting under anaerobic conditions. Fermentation was well
established within twenty-four hours, and continued for some
days. A dog's heart weighing 21 gm. caused the evolution of
•3 to -5 gm. of CO2 per day for the first three days, and 1*97 gm.
in all during ten days. The alcohol formed at the same time
amounted to 2*09 gm., so a normal alcoholic fermentation seemed
to have occurred. In that gelatin and bouillon cultures failed
* Buchner and Meisenheimer, Ber,^ 39, p. 3204, 1906.
^ Blumenthal, Zeitf, didt u,phys, Tkerap.^ vol. ii.
3 Umber, Zeit.f. klin, Med,y 39, p. 13.
* Herzog, Hofmeistet's Beitr,y 2, p. 102, 1902.
* Stoldasa and Czerny, Centralb, f. physioL^ 16, p. 652, 1903; Stoklasa,
Jelinek, and Czerny, Wid,^ 16, p. 712 ; Stoklasa, Jelinek, and Vitek, Hof-
tneistet^s Beitr,^ 3, p. 460, 1903.
102 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
to show the presence of bacteria or hyphomycetes, Stoklasa
concluded' that the fermentation of the glucose was effected by
an intracellular zymase.
To isolate the enzyme, Stoklasa adopted the method used by
Buchner for yeast zymase. A mixture of alcohol and ether was
added to the press juice obtained from the minced tissue, and
the precipitate was quickly washed with ether, dried, and
powdered. In one experiment 9-64 gm. of the powder obtained
from muscle juice was placed in 15 per cent glucose solution at
37°, and in eighteen hours it formed 73 gm. of COg and 78 gm.
of alcohol. In another experiment 10 gm. of ox lung powder
gave I'lg gm. of CO2 during the first twelve hours of fermen-
tation, and 3'09 gm. of COg, together with 3-20 gm. of alcohol, in
fifty hours. Stoklasa subsequently found that considerable
quantities of lactic acid were formed, as well as alcohol and COy
For instance, 10 gm. of muscle powder, placed in 50 cc. of
IS per cent glucose at 37^ formed 27 gm. of CO2, 2-8 gm. of
alcohol, and 17 gm. of lactic acid. Liver and lung powder gave
a similar result, and even the alcohol-ether precipitate of blood
induced a moderate amount of alcoholic fermentation in glucose,
but it formed only a small amount of lactic acid. Most active of
all in producing alcoholic fermentation was pancreas powder.
Simicek^ found that -164 gm. of this powder, placed in 15 per
cent glucose solution at 37°, gave -42 gm. of COg and i'i2 gm.
of alcohol in four days. It could likewise ferment cane-sugar,
maltose, and lactose. For instance, 5 gm. of pancreas powder,
placed in 50 cc. of 30 per cent, lactose at 36° for seventy-two
hours, gave •846 gm. COg, -122 gm. alcohol, and had an acidity
corresponding to i-o8 gm. of lactic acid.
All of these experiments were made under anaerobic con-
ditions, and, according to Stoklasa and his colleagues, in the
entire absence of bacterial infection. Could, we accept them
unreservedly, they would be of great service in helping us to
understand, not only the processes of glycolysis in the body,
but also the processes of tissue respiration. Unfortunately we
are unable to put our faith in them at present, as most other
investigators have been unable to confirm them. Maz6* found
^ Simdcek, Centralb,/, PhysioLy 17, pp. 3 and 209, 1903.
2 Maze, Ann, de PInsL Pasteur^ 18, p. 378, 1904.
ZYMASE IN ANIMAL TISSUES 103
that the alcohol-ether precipitate from the expressed juice of ox
lung, and that from pounded peas, caused a vigorous fermenta-
tion in glucose solution with the formation of CO^, alcohol, and
lactic acid, but bacteria were always present. Also the first
portions of the gas evolved consisted largely of hydrogen.
Battelli ^ found that if only sufficient antiseptic were present, not
a trace of fermentation resulted. Portier^ found that the
precipitate from the expressed juice of various organs of the dog,
pig, and horse, when placed in glucose solution containing
I per cent NaF, did not produce any glycolysis in two days at
36°. Also he points out that the fermentation of cane-sugar and
lactose by pancreas juice, which Simdcek observed, implies the
presence of invertase and lactase ferments in the pancreas. As
was mentioned in the previous lecture, it is practically certain
that no lactase whatever exists in this organ, and it is very
doubtful if any invertase does either.
Hence until more satisfactory evidence is brought forward,
we are not justified in assuming that animal tissues contain
enzymes producing alcoholic fermentation. The fact that
Stoklasa and his co-workers could not detect bacteria by means
of their cultures does not necessarily prove that they did not
exist As far as one can gather from their papers, the cultures
were as a rule made under aerobic conditions, though the
fermentations were anaerobic. Hence the cultural conditions
may not have been favourable for growth of the bacteria which
induced the alcoholic fermentation.
On the other hand, there is a good deal of evidence that
glycolytic enzymes of some sort exist in animal tissues. The
evidence of Blumenthal has already been quoted. Arguing from
the well-known discovery of v. Mering and Minkowski that
extirpation of the pancreas causes diabetes, Cohnheim'
endeavoured to prove that the glycolytic power of the muscles is
dependent on an internal secretion of the pancreas. He found
that if glucose were incubated with muscle press juice or with
pancreas press juice, little or none of it disappeared. If, on the
other hand, fresh muscle and pancreas were minced together, the
^ Battelli, Campus Rendus^ 137, p. 1079, 1903.
* Portier, Ann. de PInst PcLsteur^ 18, p. 633, 1904.
3 Cohnheim, Zfit,f,physioL Ckem,^ 39, p. 336, 1903.
104 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
mixed juice squeezed out from the two tissues possessed distinct
glycolytic power. For instance, samples of the mixed juice
when incubated for a considerable time with about 2 per cent,
of glucose induced a destruction of -35 to -84 p^r cent, of
the sugar, whilst the juice of muscle alone, kept with glucose
under similar conditions, destroyed o to -026 per cent.,
and the juice of pancreas alone destroyed no sugar at
all. Toluol was added to prevent bacterial action, whilst the
acidity of the juice was neutralised by sodium bicarbonate.
Subsequently Cohnheim^ showed that the activating power of
the pancreas is not dependent on an enzyme, in that aqueous or
alcoholic extracts of boiled pancreas were equally efficient. In
order to obtain the best results, only a moderate amount of
pancreatic extract or pancreas juice must be added to the muscle
juice, as an excess of it exerts an inhibitory influence. It was
found, for instance, that muscle juice alone destroyed -034 gm.
of glucose. On addition of 10 c.c. of aqueous extract of pancreas
to 40 C.C. of juice it destroyed -115 gm. ; on addition of 20 cc, it
destroyed -174 gm. ; of 28 cc, -093 gm. ; and of 50 cc, none
whatever. The activating body is present in the blood as well as
in the pancreas, so that if the muscles are not thoroughly washed
free of blood, their juice has considerable glycolytic power.
These very interesting experiments have been repeated by
Claus and Embden,^ but they found little or no glycolysis unless
bacteria were present. Cohnheim attributes their non-success
to the fact that they used sodium chloride in preparing their
muscle juice, and added too much pancreatic extract. De
Meyer' concluded that a glycolytic enzyme is present in the
blood and lymph alone, and does not occur in the tissue cells.
It is formed, he considers, by the interaction of an activating
body (an amboceptor) from the islands of Langerhans of the
pancreas with a zymogen secreted by the leucoc3^es of the
blood De Witt* ligatured the duct of a portion of the cat's
pancreas, and 49 to 197 days later, when all but the islands of
» Cohnheim, Ztit. / pkysiol. Cksm.^ 42, p. 401, 1904 ; 43> P- 547, 19^5 5
47, p. 253, 1906.
2 Claiis and Embden, Hofmeister^s Beitr,^ 6^ pp. 214 and 343, 1906.
s De Meyer, Ann. Soc. R<^. d$ Sa. 4 Bruxflhy 1906 ; Abstract in
Centralb.f, Physipl,, 20, p. 343.
♦ Pe \N\xXjJoum, of Exp. M€d.y 8, p. 123, 1906.
GLYCOLYTIC ENZYMES 105
Langerhans had undergone atrophy, made extracts of it and
of the healthy portions of the gland. These extracts, sometimes
boiled, sometimes not, were mixed with muscle extract and -7
to 4 per cent, of glucose, and in twenty-four hours 'i to -9 per
cent, of this sugar disappeared. In that the extracts of atrophied
gland conferred just as much glycolytic power on the muscle
extract as those of healthy gland, De Witt concluded that the
activating body is present in the Langerhans islands alone. The
experimental evidence is not sufficiently complete, however, for
one to accept it unreservedly.
A careful repetition and full confirmation of Cohnheim's
work has been made by Hall.^ Press juice of muscle and of
pancreas, and alcoholic extract of boiled pancreas were
employed, and were allowed to act singly or in combination
upon 2 to 4 per cent, glucose solution for fifteen to
seventy-two hours at 37° in presence of toluol. Reckoning the
total sugar present as 100, Hall found that on an average the
pancreas juice alone destroyed -3 per cent, of it, muscle juice
alone i-6 per cent, pancreas plus muscle juice 4*4 percent,
and alcoholic extract of pancreas plus muscle juice no less thjm
1 8* 3 per cent These results strongly support Cohnheim*s, for
the negative results always obtained with pancreas juice alone,
the consistently low results obtained with muscle juice alone,
and the consistently high ones with pancreatic extract and
muscle juice, render the chance of bacterial infection very
improbable. Also in a number of cases both aerobic and
anaerobic cultures were made, and all but one proved sterile.
The glycolytic power of extract of pancreas and muscle juice
upon fructose, lactose, and arabinose was tested, but with
negative results.
Several observers have found that glycolytic power is by no
means confined to the muscles and pancreas. Hirsch* stated
that when the liver underwent autolysis in the presence of
antiseptics, some of its carbohydrate disappeared, or if sugar
were added, this diminished likewise. The glycolysis was
greatly increased if minced pancreas were added, though the
pancreas alone had no glycolytic power. Arnheim and
' Hall, Amer, Joum, PhysioL^ 18, p. 283, 1907.
2 Hirsch, Hofmeister^s Beitr.^ 4, p. 535, 1904.
O
106 ZYMASE AND OTHEH GLYCOLYTIC ENZYMES
Rosenbaum^ made most of their observations with dried
powders prepared from the tissue juices by precipitation with
acetone, and subsequent washing with ether. A gramme of
powder was kept with 20 c.c. of 4*6 per cent, glucose solution
for twenty-four hours at 37° in presence of chloroform, and the
loss of sugar during this period estimated by polarimeter. The
mean percentages of sugar destroyed were the following : —
Muscle Powder alone destroyed 10 per cent.
Liver „
27
Pancreas „
u 36
Pancreas + Muscle
65 ,
Pancreas + Liver
57
Liver + Muscle
»» 16 „
As in Cohnheim's observations, the pancreas powder greatly
increased the glycolytic power of muscle. It increased that of
liver as well, but contrary to Cohnheim and to Hall, the pancreas
itself had a considerable glycolytic power.
In other observations Arnheim and Rosenbaum determined
the weight of CO2 evolved in twenty-four hours by an incubated
mixture of i or 2 gm. of powder with 30 c.c. of 10 per cent,
glucose solution. The data show that there was a small evolu-
Pancrea^ Powder alone + Glucose Solution lost •04, •06, *I4, 'iB, ao gm. COg
Muscle „ „ • « M -Hgm.
Pancreas + Muscle .„ „ „ „ -66, -24 gm. . .
Pancreas + Liver „ „ „ „ -44, '62, loi gm.
tion of CO2 from the pancreas or muscle alone, but quite a
considerable one from the pancreas plus muscle and pancreas
pius liver. These experiments are not directly comparable with
those of the previous series, but they bear a similar relationship
to one another, and show that a large proportion of the decom-
posed glucose was converted into COg.
In almost all cases the absence of bacterial infection was
proved by cultures on gelatin and agar, made under both
aerobic and anaerobic conditions, and antiseptics such as toluol
or chloroform were always added : hence one may at least
provisionally accept the results. It is by no means unlikely
that there was a similar evolution of COg in Cohnheim's and
* Arnheim and Rosenbaum, Zeitf.physioL Chem^ 40, p. 220, 1903.
ALCOHOL IN ANIMAL TISSUES 107
Hall's experiments, but no direct observations on the point were
made.
The glycolytic power of the press juice of pancreas, liver,
and muscle, and also of their alcohol-ether precipitates, has been
observed by Feinschmidt.^ Even in the presence of -9 per cent.
NaF a considerable glycolysis occurred. Large quantities of
COj were evolved, and a considerable acidity developed in the
digestion liquid. Alcohol was shown to be present by qualitative
tests, but the amount was so small that Feinschmidt considers
that the fermentation should not be spoken of as an alcoholic
one.
Evidence of quite another character in favour of an alcohol-
producing enzyme in blood and animal tissues has been brought
forward by Ford.^ As long ago as 1858 Ford stated that traces
of alcohol are normally present in blood and the tissues. To
demonstrate its existence, the blood was obtained fresh from the
slaughter-house, and immediately subjected to distillation. The
organs were similarly treated after being chopped up. The first
distillate was purified and concentrated by successive distilla-
tions, often twelve or more in number, until a final distillate of i
to 3 gm. was obtained. The alcohol in this was estimated
quantitatively by a specific gravity determination, and qualita-
tively by the chromic acid test, and ignition of the vapour of the
boiling alcohol. Samples of blood varying from 6970 to 36,300
c.c. were analysed, and were found to contain -0020 to 'Oisegm.
of alcohol per kilogram. Probably a good deal of the alcohol
originally present in the blood disappears as the result of
post-mortem oxidation, for blood to which a strong solution
sulphuretted hydrogen was added, so as to abolish this oxidation,
gave nearly double the yield of alcohol Fresh ox liver gave
only 0017 gm. of alcohol per kilogram, or no more than would be
present in the blood of the organ. Pancreas and lung tissue
likewise contained traces of alcohol.
The glycolytic power of the blood is well known. Claudie
Bernard found that the blood of a dog when fresh contained
• 107 per cent, of sugar. After standing for thirty minutes at 15*",
it had dropped to 081 per cent ; after five hours, to -044 per
1 Feinschmidt, Hofmeisiers Beitr.^ 4, p. 511, 1904.
* YoT^Journ, PhysioLy 34, p. 43if 1906.
108 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
cent ; whilst after twenty-four hours it had entirely disappeared.
Subsequent observers have confirmed Bernard's result, and they
attribute the glycolysis to the action of an enzyme. According
to Arthus^ this enzyme arises from the leucocytes, on their
post-mortem disintegration. It is not present in blood plasma.^
It is generally assumed that this enzyme in an oxidase, and
Seegen * states that its action is favoured by aeration of the blood.
In any case it is possible that the sugar is in part converted
into alcohol Oppenheimer* endeavoured to obtain evidence of
zymase in blood, and he found that if fresh blood were allowed
to stand with sugar, and were distilled, small quantities of a
substance were obtained which gave the iodoform test, and which
was not acetone. The fresh blood also gave a feeble iodoform
test, so it seems very probable that alcohol was originally
present, and that more was formed in the kept blood.
The presence of alcohol in the distillate from brain, muscle,
and liver of the rabbit and horse was demonstrated by Rajewsky ^
in 1875. The distillate readily gave the iodoform test, and
in the presence of platinum black formed aldehyde, but no
quantitative estimations were attempted. Again, Kobert®
found that if the yolk of tortoise eggs, or sea-urchin's ova, were
ground up into an emulsion with i per cent, sodium fluoride
solution containing toluol, and the mixture were kept in an
incubator with glucose for four or five days, and then distilled,
the distillate contained appreciable quantities of alcohol.
Kobert tested for it by the iodoform and chromic acid tests^
but did not estimate it quantitatively. He also found that if
Ascarts were ground up with kieselguhr, and the mixture
incubated for sixteen hours with sodium fluoride and toluol
solution, it gave an alcohol-containing distillate, A similar
result was obtained with earthworms, and hence Kobert con-
cludes that these organisms, and also the ova, contain zymase.
Though for the present we cannot definitely accept or reject
the presence of an enzyme of alcoholic fermentation in animal
1 Arthus, Arch, dephysiol. (5), 3, p. 425, 1891 ; (S), 4, p. 337, 1892.
* Doyen and Morel, C, /?. Soc. BioLy 55, p. 215, 1903.
' Seegen, Centralb.f, PkysioLy 5, pp. 821 and 869, 1891.
* Oppenheimer, Die Fermente^ 2nd ed., Leipsig, 1903, p. 320.
* Rajewsky, Pfliiget^s Arch,,^ 11, p. 122, 1875.
* Kobert, PfiUget^s Archiv, 99, p. 116, 1903.
ZYMASE IN VEGETABLE TISSUES 109
tissues, we can speak with greater confidence with regard to
vegetable tissues. It was shown by Rollo ^ that the higher plants
formed alcohol if they were were kept in absence of oxygen, and
Pasteur 2 and many other investigators confirmed this observa-
tion. Hence it has been suggested that the anaerobic respiration
of plants is no more or less than alcoholic fermentation. In
support of this hypothesis Polzeniusz and Godlewski * showed
that the anaerobic respiration of pea seeds corresponded to the
equation :
CeHigOg = 2C02 + 2C2HeO
whilst Godlewski* observed the same thing for lupin seeds.
Nabokich ^ repeated the experiments, and found that the ratio
of CO2 to alcohol did not always agree with that of alcoholic
fermentation. Sometimes the alcohol amounted to only 50 per
cent, of the theoretical.
Recently Palladin and Kostytschew® investigated and com-
pared the anaerobic respiration of living and dead seeds and
plants. The seeds were killed by placing them in a U-tube, and
cooling them to a temperature of — 20° to — 3° for twenty-four
hours. A current of hydrogen was then led through, and the
COg given off collected in baryta water. It appeared that
though the anaerobic respiration of living lupin seeds, germinat-
ing or otherwise, corresponded fairly closely with alcoholic
fermentation, that of the dead seeds did not. They continued
to evolve CO2 for a good many hours, but formed little or no
alcohol. For instance, 100 gm. of living lupin seeds in twenty-
four hours at 20*" evolved -160 gm. of COg and '145 gm. of
alcohol, whilst 100 gm. of previously frozen seeds evolved '083
gm. of CO2, but no alcohol whatever. In contrast to lupin
seeds, castor^oil and pea seeds, and germinating wheat, showed
a considerable formation of alcohol whether they were dead or
alive. Living castor-oil seeds formed COg and alcohol in the
' Rollo, cited by Oppenheimer, Die Fermente^ p. 319.
2 Pasteur, Comptes Rendus, 75, p. 1056, 1872.
3 Polzeniusz and Godlewski, Bull, de PAcad, d. Set, d, Cracovie^ 1897,
p. 267 ; 1901, p. 227.
^ Godlewski, ibid^ 1904, p. 115.
^ Nabokich, Ber. d. botaru Ges,y 21, p. 467, 1903.
^ Palladin and Kostytschew, Zeitf,physioL Chem,^ 48, 214, 1906.
no ZYMASE AND OTHER GLYCOLYTIC ENZYMES
proportion of i.oo to 60, and dead ones, in the proportion of 100
to 59, hence the respiration was not a typical alcohoh'c one in
either case. Dead stalk tips and leaves of the vetch ( Viciafabd)
evolved COg and alcohol in the proportion of 100 to 17; dead
pea seeds, in the proportion of 100 to 75 ; and dead germinating
wheat in the proportion of 100 to 93. It seems probable,
therefore, that anaerobic respiration is in all cases similar to
alcoholic fermentation, but that frequently other processes are
at work which convert the alcohol first formed into some other
products. In living organisms these products are COg and
water, provided that sufficient oxygen is available. Thus
living pea seeds, in absence of oxygen, gradually accumulate
alcohol, but in presence of oxygen, oxidise it completely.
Frozen pea seeds accumulate a good deal of alcohol even in
the presence of oxygen, so in their case the oxidising mechanism
has been weakened or destroyed by the low temperature.
As zymase is almost certainly present in many seeds and
plants, if not in all, one would expect that it could be isolated
like yeast zymase. Stoklasa, Jelinek, and Vitek ^ find that the
juice expressed from beetroot, if kept in hydrogen in presence
2 per cent, of potassium metarsenite, undergoes a very slow
alcoholic fermentation. For instance, 500 cc. of the juice, at a
temperature of 2^'', gave '179 gm. of COg and -120 gm. of
alcohol in six days. The precipitate thrown down by addition
of alcohol plus ether to the juice was more active, as 6 gm. of
it caused immediate fermentation in the 100 cc. of 15 per cent,
glucose solution to which it was added, and in forty-eight hours
at a temperature of 30° formed '64 gm. of CO2 and .94 gm. of
alcohol. Subsequently Stoklasa, Ernest, and Chocensk^ ^ found
that the alcohol-ether precipitate prepared from the juice of the
root and leaves of the beet induced lactic acid fermentation.
Thus 10 gm. of precipitate, kept in 15 per cent, glucose solution
together with i or 2 per cent, of salicylic acid for forty-eight
hours at 20°, gave -08 to •36 gm. of lactic acid, -28 to -86 gm. of
CO2, and -21 to -80 gm. of alcohol The digestion liquids were
tested and found to be sterile, but until confirmatory evidence
* Stoklasa, Jelinek, and Vitek, HofmeUtet^s Beitr.^ 3, p. 460, 1903.
2 Stoklasa, Ernest, and Chocenskj^, Zeit /. physioL Chim.^ 5CS p. 303,
1907.
AUTOLYtIC ACIDS 111
has betn obtained by other observers it is best not to accept
this apparent isolation of plant zymase as proven. In the
only other observations upon plant juices of which I find a
record, an enzyme of alcoholic fermentation seemed to be
lacking. Hahn^ pounded up the spadices of the Cuckoopint
(Arum maculatum) with sand and kieselguhr, and pressed out
the juice. It contained a good deal of reducing sugar, and on
standing a few days this entirely disappeared. For instance,
20 C.C. of juice obtained from the upper club-shaped part of the
$padix contained 'ip gm. of sugar, but after six days at 25°,
none whatever; whilst 20 c.c. of juice from the lower flower-
carrying part of the spadix contained -364 gm. of sugar, but
after two days, only -092 gm. This glycolysis was accompanied
by some evolution of CO2, but not enough to account for the
sugar lost. A good deal of acid was formed, but never any
alcohol.
It has been stated incidentally that in autolyses of both
animal and plant tissues considerable acidity is developed. In
most cases the nature of the acids formed, and their amount,
have not been studied in detail. But Magnus-Levy ^ has done
this for autolyses of dog and ox liver, and has obtained some
very noteworthy results. In some experiments large pieces
of liver (150 to 900 gm.) were kept under aseptic conditions
for some hours or days at 38°, and in others the liver was
minced and kept with twice its volume of saline, and an
antiseptic. In the former series, the asepsis was verified by
aerobic and anaerobic cultures. The aseptic liver became
strongly acid in twenty-four hours, and formed a semi-fluid
mass. It contained considerable quantities of non-volatile acids
such as lactic and succinic acids, and of volatile acids such as
formic, acetic and butyric The data given in the table show
the amounts of normal sodium hydrate solution required to
neutralise the acids in 100 gm. of the gland substance. We
see that after twenty-four hours' autolysis the ox liver needed
160 C.C. of alkali to neutralise its non-volatile acids, an amount
corresponding to the presence of i'44 gm. of lactic acid Its
volatile acids were much smaller, but dog's liver showed an
1 Hahn, Ber,, 33, p. 3555, 1900.
2 Magnus-Levy, HofmeisUr^s B^iir,^ 2, p. 261, 1902.
112 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
inverse relationship, and formed much more volatile acids than
non-volatile. The ratio of V to N-V was invariably low in ox
liver autolyses, and high in dog liver autolyses. The data
Tissue.
Non- volatile
Acids.
Volatile
Acids.
V-i-N.V.
Ox Liver, i day aseptic .
„ 9 days „ . . .
Dog's Liver, l day aseptic
„ 6 days „ . . .
l6o
I9-I
2-2
2-7
2-8
4-8
8.9
I8-0
•18
•25
67
show that most of the acid formation occurred during the first
twenty-four hours. Another experiment showed that very
little occurred in the first six hours, hence there must have
been a rapid disintegration between these two periods.
In comparison with aseptic autolyses, antiseptic ones occur
extremely slowly ; so much so that there may be less decom-
position in six months than in a single day of aseptic autolysis.
The following experiment is a case in point : —
Tissue.
Non-volatfle 1 Volatile
Acids. Acids.
Ox Liver, i day under aseptic conditions
„ 2^ months with Chloroform
„ 2^ „ with Toluol
»i 6 i» »» ...
160
4-3
77
8-3
2*8
2-0
In addition to these acids, the autolysing liver gave off a
good deal of gas. In one experiment 45 gm. of rabbit's liver,
under aseptic conditions, gave off 100 cc of gas in two days.
Two-thirds of this gas was COg, and the remainder hydrogen.
This considerable evolution of hydrogen suggests bacterial
action, and Magnus-Levy realised that micro-organisms may
have been present which were not revealed by his cultures.
The fact that Lane-Claypon and Schryver^ observed scarcely
any more proteolysis when minced liver was kept in saline at
37° for twenty-four hours without an antiseptic than when it was
kept in presence of toluol supports this view of bacterial infection.
^ Lane-Claypon and Schryvety Joum, PhystoL^ 3i> P- 169, 1904.
AUTOLYTIC ACIDS
113
On the other hand, Levy found that the acids formed in the
aseptic autolyses were of the same character as those in the anti-
septic ones, and so it looks as if they were both formed in the
same way, viz, by enzyme action.
This autolytic acid formation is by no means confined to the
liver, as the following data show : —
Organ.
Duration of
Autolysis.
Non-Tol«tUe
Acidi.
VoUtUe
Acids.
Spleen of Ox .
Muscle of Horse
Salivary gland of Ox
Thymus of Ox .
Heart muscle of Calf
Kidney of Dog .
Testis of Bull .
Lymph glands of Ox
Pancreas of Dog
Lung of Calf .
Ovary of Ox .
Months.
5
5
4
Si
5
6
5
^6^
4i
4»
7-6
3-0
5-6
2-6
1-6
1-3
1-4
5-6
3-3 .
3'5
1-6
.6
1*0
.6
1.2
.8
.6
•2
These tissues were kept under antiseptic conditions at 38°.
It will be seen that only spleen and muscle developed as much
acidity as ox liver.
The origin of these acids is very uncertain. Neumeister ^
and Asher and Jackson,^ arguing more especially from the
great excretion of lactic acid observed by Minkowski in geese
with extirpated liver, think that the lactic acid is formed from
proteins, Magnus- Levy, in common with many other physio-
logists, attributes their origin to carbohydrates. He found that
during the aseptic autolysis of dog and ox liver a good deal of
the glycogen and sugar disappeared, and that the actual loss
corresponded fairly well with the increase of acidity. In making
the calculation, he assumed that the acids formed were all pro-
duced from lactic acid, one molecule of acetic acid arising from
one molecule of lactic acid, and one of butyric acid from two
molecules of lactic acid, just as they seem to be in acetic and
butyric fermentations. They could scarcely have been formed
from the higher fatty acids of the liver fat, as Siegert ^ has shown
* Neumeister, Lehrbuch d.physiol, Ckem,^ 2nd ed., p. 313.
2 Asher and Jackson, Zeitf, BioL^ 41, p. 393, 1901.
3 S'legtrty jffb/meisUr^s Beiir,^ i. p. 114, 1902.
P
114 ZYMASE AND OTHER GLYCOLYTIC ENZYMES
that during liver autolysis the quantity of these acids remains
unchanged.
The lactic acid present in fresh tissues is almost always the
dextro-rotatory acid, whilst Levy found that that formed by
autolysis is chiefly the inactive form. At least in antiseptic
autolyses only lo per cent of it consisted of the dextro acid, but
in aseptic autolyses as much as 40 per cent, was of this kind.
Mochizuki and Arima ^ allowed minced bulls* testes to digest at
38° with toluol and chloroform, and they found that the dextro
acid was the only one formed. The testes contained 045 per
cent, of the acid originally, and after two to fifteen days' autolysis
it increased to • 1 26 to -23 1 per cent. Also Kikkoji ^ observed a con-
siderable formation of dextro acid in the autolysis of ox spleen.
From 500 gm. of the fresh organ he isolated -8 gm. of lactic acid.
It will be remembered that the lactic acid produced by the
action of an acetone preparation of B. Delbtiicki on glucose was
found by Buchner and Meisenheimer to be the inactive body,
hence one must conclude that intracellular enzymes exist which
can give rise to both forms of the acid.
' Mochizuki and Arima, Zeit, f. physioL Chem,^ 49, p. 108, 1906.
2 Kikkoji, ibid,y 53, p. 415, 1907.
LECTURE V
OXIDISING ENZYMES
Oxygenases or aldehydases and their various activities. Peroxidases and
their estimation. Doubtful enzymic nature of oxidases. Tyrosinases,
laccase, and alcohol-oxidase. Catalases : their estimation, mode of
action, and relation to functional capacity. Inorganic ferments.
Respiration in dead animal and plant tissues, before and after disinte-
gration, and its relation to respiratory enzymes. Intramolecular oxygen.
Respiratory processes in biogens.
The. processes of oxidation which are continuously taking
place in all living tissues are even more important than those
of hydrolysis, in that they are chiefly responsible for the
production of heat and other forms of energy. Though our
knowledge of the subject is at present very fragmentary, it
seems probable that these oxidations are brought about by
intracellular oxidising enzymes. Hence the study of such
enzymes is of great interest and importance. As long ago as
1863 Schonbein made a number of observations upon them,
but since his time, and until the last few years, they have
been almost completely neglected. Even now much of our
information is very inexact and contradictory.
The oxidising enzymes have been separated by Bach and
Chodat ^ into three main classes, and the scheme of classification
suggested by them is generally accepted as a convenient and
correct one. Members of the first class, the true oxidases^
oxygenases^ or cddehydaseSy possess the power, when in the
* Bach and Chodat, Ber,^ 35, pp. 1275, 2467, 3943, 1902 ; 36, pp. 600,
606, 1756, 1903 ; 37, pp. 36, 1342, 2434, 3785, 3787, 1904 ; 38, p. 1878, 1905 ;
39, pp. 1664, 1670, 2126, 1906 ; 40, p. 230, 1907 ; Biochem, Centralb,^ i, pp.
417 and 457, 1903-
115
116 OXIDISING ENZYMES
presence of oxygen, of oxidising aldehydes such as * salicyl-
aldehyde and formic aldehyde to their corresponding acids,
or of oxidising tincture of guiacum resin to a blue colour. The
second class of oxidases, the peroxidases^ turn guiacum tincture
blue only if hydrogen peroxide be present. The third class,
the catalasesy are probably not true oxidising enzymes at all,
as they cannot turn guiacum blue either in presence or absence
of hydrogen peroxide, but they have the power of liberating
oxygen from hydrogen peroxide solution.
The guiacum test depends on the oxidation of guiaconic
acid, C20H24O5, to guiacum blue, CgoHggOg (Doebner), so it is
best carried out by using an alcoholic solution of pure guiaconic
acid. Inorganic substances, such as iodine, chlorine, bromine,
nitric acid, chromic acid, ferric chloride, copper salts, and many
peroxides, likewise effect this oxidation. Besides this test
several other colour reactions have been used for investigating
the oxidases. Rohmann and Spitzer ^ showed that if a dilute
alkaline solution of a-naphthol and paraphenylene-diamine are
added directly to tissue pulp in presence of air, a blue-violet
colour develops owing to the absorption of oxygen and the
formation of indophenol. PohP .found that extracts of the
tissues gave a similar reaction. Bourquelot* observed that
oxidases turn guiacol red. Kastle and Shedd* found that
phenolphthalin is changed by them to phenolphthalein ; that
paraphenylene-diamine is changed to a dark brown colour,
and that the colourless reduction products of indigotin and
methylene blue are re-oxidised. Whether all these reactions are
brought about by true oxidases, or can to some extent be induced
by peroxidases and other bodies, has not yet been properly
investigated. The evidence, such as it is, Suggests that several
of the different reactions are brought about by different enzymes,
or that a number of oxidases exist. Thus Rosell ^ precipitated
the oxidases from various tissue extracts by means of uranyl
^ Rohmann and Spitzer, Ber,^ 28, p. 567, 1895.
« Pohl, Arch.f, exp. Path,, 38, p. 65.
3 Bourquelot, C, R. Soc. Btol,^ 46, p. 896, 1896.
* Kastle and Shedd, Atner, Chem, Journey 26, p. 527, 1901.
* Rosell, "Dissertation," Strassburg, 190 1, quoted from Ergebnisse der
Physiol., I., i., p. 233.
OXYGENASES
117
acetate, and investigated the action of solutions of the precipi-
tates* In no case did they give the guiacum reaction, whilst
in every case they liberated oxygen from hydrogen peroxide.
To the salicylaldehyde and indophenol tests, however, they
reacted very differently- Some extracts gave a positive result
in both cases, others a negative result in both cases, and others
one negative and one positive result, as the following data
show : —
Tissue.
Aldehydase.
Indophenol
Oxidase.
Salivary glands
Thymus ,
Spleen .
Lungs .
Brain . •
Suprarenal glands
Testis .
Kidnev .
Lymph glands
Pancreas
Bone marrow .
Mammary glands
Muscle ,
+
+
+
+
+
+
+
+
+
+
+
+
Abelous and Biarn^s^ found that the minced tissue of
most organs contained aldehydase, but did not give the
indophenol reaction. Again, Kastle and Shedd^ found that
extracts of sheep's liver and testis — which are known to contain
aldehydase — did not give either the guiacum reaction or oxidise
phenolphthalin to phenolphthalein. On the other hand, extracts
of most vegetable tissues, such as potato and maize, did both,
and there was a roogh parallelism between the depth of the
two reactions given by each tissue.
The aldehyde-oxidising power of the tissues was first
investigated by Schmiedeberg,* who found that if oxygenated
blood containing benzyl alcohol or salicylaldehyde were
perfused through a freshly excised liver or lung, a small amount
of the corresponding acid was formed. Jaquet * repeated these
^ Abelous and Biarn^s, Arch, de PhysioL^ 1895, pp. 195 and 239.
2 Kastle and Shedd, loc. cit
3 Schmiedeberg, Arch,f, exp. Path,^ 14, pp. 288 and 379, 1881.
* Jaquet, /feV/., 29, p. 386, 1892.
118
OXIDISING ENZYMES
experiments, and showed that even after death the tissues still
retained their oxidative power. A lung previously frozen or
kept with 2 per cent phenol solution for forty-eight hours still
possessed half or more of its original oxidising capacity. A
kidney or half a lung of a horse was kept for twelve days in
75 per cent, alcohol, and was then perfused with saline or blood
containing salicylaldehyde or benzyl alcohol for two-and-a-half
to five hours at 37°. From -032 to -053 gm. of the corre-
sponding acid was formed. Even after the organs were minced
up they preserved a good deal of their activity. Two kidneys
(of the horse) were minced, hardened with alcohol, and dried,
and the product kept, with frequent shaking, with i litre of
blood and i gm. of salicylaldehyde for twenty-four hours at
25° to 30°. The salicylic acid formed amounted to • 13 gm., whilst
1 kg. of horse muscle, similarly treated, gave only '02 gm. of
the acid. Again, the juice expressed from half a lung (horse)
and kept for five hours at 35° to 40° with blood and salicylaldehyde,
gave only '023 gm. of salicylic acid. The oxidation was due at
least in part to a soluble enzyme, as a filtered extract of two
alcohol-hardened kidneys, when kept with the aldehyde, yielded
•012 to -036 gm. of salicylic acid.
It will be seen that the salicylic acid formed was in every case
very small in amount, considering the very large weight of lung
or kidney taken. Abelous and Biarnes,^ who used fresh tissues,
obtained somewhat better results. They added 100 gm. of
tissue to I litre of saline containing -5 per cent, of NagCOg and
2 C.C of salicylaldehyde, and kept the mixture for twenty-four
hours at 38* in a current of air. The following amounts of
salicylic acid were formed : —
Tissue.
No Antiseptic.
1 per cent. NaF.
gm.
gm
Spleen ....
.252
Lung
.146
.142
Liver
:^l
•139
Thyroid .
Kidney .
•062
•077
Th)rmus .
.061
060
Suprarenal gland
K>6o
...
Testis ....
.023
••'
Abelous and Biam^, loc, cit
ALDEHYDASE lid
It will be seen that the oxidation was h'ttle if at all diminished
by the presence of i per cent, of sodium fluoride. Three of the
tissues worked with, viz. pancreas, muscle, and brain, effected
no oxidation at all, either with or without NaR It will be
remembered that Roseli likewise found no aldehydase in
pancreas and muscle. Blood contained a small amount of the
enzyme, as i kg. of defibrinated ox blood, kept for twenty-four
hours at 38** in a current of air with salicylaldehyde, formed
•176 gm. of salicylic acid, and i kg. of pig's blood formed
•060 g*m. of acid* Abelous and Biarnfes confirm Jaquet*s
conclusion that the oxidase is a soluble ferment, but they found,
as he did, that a good deal of it is destroyed in the process of
coagulating the tissue with alcohol, drying, and extracting it.
Salkowski and Yamagiwa,^ like Abelous and Biarnes, found that
spleen tissue possessed the most active oxidasic power, whilst
liver was only a little inferior. The kidney, on the other hand,
formed only a tenth to a twentieth as much salicylic add as the
spleen. They did not find pancreas and muscle to be entirely
lacking in enzyme, but pancreas formed only a twentieth to a
hundredth as much salicylic acid as the spleen, and muscle not
a hundredth as much. Zanichelli,^ on the other hand, found
that the pancreas possessed a good deal of oxidising power.
The oxidising action of the tissues on formic aldehyde was
studied by Pohl.^ It proved to be very weak, for an extract of
200 gm. of ox liver gave only '0068 gm. of formic acid in one
hour, and -033 gm. in fifteen hours, whilst aqueous chloroform
or sodium fluoride extracts had no oxidative power whatever.
The solubility and precipitability of aldehydase has been
studied by Jacoby.* An extract of minced ox liver was
fractionally precipitated by ammonium sulphate, and it was
found that whilst very little of the enzyme was thrown down
by 33 per cent, saturation with the salt, all of it was thrown
down by 60 per cent, saturation. This precipitate could be
dissolved up again in water, re-precipitated with dilute alcohol,
and when re-dissolved again furnished an active oxidase solution.
^ Salkowski and Yamagiwa, Centralb. med, Wiss,^ 32, p. 913, 1894.
2 Zanichelli, Arch, di FanHacoLy 3, p. 8.
3 Pohl, Arch,/, exfi. Path., 38, p. 65.
* Jacoby, Zeit. f. physiol. Chem.y 30, p. 135, 1900.
120 OXIDISING ENZYMES
Bach and Chodat^ fractionally precipitated the enzymes in the
juice of the fungus LactariuSy and they found that 40 per cent,
alcohol threw down all the true oxygenase, whilst much of the
catalase was still left in solution.
Peroxidases, — We have seen that true oxidases, though of
wide distribution, are not present in all tissues, or if present,
they are in such small amount that they cannot be demonstrated.
Peroxidases, on the other hand, are stated by several investi-
gators to be present in all living tissues. However, Loew^
found that aqueous extracts of certain tobacco plants, though
they had ah energetic action on hydrogen peroxide, were unable
to turn guiacum tincture blue in its presence. Kastle and
Loevenhart* were unable to obtain the reaction with onion
bulb: but as they point out, it is to be remembered that
Hunger * showed that the reaction is sometimes masked by the
presence of glucose and other powerful reducing substances.
The evidence of the almost universal presence of peroxidases
is, in the case of animal tissues at least, to be accepted with
reserve, because the guiacum and hydrogen peroxide test is
given by haemoglobin. As Czyhlarz and v. Fiirth^ point out,
it is almost impossible to remove the haemoglobin thoroughly
from the tissues by perfusion, and hence the method is un-
reliable. A better test for peroxidase depends on its power of
hastening the liberation of iodine from acidified potassium iodide
solution in presence of hydrogen peroxide, for this reaction is
not influenced by haemoglobin. A quantitative measure of the
action can be obtained by titrating the free iodine against
sodium thiosulphate solution (Bach and Chodat). Even this
iodine method is very imperfect, for the reaction may be
completely stopped if much protein or other iodine-binding
tissue constituents are present, and hence only a positive result
is of significance. Czyhlarz and v. Fiirth find that leucocytes,
bone marrow, spleen, lymph glands, and spermatozoa give the
reaction. The enzyme is contained in the cell constituents, and
* Bach and Chodat, Ber.y 36, p. 606, 1903.
2 Loew, Report 6^^ U. S, Dept, of Agriculture^ 1901.
3 Kastle and Loevenhart, Atner Ckem. Joum,^ 26, p. 539, 1902.
* Hunger, Ber, d, bot Ges.^ 19, p. 574.
* Czyhlarz and v. Fiirth, Hofmeister's Beitr^ 10, p. 358, 1907.
PEROXIDASE ESTIMATION J 21
not in the surrounding liquids, though it can be partially ex-
tracted from the cells by salt solutions. For the quantitative
estimation of tissue peroxidases, Czyhlarz and v. Fiirth use a
spectrophotometric method, dependent on the oxidation of the
leuco base of malachite green to the actual blue-green pigment.
They find that with true tissue peroxidases the rate of oxidation
is at first proportional to the time, but that as the reaction
proceeds it slows down more and more, and after an hour or so
stops altogether. The amount of oxidation so induced is
directly proportional to the amount of enzyme present. Solu-
tions of haematin also bring about the oxidation, but in their
case the rate of oxidation is strictly proportional to the time
throughout, and shows no tendency whatever to slow down.
Also this haematin reaction is greatly affected by variation in
the concentration of the catalysing pigment and the hydrogen
peroxide, but only slightly by variation in the leuco base, whilst
the peroxidase reaction is much more dependent on change in
the concentration of the leuco base than on that of the peroxide.
Buckmaster^ has shown that haemoglobin induces the reaction
in the same way as haematin, but that haematoporphyrin and
baematoidin do not Hence the presence of iron in the molecule
is essential, just as it is essential for the guiacum reaction.^
In that numerous inoi^anic oxidising agents can effect the
conversion of guiaconic acid into guiacum blue, of salicylaldehyde
into salicylic acid, and the other reactions held to indicate the
presence of oxidases, it might reasonably be questioned whether
these oxidases are enzymes at all, and are not merely unstable
organic bodies, such as organic peroxides, which possess feeble
oxidising power. The chief justification for classing them with
the enzymes seems to lie in their extreme instability, and in the
fact that their precipitability by salts roughly corresponds to
that of other enzymes. On the other hand, they resist a
considerably higher temperature than other enzymes, for
Czyhlarz and v. Fiirth found that peroxidase solutions could
be heated nearly to boiling point without losing all their activity.
Again, the oxidases do not seem to possess what is the most
important and distinctive property of enzymes, viz. that of
* Buckmaster, Joum, Physiol,, 37, p. xi., I908.
2 Buckmaster, ilnd,^ 3S, p* xxxv., 1907.
122 OXIDISING ENZYMES
acting as a catalytic agent, or of being able to induce an
indefinitely large amount of chemical change without themselves
undergoing destruction. The extremely small production of
salicylic acid from salicylaldehyde has already been commented
on, and when we remember that some enzymes, such as
invertase, can hydrolyse at least 100,000 times their weight of
the substance upon which they are exerting their specific
activity, we are forced to conclude, either that the tissues
contain excessively small amounts of the oxidases, or that
these bodies are not true enzymes in the ordinarily accepted
sense. The latter conclusion is supported by the action of
peroxidase on the malachite green base, for we saw that the
final amount of oxidation effected by it was proportional to the
amount of enzyme present, and that it very soon came to
an end.
But what is the probable nature and mode of action of these
pseudo-enzymes ? Bach ^ considers that the oxidases of the
blood are simply readily oxidisable substances which have a
special capacity for forming peroxides. Kastle and Loevenhart ^
agree with him, and extend his view to the oxidases of the
tissues. They think that organic peroxides, ue, oxidases, are
not present in the intact cells as such, but are in the form of
readily oxidisable substances which in the presence of air or
oxygen unite with the oxygen to produce the peroxide. These
peroxides can then transfer their oxygen to other less readily
oxidisable substances such as salicylaldehyde or guiaconic acid.
A concrete instance of an organic peroxide of this character is
found in benzaldehyde, for Baeyer and Villiger* have shown
that this body, when exposed to atmospheric oxygen, is oxidised
to benzoyl hydrogen peroxide, QHg. CO — O — OH. Hence a
mixture of benzaldehyde and water, in presence of air, turns
guiaconic acid blue directly. Benzoyl hydrogen peroxide reacts
with reducing agents such as indigo, guiaconic acid, etc., to form
benzoic acid and an oxidation product, but as it is itself oxidised
when acting as an oxygen carrier, its activity comes to an end.
It is not a true catalytic agent, therefore, and Kastle and
^ Bach, Comptes Rendus^ 124, p. 951, 1897.
2 Kastle and Loevenhart, Amer, Chenu Joum.^ 26, p. 539, 1902.
2 Baeyer and Villiger, Ber,y p. 1569, 19CX).
TYROSINASE 123
Loevenhart think that the so-called oxidising enzymes of the
tissues are peroxides of a similar non-catalytic character.
As regards the peroxidases, Kastle and Loevenhart suggest
that their blueing action upon guiacum H-HgOg depends on
the HgOg first reacting with one or more organic substances
present in the plant or animal extract to form an organic
peroxide. This peroxide then oxidises the guiaconic acid to
guiacum blue. A concrete instance of such a reaction is found
in acetic peroxide, a highly unstable body which Baeyer and
Villiger have prepared by the action of HgOg on acetic an-
hydride. This body reacts with guiaconic acid to form guiacum
blue and acetic acid.
Even if further research proves that the oxidases are true
catalytic agents, they may still be of the nature of unstable
organic peroxides, only they may have the property of taking
up oxygen and passing it on to oxidisable substances without
themselves undergoing further change.
In addition to the oxidases and peroxidases mentioned,
several other oxidising enzymes have been recorded in animal
tissues. It will be remembered that the liver contains an oxi-
dase which can convert xanthin and hypoxanthin into uric acid.
Whether this oxidation is the work of specific enzymes, or is
dependent on the aldehydase of the liver, we do not know.
Tyrosinase, — Another oxidase, which is apparently specific,
is the tyrosin-oxidising enzyme tyrosinase. This body was
discovered by Bertrand^ in plants in 1896, whilst two years later
Biedermann ^ showed that a similar enzyme is present in the
intestinal juice of the meal-worm ( Tenebrio molitor), v. Fiirth and
Schneider^ found it in the body fluids of certain Lepidoptera.
They showed that the darkening which the haemolymph under-
goes on exposure to air is due to a tyrosinase. A preparation
of the enzyme was obtained by precipitating the press juice of
the pupae with half-saturated ammonium sulphate. A solution
of the precipitate in -05 per cent. NagCOg, when shaken in air
with tyrosin solution, changed to a violet and then to a black
colour, and finally yielded a precipitate of melanin. This
^ Bertrand, Bull, de la Soc. Chem, (3), 15, p. 793, 1896.
* Biedertnann, Pfluger's Arch,^72^ p. 105, 1898.
3 V. Fiirth and Schneider, Hofmeistet^s Beitr.^ i, p. 229, 1901.
124 OXIDISING ENZYMES
melanin contained 137 per cent of nitrogen, and smelt of indol
when heated with caustic soda. In addition to its action upon
tyrosin, tyrosinase gives the indophenol reaction and will con-
vert other aromatic bodies such as pyrocatechin, hydroquinone
and suprarenin into melanins* It does not give a typical
guiacum reaction.
Tyrosinase seems to be widely distributed in the animal
kingdom. It is present in the crayfish, in Cephalopods {Sepia
officinalis) and in sponges, and Miss Durham^ has shown that it
is present in the skin of foetal and new-bom rats and rabbits.
An aqueous extract of the ground-up skin, if mixed with
tyrosin and a drop of ferrous sulphate to serve as activator,
gradually takes on a dark colour and forms a black precipitate.
An extract of the skin of red-skinned guineapigs gives under
similar conditions an orange-coloured pigment.
Vegetable tyrosinase is probably as widely distributed as
animal tyrosinase. Bertrand found that if the fungus Russula
nigricans is boiled with alcohol and extracted with boiling
water, some of its chromogen, which is a tyrosin-like body,
passes into solution. If this solution is mixed with a cold-
water extract of the fungus, it turns red and subsequently black.
If the extract is added to a solution of tyrosin> it acts in the
same way and changes it first red, then black, and then forms a
black precipitate. The conversion is dependent on the presence
of air, and an absorption of oxygen accompanies it. The juice
expressed from the roots of the beet and the dahlia, and from
the tubers of the potato, likewise contains tyrosinases which
convert the tyrosin-like bodies present into melanins and act
similarly upon added tyrosin. Bourquelot* found that tyrosinase
also acts upon numerous other aromatic substances such as the
cresols, resorcinol, guiacol, thymol, and naphthol.
Vegetable Oxidases, — Most of the observations recorded
above, except those in the last paragraph, concern the oxidases
of animal tissues. Similar classes of enzymes are found also
in vegetable tissues. True oxidases, capable of blueing guiacum
without the addition of hydrogen peroxide, are more widely
^ Durham, Proc, Roy. SoCy 7A^p* Zio.
^ Bourquelot, Connies Rendus^ 123, pp. 315 and 423^ 1896; Bull, de la
Soc. MycoL de France^ 13, p. 65, 1897.
LACCASE 125
distributed in plants than in animals. They are especially
abundant in potatoes, and in apples and other fruit, just beneath
the skin. They give the indophenol reaction, and oxidise
phenolphthalin to phenolphthalein, but their action on salicyl-
aldehyde has scarcely been investigated at all In addition to
the oxygenases and peroxidases mentioned, other specific oxidis-
ing enzymes have been described. One of these, laccase, is the
first oxidase definitely recognised as such. In 1883 Yoshida^
showed that the production of lacquer varnish from the sap of
the lac tree of South-East Asia is dependent on this enzyme.
The fresh lac juice is nearly white, but on exposure to air it
rapidly changes to 'brown, then black. It dries with a brilliant
black lustre, owing to its chief constituent, urushic acid, Ci^HigOg,
being converted into oxyurushic acid, C14H18O3, by the action of
the laccase. Bertrand^ has shown that laccase can oxidise
hydroquinone to quinone, and pyrogallol to purpurogalline,
and can act similarly upon other polyphenols. He finds that
enzymes with the same action as laccase are present in many
other plants, such as the roots of the beet, carrot, and turnip :
in the potato, apple, and pear : in the vegetative parts of clover
and asparagus ; in the flowers of Gardenia^ and in many fungi,
whilst Rey-Pailharde has found it in germinating seeds. Argu-
ing from its distribution, one would imagine that laccase is one
and the same enzyme as the guiacum-blueing oxygenase, and
indeed Bertrand used the guiacum test to some extent for its
identification. On the other hand, the activity of laccase seems
to be associated with the presence of manganese. Its ash
always contains traces of the metal — sometimes over i per
cent, of it — and Bertrand found that the activity of an enzyme
preparation is proportional to the manganese present
Laccase is distinct from tyrosinase, but Bourquelot^ found
that all of the many fungi examined by him contain both
enzymes in varying proportions. If a solution of the two
1 Yoshida, Jaurn, Chem, S&c,, 43, p. 472, 1883. See also, Reynolds
Green's SolubU Ferments and Fermentation^ from which this brief account
is mainly drawn.
^ Bertrand, Comptes Rendus^ 118, p. 121 5, 1894 ; 120, p. 266, 1895 ; 121, p.
166, 1895 ; 122, p. 1 132, 1896 ; 123, p. 463, 1896 ; 124, pp. 1032 and 1355, 1897.
* Bourquelot, loc. cit.
126 OXIDISING ENZYMES
enzymes be heated to 70°, the tyrosinase is. destroyed, whilst
the laccase remains.
Akofiol-oxidase, — More interesting, perhaps, than any of the
above described vegetable oxidases is the alcohol-oxidase of
acetic acid fermentation. Buchner, working in conjunction
with Meisenheimer 1 and subsequently with Gaunt,^ endeavoured
to show that this fermentation is the work of an oxidising
enzyme. To obtain sufficient bacteria of acetic acid fermenta-
tion, beer wort to which 4 per cent, of alcohol and i per cent,
of acetic acid had been added was used as a culture medium,
and was inoculated with bacteria from pure cultures. The
bacteria were separated by centrifugalisation, dried fifteen to
twenty hours on a porous clay plate, rubbed up for fifteen
minutes with 20 volumes of pure acetone, washed two or three
times with ether on a filter, and dried in vacuuo over sulphuric
acid. A sample of 20 gm. of this " Acetondauerpraparat " was
placed in a litre flask with about 250 c.c. of 4 per cent, ethyl
alcohol, 8 C.C. of toluol, and excess of calcium carbonate to
neutralise the acid formed, and was kept in a current of air
for three days at 28°. The best experiment showed a yield
of 4 gm. of acetic acid per 100 gm. of dried bacteria (equivalent
to about 1000 gm. of moist living bacteria), so the oxidation was
small compared with that effected by living bacteria. Press juice
from the bacteria had no oxidising power at all, so Buchner and
Gaunt suggest that the oxidase may have been destroyed during
the process of extraction, or that it is an insoluble body which
does not pass out in the juice. But unfortunately one is com-
pelled to entertain still a third alternative, viz. that the oxidation
effected by the acetone preparation was due solely to the
presence of living bacteria. Buchner and Gaunt tested their
preparations upon sterile beer wort, and found that a consider-
able proportion of them contained living bacteria. They think
that these living bacteria formed only a very small fraction of
the whole, but there is no exact evidence on the point. Living
bacteria, if placed in beer wort in presence of toluol, produced
very much less acetic acid than in absence of toluol, but they
still formed five or ten times more acid than an equal amount of
^ Buchner and Meisenheimer, Ber.^ 36, p. 634, 1903.
^ Buchner and Gaunt, Annalen^ 349, p. 140, 1906.
CATALASES 127
acetone bacteria. This shows, according to Buchner and Gaunt,
that the acetone treatment destroys a good deal of the oxidase,
but one might reasonably argue that it killed all but a fifth
or a tenth of the bacteria, and that this small fraction was
responsible for the whole of the acetic acid formation. Until
more adequate proof has been afforded, therefore, we cannot
accept the existence of an alcohol-oxidase as proven.
Catalases. — The third class of oxidising enzymes, the cata-
lases, is the most widely distributed of all, for every animal and
vegetable tissue or fluid which gives the peroxidase reaction
with guiacum and hydrogen peroxide can also decompose the
peroxide, whilst certain of them which give the latter reaction
are unable to effect the former. The presence of catalase but
absence of peroxidase in aqueous extracts of certain tobacco
plants has already been noted. Senter ^ prepared a solution of
a "haemase" from blood which energetically decomposed
h)'drogen peroxide, but did not give the peroxidase reaction.
Schonbein^ found that all the enzymes tested by him had
the power of blueing guiacum ^/«j hydrogen peroxide, and also of
decomposing this peroxide, so he thought that both these
properties were specific for enzymes. Jacobson^ showed that
this was not the case, for emulsin, if heated to 69**, loses its
power of decomposing hydrogen peroxide, but still retains
its action on amygdalin. Also the catalases are very unstable,
so that fresh tissue extracts when kept for some time, or
extracts of dried tissues, lose their power of decomposing HgOg,
though still preserving their other activities.* If a large excess
of H2O2 be added to an enzyme solution, its catalytic power
upon the peroxide may be lost, but not its sffccific fermentative
power.
The first attempt at quantitative comparison of the catalytic
power of various animal tissues was made by Spitzer,^ who
placed I or 2 gm. of the fresh blood-free tissue in a flask with
10 C.C. of 2 or 3 per cent. HgOg solution, and measured the rate
* Senter, Proc, Roy. Sac,y 74, p. 201, 1904.
2 Schbnhtm, Joum, f, prakt Chem,^ 89, p. 323, 1863.
3 Jacobson, Zeit f, physioL Chent.^ 16, p. 340.
* Kobert and Fischer, P Auger's Arch.^ 99, p. 123, 1903.
^ Spitzer, ilnd,^ 67, p. 615, 1897.
128
OXIDISING ENZYMES
of evolution of oxygen. In all cases the oxygen came off most
rapidly at first, and then gradually dwindled down, but the
initial rate of evolution was much greater with some tissues
than with others. Spitzer arranged them in the following order,
according to their catalytic activity: blood, spleen, liver,
pancreas, thymus, brain, and muscle. Of these organs the
thymus and pancreas were taken from the ox, and the
remainder from the dog. Several other investigators have
repeated t|iese observations, and have found a more or less
similar order of activity. For the various tissues of the calf
Abelous ^ found it to be liver, kidney, pancreas, spleen, heart,
lung, thymus, brain, and striped muscle, whilst for those of the
ox Rosenbaum ^ gives it as liver, pancreas, spleen, muscle, and
brain. Much more complete series of observations have been
made by Battelli and Stern.* They point out that Spitzer used
far too much tissue for the peroxide taken, and in their
experiments they took care that there was always a consider-
able excess of peroxide. The oxygen liberated in ten minutes
by the addition of s c.c. of dilute emulsion of the fresh tissue
Animal.
1
1
1
1
1
1
S
i
Gnineapif
5800
480
490
350
260
99
34
20
Rabbit .
370
390
460
148
98
65
16
10
Dog. .
624
210
51
33
52
15
9
7
Cat .
1390
180
540
150
250
32
21
12
Pieeon
Adder .
14S0
340
14
66
...
460
200
1390
...
355
237
59
19
Frog
3S70
483
60
130
195
31
7
53
Fish (^Leuciscus)
2540
...
55
...
...
25
6
29
to 30 cc. of I per cent. HgOg was measured, and the data in the
table show the volumes of gas liberated per decigram of tissue.
In most animals the liver possessed the greatest catalytic
activity, and guineapig's liver set free no less than 5800 c.c. of
oxygen per decigram, or in the actual experiment, -001 gm. of
liver liberated 58 c.c. The next most active guineapig tissue.
1 Abelous, C. R. Soc. Btol, 1899, p. 328.
2 Rosenbaum, Festschrift f, Salkowski^ Berlin, 1904.
3 Battelli and Stern, Arch, di Fisiol.y 2, p. 471, 1905.
CATALASE 129
the kidney, liberated only a twelfth as much oxygen, whilst the
least active tissue, the brain, liberated only about a three-
hundredth as much. The relative order of activity of the
several tissues examined differs somewhat in different species
of Animals, though in every case but two the liver proved to
be the most active organ, whilst muscle and brain were always
the least active. The greatest abnormalities were found in the
liver of the rabbit, which was less active than the kidney and
blood, and in the blood and tissues of the adder. The adder's
blood liberated nearly three times more oxygen than any
mammalian blood, and one hundred times more than pigeon's
blood, whilst adder's lung and cardiac muscle liberated con-
siderably more oxygen than the corresponding tissues in other
animals. It might be thought that these erratic values were
largely dependent on chance variations, or experimental error,
but Battelli and Stern say that for animals of the same species
the values obtained for each tissue are remarkably constant,
and do not differ from one another by more than 30 per cent
Again, Lesser ^ has quite independently obtained equally wide
differences with the blood of various animals. He found that
100 cc. of -3 per cent, solution of blood decomposed the
following weights of hydrogen peroxide in ten minutes at 38° :
rabbit, 754 gm.; dog, .157 gm. ; pigeon, -015 gm.; frog,
•on gm.
It will be seen that the tissues of warm-blooded animals
were no more active than those of cold-blooded ones, and that
allied species of animals such as the rabbit and the guineapig
showed no more resemblance to one another than to any other
vertebrate animal. It might seem hopeless, therefore, to
attempt any deductions as to the possible functions of catalase
from such apparently incompatible data as these; but other
experimental results obtained by Battelli and Stern indicate
that there is a close relationship between the catalytic power
of a tissue and its functional activity. The table shows the
oxygen liberated by the tissues of guineapigs of various ages,
and it will be seen that with the growth of the embryo and of
the new-born guineapig for the first week after birth, there was
an enormous increase in the catalytic activity of the liyer and
1 Lesser, Ztitf, BioL^ 48, p. i, 1906.
130
OXIDISING ENZYMES
kidney, and a slight one in that of the other tissues. As already
mentioned in a previous lecture, I observed a similar large
increase in the ereptic power of the liver and kidney of guinea-
pigs, rabbits, and cats during intra-uterine growth and for the
first week of post-natal existence. I also found a distinct
though less considerable increase in the ereptic power of brain
and muscle.
Liver.
Kidney.
Blood.
Spleen.
Lung.
Muacle.
Brain.
5 gm. embyro •
aso
30
»••
...
12
17 .,
450
70
...
230
...
IS
12
36 „
780
no
390
290
...
19
12
Foetus at term *
1200
150
40s
300
180
24
16
2-day Guineapig .
2000
240
510
330
360
24
14
7-<iay „
Adult „
5400
410
480
285
360
24
16
5800
480
490
350
260
34
20
Starvation of rats for four to eight days, or of frogs for
several months, did not influence the catalytic power of the
tissues, neither did fatal poisoning with KCN or phosphorus:
but doses of phosphorus which were not sufficient to kill, but
which produced fatty degeneration of the liver, caused the
catalytic power of this organ to dwindle to half or a third its
normal value. On the other hand, the kidney, blood, lung,
muscle, and brain showed a considerable increase in their
catalytic power. Jolles and Oppenheim^ found that the
catalytic power of human blood is greatly diminished in certain
diseases such as tuberculosis, nephritis, and carcinoma.
In its physical properties catalase appears to resemble other
enzymes. Senter^ found that it could be precipitated from
laked blood by alcohol, and extracted from the precipitate with
water. From 30 to 40 per cent, of the total enzyme originally
present was thereby obtained in solution, and this solution, if
kept at 0°, preserved its activity for some weeks. Loew* used
ammonium sulphate as a precipitating agent, and from aqueous
extracts of leaves of the tobacco plant he isolated two catalases ;
one, a-catalase, was soluble only in dilute alkalis, and was
} Jolles and Oppenheim, Munckener med, IVocA., N. 47, 1904.
* Senter, Zeitf, physik, Chem,^ 44, p. 257, 1903.
^ Loew, loc, cit.
CATALASE 131
decomposed by it with formation of j8-catalase. It appeared
to consist of catalase in combination with a nucleoprotcin,
whilst )8-catalase had the properties of a proteose.
There is no good proof that the nucleoprotein a-catalase is
a genuine entity. More probably it consisted of certain nucleo-
protein constituents of the plant tissues which had adsorbed
some catalase. A similar explanation may hold for Spitzer's ^
results. Spitzer stated that the nucleoproteins of the liver,
pancreas, kidney, testis, and blood decomposed hydrogen
peroxide as energetically as the tissues themselves, whilst acids,
alkalis, heat, and protoplasmic poisons as potassium cyanide
and hydroxylamine had an enfeebling or arresting action on
these nucleoproteins just as they had on the tissues themselves.
But Spitzer did not purify his nucleoprotein preparations very
thoroughly, and in all his experiments he added such a large
excess of nucleoprotein to the hydrogen peroxide that the
oxygen liberated may have been due entirely to adsorbed
catalase. Spitzer pointed out that these nucleoproteins contain
iron, and he suggested that this iron plays an essential part in
the oxidation phenomena of tissues and extracts. However,
Senter showed that his blood catalase contained no iron.
The mode of action of catalase has been discussed at length
by a number of investigators, but lack of adequate experimental
data precludes any finality in their conclusions. It is important,
in the first place, to decide whether catalase is a true oxidising
ferment or not. Loew thinks that it is, as he found that his
preparations of )3-catalase, obtained from tobacco leaves and the
juice of the poppy seed, were able to oxidise hydroquinone to
quinone, though they did not give any of the other oxidase
reactions. Shaffer ^ thinks that this quinone formation was
undoubtedly due to the presence of some enzyme other than
catalase, for he and Buxton ^ found that embryonic and adult
animal tissues always contained catalase, but frequently
possessed no power of oxidising hydroquinone. Again, we
know that anaerobic organisms such as intestinal worms and
certain micro-organisms contain catalase, which in these instances
J Spitzer, P Auger's Arch.^ 67, p. 615, 1897.
* Shaffer, Amer.Joum, PhysioL^ 14, p. 299, 1905.
3 Shaffer and Buxton, Joum, Med, Research^ 8, p. 543, 1905.
l32 OXiDlSING teNZYMKS
cannot possibly escert an oxidising function. On the other
hand, Ewald ^ found that if a small quantity of blood catalase is
added to haemoglobin solution, the mixture is reduced by
ammonium sulphide two or three times more rapidly than if no
catalase is added. Hence he thought that the catalase acted as
an oxygen carrier from the oxyhaemoglobin to the ammonium
sulphide. Czyhlarz and v. Fiirth ^ repeated this experiment, but
they found that the addition of unboiled catalase extract to
haemoglobin and ammonium sulphide did not lead to any more
rapid reduction than the addition of boiled extract, hence they
conclude that the catalase has no direct oxidising action.
Inorganic ferments, — It has long been known that hydrogen
peroxide is decomposed by many inorganic substances, as well
as organic, and Schonbein^ found that hydrocyanic acid not
only prevented its decomposition by various animal and plant
enzymes, but also inhibited the action of platinum black upon it.
Hence he concluded that the catalytic influence of the organic
and inorganic substances is of similar character. In recent
years Bredig* and his co-workers have brought forward fresh
evidence in support of this view, and have shown that close
analogies exist between the action of colloidal gold and platinum
upon hydrogen peroxide, and that of catalase. So much so that
Bredig speaks of these inorganic colloids as " inorganic ferments,"
and refers to the inhibitory effect upon their action produced by
traces of hydrocyanic acid, sulphuretted hydrogen and other
substances as a "poisonous" influence. He finds that just as
HCN is the strongest poison for blood catalase, so it is the
strongest poison for colloidal platinum. For instance, the
presence of -0014 mg. of HCN per litre was sufficient to
reduce the activity of a colloidal platinum preparation to half its
original value, whilst other blood poisons such as cyanogen
iodide, corrosive sublimate, phosphorus, and carbonic oxide,
behaved similarly towards the platinum. Kastle and Loeven-
* Ewald, P Auger's Arch.y 116, p. 334, 1907.
2 Czyhlarz and v. Furth, loc, cit,
^ Schonbein, ZeiLf, BioLy 3, p. 140, 1867.
* Bredig and v. Berneck, ZeiLphysik, Cltem.^ 3i> P- 258, 1899; Bredig
and Ikeda, ibid,^ 37, p. i, 1901 ; Bredig and Reinders, ihid.y 37, p. 323, 1901 ;
Bredig, " Anorganische Fermente,'- Lcipsig, 1901.
INORGANIC CATALYSTS 133
hart ^ wholly disagree with this analogy, and say that when it
holds at all it is a mere coincidence. They find that many
substances act altogether differently on the two orders of
catalysers. Potassium bromide considerably retards the action
of liver catalase upon hydrogen peroxide, and it similarly
retards the action of finely divided platinum. On the other
hand, it accelerates the action of finely divided copper and iron,
but completely inhibits the action of silver and thallium;
Hydroxylamine doubles the action of silver upon the peroxide,
does not influence the action of platinum, but reduces that of
catalase to a twenty-fifth its original value. Thio-urea acceler-
ates the action of liver catalase, but reduces that of silver and
platinum to a tenth their normal values.
Kastle and Loevenhart say that the effect of any particular
substance upon an inorganic catalyst can be explained, at any
rate in the majority of cases, on purely chemical grounds. The
inhibitory substance may form a thin insoluble protective film
over the surface of the catalytic metal.
The mode of action of catalases is probably of an essentially
different character from that of inorganic catalysts. As Shaffer ^
points out, the oxygen liberated by catalase from hydrogen
peroxide is only molecular oxygen, whilst inorganic catalysts
such as platinum black can absorb molecular oxygen, or oxygen
from H2O2, and render it active. The difference in the char-
acter of the oxygen is shown by the fact that colloidal platinum
can turn guiaconic acid blue (Bredig^), and liberate iodine from
potassium iodide (Liebermann *), whereas catalase cannot
Hence Liebermann's hypothesis, that the action of catalase upon
H2O2 takes place in the following two stages —
Ferment + HgOg = Ferment . O + H2O
Ferment . O + H2O2 = Ferment + Og + HgO
— is probably incorrect, though it is very likely that the action of
platinum black takes place in accordance with a similar scheme,
which may be represented thus—
Pt + HgOj = PtO + HgO
PtO + HgOg = Pt + 02-fH20.
^ Kastle and Loevenhart, Amer, Chem. Journ,^ 29, pp. 397 and 563, 1903.
* Shaffer, Amer.Journ. PhysioL^ 14, p. 299, 1905,
^ Bredig, "Anorganische Fermente," Leipsig, 1901.
* Liebermann, Pftuger's Arch,^ 104, p. 670, 1904.
134 OXIDISING ENZYMES
As regards the function of the catalases in the living tissues
we are entirely in the dark. It has been suggested by Loew
that in the processes of tissue respiration hydrogen peroxide is
constantly being formed, and as it is a violent poison, catalase
must be present to prevent its accumulation. This hypothesis
is improbable for several reasons. In the first place, hydrogen
peroxide is not a violent poison, as Bach and Chodat^ have
cultivated certain plants in a medium containing -68 per cent
of it. But even if it were a poison, it could not be formed at
all in anaerobic oi^anisms, yet these organisms, as stated above,
contain catalase just like aerobic ones. Then if hydrogen
peroxide were formed during respiration, one would expect
the catalase content of a tissue to be a measure of its respiratory
activity. But a reference to the table on p. 128 shows that this
cannot be the case. The respiratory activity of guineapig's
liver cannot be sixteen times greater than that of rabbit's liver,
or 290 times greater than that of guineapig's brain. In fact, it is
possible that the catalytic power of a tissue is a measure of its
reducing power rather than of its oxidising power. Pozzi-
Escot^ considers that catalase is identical with the "hydro-
genase " of Rey-Pailharde,* and called by him philothion, from
its power of converting free sulphur into sulphuretted hydrogen.
Bach and Chodat* do not admit the identity of these two
ferments, but the fact of its being suggested shows how
supremely ignorant we are of the function of catalases. That
such a function does exist, seems to be proved by the variations
in the catalytic power of the tissues with functional activity.
Of course it is possible that the power of decomposing H2O2 is
a chance property of certain organic substances formed in the
tissues, but even if this is the case it is important for us to
elucidate the meaning of the reaction, as it would give us a
convenient measure of the amount of such hypothetical
substances in the tissues, and the variations they undergo in
different phases of cellular activity.
1 Bach and Chodat, Biochem, Centrcdb.^ 1903, p. 4i7«
2 Pozzi-Escot, Comptes Rendus^ 134, p. 66, 1902 ; Amer. Ckem, Joum.y
p. 29, 517, 1903.
3 Rey-Pailharde, Comptes Rendus^ 106, p. 1683, and 107, p. 43, 1888 ;
C, R. Sac. Biol.y 46, p. 455, 1894.
^ Bach and Chodat, loc. cit.
TISSUE RESPIRATION 135
Tissue Respiration, — Before discussing the probable or
possible part played by oxidases and peroxidases in tissue
respiration, it will be well to adduce evidence to show that the
respiration is probably due to enzymes of some kind or other.
I endeavoured^ to obtain some proof of it by measuring the
gaseous metabolism of a convenient organ, viz, the mammalian
kidney, under various abnormal conditions. The gaseous
exchange was determined by perfusing it continuously with
oxygenated Ringer's solution for about twelve hours. The
gases were boiled off in vacuuo from samples of the saline
solution before and after perfusion, and analysed, and so the
intake of oxygen and output of COg were measured. A kidney
kept for twenty-four hours at a temperature of —8° to — 1°
(whereby it was frozen hard) had about a third the gaseous
metabolism of normal kidneys, whilst another kidney frozen to
— 8° and thawed again three times on three successive days had
about half the normal metabolism. In that Pictet * found that
frogs survived exposure to a temperature of —28°, it m^ht
reasonably be held that this freezing did not kill the tissues, so
in another series of experiments the kidneys were heated for
half an hour to various temperatures up to 60° before they were
perfused. It has been found by Halliburton * that the kidney
contains a cell globulin coagulating at 50° to ^s""^ and it has
been shown by Brodie and Richardson,* and by myself,^ that
muscle when gradually heated undergoes shortening in several
steps, at temperatures which roughly correspond with those at
which the muscle proteins undergo coagulation. So presumably
exposure of a kidney to a temperature of over 50° would cause
some protein coagulation, or would precipitate one of the
constituents of the protoplasm of the cells, and would thereby
destroy the vitality of these cells. Yet I found that a kidney
retained about a fifth of its normal gaseous metabolism after
being heated to 53°, whilst kidneys heated to 55° and 60"* retained
a tenth of their metabolism. Again, perfusion of a kidney with
* Vernon^ Jaum. Physiol.^ 35, p. 53, 1906.
* Pictet, Rev, Scienty 52, p. 577, 1893.
3 Halliburton, /^wm. Phy^L^ 13, p. 810, 1892.
* Brodie and Richardson, PhiL Tram, Roy, Soc.^ 191 B, p. 127, 1899.
^ Vernon, /^wr«. Pkysiol,^ 24, p. 239, 1899.
136 OXIDISING ENZYMES
I per cent, sodium fluoride or i per cent arsenious acid solution
caused the tissue respiration to fall to less than a third the
normal during the next few hours, but it did not absolutely
stop it even in three days. In all of these experiments the
respiratory quotient kept at about -85, or the ratio of CO2
production to oxygen absorption was the same as in the normal
living kidney.
Upon plants numerous observations have been made by
Palladin^ and his co-workers. The vegetable organs under
observation were killed by freezing them at a temperature of
— 20° to — s"" for twenty hours. A current of hydrogen was then
drawn over them at room temperature, and the CO2 outflow
determined. From etiolated leaves of the vetch the CO2 came
off at the rate of '028 gm. per hour per 100 gm. of tissue during
the first four hours ; at '009 gm. per hour during the next four
hours, and at -0024 gm. per hour during the next fifteen hours.
Palladin suggested that this CO2 was formed by a hypothetical
*' carbonase " enzyme. On replacing the hydrogen by an air
current, a fresh outflow of CO2 began, and continued at the
rate of -007 to -005 gm. per hour for the next forty hours or
more. Its formation was supposed by Palladin'to be due to an
oxidase enzyme. The amounts of CO2 evolved from germinating
wheat and etiolated vetch leaves varied between the following
limits. They are calculated for 100 gm. of plant substance : —
Plant.
In Hydrogen Current
(Carbonase).
In Air Current
(Oxidase).
Germinating Wheat ....
Etiolated Leaves of Vetch .
1025 to 1*282 gm.
•100 to '185 gm.
•142 to •245 gm.
No direct observations upon the oxygen intake were made
in these particular experiments, but on keeping other
(previously frozen) etiolated vetch leaves in a closed volume
of air over mercury, and analysing samples from time to time,
it was found that the oxygen was absorbed in rough proportion
1 Palladin, Zeif. /. physioL Chem,, 47, p. 407, 1906 ; Palladin and
Kostytschew, /^V/., 48, p. 214, 1906; Maximow, 5^r. d. Deutsch. bot, Ges.^
22, p. 904 ; Tschemiajew, ibid., 23, 1905 ; Palladin, ibid., 23, 1905 ; 24, 1906 5
Krasnosselsky, ibid., 1906.
VEGETABLE RESPIRATORY ENZYMES 137
to the COg given out. The actual respiratory quotients
obtained varied froni 2*o to '3.
It might naturally be asked if there is any proof of the
existence of these carbonases and oxidases which are supposed
by Palladin to be responsible for the CO2 formation. No
attempts were made to isolate them, and in fact Palladin lays
stress on the fact that any injury to the anatomical structure
of the dead plant acts destructively on the activity of the
respiratory enzymes. Hence the proof of their existence
depends entirely on this output of CO2 from the dead plants.
The COg evolved in a hydrogen current is very probably due
to an intracellular zymase, whilst that subsequently evolved in
an air current may with considerable probability be referred to
an oxidase. Palladin found that there was a further output of
CO2 if the plant leaves were rubbed up in a mortar with
pyrogallic acid solution, and the mixture kept in an air current :
but probably this COg is not of enzymic origin, as Stoklasa,
Ernest, and Chocensk^ ^ found that if vegetable tissues such as
the leaves and root of the beet were slowly dried, powdered,
and then heated to 1 50° for fourteen hours, so as to destroy all
the enzymes, they still yielded about as much COg when treated
with pyrogallic acid as the fresh tissues did.
The great instability of the respiratory enzymes is shown
by Palladin and Kostytschew's observations upon peas. As
was stated in the previous lecture, living seeds of the pea and
other plants form COg and alcohol when kept in a hydrogen
atmosphere. But living seeds kept in air show no accumulation
of alcohol, />. they are able to oxidise it as it is formed. Frozen
pea seeds, on the other hand, accumulate a considerable amount
of alcohol in presence of full oxygen supply, so the freezing
seems to destroy their respiratory oxidases more than their
zymase. For instance, 200 frozen pea seeds, kept at 20° for 98
hours in a current of air, formed 1-482 gm. of COg and 1-013 gm.
of alcohol, or in proportion of 100 to 68. An equal quantity of
frozen seeds kept for the same time in a current of hydrogen,
formed -775 gm. of COg and -553 gm. of alcohol, or in proportion
of 100 to 71.
Palladin's conclusion that the respiration of plants is
^ Stoklasa, Ernest, and Chocensky, Zeitf, physioL Chem,^ 50, p. 303, 1907.
S
138
OXIDISING ENZYMES
dependent on their structural integrity might be thought to
apply to animal tissues as well, but this is very far from being
the case. Thunberg ^ pounded up the muscles of one leg of a
frog in a mortar for 15 to 30 minutes, and compared their
respiration with that of the uninjured muscles of the opposite
limb by means of his microrespirometer. During the first hour,
at room temperature, the pounded muscles absorbed 3*07 c.c. of
oxygen per 100 gm., whilst the uninjured muscles absorbed
5-94 C.C., or nearly twice as much. In succeeding hours the
oxygen absorption of the uninjured muscles remained fairly
constant, whilst that of the injured muscles rapidly deteriorated,
so that in the fourth hour the former absorbed 4-86 c.c. of
oxygen, and the latter 1-22 c.c, or a fourth as much. Lussana^
crushed fresh tissues of the rabbit in a metallic sieve, and found
that 100 gm. of liver, kept in a closed vessel of air at a tempera-
ture of 35° to 40° for four hours, absorbed 39' 5 c.c. of oxygen and
gave out 44-8 c.c. of COg, whilst muscle under similar conditions
absorbed 12-2 c.c. of oxygen and gave out 87 of COg.
Much more striking are the results obtained by Battelli and
Stern.^ These observers minced up the tissues finely, and put
40 gm. of them with 100 c.c. of blood or other liquid in a
600 C.C. flask full of oxygen. The flask was kept in a water-
bath at 38°, and was rapidly shaken by mechanical means.
After half an hour or an hour the change in the volume of the
gas in the flask was measured and a sample of it was analysed.
The following data show the volumes of oxygen absorbed by
100 gm. of tissue in half an hour. The organs were removed
from the dog and minced within 20 minutes of death : —
Kidney
Heart muscle
Skeletal muscle .
Liver ....
242 C.C.
194 i^
184 »
176 „
Brain ....
Pancreas
Thyroid
Spleen.
82 C.C.
76 „
26 „
19 M
As a rule the liver was somewhat more active than skeletal
muscle. Otherwise these data correspond with the average
results. It will be seen that they show an extremely large
^ Thunberg, Hammarstetis Festschrift^ Upsala, 1906.
2 Lussana, Arck, di FisioL^ 3, p. 113, 1906.
' Battelli and Stern, Joum, dephysioL^ 9, pp. i, 34, 228, and 410, 1907.
TISSUE RESPIRATION 139
oxygen absorption, much larger, in fact, than that found in
living animals. Regnault and Reiset and others found that the
dog absorbs 700 to 1 300 c.c. of oxygen per kilogram per hour,
ue. 35 to 65 cc per 100 gm. per half-hour, or very much less
than the majority of these minced tissues. Again, Barcroft^
found that individual organs of the dog, perfused with oxygen-
ated blood under normal conditions, absorb the following
volumes of oxygen per 100 gm. per half-hour when in a resting
condition ; pancreas, 1 50 c.c ; kidney, 90 cc ; muscle of leg,
6 cc. It would seem, therefore, that the mechanical injury to
the tissues for the time being heightens their oxygen absorp-
tion powers considerably. It increases their COg production
in similar proportion, for the quotients obtained varied, as a
rule, from 7 to 1-4. After the first few minutes, the gaseous
metabolism rapidly diminished, and in some cases ceased alto-
gether after an hour or so. For instance, minced horse muscle,
placed in saline, absorbed oxygen at the following rates per
hour: 195 cc. during the first 10 minutes; 240 cc. in the next
20 minutes ; 70 cc in the next 30 minutes ; and 6^ cc in the
next 30 minutes. Some rabbit's muscle ceased absorbing oxygen
after an hour and some horse's liver almost ceased absorbing
after 40 minutes. But the results are extremely variable and
irregular, and the method used by Battelli and Stern is
undoubtedly a rough one and liable to considerable error.
When the minced tissue and blood were shaken up with air
instead of oxygen, the oxygen absorption was diminished by
about 50 per cent, in the case of muscle, but only by about 20
per cent, in the case of other tissues. Again, the oxygen
absorption was much smaller if the minced tissue were shaken
up with saline instead of with blood. For instance, ox muscle
in saline absorbed 50 cc of oxygen per hour : in a mixture of
2 of saline and 3 of blood, 132 cc. per hour, and in blood only,
177 cc. per hour. These differences were not due to any
appreciable absorption of oxygen by the blood itself, but were
presumably owing to the haemoglobin of the blood acting as a
more efficient oxygen carrier to the tissues.
Other observations were made upon the vitality of the
tissues after death of the animal. Muscle of the dog, kept at
^ Barcroft, Biochem* Jiwm,, i, p. 6. 1906.
140 OXIDISING ENZYMES
o° for seventy minutes after death, and then minced and placed
in I per cent, disodium phosphate solution, absorbed 196 c.c of
oxygen in half an hour, whilst muscle kept eighteen hours at o**
before mincing absorbed 58 c.c. Some of the same muscle kept
for seventy minutes at 30° before mincing, absorbed 247 c.c. of
oxygen, and when kept for seven hours at 30°, it absorbed only
6 cc. Antiseptics greatly diminished the respiratory powers of
the tissues. For instance, some rabbits* liver, when placed in
blood and sodium phosphate solution, absorbed 118 c.c. of
oxygen in half an hour, but when i per cent of NaF was added
it absorbed 23 c.c ; with -oi per cent. KCN it absorbed 21 c.c. ;
and with -02 per cent, of arsenite, only 6 c.c.
As Battelli and Stern point out, the minced tissues used in
their experiments undoubtedly contained many intact and still
living cells, and hence one is not justified in assuming their
respiration to be entirely the work of respiratory enzymes.
Still it is very remarkable that the gaseous metabolism should
have been so large. The experiments, when repeated, should
be made with tissues minced to various degrees of fineness, and
their respiration should be measured over considerably longer
periods. Also it would be of great interest to measure the
gaseous metabolism of the expressed juice of tissues, free of all
solid constituents, with a view to determining if respiratory
enzymes are soluble bodies. Battelli and Stern ^ found that if
freshly minced muscle were shaken up for five minutes with
i^ volumes of water or saline, and the extract were quickly
strained off through a linen cloth, neither it or the solid
residue of minced muscle had much respiratory activity when
kept in a flask of oxygen on the water-bath ; but if extract and
residue were mixed, the mixture had more than three times the
gaseous metabolism of either single constituent For instance,
30 gm. of the solid residue of minced ox diaphragm muscle,
mixed with 10 to 70 cc. of extract, absorbed about 30 c.c of
oxygen in half an hour. The experimental results were very
irregular, but a careful repetition and extension of them by more
exact methods may be of great value to us in elucidating the
mechanism of tissue respiration. As they stand, they seem to
show that respiration is chiefly carried out by the interaction of
^ Battelli and Sitm, /oum, de PhysioLy 9, p. 737, 1907.
TISSUE RESPIRATION 141
a soluble constituent of the tissues with some insoluble con-
stituent of the cellular framework. But it is impossible to make
such a deduction with any safety, as the extraction period of the
minced muscle was so short.
Though in most observations on gaseous metabolism the
respiratory quotient is found to approach unity, there is plenty
of evidence to show that the CO2 output is not directly
dependent upon a contemporary oxygen intake. As long ago
as 1803 Spallanzani showed that a frog continued to exhale CO2
even when entirely deprived of oxygen. Pfliiger,* and sub-
sequently Aubert,^ found that frogs kept in nitrogen containing
no trace of oxygen continued to give out COg for some hours at
a rate but little inferior to that exhibited by frogs kept in air.
They gave it out rapidly and for a brief period if kept at a high
temperature, and slowly and for a long period if kept at a low
one ; but the total volume of CO2 exhaled was practically the
same in all cases, amounting to about 200 c.c. per kilogram
body weight. Thunberg^ found that a snail, Limax agrestiSy
when kept in a nitrogen atmosphere, exhaled niore than 11 00
C.C. of CO2 per kilogram, whilst the mealworm, Tenebrio molitor,
exhaled about 1000 c.c. The writer* found that the mammalian
kidney, when perfused with boiled (oxygenless) saline, gave out
about 100 cc. of CO2 per kilogram.
It is generally assumed that this CO2 is formed at the
expense of a supply of intramolecular oxygen which is stored
up in the tissues, though as Winterstein ^ and the writer « point
out, there is at present no completely satisfactory proof of its
existence. But admitting the probability of its presence in the
tissues, in what form is it stored up ? Verworn,^ arguing from
the fact that increased functional activity of muscles and other
organs has no effect whatever on nitrogenous metabolism,
though it may increase their CO2 output and oxygen intake
1 Pfluger, Pfluget's Arck,, 6, p. 43, 1871 ; 10, p. 251, 1875 ; M, p. 5, 1878.
* Aubert, ibid.^ 26, p. 293, 1881.
2 Thunberg, Skand. Arch./. Physiol.^ 17, p. 133, 1905.
* Vtmon^Joum. Physiol.^ 35, p. 53, 1906.
^ Winterstein, Zeii.f. allgem. Physiol. ^ 6, p. 315, 1907.
" Vernon, Set. Progress^ 2, p. 160, 1907.
' Verworn, Arch. / (^AnaL u.) PhysioL^ 1900, Suppl. p. 152 ; also, "Die
Biogenhypothese," Jena, 1903.
142 OXIDISING ENZYMES
tenfold, supposes that only non-nitrogenous groupings or side-
chains of the "biogen molecules" of the protoplasm are
concerned in the ordinary processes of tissue respiration. He
suggests that these side-chains may be carbohydrate groupings
of an aldehyde character, that the intramolecular oxygen is
stored up in the biogens in the form of an NOg grouping
attached to a benzene ring, and that one atom of oxygen from
the NO2 oxidises the — CHO and — CHOH groups of the
carbohydrate molecule to CO2 and water. This hypothetical
NOg compound is comparable to the organic peroxide structure
which Kastle and Loevenhart attribute to the oxidases of tissue
extracts, and though at present neither they or Verworn have
any direct experimental evidence in support of their views, it is
quite possible that the tissue oxidases may ultimately prove to
be compounds of this nature which have broken away from
the cellular protoplasm in which they functioned as oxygen
carriers.
The hypothesis that the respiratory processes of tissues are
carried out by non-nitrogenous side-chains is supported by some
curious and unexpected results which I obtained when investi-
gating the gaseous metabolism of the mammalian kidney by the
previously mentioned method. I found that the protoplasm is
in a state of such instability that it is liable to undergo sudden
disintegration, whereby as much as 9 to 17 per cent, of the total
protein present in the kidney tissues is washed out during the
course of an eleven-hour perfusion. Yet the respiratory powers
of these kidneys were just as great as those of other kidneys in
which there had been little or no tissue disintegration. The
proof is not a complete one, however, as in all experiments the
gaseous metabolism had dwindled to a half or less of its initial
value by the end of the perfusion, and hence it is possible that
the protein which broke away from the tissues consisted in part
of the no longer functioning respiratory side-chains.
The carbohydrate-like nature of the side-chains concerned in
tissue respiration is supported by evidence quoted in the previous
lecture. We saw that the tissues, either alone or in combination
with one another, had considerable glycolytic power. The
existence of an enzyme of alcoholic fermentation, though not
properly substantiated for animal tissues, seemed fairly well
TISSUE RESPIRATION 143
established for vegetable ones, whilst Magnus-Levy sholved
that the liver and other tissues, when kept under aseptic
conditions, rapidly formed considerable quantities of lactic and
other acids. Though there is no actual proof that these acids
were formed from carbohydrate, probability is all in favour of it.
It is probable that the lactic acid and other intermediate bodies
formed by the decomposition of carbohydrates and other sub-
stances in the tissues are subsequently oxidised to COg and
water by an intracellular oxidase or peroxide which takes up
oxygen from the blood and transfers it to them. In the absence
of an oxygen supply, these intermediate products accumulate
more and more, whilst the COg into which they would normally
have been oxidised rapidly decreases. Thus Fletcher and
Hopkins^ found that absolutely fresh frog's muscle contained
only a trace of lactic acid (-oi 5 per cent, or possibly less), but if
it were placed in an atmosphere of hydrogen or nitrogen, the
acid gradually increased till by the time the muscle passed into
rigor mortis it amounted to -24 to -40 per cent. A muscle
which had accumulated a certain amount of acid as the result of
fatigue or insufficient oxygen supply was able to oxidise and
destroy it, or at least a part of it, if placed in an oxygen
atmosphere. This oxidation may have been the work of an
intracellular oxidase, but if so the enzyme is a very unstable
body ; for it was found that chopped-up muscle, when kept in
oxygen, is unable to oxidise and destroy its lactic acid.
However, the oxidising power of the tissues can be destroyed
by less violent methods than mechanical disintegration, for I
found that if a kidney were perfused with saline containing
•06 to -ID per cent, of lactic acid, or '00$ to '025 per cent of free
ammonia, it somewhat rapidly lost its oxygen-absorbing power,
but it still more rapidly lost its COg-producing power, so that
after four to eight hours' perfusion its respiratory quotient
dropped to -46 to -36 : i.e. only half the usual proportion of oxygen
absorbed was being used to oxidise tissue constituents to COg.
It will be seen that at present we are supremely ignorant as
to the connection of the oxidases and peroxidases with tissue
respiration. Many of the isolated experimental results above
described are of great interest in themselves, and they suggest
^ Fletcher and Hopkins, yi7«r«. PhysioL^ 35, p. 247, 1907.
144 OXIDISING ENZYMES
that the connecting links may be discovered in course of time.
But much further investigation will first be required, not only to
elucidate these links, but to sweep away a good deal of the
inexact and careless experimental work with which this
particular problem is especially encumbered.
LECTURE VI
THE CONSTITUTION AND MODE OF ACTION OF ENZYMES
Preparation of pure pepsin. Its protein-like nature. Influence of proteins
on stability of enzymes. Precipitability of enzymes. Relation of
rennin to pepsin, trypsin, and other proteolytic enzymes. Adsorption
of enzymes and of dyes. Slight diffusibility of enzymes. Optical
activity of enzymes. Chemical combination of enzyme with substrate.
Velocity of enzyme action, and its deviations from law of mass action.
Sufficient evidence has been adduced in previous lectures to
show that many if not most of the chemical processes of living
tissues are dependent upon enzymes, and hence if we are ever
to understand the inner mechanism of these processes, it is
essential for us to understand the chemical constitution and
mode of action of enzymes. Though a large amount of work
has been done upon both of these problems, the positive results
obtained in answer to the former one have hitherto been very
limited. And this for two reasons. Firstly, the enzymes are
such extremely unstable bodies that it is impossible to purify
them without destroying a great deal of their activity; and
secondly, we have no absolute criterion as to how much of the
product obtained is actual enzyme, and how much impurity.
In attempting to isolate an enzyme from a solution, therefore,
it is important that the methods of purification should be as
gentle as possible, and that at each stage of the purification or
concentration a quantitative estimation should be made of the
total amount of enzyme present, so as to determine how much
of it, if any, has been destroyed. In fact, the ideal method
would be to follow that used by Madame Curie in isolating
radium from pitchblende, when the unknown element was
146 J
146 CONSTITUTION AND MODE OP ACTION OF ENZYMES
traced by its radio-activity, and by various purification processes
obtained in greater and greater concentration, till the prepara-
tion of maximum radio-active power was found to be the pure
radium salt. Probably such a method would be impossible with
enzymes, unless a temporary stability could be artificially
induced, but there seems to be no other way of proving the
purity of an enzyme preparation.
Practically all the attempts hitherto made to isolate a pure
enzyme have been upon exoenzymes, but we have every reason
for thinking that the endoenzymes closely resemble these
bodies both in chemical and physical properties, and that what
holds for the one class holds almost equally well for the other.
Of the proteolytic exoenzymes, pepsin and to a less extent
trypsin have received the most attention, but I do not intend
to describe in detail the elaborate methods used by the earlier
observers such as Briicke ^ and Kiihne to isolate these enzymes,
as they could by no possibility have yielded anything like a
pure product. Briicke, for instance, allowed pig's stomach to
digest Itself for several days in presence of phosphoric acid —
whereby a very large amount of the enzyme must have been
destroyed — and then threw down a precipitate of calcium
phosphate by the addition of lime water. The colloidal pepsin
was adsorbed by this precipitate and carried down with it, but
so was much of the protein impurity. The precipitate was
dissolved in dilute HCl, and a second precipitate thrown down
by the addition of more lime water. Each of these precipita-
tions and re-solutions would undoubtedly destroy a large
amount of the enzyme, though Briicke did not attempt to
determine whether this was the case or not. The pepsin was
then precipitated still a third time with cholesterin, and after
various washings the cholesterin was extracted with ether, and
a small quantity of slimy substance was left This, when
dissolved in -i per cent HCl, was able to digest a flake of fibrin
in about an hour ; but a few drops of it diluted with 5 cc of
•I per cent HCl dissolved fibrin equally quickly, so it must
undoubtedly have contained some impurity which checked its
activity when in concentrated solution. The solution did not
seem to contain a trace of protein, as it failed to give a cloud
^ Briicke, Sitzungsb. d, k. AkcuL d. Wiss. Wim.\ 4S> P- 601, i86i.
ISOLATION OF PEPSIN 147
with nitric acid, tannin, or mercuric chloride. Hence Briicke
concluded that pepsin cannot be a protein body.
It is probable, though there are no quantitative data bearing
on the point, that the method of producing an inorganic
precipitate in an enzyme solution, in the hope that it will carry
down the enzyme with it but not the impurities, is a futile one,
and calculated to destroy a larger proportion of enzyme than
it removes of impurity. Salting out with ammonium sulphate
or other salt is probably a much better method, as it is less
violent, whilst fractional precipitation with alcohol may in some
cases be a usefur method, though it should always be accom-
panied by- quantitative enzyme estimations.
The methods used by re'cent workers for the isolation of
pure pepsin do not suffer from many of the disabilities of the
older ones. In the first place, it is possible to obtain a very
active pepsin solution containing but few of the protein and
other impurities which must be present in every extract of
gastric mucous membrane. This is done by giving a fictitious
repast to a dog in which oesophageal and gastric fistulae have
been made in accordance with Pawlow's mdthod. In this way
several hundred cubic centimetres of pure gastric juice are
obtained, uncontaminated by any food material. Schoumow-
Simanowsky^ found that if this juice were cooled to o°, a
fine powdery precipitate was thrown down which seemed to
consist of pure pepsin. Saturation with ammonium sulphate
threw down a precipitate of very similar composition, as can
be seen from the data given in the table below. These figures
show that the precipitated product had the composition of
proteins with the addition of -So to 1-17 per cent, of chlorine
(present as hydrochloric acid). No proof was furnished of the
identity of the precipitates with pepsin, other than similarity of
composition and their very great digestive activity.
Nencki and Sieber ^ endeavoured to obtain a purer pepsin
from gastric juice by the method first adopted by Pekelharing.^
This depends on the fact, first noted by him, that if an extract
of pig's stomach containing pepsin and hydrochloric acid be
* Schoumow-Simanowsky, Arch,f,exp, Path,^ 33>:P» 33^, 1894.
2 Nencki and Sieber, Zeit f, physioL Chem,^ 32, p. 291, 1901.
3 Pekelharing, ibid,^ 22, p. 233, 1897.
148 CONSTITUTION AND MODE OF ACTION OF ENZYMES
dialysed for fifteen to twenty hours, all but -02 per cent, of the
HCl dialyses away, and a precipitate is thrown down. This
precipitate, when dissolved in dilute hydrochloric acid, digests
proteins very vigorously, and was thought by Pekelharing to
be pepsin mixed with a variable amount of a phosphorus-
containing body as impurity. Nencki and Sieber found that
the product thrown down from dialysed gastric juice had an
elementary composition similar to that found by Schoumow-
Simanowsky, except that it contained only about half as much
chlorine. It also contained •073 to -148 per cent of phosphorus,
and -11 to -18 per cent of iron. Hence they concluded that
pepsin is a nucleoprotein containing iron, and that it is bound
up with HCL They found by qualitative tests that small
quantities of lecithin were present, and they thought that it
was in chemical combination with the pepsin, and not merely
mixed with it
Pepsin preparod by
C.
H.
'n.
S.
01.
CooliDg Juice to 0' (Schoumow-Simanowsky)
Precipitating with (NH4)2S04 „
Dialysing Juice fNencki and Sieber) ,
„ „ (Pekelharing)
Precipitating with (NH4)2S04 (Pekelharing)
50-73
50-37
51-26
51-99
52-16
7-23
6.88
6.74
7-07
7-09
1477
14-33
14.44
14.70
.98
1.29
1.83
IK)9
.84
-475
•49
Nencki and Sieber were mistaken in ascribing such a highly
complex structure to pepsin, as Pekelharing,^ who subsequently
prepared pepsin by the same method, obtained a colourless
product which contained only -oi per cent of phosphorus, whilst
by ammonium sulphate precipitation he obtained a pepsin con-
taining no phosphorus whatever. It could not possibly be a
nucleoprotein, therefore, and the small quantity of phosphorus
found by Nencki and Sieber, and by himself in his earlier
preparations, must have been due to impurity. He took care
to filter his gastric juice before dialysing, which Nencki and
Sieber did not do, and this may have removed small quantities
of nucleoprotein-containing mucus and cell debris shed from the
gastric mucous membrane. Pekelharing did not make any
analyses of the iron content of his preparations, but there can
1 Pekelharing, Zeitf, physioL Chem,^ 35> P- h 19^*
COMPOSITION OF PEPSIN 149
be little doubt that it is an impurity, even though Nencki and
Sieber found it was always present in fairly constant amount
Supposing the pepsin molecule contained only a single atom
of iron, its molecular weight would come to about 50,000 if
only •!! per cent, of iron were present; but such a high
molecular weight is improbable, as in its physical properties
pepsin closely resembles other proteins of lower molecular
weight It is precipitated by half saturation with ammonium
sulphate, and is coagulated and destroyed at about the same
temperature as they are. Thus a solution heated to 65° for two
minutes becomes faintly opalescent, and loses a little of its
digestive power ; heated to 70° it becomes opalescent, and loses
most of its digestive power; heated to 75° it forms a white
cloud, and loses the whole of its digestive power.^
Hence we may provisionally conclude that pepsin is a protein
body containing neither iron or phosphorus. FriedenthaP
found that it contains a pentose group, capable of yielding
an osazone with phenylhydrazine, and Pekelharing confirms
this conclusion. Pekelharing found almost exactly the same
percentage of chlorine as did Nencki and Sieber, and he agrees
with them in thinking that this chlorine is a constituent of the
protein molecule. When the pepsin was well washed with 96
per cent alcohol, -29 per cent of this -48 per cent of chlorine
present was removed. The enzyme at the same time completely
lost its digestive activity, so perhaps this is definitely dependent
upon the presence of the chlorine.
If pepsin is a protein-like body, how is it that Briicke and
other observers have obtained purified preparations of the
enzyme, which according to them showed considerable digestive
activity but which gave few or none of the protein reactions ?
The explanation of this apparent contradiction probably
depends on the fact that the digestion test for pepsin is a
very much more delicate one than any known protein test,
and in their efforts to purify their pepsin these observers
obtained a solution too dilute to give aught but the digestion
test. Hofmeister ^ states that the biuret test is not yielded by
^ Pekelharing, ZeiL f, physioL Chem,^ 22, p. 242, 1897.
2 Friedenthal, Arch,/, {Anat u.) Physiol,^ 1900, p. 186.
3 Hofmeister, Zeit f, physioL Chent,^ 2, p. 291, 1879.
160 CONSTITUTION AND MODE OF ACTION OF ENZYMES
a I in 10,000 protein solution. Nitric acid gives a cloud with a
I in 20,000 solution, and Millon's test also gives a positive result
at this dilution, but not at still greater dilutions. Potassium
ferrocyanide and acetic acid give a distinct cloud with a i in
50,000 solution, but not at double the dilution. Tannic acid,
phosphotungstic acid, and Brlicke's reagent are the most delicate
tests of all, as they give a reaction with a i in 100,000 solution
of protein. However, Pekelharing states that -000,001 gm. of
his pepsin, dissolved in 6 c.c. of -2 per cent. HCl, dissolved a
flake of fibrin in some hours, so sixty times this quantity of
enzyme would yield a digestive solution of considerable activity.
Yet even then it would form only a i in 100,000 protein solution,
or would give none of the usual protein reactions except the last
three cited.
The evidence concerning the preparation of pure pepsin
has been described at some length, as it seems to me that in
this one case the probabilities are in favour of a nearly pure
product having been obtained. The method of preparation is
so simple that there can have been very little destruction of
enzyme during its progress. The same cannot be said of the
processes employed with any other enzyme, and hence it will
not be necessary to describe them in detail. Trypsin, for
instance, has probably never been obtained in a condition
of even approximate purity. It has hitherto been prepared
from pancreatic extracts, which must contain a large
amount of protein impurity, as well as erepsin and
other intracellular enzymes. Pancreatic juice would afford
no more suitable a source of the eqzyme, as it contains
large amounts of protein and of other enzymes, and more-
over it would have first to be activated by the addition of
enterokinase.
For a long time past it has been urged that enzymes are
nucleoprotein bodies, and though this cannot be true of pepsin,
there is no reason why it should not hold for other enzymes.
In the case of fibrin ferment, for instance, a large amount of
evidence has been adduced in favour of this view, for it is
found that snake venoms and extracts of tissues and of alcohol-
coagulated blood serum contain nucleoproteins, and can also
coagulate fibrinogen. But no adequate proof has yet been
ISOLATION OF INVERTASE 151
affibrded that the clotting is due to the nucleoprotein and not
to a thrombin adsorbed by it
Again, it is possible, though not probable, that some
enzymes are not protein bodies at all, 0*Sullivan and
Tomson^ isolated the invertase from aqueous solutions of
yeast by the gradual addition of 70 per cent, alcohol. After
standing two days, the precipitate thrown down was wzished in
alcohol of 47 per cent concentration, and was then re-dissolved
in water. By no means all of the precipitate passed into
solution, but almost all of the invertase must have done so, as
only 12 per cent of the original inverting power was lost The
enzyme preparation contained some ash, most of which could
be removed by dialysis, but otherwise O'Sullivan and Tomson
think that it was fairly pure. It contained 46-4 per cent of
carbon, 6-63 per cent, of hydrogen, and 3-69 per cent of
nitrogen, and may have been a combination of protein with a
carbohydrate. However, there is no proof that the enzyme
was at all pure, as on attempting to purify it by fractional
alcohol precipitation it quickly lost its activity. In fact, the
purer the enzyme the sooner did it lose its activity and the
more readily was it destroyed by alcohol. The stability of the
enzyme was found to be greatly increased by the presence of
cane-sugar. When a solution of the purified enzyme was
quickly heated to 45°, and cooled, no less than 70 per cent, of
its activity was lost, and when heated to 50°, 98 per cent was
lost. But when cane-sugar was present it could be heated to
60° without losing any activity at all, and on heating to 70° it
lost only 66 per cent, of its activity. This protective influence
of the sucrose is due, in all probability, to the enzyme entering
into a loose combination with it
Purified preparations of invertase and other enzymes are
more unstable than impure ones, largely because of the absence
of protein impurity. Any excess of alkali, acid, salt, or other
substance present which may tend to act harmfully upon the
enzyme then reacts and enters into loose combination with
the protein, and to a corresponding degree spares the protein-
like enzyme. Thus Falk^ found that the retardation exerted
1 O'SuUivan and Tomson, /<?«r«. Chetn. Soc, Trans.^ 1890, p. 834..
2 Falk, Virchov^s Arch,, 84, p. 119, i88i.
152 CONSTITUTION AND MODE OF ACTION OF ENZYMES
by a small amount of hydrochloric acid upon the amylblytic
action of saliva was much diminished if peptone were present
Chittenden and Ely^ found that peptone would also prevent
sodium carbonate from exerting its retarding action upon
saliva to a large extent. Langley and Edkins ^ observed that
the destructive action of sodium carbonate upon pepsin is
diminished by the addition of proteins. This was probably
due, they thought, to the alkali combining with the protein,
" for the greater the amount of sodium carbonate present, the
greater must be the amount of protein to lessen appreciably
the destruction." Biernacki^ found that proteoses and peptones
exert a considerable protective influence upon trypsin, for the
trypsin of pancreatic extracts was completely destroyed when
kept for five minutes at 50° with from -25 to -5 per cent of
Na2C03, but if proteoses or peptones were added, it had to be
heated to 60° before undergoing a similar rapid destruction.
Even at 37^ trypsin is rapidly destroyed by sodium carbonate,
for I found* that in one hour -4 per cent. NagCOg destroyed
from 55 to 75 per cent of the enzyme in a fresh preparation.
If any protein, proteose, or peptone were present, however, it
protected the enzyme in proportion to its concentration. Thus
in presence of -4 per cent of protein I found ^ that on an
average 45 per cent of the trypsin was destroyed in an hour ;
with I per cent of protein, 27 per cent was destroyed ; with
2 per cent of protein, 12 per cent was destroyed; and with
4 per cent of protein, only 7 per cent was destroyed. When
no protein at all was added, 56 per cent of the trypsin was
destroyed in the hour. Protein decomposition products could
protect the trypsin as well as protein itself, and in fact the
protective influence of a substance seemed to depend entirely
upon its power of neutralising the sodium carbonate. Amino
acids such as glycin, taurin, leucin, aspartic acid, and hippuric
acid acted as efficiently as proteins, whilst creatin, urea, glucose,
and maltose, which are unable to combine with alkali, exerted
* Chittenden and Ely, Amer. Chem, Joum,^ 4, p. 107, 1882 ; Joum.
Physiol, 3, p. 327, 1882.
2 Langley and Edkins, /<9«r«, PhysioL, 7, p. 371, 1886.
3 Biernacki, Zeitf, Biol., 28, p. 49, 1891.
* Vernon, /oum, PhysioL, 27, p. 269, 1901 ; and 28, p. 448, 1902.
* Vernon, ibid., 31, p. 346, 1904.
PRECIPITABILITY OF ENZYMES 153
no protective effect whatever. It seems highly probable,
therefore, that trypsin, pepsin, and other enzymes resemble
proteins in being able to act as weak acids when in alkaline
solution, and as weak alkalis when in acid solution. The
protective influence of proteins and their decomposition
products upon them is chiefly one of mass action, as they
can likewise act as pseudo-acids or pseudo-bases, and combine
with the destructive alkali or acid present.
Though we are justified in regarding enzymes as protein-
like bodies, it is probable that they differ as much from one
another in their chemical constitution and physical properties
as do the proteins from which they may be derived. For
instance, they differ a good deal in their precipitability.
Danilewsky^ found that if collodion solution were added to
pancreatic extracts, a precipitate was thrown down which when
extracted with water gave a solution containing trypsin but no
diastase. The filtrate from the precipitate, however, contained
a large amount of diastase, but only a little trypsin. These
results have been partially confirmed by Lossnitzer.^ Also
Dastre^ found that trypsin is only slightly soluble in 50 per
cent alcohol, whilst pancreatic diastase is slightly soluble in
65 per cent, alcohol. I have carried out * systematic fractional
precipitations of diluted glycerin extracts of pancreas with
alcohol, and have estimated the amounts of tryptic, rennetic,
and diastatic enzymes still left in solution in the filtrates, and
those present in solutions of the precipitates. As can be seen
from the data in the table, increasing strengths of alcohol threw
down increasing but proportionate amounts of trypsin and
rennin, so that the ratio of the one ferment to the other was
constant both in the filtrates and in the solutions of the
precipitates. In confirmation of previous observers, I found
that the diastase was much less readily precipitable. The
filtrate from a mixture of i part of extract with 2-5 parts of
alcohol contained more than four times as much diastase,
relative to trypsin, as the original extract In every case the
* Danilewsky, VirchoTt^s Arch,^ 25, p. 279, 1862.
^ Lossnitzer, Arch, d, Heilk,^ 5, p. 556, 1864.
3 Dastre, Arch, de Physiol,^ 8, p. 120, 1896.
* Vernon, /<?i/r«, Physiol.,^ 29, p. 302, 1903.
U
154 CONSTITUTION AND MODE OF ACTION OF ENZYMES
processes of precipitation and re-solution of the enzymes
destroyed about 45 per cent, of the trypsin and rennin present,
but with the greater strengths of alcohol (2 to 3 vols.) no less
than 76 to 86 per cent, of the diastatic enzyme was destroyed.
Other experiments showed that with more concentrated
glycerin extracts the addition of three volumes of alcohol
might destroy 99 per cent, of the diastatic enzyme in three
hours. Hence fractional alcohol precipitation is obviously not
Filtrate.
Precipitate dissolved
in Water.
•2 J
|l
•1-
Is
•1-
is
Glycerin Extract alone ,
I of Extract to '8 of Alcohol .
I M I .. .
I „ 1-5 .,
I » 2 „ .
I M 2.5 „
I „ 3 V .
317
290
26.6
20*8
9.4
2.2
•0
95-5
79.6
72.7
S5-9
28.3
...
30
2-7
2-7
2-7
3-0
89-3
8l-2
79.8
680
56.6
26.1
2.6
2.8
2-8
30
3-3
60
11.9
20
4.2
8.6
15-2
22-1
24.6
I7'2
31-3
45-2
52-1
20
2-1
2-0
2'I
4.6
6.4
7-4
7-7
9-7
12a
a suitable method for the purification of these particular
enzymes.
The fact that trypsin and rennin have identically the same
precipitability by alcohol raises a point of some interest, and
one which has received a good deal of attention of recent years.
Nencki and Sieber^ found that the pepsin they obtained by
dialysing gastric juice had a milk-coagulating function as well
as a peptic one, and they suggested that one and the same
molecule might have different enzyme actions. Just as Ehrlich
in his side-chain theory considers that these chains have
different configurations and functions, so they think that the
pepsin molecule may exert a hydrolytic function by one of its
side-chains, and a milk-coagulating one by another. Still they
admit that their pepsin-rennin preparation may have been a
mixture, as they made no attempt to separate the two enzymes.
However, Grutzner,^ and subsequently Winogradow,^ found that
* Nencki and Sieber, Zeitf, Physiol, Chem.^ 32, p. 291, 1901.
2 Griitzner, Pfliiger^s Arck., 16, p. 119, 1878.
3 Winogradow, /toT., 87, p. 120, 1901.
PEPSIN AND RENNIN 155
the quantities of peptic and of rennetic ferments in the gastric
mucous membrane at various times after a meal run completely
parallel. Pawlow and Parastschuk ^ fed dogs, in which a small
side stomach had been made, with milk, meat, and bread, and
found that the juice collected from the stomach during
successive hours after a meal showed parallel changes in its
protein-digesting and milk-coagulating power with the different
diets. Also the acid juice, when kept in an incubator and
tested from day to day, was found to deteriorate to an equal
extent in respect of both these activities, and the same thing
wsis observed when the juice was heated for a short time to a
temperature of 50° to 60°. The same parallel between proteo-
lytic and milk-coagulating power was observed with activated
pancreatic juice. Hence Pawlow and Parastschuk conclude that
both these activities are due to one and the same enzyme, and
they show that Hammarsten was mistaken in supposing that he
had been able to separate the pepsin and rennin in gastric juice.
It Weis stated by Bang ^ that the rennet ferment in extracts
of calf stomach differed from the ferment in extracts of pig's
stomach in that it was differently affected by the addition
of calcium chloride and of alkali, and was more rapidly
destroyed when heated to 70°. Gewin'^ found that these
differences were due to the presence of impurities, and that
the enzymes differed less and less from one another the more
they were purified. He also found that minced coagulated egg
albumin adsorbed pepsin and rennin equally from a solution of
the two ferments, and that half saturation with ammonium sul-
phate precipitated them equally. Hence he agrees with Pawlow
and Parastschuk as to the identity of pepsin with rennin.
On the other hand, I have made a number of observations
upon pancreatic extracts which seem to show that Nencki and
Sieber's hypothesis is the correct one, and that the proteolytic
and milk-coagulating powers are due to different side-chains of
a single enzyme molecule. Thus I found* that if dilute
alcholic and saline extracts of pancreas were kept for some
months and tested from time to time, their tryptic and rennetic
1 Pawlow and Parastschuk, Zeit f. physioL Chem,^ 42, p. 415, 1904.
2 Bang, Pfliiger^s Arch,^ 79, p. 425.
^ Gewin, Zeit f. physiol. Chem,^ 54, p. 32, 1907.
* VexnoTiy Journ. Physiol,^ 27, p. 269, 1901.
156 CONSTITUTION AND MODE OF ACTION OF ENZYMES
powers varied independently, so that at one time the rennetic
power, relative to the tryptic, might be seven times greater than
at another. As a rule the trypsin is more unstable than the
rennin, and when in two experiments the minced pancreas was
kept for twenty-five and sixty-three hours respectively before
the addition of the extracting liquid, it was found that 83 and
75 per cent, respectively of the tryptic activity was lost, whilst
in the first experiment only 30 per cent, of the rennetic power
was lost, and in the second experiment, none whatever of it.
Still the tryptic and rennetic enzymes of an extract, though
they can vary independently and be to some extent destroyed
independently, can never be separated from one another. We
saw above that their precipitability by alcohol was identical,
and I found 1 that if minced pancreatic tissue were fractionally
extracted, they passed into solution at the same rate. The
minced pancreas was shaken for one or two hours with twice its
volume of the extracting medium (slightly diluted glycerin or
dilute alcohol), and was then separated from it by centrifugali-
sation. Two more volumes were added, and these were
separated in the same way after about twenty hours' extraction.
The next extraction lasted four days, the next eight days, and
the next twenty-five days. The amounts of trypsin, rennin, and
diastase in these several extracts were estimated, and it was
found that the ratio of rennin to trypsin was practically constant
throughout. For instance, in the five glycerin extracts of a pig's
pancreas, the rennetic values were 2-8, 2-4, 2-7, 3-1, and 3-1 times
the respective tryptic values. On the other hand, the ratio of
diastase to trypsin varied greatly, and in one experiment it sank
from 3-9: I for the first extract, down to -34: i for the last
It seems probable that a rennetic enzyme invariably
accompanies every proteolytic one, even though it may be
impossible for it ever to exert its milk-coagulating power. Thus
it is present in the stomach of fishes, and the pancreas of many
animals : in the fruit, seeds, and leaves of many plants, and in
many micro-organisms. Edmunds^ found that it is present in
small quantity in the liver, spleen, kidney, thyroid, thymus, lung,
muscle, brain, small intestine, testis, and ovary, or is as uni-
* Vernon, Jourtu PhysioL^ 28, p. 448, 1902.
* Edmunds, ibid,^ 19, p. 466, 1895.
CONSTITUTION OF ENZYMES 157
versally present as /8-protease and erepsin. It may not always
be found in sufficient quantity to curdle milk, for I have shown ^
that the proteolytic ferment is antagonistic to the milk-curdling
action, in that it tends to dissolve the curd as fast as it is
formed. But the presence of the rennin can always be
demonstrated by means of Roberts' "metacasein" reaction.
Because of its universal occurrence, it seemed to me possible,
if not probable,^ that rennin is not a genuine ferment of
functional significance, but is merely a by-product in the
formation of proteolytic enzymes, which chances to possess the
property of curdling milk.
The enzymes, though protein-like bodies, must evidently
possess certain groupings which are not present in the inert
protein molecule, and which confer upon them their lability and
their catalytic powers. What these special groupings consist of
we are entirely ignorant Loew* suggests that they may be
ketone groups, in that hydrocyanic acid, which readily forms
additive compounds with ketones, paralyses the action of many
enzymes. If the hydrocyanic acid is removed, the enzyme
recovers its activity, so that its combination with the ketone
groups must be a loose one, easily dissolved. Again, hydrazine
and hydroxylamine, which in i per cent solution at 40° destroy
the activity of pepsin, trypsin, and diastase in two to four hours,
likewise react readily with ketone groups to form hydrazones
and oximes respectively. Loew thinks that the lability of
enzymes is heightened by the presence of amino groups. Thus
nitrous acid, which even in dilute solution readily acts upon
amino groups, destroys enzymes very rapidly. Formaldehyde
behaves similarly, though greater concentration is necessary.
But in any case there can be but little doubt that enzymes,
in virtue of their protein-like nature, contain amino groups.
The lability of some enzymes seems to be a variable
factor, quite apart from the presence or absence of protein or
other substances conferring stability upon them. As already
mentioned, I found * that the tryptic enzyme in fresh pancreatic
1 Vernon, /M^m. Physiol.^ 27, p. 176, 1901.
2 Vernon, ibid,, 28, p. 470, 1902 ; 29, p. 331, 1903.
* Loew, Pfliiget^s Arch., 102, p. 95, 1904.
* Vernon, Joum, PhysioL, 26, p. 405, 1901 ; 27, p. 269, 1901 ; 30, p. 350,
1903.
158 CONSTITUTION AND MODE OF ACTION OF ENZYMES
extracts is so unstable that 55 to 75 per cent, of it is destroyed
on keeping them for an hour at 37° with -4 per cent NagCOy
But extracts which had been kept for some months at room
temperature, or for twenty-four hours at 38° with NagCOg, and
which had in consequence deteriorated in activity, were more
and more stable the greater the deterioration, so that very weak
extracts suffered a destruction of only 3 to 7 per cent of their
proteolytic activity when kept for an hour with -4 per cent
NagCOy Presumably this was due to the most unstable
trypsin groupings first undergoing destruction, whilst the more
and more stable ones remained. It was natural to conclude
that the extracts contained a number of trypsins of various
degrees of stability, but I found ^ that it was impossible to
separate stable and unstable trypsins by fractional alcohol
precipitation, so it is probable that the tryptic-rennetic molecule
contains a number of tryptic side-chains of various degrees of
stability, which can be destroyed one at a time independently
of one another. The rennet ferment of the pancreatic extracts
behaved similarly to the tryptic,^ so the tryptic-rennetic enzyme
must likewise contain a number of rennetic side-chains of
various degrees of stability. The erepsin of fresh pancreatic
and intestinal extracts also reacted in the same way," so it may
be a property of many enzymes, intracellular no less than
extracellular, to possess numbers of side-chains of different
degrees of stability. It is not a property of all enzymes,
however, as the diastatic enzyme of the pancreas showed no
such variations of stability.* That is to say, fresh and active
extracts were no more unstable than old, feebly-acting extracts,
or the extracts, when kept in dilute solution in an incubator,
showed no more rapid deterioration of ferment activity during
the first hour of incubation than in subsequent hours.
Nevertheless it is possible that the pancrezis contains more
than one tryptic enzyme; for Fermi ^ has shown that the
addition of dilute solutions of mercuric chloride, phenol, salicylic
and hydrochloric acids to an extract may destroy its power
of digesting fibrin, but not that of digesting gelatin. Also
* Vtmon^ Joum, PhysioL^ 29, p. 302, 1903. 2 /^y;^ 27, p. 195, 1901.
3 Ibid,, 30, p. 330, 1903. * IbiiL, 27, p. 197, 1901.
* Fermi, Arch./. Hygiene^ 10, p. i, 1890.
CONSTITUTION OF ENZYMES 159
PoUak ^ found that if pancreatic extract were acted upon with
hydrochloric acid for a few minutes, and then neutralised, it
might entirely lose its power of digesting serum proteins, but
still retain two-thirds of its original gelatin-digesting power.
He supposed that the acid destroyed the trypsin, but left a
" glutinase " enzyme comparatively unharmed. But perhaps it
is due to the stable tryptic side-chains being more especially
suited for the digestion of gelatin, whilst the unstable ones
chiefly digest native proteins.
Modern work on immunity, especially the evidence obtained
from precipitin formation, has shown us that the serum proteins
of one animal differ from those of other animals of different
species, and that this difference is the greater the further apart
they are genetically. Again, Abderhalden and others have
shown that differences of chemical composition exist between
the bloods of Carnivora and Herbivora, whilst there is a
similarity between the blood of the sheep and ox. It is almost
certain, therefore, that the enzymes differ likewise in compo-
sition, and that the pepsin of the gastric mucous membrane of
man differs slightly from that of the ape, more widely from that
of the dog or pig, and more widely still from vegetable pepsins.
Correlated with differences of chemical constitution and con-
figuration, we should expect to find differences of action. As
far as I am aware, this point has not been tested for proteolytic
ferments, but I found ^ that the diastatic ferments of the
pancreas of various animals differ considerably. The most
convenient method of testing the course of their hydrolytic
action upon starch paste is by means of the reaction with
dilute iodine solution, and I determined the times required by
the digests to reach a definite violet stage, a brown stage, and
the achromic point. With an achromic point time of lo minutes,
I found that the extracts of dog's and pig's pancreas took
approximately 2| minutes to digest the starch to the violet
stage, and 7 minutes to the brown stage. Extracts of sheep
and ox pancreas took i-i minute for the violet stage, and 4-3
minutes for the brown stage, whilst extracts of human pancreas
took 20 minutes for the violet stage and 63 minutes for the
* PoUak, Hofineistet^s Beitr.^ 6, p. 95, 1906.
2 V tmoTif Journ, PhysioLy 28, p. 156, 1902.
160 CONSTITUTION AND MODE OF ACTION OF ENZYMES
brown stage. What these particular colour stages correspond
to as regards dextrin formation we do not know, but I found
that the rate of formation of maltose was likewise different with
the different extracts. The achromic point stage corresponded
to the hydrolysis of approximately 58 per cent, of the starch to
maltose in all cases, but previous to this point being reached,
the pig's pancreas extract formed maltose much more slowly
than the sheep and ox pancreas extracts. These extracts, in fact,
gave identical results in the progress of their maltose formation.
As might be expected, malt diastase gave the most divergent
results of all, both in the formation of maltose, and in the course
of the colour reactions. With an achromic point time of 10
minutes, it took only -5 minute to bring the starch to the
violet stage, and 2-5 minutes to bring it to the brown stage.
Of course it is possible, as was suggested to me by Dr Bayliss,
that these results are due to the presence of different amounts
of independent dextrin-forming and maltose-forming enzymes
in the various extracts.^
Adsorption. — The precipitability of enzymes by alcohol and
salts, and their ready coagulability and destruction on exposure
to high temperature, is dependent on their colloidal nature.
But they possess other properties typical of colloids, and one
of the easiest to demonstrate is the property generally known
as adsorption. As long ago as 1872 v. Wittich^ pointed out
that fibrin, when placed in a dilute pepsin solution, was able to
absorb a good deal of the enzyme, and to fix it so firmly to
itself that it was not removed — or at least only in part — by
subsequent washing. Other observers have shown that fibrin
can similarly absorb trypsin, papain, ptyalin, malt diastase,
invertase, and maltase, so probably it can absorb all enzymes.
V. Wittich thought that the pepsin chemically combined with
the fibrin, but subsequent research has shown that the process
is more a physical than a chemical one, and is due to the
condensation of a surface layer of enzyme molecules upon the
porous fibrin structure. This property of adsorbing dissolved
substances is possessed by a large number of bodies in varying
degrees. Pepsin is adsorbed in considerable amount by animal
^ Cf. Frankel and Hamburg, Hofmeister's Beitr,^ 8, p. 389, 1906,
2 V. Wittich, Pflilget's Arch.^ 5, p. 443, 1872.
ADSORPTION 161
charcoal, kieselguhr, powdered brick ; freshly precipitated
barium sulphate, calcium phosphate, magnesium carbonate;
lead, copper, and uranium salt precipitates; cholesterin and
fatty acids. Dauwe^ found that coagulated serum and egg
proteins, casein, raw and cooked meat, and also gelatin, agar,
chondrin, and haemoglobin energetically adsorbed it, but that
clay, quartz sand, glass powder, talc, and magnesium phosphate
adsorbed it little if at all. The adsorptive power of a substance
depends very largely upon its state of division, for the amount
of surface it offers for the molecules of enzyme to condense
upon varies inversely as the diameter of the constituent
particles. If a particle of a substance i mm. in diameter be
split up into particles I/jl in diameter, the total surface area is
increased a thousandfold.
The physical process of adsorption is often complicated by
a certain amount of chemical combination, i.e. of the interaction
of atoms in accordance with their definite combining weights :
but the compounds so formed may be so unstable and easily
dissociated that though they are true chemical combinations
they may appear to be the result of adsorption processes. In
the reaction of a strong acid such as HCl with an equivalent
amount of a weak base such as ammonia, one finds that the
whole of the acid and base in solution combine together, but
as Arrhenius and Madsen ^ point out, this is by no means the
case if a weak acid sych as boracic acid be allowed to react with
the ammonia In a solution containing equivalent quantities of
acid and base, only half of the acid is chemically combined with
half the base, and the other halves remain free. If an excess of
acid be added, more and more of the base is fixed, but even with
five equivalents of acid to one of base, 17 per cent, of the base still
remains uncombined. Hence in many cases of absorption of a
dissolved substance by a solid it may be impossible to state how
far the process is a physical one of adsorption, and how far one
of weak chemical combination. In fact, every kind of transi-
tional stage between the two extremes is met with.
The adsorption phenomena of enzymes, closely resemble
* Dauwe, HofmHster's Beitr.^ 6, p. 426, 1905.
* Arrhenius and Madsen, see Arrhenius, ImmunO'Chemistryy New York,
1907, p. 174.
X
162 CONSTITUTION AND MODE OF ACTION OF ENZYMES
those of dyes, and most of our exact information is obtained
from observations upon these latter substances.^ What may
be called the "Law of Adsorption "^ seems to hold equally
for both. According to this law we find that if a suitable
adsorbent substance is placed in solutions of a dye of pro-
gressively diminishing concentration, the amount of dye taken
up is relatively larger and larger the more dilute the solution.
For instance, Bayliss found that equal amounts of filter paper
placed in dilute alcoholic solutions of congo red adsorbed the
following proportions of the dye : —
Concentration of
Soliition.
Proportion of Dye
£solntion.
Proportion of Dye
in Paper.
•014
K)I2
010
008
006
•004
Per cent.
40
20
9-3
4
1-3
trace
FeroMit.
60
80
practically all
In a similar manner pepsin is adsorbed by fibrin to a rela-
tively much greater extent from dilute solutions than from
more concentrated solutions (v. Wittich, Dauwe).
A case of adsorption conjoined with true chemical combin-
ation has recently been described by Barratt and Edie.'
Cotton wool was kept with methylene blue solution of known
concentration for two to thirty-one days at 45**, and the amount
of dye taken up was estimated. The mean results obtained
were the following : —
in OottoB Wool.
Percentage of J>we
Percent.
4-061
•292
•0266
•0153
•0028
Peroent.
.76
.72
•58
•53
•49
Peroent.
..29
•25
•II
.06
02
^ For full literature and discussion of the theory of staining reactions
see G. Mann, Methods and Theory of PhysiologUal Histology^ Oxford, 1902,
pp. 330-370. * Cf, Bayliss, Biochem. Joum,^ i, p. 175, 1906.
^ Barratt and Edie, ihid.^ 2, p. 443, 1907,
ADSORPTION 16S
Barratt and Edie conclude — for reasons stated fully in their
paper — that in every case the larger part of the dye, viz.
•47 per cent, was in chemical combination, so that in dilute
solution only a very small proportion of it was adsorbed.
Similar cases of adsorption complicated by chemical
combination are met with in the case of enzymes. When
an enzyme is taken up from solution by a substance upon
which it can act, it seems probable — for reasons stated below —
that some of it enters into loose chemical combination with the
substance. But it is as a rule impossible to prove whether the
process is one of adsorption or of chemical union. For instance,
it was- pointed out by W. A. Osborne that calcium casein-
ogenBte does not pass through a porous clay filter, whilst
trypsin does. Bayliss^ found that if solutions of trypsin and
calcium caseinogenate were mixed together and filtered, no
trypsin came through ; so one might presume that the trypsin
was to some extent chemically combined with the caseinogen
substrate. However, a mixture of caseinogen and malt diastase
likewise yielded no enzyme-containing filtrate, though in this
case the caseinc^en can only have adsorbed the enzyme.
A curious case of adsorption which has the appearance of
chemical combination was noted by Hedin^ for the intra-
cellular proteases of the spleen. Hedin found that whilst
charcoal adsorbed both a- and /3-proteases in the same propor-
tion, kieselguhr adsorbed much more of the a-protease. In fact,
it was doubtful whether it adsorbed any of the /8-protease at all.
This specific adsorption must have been a physical phenomenon,
as there could scarcely have been a chemical union between the
kieselguhr and the a-protease.
The adsorbent power of some substances is very large. For
instance, Hedin * mixed 80 cc of trypsin solution with •$ gm. of
animal charcoal, and found on filtration twenty-four hours later
that the whole of the enzyme had been adsorbed. A similar ex-
periment with • i gm. of charcoal gave 3-75 units of enzyme in the
filtrate, and one with -05 gm. of charcoal, 2575 units, whilst
the trypsin solution originally contained no less than 750
units of enzyme. The enzyme is somewhat firmly fixed by the
1 Bayliss, iac. cit^ p. 224. ^ Hedin, Biochem, Joum,^ 2, p. 112, 1907.
3 Hedin, ibid,y i, p. 483, 1906.
164 CONSTITUTION AND MODE OF ACTION OF ENZYMES
charcoal, for on mixing charcoal containing adsorbed trypsin
with dilute caseinogen solution, only i to 15 per cent of the
adsorbed enzyme was extracted by the caseinogen and utilised
for digestion. This extraction requires time, but at a tempera-
ture of 20** it was completed in half an hour or les&^
A more typical property than adsorption possessed by
enzymes in common with colloids is that of non-diffusibility,
but the evidence is very contradictory, v. Wittich stated that
pepsin dialysed slightly if the liquid inside and outside the
dialysis tube contained -2 per cent HCl, but neither Ham-
marsten or WolflThiigel could confirm the statement Hoppe-
Seyler found that diastase could pass through animal membranes
and parchment paper without great difficulty, but Wroblewski
could not detect any appreciable dialysis in twenty-four hours.
The question has been investigated by Chodschajew^ with great
care. The liquid inside and outside the dialyser contained
I per cent of sodium fluoride to prevent sepsis, and the integrity
of the dialysis membrane was tested thoroughly after each
experiment Chodschajew found that all the enzymes tested
by him, viz. yeast invertase, malt diastase, emulsin, trypsin, and
pepsin, dialysed to a very slight extent As a rule traces of
dialysed enzyme could be detected after thirty-six hours, and
almost always after eight days' dialysis. Again, Frankel and
Hamburg* state, though without giving any experimental details,
that malt diastase is diffusible. They find that the diastase is a
mixture of starch-liquefying enzymes and of enzymes for con-
verting starch into sugar. The latter enzymes dialyse through
parchment paper, but the former do not Hence their molecules
must be of different size.
On the whole, therefore, we are justified in supposing that
enzymes can diffuse to an extremely slight extent, but probably
a good deal less than proteoses, though more than native pro-
teins. On these grounds it seems probable that enzymes have
if anything smaller molecules than native proteins. If the per-
centage of chlorine in pepsin found by Nencki and Sieber and
by Pekelharing be taken as correct, and if it be assumed that
^ Hedin, Btockem, Joum,y 2, p. 81, 1907.
' Chodschajew, Arch, de PhysioL^ 10 (S)> P- 241, 1898. Literature here
quoted.
3 Frankel and Hamburg, Hofmeistet^s Beitr,^ 8, p. 389, 1906.
OPTICAL ACTIVITY OF ENZYMES 165
the pepsin molecule contain only a single atom of chlorine, its
molecular weight works out at about 7400. This is a somewhat
higher figure than the minimum value assigned to some pro-
teins, but the whole evidence is so doubtful that it scarcely
warrants discussion.
Upon the optical properties of enzymes no direct determina-
tions of much value have been made. Pekelharing ^ states that
his pepsin had a laevo^rotation, for yellow light, of about 50"*, or
a value similar to that of proteins, but he did not attempt to
determine it at all exactly. Much more interesting evidence
than this has been obtained by an indirect method. Fischer
and Bergell ^ observed that if trypsin were allowed to digest the
optically inactive racemic body carbethoxyglycyl-dl-leucin for
some days, the solution became dextro-rotatory. It contained
an oil which was probably carbethoxyglycyl-d-leucin, and
leucin, which was isolated and found to consist for the most part
of the laevo-rotatory body* That is to say, the trypsin had
hydrolysed one of the two optically active components of the
racemic leucin compound more rapidly than the other, and so
was itself presumably an optically active substance. Dakin^
tested the action of the intracellular lipolytic enzyme of the liver
upon several of the esters of optically inactive mandelic acid
with a similar result. The methyl, ethyl, iso-amyl, and benzyl
esters of mandelic acid, C6H5.CH(OH).COOH, were partially
hydrolysed by means of the enzyme, and the mandelic acid
liberated was strongly dextro-rotatory, whilst the residual
ester was correspondingly laevo-rotatory. The free acid never
contained more than 38 per cent of the dextro-rotatory body,
over and above that required to neutralise the laevo acid
liberated, so even in the earliest stages of hydrolysis a good
deal of the laevo ester was acted upon by the enzyme. If the
hydrolysis was complete, the mandelic acid formed was optically
inactive : Le, it consisted of equal parts of the opposite optical
isomers. If the esters are hydrolysed with symmetrical reagents
such as acid or alkali, the two optical isomers are split up at the
same rate, and the mandelic acid separated at any stage of the
^ Pekelharing, ZeiL f, physiol, Chem.^ 35, p. 27, 1902.
2 Fischer and Bergell, Ber.y 36, p. 2592, 1903.
3 Dakin, Journ, PhysioLy 32, p. 199, 1905.
166 CONSTITUTION AND MODE OF ACTION OF ENZYMES
hydrolysis is always inactive. Dakin suggests that in the
hydrolysis by lipase the dcxtro and laevo components of the
inactive ester first combine with the enzyme, but in that this is
an optically active asymmetric body, they do so at dtiTerent
rates. The compound enzyme plus ester is then hydfx)iysed,
but since the compound enzyme plus d-«ster is not the optical
<^)posite of enzyme plus l-ester, die rate of diange in the two
cases is again different. In the present instance, the enzyme
plus d-e^er is probably formed more rapidly and hydrolysed
more rapidly than the other compound.
On the hypothesis that the hydrolysis is influenced by the
configuration of the complex enzyme plus d- or l-ester molecules,
one would naturally expect that the acid liberated from the
closely related esters would be of the same sign. As already
stated, the dextro-rotatory acid was always formed from the
mandelic estera From the methyl and ethyl esters of phenyl-
chlbracetic acid and phenyl-bromacetic acid, however, Dakin ^
found diat the laevo acid was always liberated first by the
action of lipase.
The assumption made by Dakin that the hydrolysis of the
substrate is preceded by a combination of the enzyme with
it is supported by other evidence. As previou^y stated,
O'Sullivan and Tomson found that invertase required a
temperature fully 2^ higher to destroy it when cane-sugar
was present than when it was absent This was presumably
due to the enzyme forming a loose combination with the sugar,
and being protected thereby. Again, certain of the numerous
observations made upon the velocity of enzyme action point
to a similar conclusion. Enzymes are regarded as catalysts
which accelerate, or sometimes retard, the velocity of chemical
reactions in the same way as inorganic catalysts. Their action
should accordingly conform to the law of mass action, or the
amount of chemical change effected by them in a substance
in a given time should always bear a constant ratio to the mass
of that substance remaining unchanged. Hence die course of
the change should be capable of expression as a Ic^arithmic
curve, or for unimolecular solutions the expression — log
* Dakin, /Mirfw, PhysioLy 32, p. 199, 1905.
VELOCITY OF ENZYME ACTION
167
(where x is the amount of chemical change induced in the time
/) should be constant This constant, K, is known as the
velocity co-eflficient, or velocity constant. When this formula
is applied to the experimental data obtained with various
enzymes, it is found that though it holds for part of the
reactions, it does not apply to the initial and final stages. For
instance, Adrian Brown ^ found that when invertase was allowed
to act upon cane-sugar in solutions of different concentrations,
the amount hydrolysed did not bear a constant proportion to
the total amount of sugar present, but that a practically constant
weight was inverted in every case. Thus :
InlOOcc. ^
GranuofCMM-Sagitf
inverted in 60 minutes.
FxAcikmofOMe-Sagwr
Inyerted in 60 minutes.
K.
4.89
9-85
19.91
29-96
40-02
1.230
1-355
1-355
1076
Percent.
25.2
13-8
6.8
4-1
2.7
<902IO
00107
•000$ I
.00031
*00020
On the other hand, in dilute solutions of cane-sugar, when the
proportion of sugar to enzyme fell below a certain maximum.
Brown found that the amount of sugar hydrolysed was directly
proportional to the amount present
Gmms of Oft&e-Sngftr
per 100 o.e.
Gnmi of CSuie-Sagar
inyerted in 60 minutes.
K.
20
10
•5
-25
.308
.249
•129
K>6o
00219
•00239
00228
These data show that with i per cent, or less of cane-sugar K
was constant.
Horace Brown and Glendinning* obtained somewhat
similar results for the action of malt diastase upon starch,
and they lay stress on the fact that in the initial stage of the
reaction equal amounts of starch are hydrolysed in equal
times, or in other words, the reaction is linear, not logarithmic.
* A. Brown, /<?««». Chim, Soc. Trans.^ 1902, p. 373.
3 H. Brown and Glendinning, tbid^ 1902, p. 3B8.
168 CONSTITUTION AND MODE OF ACTION OF ENZYMES
E. F, Armstrong ^ investigated the action of lactase and maltase,
and he found that in addition to an initial linear stage and
subsequent logarithmic stage, there is a final stage in which
the velocity constant diminishes owing to the influence of the
products of action. Bayliss^ observed the same thing as
regards the action of trypsin on caseinogen.
Victor Henri,' arguing from his investigations upon the
action of invertase, came to the conclusion that part of the
enzyme remained free, part combined with the cane-sugar,
whilst still another part combined with one of the products of
reaction, viz. fructose. Thus he showed that fructose retarded
the action of the enzyme, whilst glucose did not Brown and
Glendinning, following the somewhat similar views expressed
by Adrian Brown, assume that the hydrolysis is preceded by
a combination of the enzyme with the substrate. In the initial
stage, when the sugar (or other substrate) is in large excess,
the whole of the enzyme is combined with sugar, but the
amount of sugar so combined forms only a small fraction of
the whole quantity of sugar present. Consequently the enzyme
will hydrolyse equal quantities of sugar in equal times. As
the concentration of the sugar diminishes, the amount of it at
any time in active association with enzyme will be proportional
to this concentration, or the rate of change will follow the law
of mass action.
Deviations from the law of mass action are brought about,
not only by the accumulation of products of action, but by
another factor closely related to the retardation so produced,
viz. the reversible nature of these reactions. This subject is
discussed in detail in the next lecture, together with evidence
in favour of a definite union between enzyme and substrate.
Still another cause of deviation from the law is found in the
gradual destruction of the unstable enzyme which frequently
occurs throughout the hydrolysis.*
» E. F. Armstrong, Proc. Roy, Soc.^ 73, p. 500, 1904.
2 Bayliss, Arch. ii. Set. Biol., 11, SuppL, p. 261, 1904.
9 Henri, Zeitf.physik. Chem.^ 39, p. 194, 1901 ; also, Lois ginirales de
P Action des Diastases^ Paris, 1903.
^ For a more detailed account of the velocity of enzyme action, see
Moore, Recent Advances in Physiology^ e4. by L. Hill, London, 1906 ; also,
Bayliss, Sci, Progress^ 1906, p, 381.
LECTURE VII
REVERSIBLE ENZYME ACTION
Stereoisomeric sugars and their corresponding enzymes. Stereoisomeric
polypeptides and proteolytic enzymes. Retardation exerted by
products of action. • Interaction of organic acids, alcohols, esters, and
YfaXex. Reversible action of sucroclastic enzymes. Synthesis of
maltose, revertose, isomaltose, isolactose, cane-sugar, emulsin.
Synthesis of ethyl butyrate, glycerin triacetate, methyl oleate,
mono<olein and triolein by lipolytic enzymes. Action of organic and
inorganic catalysts compared. Synthetic action of proteolytic enzymes.
Formation of plastein. Energy relations of reacting systems. Trans-
formation of radiant energy of sun into chemical energy by catalytic
agents. Synthesis in plants and animab.
We saw in a previous lecture that each of the three bioses, cane-
sugar, maltose, and lactose, is hydrolysed by a special enzyme.
Also the trehalose of various fungi is hydrolysed by a specific
trehalase enzyme, and the hexatriose sugar raffinose by a
specific rafHnase. There is evidently, therefore, some close
relationship between the structure of a sugar and that of the
enzyme which can hydrblyse it In 1894 Emil Fischer,^ by
his classical researches upon artificial and natural glucosides
and their related enzymes, largely extended our knowledge in
this field. D-glucose (dextrose) is known to exist in two stereo-
isomeric forms, and Fischer found that when it is dissolved in
methyl alcohol containing hydrochloric acid, two stereoisomeric
methyl glucosides are formed, according to the equation : —
C^H^p. + GHjOH = QHiiOe-CHg + Hp.
1 Fischer, B^k^ 27, pp. 2985, 3479, i894 ; 28, pp. 1429, 1508, 1145, 1895.
Summary in Zeit f, physioL Chetn,^ 26, p. 60, 1899
170
REVERSIBLE ENZYME ACTION
According to Fischer these glucosides have the following
formulae, which are identical except as regards the spatial
position of the — O.CH3 and the — H radicals attached to the
first carbon atom. This difference of configuration is quite
sufficient to determine their reaction with enzymes, for Fischer
found that one of them, which he called the a-methyl-glucoside,
-O . CH,
CHg . O— C— H
H— C— OH
HO— C— H
was readily split up by yeast maltase into glucose and methyl
alcohol, whilst the stereoisomeric j8-methyl-glucoside was not
attacked at all Emulsin, on the other hand, split up the
j8-glucoside, but not the a-glucoside. The corresponding a- and
^-ethyl-glucosides reacted in the same way with the two
enzymes, but none of the glucosides of the pentoses and heptoses
prepared by Fischer were attacked by either enzyme. This is
a somewhat remarkable fact, for o- and j8-methyl-xylosides, for
instance, have the following formulae : —
-C— O . CH3
CH3 . O— C— H
CHgOH
H— CH2OH
or differ from the corresponding methyl-glucosides only by the
lack of an H-C-OH grouping. Yet this is sufficient to
prevent the enzymes from reacting with them, and splitting off
methyl alcohol. It would seem, therefore, that when an enzyme
acts upon a molecule of a sugar, it comes into contact with it
ACTION OF ENZYMES ON GLUCOSIDES 171
at a number of different points, perhaps at each carbon atom,
and that an alteration in the spatial arrangement of the
groupings attached to any one of these carbon atoms may be
sufficient to prevent the enzyme from combining or entering
into sufficiently close relationship with the sugar molecule to
hydrolyse it On the basis of these results, Fischer suggested
that the molecules of enzyme and sugar must be mutually
related to one another in the same way as a key is related to
the lock which it alone is able to unfasten.
All the natural glucosides hitherto investigated seem to be
related to the artificial jS-glucosides, as they are hydrolysed by
emulsin, and not by maltase. At least this is the case as
regards salicin, helicin, aesculin, arbutin, coniferin, and syringin.
Saponin, phloridzin, phillyrin, and apiin are not hydrolysed by
either enzyme, whilst amygdalin is hydrolysed by both. The
action of the two enzymes differs considerably, however, as
emulsin splits up amygdalin into benzoic aldehyde, hydrocyanic
acid, and glucose, thus :
C20H27NO11 + 2H2O = HCN + CeH5.CHO + 2CeHi20e»
but maltase is only able to split off a single glucose group, and
leaves the remaining nitril-glucoside of mandelic acid untouched.
Maltose, in that it is hydrolysed by maltase, may be looked
upon as glucose-a-glucoside, whilst according to E. F.
Armstrong ^ isomaltose, in that it is hydrolysed by emulsin and
not by maltase, is presumably the stereoisomeric glucose-/8-
glucoside.
However, the action of enzymes is not invariably specific.
Thus emulsin can hydrolyse /8-methyl-glucoside, j8-methyl-
galactoside, milk sugar and amygdalin, but Fischer does not
on this account consider that it contains four distinct enzymes^
Again, beer yeast, which ferments mannose, glucose, fructose,
and galactose (the d- forms), probably contains only a single
zymase. On the other hand, Fischer and other investigators
have shown that of the eleven known aldehyde hexoses, only
three are acted upon by any of the sucroclastic enzymes, or
are fermentable, these being the naturally occurring d-glucose,
d-mannose, and d-galactose. The stereoisomeric hexoses prp-
* E. F. Armstrong, Proc, Ray. Soc,^ B. 76, p. 592, 1905.
172 REVERSIBLE ENZYME ACTION
duced by artificial means cannot be fermented or split up by
enzymes. Similarly the naturally occurring d-fructose is the
only member of the ketone hexoses which is fermentable or
acted on by enzymes.
Similar evidence of correlation between enzyme and
substrate has been obtained for proteolytic enzymes and
certain polypeptides. Fischer and Abderhalden^ found that
none of the synthetically prepared polypeptides were hydro-
lysed by pepsin, but that about half of them were attacked by
trypsin, and almost all of them by the proteolytic endoenzymes
of the liver and other organs. Such of them as were racemic
bodies were as a rule hydrolysed asymmetrically, and only a
half or a quarter of them split up. On hydrolysis of alanyl-
glycin, for instance, only the d-alanyl-glycin isomer was attacked,
and the laevo body was untouched, whilst on hydrolysis of
leucyl-glycyl-glycin the 1- body was attacked, and the d- isomer
untouched. Most interesting of all is the case of alanyl-leucin
A. This body consists of the four stereoisomers :
d-alanyl-l*leucin,
1-alanyl-d-leucin,
d-alanyl-d-leucin,
1-alanyl-l-leucin,
and only the first of these bodies was hydrolysed by trypsin.
Now it is found that the alanin formed by the hydrolysis of
native proteins is always the d- form, whilst the leucin is always
the 1- form. Hence it follows that trypsin can only attack poly-
peptides of which the amino acid constituents have a similar
confiiguration to those present in native proteins.
Probably enzymes enter into almost as intimate a relationship
with certain products of their action as with the substrate they
have split up. Tammann ^ showed that the hydrolysis of salicin
and amygdalin by emulsin is considerably retarded by the
addition of any one of their respective products of action ; but
Henri found that as regards invertase the retarding effect is
practically confined to one of the products of action, viz. fructose,
* Fischer and Abderhalden, Zeitf.physiol, Chem,^ 46, p. 52, 1905. Sec
also, Abderhalden and Teruuchi, ibid,^ 47, pp. 159 and 466, 1906 ; 49, p. i,
1906 ; Abderhalden and Hunter, iHd,y 48, p. 537, 1906.
^ Tammann, ibid,^ 16, p. 291, 1892.
ENZYME AND HYDROLYTE
17S
and that glucose has little or no influence. E. F. Armstrong^
examined the influence of glucose, galactose, and fructose upon
the action of each of the four enzymes lactase, emulsin, maltase,
and invertase, and tabulated his results as follows : —
Bnzjme.
CoRMiponding Hydrolyto.
BUbot of HexoM on rate of
change.
Glucose.
Oalaetose.
Pructoae.
Lactase .
Emulsin .
Maltase .
Invertase
rj9-galactosides {ij. Milk Sugar,
\ i^-alkylgalactosides)
r/3-glucosides \0, most natural
\ fi;lucosides^ : /S-galactosides
/a-glucosides (i>. maltose, a-alkyl-
\ glucosides): a-galactosides
/Fructosides (m. Cane-Sugar, Raf-
\ finose and Gentianose)
No ^
influence j
Retards
considerably
Retards
considerably
No
influence
Retards
Retards
slightly
Retards
slightly
\
/No
linfluence
No
influence
No
influence
Retards
The substances recorded in the second column of the table
are those which, according to Emil Fischer, are alone hydrolysed
by the particular enzymes given in the first column. The table
shows us that the action of lactase is retarded by only a«^ of its
products of action, viz. galactose, and that the other product,
glucose, does not affect it any more than it affects the action of
invertase. We see from the second column that emulsin and
maltase can hydrolyse galactosides, but they do not attack them
so readily as they do glucosides, just as galactose is fermented
less readily than glucose. Galactose differs from glucose merely
in a reversal of the — H and —OH radicals attached to the
fourth carbon atom, so this difference of spatial arrangement
is not sufficient to prevent enzyme action, though it retards it
In a corresponding manner the addition of galactose retards the
action of emulsin and of maltase much less than the addition of
the more closely correlated glucose.
These results prove without doubt that an enzyme enters
into an intimate relationship of some sort with one or more
of the products of its activity. It is thereby withdrawn from the
sphere of action, and so the hydrolysis undergoes an increasing
degree of retardation the further it proceeds. Looked at in
another way, we may say that the retardation is produced by a
1 E. F. Armstrong, Proc. Roy, Soc., 73, p. 516, 1904.
174 REVERSIBLE PNZYME ACTION
tendency to reverse action. Just as the enzyme enters into
intimate relationship with the substrate it is hydrolysing, so
it enters into intimate relationship with one or more of the
products of its hydrolysis, and in virtue of this intimate relation-
ship tends to bring about a union of the cleavage products.
E. F. Armstrong ^ does not believe that the retardation effected
by products of action is due to a tendency to reverse action, in
that the action of emulsin upon milk sugar is retarded chiefly by
glucose, whilst that of lactase upon milk sugar is retarded by
galactose. That is to say, the enzyme does not appear to enter
into intimate relationship with both of the cleavage products
formed by its activity. But there is no reason for assuming
that such a relationship is essential for the synthesis, as the
lactase, for instance, might attach a molecule of galactose to
itself, and then combine it with a glucose molecule, without enter-
ing into intimate relationship with this molecule. Similarly when
lactase hydrolyses lactose, there is no good reason for assuming
that it enters into intimate relationship with more than the
galactose half of its carbon atoms.
The retardation exerted by products of action, and the
tendency to reverse action induced by them, was observed more
than forty years ago by Berthelot and Pean de Saint Gilles ^ in
their investigations upon the conditions of etherification. They
showed that when organic acids and alcohols are allowed to
react together, or the corresponding esters and water, the
final condition of equilibrium depends only upon the mass of
reacting substances, and is not affected by the particular com-
bination in which the substances are broi^ht together. For
instance, when ethyl alcohol is heated with an equivalent
quantity of acetic acid, the following reaction occurs : —
CHj-COOH + CgHs-OH = CHa-CO-O-CgHj + HgO;
but the ethyl aeetate and water formed react with one another
to form ethyl alcohol and acetic acid again, and these two
reactions proceed simultaneously at different rates until they
reach an equilibrium point, i>. a point at which they both
> E. F. Armstrong, Proc. Roy. Soc^ 73, p. 516, 1904.
« Berthelot and Saint Gilles, Ann, Chem, Phys, (3), 65, p. 385 ; 66, p. 5,
1862.
ESTKRIFICATION
176
proceed at the same rate. In the case of molecular proportions
of these bodies reacting at a temperature of 1 54", Menschutkin
found that this equilibrium point is such that two-thirds of the
acetic acid and alcohol present have combined to form ethyl
acetate, and one-third remains uncombined ; or, if the ethereal
salt and water be kept together at a like temperature, theii the
reverse action occurs, and a third of the salt is hydrolysed.
With increasing amounts of water, the mass action causes the
equilibrium point to approach nearer and nearer to the
alcohol plus acid end of the reaction, as the following data
show : —
Molecules of
Acetic Acid.
Molecules of
Alcohol.
Molecules of
Wftter.
Per cent, of Acid
Esterified.
I
I
I
I
I
I
I
2
3
23
98
66.5
407
II.6
7-3
The equilibrium point varies with different alcohols and acids.
Secondary alcohols undergo a smaller amount of esterification
than primary alcohols, and tertiary alcohols very much less still.
In the presence of catalysts such as acids, the velocity of esteri-
fication is greatly increased, and the stronger {i,e. the more
dissociated) the acid the more it accelerates the velocity of
action. Thus Ostwald found that methyl acetate is hydrolysed
300 times more rapidly by hydrochloric acid than by acetic
acid. The velocity of hydrolysis of the ester is accelerated by
the presence of acids in like proportion, so the equilibrium
point is not changed. That is to say, the added acid is purely
a catalytic agent, which undergoes no alteration itself, and by
its presence neither adds energy to the reacting system, or takes
it away.
As far as we know, every enzyme is retarded to a greater or
less extent by the accumulation of its products of action, and
hence it seems highly probable that no decomposition produced
by an enzyme acting in the presence of such products is able to
proceed to absolute completion, but reaches an equilibrium
point which falls short of it to a greater or less extent. Arguing
from the analogy of chemical reactions such as that above
176 REVERSIBLE ENZYME ACTION
referred to, it seems to follow that every enzyme which can
hydrolyse a substance into two or more cleavage products is
likewise able to dehydrate them back to the original substance.
The synthetic power may be so small as to be undetectable by
direct experimental methods, but it must always exist to a
greater or less degree.
The first definite proof of the capacity of enzymes to induce
reverse or synthetic action was obtained by Croft HilP in 1898.
An aqueous extract of dried and pounded yeast containing
maltase and other enzymes was allowed to act upon glucose.
The dehydration induced was estimated by determinations of
the cupric reducing power and the specific rotatory power, and
Hill found that when the yeast extract was allowed to act upon
40 per cent, glucose solution, the reducing power gradually
diminished, whilst the rotatory power correspondingly increased.
On the supposition that the synthetically formed biose consisted
of maltose, he calculated that after thirteen days at 30° 7-5 per
cent of maltose was formed; after forty days, 13-5 per cent;
and after sixty-eight days, 1 5 per cent Judging from the small
amount of dehydration effected in the last twenty-eight days,
the equilibrium point must nearly have been reached. Hill
endeavoured to show that practically the same equilibrium
point was reached, but from the opposite direction, when
maltase was allowed to hydrolyse 40 per cent maltose solution,
but further research has shown that the processes of synthesis
and hydration occurring in a mixture of yeast extract, maltose,
and glucose are not of the simple character at first supposed,
and so the complete analogy of this particular type of enzyme
action with chemical reactions such as esterification has not yet
been established.
just as the equilibrium point in the alcohol, acid, ester, and
water system approaches nearer and nearer to the alcohol plus
acid end of the reaction, Le. to that of complete hydrolysis,
the greater the proportion of water present, so is it in the
case of enzyme actions. Or, otherwise expressed, the synthetic
power of an enzyme is smaller and smaller the greater the
dilution. Hill calculated that in 20 per cent solution of glucose,
maltase would form at most 9-5 per cent of maltose ; in 10 pdr
* Croft Hill,/i?tfr/r. Chent. Soc. Trans,^ 1898, p. 634.
ACTION OF YEAST ENZYMES 177
cent solution, at most 5*5 per cent of maltose; In 4 per cent
solution, 2-0 per cent, and in 2 per cent solution, I'O per cent
Nevertheless, however dilute the solution, there must always be
a certain amount of synthetic action.
From the synthetic product formed by the action of maltase
upon glucose. Hill prepared a small quantity of maltosazone
crystals, and he thought that maltose was the only biose present
Emmerling,^ who repeated Hill's experiments, concluded that
the sugar formed was isomaltose ; but Hill,* after re-examination
of the question, still maintained that maltose was formed,
though he found that by far the larger portion of the synthetic
product consisted of a hitherto unknown biose, which he named
revertose. Whatever be the exact nature of the polymerisa-
tion, there can be no doubt that a synthesis of some kind is
effected in concentrated glucose solution by the enzymes of
yeast extract The formation of more than one synthetic
product is not irreconcilable with the doctrine of reversible
enzyme action above expressed, for as Hill points out, one has
no right to assume that maltase is the only enzyme concerned
in the synthesis. The yeast extract probably contains several
different enzymes, each exerting a different action. The
important point which he endeavoured to establish by a
number of careful experiments, is that the synthetic products
formed by the action of yeast enzymes in a concentrated
glucose solution are hydrolysed back again to glucose on
dilution of the solution. Presumably in this case the maltase
which formed the synthetic maltose is likewise responsible for
its re-hydration, whilst a "revertase" enzyme acts similarly
upon the synthetic revertose. This view is supported by some
fermentation experiments. Certain yeasts, such as Saccharo-
myces Marxianus, ferment glucose but not maltose. Conse-
quently, when added to the product obtained by the action of
yeast extract on concentrated glucose solution, it ferments the
glucose, but leaves the synthetic bioses unchanged. But if the
yeast extract plus glucose product be fermented with yeast
known to be capable of fermenting maltose as well as glucose,
then a small part of the synthetic bioses — ^presumably the
1 Emmerling, Bck^ 34, pp. 600 and 2206, 1901.
* Croft Hill,yj7«r«. Ckem. Soc, Transit 1903, p. 578,
Z
178 REVERSIBLE ENZYME ACTION
maltose and perhaps higher polymers — is fermented as well as
the glucose. However, the larger part of them, which analysis
proved to consist almost wholly of revertose, is still unattacked ;
but if the yeast extractZ/wj glucose product is first diluted, whereby
its synthetic bioses are hydrolysed back to glucose again, then it
can be fermented completely by Saccharomyces Marxianus.
In addition to yeast extract. Hill found that taka-diastase
(which contains maltase as well as amylase) has a moderate
synthetic action upon concentrated glucose solution, whilst the
enzymes of pig's pancreas extract have a slight one. The
products of the synthetic action were not identical, but in
every case it was found that on dilution they were hydrolysed
back again to glucose. The differences of action are presum-
ably due to differences in the nature of the enzymes. The much
greater synthesis observed with yeast extract may be due to
its containing several different ertzymes, each of which exerts
a more or less independent synthetic action upon the glucose,
and forms a different biose.
Though the existence of reversible enzyme action is
generally admitted, the theory of the process above indicated
has not passed unchallenged. E. Fischer and E. F. Armstrong ^
found that when a mixture of galactose and glucose was
subjected to the action of lactase, reverse action occurred, but
the biose formed was not lactose, but the isomeric isolactose.
As already stated, Emmerling found that the biose formed by
the action of maltase upon glucose is isomaltose. Armstrong
confirms this conclusion, and surmises that the revertose
described by Hill is identical with isomaltose. Still, he gives
but few details in proof of his statement, whilst Hill, on the
other hand, prepared pure revertose, and found that in its
optical rotation and other properties it differed widely from
isomaltose, so for the present we must accept his view as the
more probable.
Arguing from this synthetic formation of isolactose and
isomaltose rather than of lactose and maltose, Armstrong^
concludes that " there can be no doubt that the enzyme has a
specific influence in promoting the formation of the biose which
^ E. Fischer and E. F. Armstrong, Ber,^ 35, p. 31 51, 1902.
* E. F, Armstrong, Proc. Roy. Soc,^ B. 76, p. 592, 1905.
SYNTHETIC ACTION OF INVERTASE 179
It caimot hydrolyse." It is difficult to see the justification for
so sweeping a statement, for, as above stated, not only has Hill
shown that the synthetic products formed by the action of
yeast extract, taka-diastase, and pancreatic diastase are hydro-
lysed again on dilution of their solutions, but Fischer and
Armstrong have themselves observed a similar hydrolysis on
diluting the mixture of lactase and . sugars containing synthetic
isolactose. Again, Visser^ found that invertase iis able to
synthesize cane-sugar from a mixture of glucose and fructose,
whilst emulsin, which hydrolyses salicin to glucose and
saligenin, can dehydrate these substances back to salicin.
Still again, Pantanelli^ found that the invertase of various
moulds could synthesize cane-sugar from invert sugar. Hence
the weight of evidence, as at present adduced, is considerably
in favour of the view that an enzyme can hydrolyse in dilute
solution the synthetic products it forms in concentrated solution.
It must be admitted, however, that there are several
difficulties which need clearing up. In the first place, it was
stated above that the revertose formed synthetically by yeast
extract was not fermented by maltase-containing yeasts, hence
the extract apparently contained an enzyme which was not
present in the living yeast cells. Then Armstrong found that
emulsin, when left for two months at 25° with concentrated
glucose solution, formed a biose which had the properties of
maltose, i,e. of a sugar not fermentable by emulsin. However, he
does not appear to have determined whether the synthetic biose
in his glucose plus emulsin mixture underwent hydrolysis on
dilution. There can be little doubt that it would have done so.
In previous lectures mention has been made of one or two
cases in which abnormal optical isomers were produced by
enzyme action. Cathcart^ found that when a-protease hydro-
lysed coagulated blood serum proteins the arginin produced was
optically inactive, and was not the dextro form which is always
obtained on hydrolysis by acids and by other enzymes. Again,
Magnus-Levy* found that 90 per cent, of the lactic acid
^ Visscr, Zeit f. phystk. Chem.^ 52, p. 257, 1905.
2 PanUnelli, Rendu, d. R, Accad. d, Liucei [5"], xvi. 6, p. 419.
3 Cathcart, Jaum. PhysioL, 32, p. 299, 1905.
* Magnus-Levy, Hofmeistet's Beiir.y 2, p. 261, 1902.
180 REVERSIBLE ENZYME ACTION
produced during antiseptic autolyses of various tissues was of
the inactive form, whilst in aseptic autolyses over 60 per cent
was of this form. But Mochizuki and Arima^ found that in
the autolyses of bull's testes only the dextro-rotatory acid was
formed, and Kikkoji ^ similarly obtained the dextro acid in ox
spleen autolyse3. These observations, taken in conjunction
with those just recorded, seem to indicate that comparatively
slight changes in the conditions of action of an enzyme, such
as acidity, alkalinity, or the presence of antiseptics, are
sufficient in some cases to influence the optical character of
the products of hydrolysis. Though in many cases Fischer's
simile of lock and key seems to be rigidly applicable, yet it is
not so in all, as in the instances adduced on a previous
page, aiid in certain other reactions. Hence it may be
that an enzyme which is unable, under most conditions, to
attack some particular stereoisomer, is able to do so in the
presence of certain abnormal substances such as acids, alkalis,
antiseptics, or some related stereoisomers. Possibly maltase,
though unable to hydrolyse a pure preparation of isomaltose,
can attack it in presence of maltose, glucose, revertose, or some
other unknown substance or substances. An explanation of
the apparently conflicting results above described will perhaps
be arrived at on some such lines as these.
Several other observations have been made upon the
synthetic power of various sucroclastic enzymes. Fischer
and Armstrong found that kefir lactase could form a bihexose
when placed in concentrated glucose solution, whilst emulsin
formed one from a mixture of glucose and galactose. The
synthetic bodies were not isolated, however. Cremer* kept
glycogen-free or glycogen-poor yeast juice with 10 per cent,
or more of fermentable sugar for twelve to twenty-four hours.
The glycogen was tested for by iodine solution, and in four
cases some appeared to be formed, whilst in four others the
result was negative. A more fully substantiated synthesis is
that effected by Emmerling * with yeast maltase. On keeping
^ Mochizuki and Arima, Zeitf.physiol. Chem,^ 49, p. 108, 1906.
« Kikkoji, iHd., 53, p. 415, 1907.
' Cremer, Ber,, 32, p. 2062, 1899.
* Emmerling, iHd.^ 34, p. 3810, 1901*
SYNTHESIS OF ESTERS 181
a mixture of 30 gm. of the nitril glucoside of mandelic acid
with 18-5 gm. of glucose and 50 c.c. of aqueous yeast extract
for three months at 35°, Emmerling found that a small quantity
of amygdalin was formed. In this synthesis a molecule of
glucose condensed on to a molecule of the nitril glucoside,
with separation of a molecule of water.
The synthetic power of lipolytic enzymes has been demon-
strated by several independent observers. The hydrolytic
action of aqueous extracts of various tissues upon ethyl
butyrate and other esters was described in a previous
lecture, and under suitable conditions it is found that
synthesis of the ester is induced. Kastle and Loevenhart^
kept 1000 C.C. of pancreatic extract with 1900 c.c. of deci-
normal butyric acid and 100 c.c. of 95 per cent, alcohol for
forty hours at 25°, in presence of thymol, and on distilling
the mixture in a slow current of air, they obtained nearly a
gramme of ethyl butyrate. A control experiment, carried .out
with boiled pancreatic extract, gave no ester whatever. .Again,
Hanriot^ found that if blood serum were kept with a dilute
solution of glycerin and isobutyric acid at 37°, the acidity
diminished rapidly owing to the formation of glycerin mono-
butyrate. A somewhat different type of reaction was investi-
gated by Acree and Hinkins.^ These observers found that
the enzymes present in commercial pancreatine amylopsin,
maltase, taka-diastase, and malt diastase were all able to
hydrolyse a dilute solution of triacetylglucose (prepared by
heating glucose and acetic anhydride together at 100°). Con-
versely, on dissolving i gm. of glucose and '25 gm. of acetic
acid in 200 c.a of water, and keeping the mixture with -5 gm.
of pancreatin and toluene at 0°, the acidity diminished in four
days by 6-6 per cent This was presumably due to the forma-
tion of glucose acetates.
An ester more closely allied to true fats, viz. glycerin
triacetate, has recently been synthesised by enzyme action.
A. E. Taylor* studied the action of the lipolytic enzyme of
^ Kastle and Loevenhart, Amer, Chetn, Joum.^ 2,i^^ P* 49i) 1900.
* Hanriot, Comptes Rendus^ 132, p. 212, 1901.
' Acree and Hinkins, Amer, Chetn, Joum,^ 28, p. 370, 1902.
* A. £. Taylor, /?i#r«. BioL Chem,y 2, p, 87, 1906. See also, Arrhenius,
Immuno-chemistry^ p. 133, 1907.
182
REVERSIBLE ENZYME ACTION
the castor-oil seed upon this body, and also upon a mixture
of glycerin and acetic acid. In this latter case equilibrium
was reached very slowly, but after several months more or
less the same end-points were attained as those given by
sulphuric acid acting under similar conditions of dilution and
temperature ( 1 8°).
Concentration.
Percent, of Hydrolysb
by means of
Calcolated.
Ha804
Lipase.
per cent.
•5
2-0
88
82
78
86
79
70
88
8p.i
69.8
The last column of the table gives the values calculated
according to Guldberg and Waage's law of mass action, on
the assumption that the first value (88 per cent.) is correct.
In that both the lipase and the acid act only as catalytic
agents, the end-points ought to be identical, and to agree
with the calculated values. The cause of the discrepancies
was not ascertained, but they seem too large to be attributable
to experimental error.
Bodenstein and Dietz ^ studied the hydrolytic action of
pancreatic lipase on amyl butyrate, and its synthetic action
upon butyric acid and isoamyl alcohol. They kept a prepara-
tion of alcohol- and ether-washed pig's pancreas with mixtures
of ester and amyl alcohol containing 6-5 to 8 per cent, of water.
Per cent, of Water
present.
6.5
6.5
8
8
Free Add remaining
in mixture of
Acid + Alcohol.
Free Add liberated
flpom mixture of
Bster+ Alcohol.
Calculated
Bquilibrlum Point.
10-58
28.75
48.51
33-58
1000
27.67
44.40
32.85
10-16
27.91
45-90
32.96
* Bodenstein and Dietz, Zeit / Electrochefn,^ 12, p. 605, 1906 ; Dietz,
Zeitf.physioL Chetn,, 52, p. 279, 1907.
SYNTHESIS OF FATS
183
and found that the same equilibrium points were gradually
approached as when the ferment was kept with equally con-
centrated solutions of butyric acid and alcohol. The data in
the table show the acidity finally attained in several pairs of
experiments, and the real equilibrium points calculated by
extrapolation.
Dietz also investigated the action of inorganic catalysts on
the synthesis and hydrolysis of the ester, and the data in the
table show the equilibrium points attained when 8 per cent, of
water was present It will be seen that they are practically
identical for the two acids tried, but that they differ consider-
Kster formed
from
Acid+Aloohol.
Acid formed
fromBtter.
Equilibrium point with Hydrochloric Acid .
„ „ Picric Add .
„ „ Pancreatic Lipase .
Per cent.
85-55
85-49
about 75-0
Percent.
14-46
14-50
about 25*0
ably from those induced by the lipase. Dietz thinks that this
difference may be due to the enzyme adsorbing some of the
reacting substances, and so undergoing a physico-chemical
change which caused it to induce a different equilibrium
point.
A very complete series of experiments upon the synthesis
of true fats has been carried out by Pottevin.^ A dried
preparation of alcohol- and ether-washed pig's pancreas was
used as the catalytic agent, and in the first group of experi-
ments Pottevin kept 50 gm. of oleic acid and 5-7 gm. of methyl
alcohol with 2-5 gm. of the pancreas powder at a temperature
of 33°. The acidity of the mixtures was measured at intervals,
and from the data in the table we see that in absence of water
85 per cent, of the oleic acid was esterified. In the presence
of increasing quantities of water the esterification was smaller
and smaller, as we should expect from the law of mass action.
At each dilution the equilibrium point was nearly reached in
eight days, and completely so in ten or fourteen days.
^ Pottevin, Ann, de PJmt, Pasteur^ 20, p. 901, 1906,
184
REVERSIBLE ENZYME ACTION
Time.
Gnmines of Water present.
7
14
86
70
146
12 hours .
36 „
60 , .
4 days .
6 „ .
5 „
10 „
14 »
15
75
83
85
85
12
35
II
76
78
79
So
10
32
70
73
74
74
23
35
46
53
54
*55
17
27
32
38
40
9
II
1*3
As likewise follows from the law of mass action, the equili-
brium point was not affected by the quantity of enzyme added :
but as can be seen from the data adduced, the velocity of esteri-
fication was greatly accelerated by increasing the enzyme.
Per cent, of Oleic Acid esterifled in
Per cent, of PanereM
Powder added.
Iday.
8 days.
80 days.
I
8
56
84
2
12
66
82
5
27
66
84
10
43
74
85
The other primary alcohols, viz. ethyl, propyl, isopropyl,
butyl, and isoamyl alcohols, showed practically the same degree
of esterification as methyl alcohol, and so did secondary butyl
alcohol, but isobutyl alcohol and tertiary butyl alcohol hardly
reacted at all.
In the second group of experiments Pottevin describes the
synthesis of both mono-olein and triolein. To obtain the
former body, 100 gm. of glycerin extract of pancreas were kept
at 35"* with 100 gm. of oleic acid. After eight days the acidity
of the mixture had diminished by a third, and 27 gm. of mono-
olein were isolated from it On dissolving mono-olein in fifteen
times its weight of oleic acid, and keeping the mixture at 35°
with I per cent of its weight of pancreas powder, the acidity
gradually diminished, and after about a month reached a constant
value. This was due to the formation of triolein, for 14-5 gm.
SYNTHESIS OF PROTEINS 185
of this fat were isolated. The considerable synthetic effect
observed by Pottevin was due to the possibility of carrying
out the esterification in the presence of very little water. If no
water whatever were present, however, the esterification of the
oleic acid hardly occurred at all, as the following data show : —
40 GM. OF Oleic Acid + 3 gm. of Pancreas Powder, kept for 20 days
AT 33°, WITH:—
130 gm. Anhydrous Glycerin + gm. Water = 3 per cent, of Acid esterified.
120 ,
» »»
10
n
= 77
no ,
» n
20
»»
= 64
100 ,
» »»
30
»
= 51
64
1 ti
66
u
- 20
28
) 11
102
II
= 5
8
) II
122
II
=
The water must therefore play a definite part of some sort
in the interaction of the glycerin and oleic acid.
In that mono-olein and triolein are readily hydrolysed by
pancreas powder when in dilute solution, it follows that the
reaction induced by the lipolytic enzyme is a strictly reversible
one.
In the light of these experiments, it appears that fats and
fatty acids afford much better material for the study of rever-
sible enzyme action than carbohydrates. The reactions which
occur are simpler, in that fats do not contain asymmetric carbon
atoms, and so do not form stereoisomers. Also, the ease with
which concentrated solutions can be employed is an important
factor. In respect of both these conditions proteins are much
less suitable for study even than the carbohydrates. It is not
surprising, therefore, that very little positive evidence has as
yet been obtained of the synthesis of true proteins by enzyme
action. In that proteins are built up of numbers of amino acid
molecules linked on to one another with the formation of
— CO — NH— groupings, it seems probable that if enzymes are
found to be capable of binding two amino acid molecules to-
gether with the formation of such a grouping, there is no
reason why they should not be able to unite three or four, or in
fact almost any number of amino acid molecules, until physical
reasons such as small solubility or colloidal nature of the product
checked the synthesis. Taylor ^ attempted to form the synthetic
* Taylor, " University of California Publications," Pathology^ i, p. 65.
2 A
IM REVERSIBLE ENZYME ACTION
peptides of Fischer by the action of trypsin upon the appropriate
amino acids, whilst Abderhalden and Rona* examined the
action of the enzymes of liver juice upon them with a similar
object In both instances the results were negative, but Taylor ^
has recently described the synthesis of a protamine by the action
of a proteolytic enzyme obtained from the liver of the soft-shelled
California clam. Four hundred grammes of protamine sulphate
prepared from the Roccus lineatus were dissolved in 1 5 litres of
water, and digested with the enzyme in alkaline solution. The
sulphuric acid was removed by precipitation with barium
chloride, and the filtrate concentrated to about 5 litres. This
solution of free amino acids and their carbonates, the products
of cleavage of the protamine, was kept at room temperature
with glycerin extract of the clam livers and toluol for five
months. The mixture kept quite sterile, and after the addition
of sulphuric acid to it, and precipitation with alcohol, a final
pure product weighing i-8 gm. was obtained, which in its
solubility, precipitability by alcohol and salts, its digestibility by
trypsin and resistance to pepsin, and in its percentage com-
position, was practically identical with the protamine sulphate
originally hydrolysed. Some of the solution of protamine
cleavage products, when tested directly without previous enzyme
treatment, yielded no protamine at all, so the product isolated
in the chief experiment must have been synthesised by the
liver enzyme.
Indications of reversible action have been obtained by
Bayliss * in the case of trypsin, A 40 per cent solution of the
products of hydrolysis of caseinogen was exposed to the action
of this enzyme, and Bayliss found that the electrical conductivity
of the mixture diminished some 27 per cent in the course of
four days, and then returned again to its original value. In the
hydrolysis of caseinogen by tryptic action, the conductivity
rapidly increases owing to the splitting up of large molecules
into smaller ones, and hence a diminution of conductivity
seems to imply the reverse process.
The reversible action of trypsin is also suggested by the
^ Abd^rtmldeti and Rona, ZeiLf.physiol. Chem,^ 49, p. 31, 1906.
a Taylor, /&ufn, BioL ChetfUy 3, p. 87, 1907.
3 Bayliss, Arch, d. Set, BioLy 11, SuppL, p. 261, 1904.
INFLUENCE OF PROTEIN DECOMPOSITION PRODUCTS 187
retardation exerted by the products of action. I found ^ that
the addition of i per cent or less of proteoses and peptones to
a solution of trypsin in -4 per cent. NagCOj had very little
influence upon its fibrin-digesting powers, but with 2 per cent of
these products its activity was reduced by 12 to 21 per cent.
BeUUve Tryptio Vftlae in piwe&ce of
^
*5 p«r cent.
1 percent.
2 per cent.
Proto-proteose
Deutero-proteose ....
Witte's Peptone ....
Antipeptone (KUhne)
Witte's Peptone, 81% hydrolysed .
Glydn
Leucin
100
103
104
100
105
III
97
86
98
98
97
100
94
103
83
79
88
84
81
Witte's peptone which had been digested with trypsin and
intestinal erepsin until 81 per cent, of it had been hydrolysed
into products no longer yielding the biuret test, exerted no
more retardation than less hydrolysed products, whilst
individual amino acids such as glycin and leucin, if in i per
cent strength, exerted little or no retardation. In all these
experiments the tryptic power of the trypsin in absence of any
decomposition products was taken as 100.
More detailed experiments upon the retardation exerted by
protein decomposition products have recently been made by
Abderhalden and Gigon.^ Yeast press juice was allowed to act
upon the polypeptide glycyl-1-tyrosin, and it was found that all of
the optically active amino acids which are formed on the hydro-
lysis of proteins, so far as they were investigated, greatly retarded
the hydrolysis. In most experiments i cc of yeast juice was
allowed to act upon •! gm. of glycyl-1-tyrosin, and -i gm. of the
amino acid was added. The acids tested were 1-leucin, d-alanin,
1-serin, 1-phenylalanin, d-glutaminic acid, d-tryptophan, and
1-tyrosin. The corresponding antipodes to these bodies, so far
as they were tested, had little or no inhibitory influence upon
the hydrolysis, whilst the racemic bodies had an interpiediate
* Vvsnotiyjoum. PkysieLy 31, p. 346, 1904.
' Abderhalden ^nd Gigon, Zeii, f, physUL Clum,^ 53, p. 251, 1907.
188 REVERSIBLE ENZYME ACTION
effect, as might be expected. Glycin, which is not an optically
active amino acid, had little or no influence on the course of
action, and so presumably it does not enter into relationship
with the enzyme. But the fact that each and all of the optically
active amino acids present in native proteins retarded the
hydrolysis is extremely suggestive. When we talk of the
configuration of an enzyme being adapted to that of its related
substrate, we do not necessarily imply that there is any close
similarity of chemical structure between their molecules, or
parts of their molecules. But this work of Abderhalden and
Gigon suggests firstly that the proteolytic enzyme of yeast juice
is a protein-like body containing each and all of the constituent
amino acid groups normally present in proteins, and secondly
that the correlation between enzyme and related substrate is
due to their molecules containing one or more groupings of
similar or identical chemical structure. That is to say, it
suggests that the active portion of an amylolytic enzyme which
comes, into direct relationship with the carbohydrate it is
hydrolysing, has itself a carbohydrate-like structure : that the
active portion of a lipolytic enzyme has an ester-like structure,
and so on. However, it must be remembered that these views
are suggestions only, and very far from being proved.
Synthetic powers have been attributed to the rennin or
pepsin of gastric juice by several observers. In 1886 Dani-
lewsky ^ noted that if rennet ferment were allowed to act upon
a solution of proteoses and peptones, it gave rise to a flocculent
precipitate. Sawjalow^ named this product plastein, and he
and others have investigated its properties, and consider it to
be a dehydration product of proteoses. The evidence con-
cerning it is extremely contradictory, for Sawjalow first found
that a plastein could be prepared from hetero-proteose and
from proto-proteose, and to a less extent from deutero-proteoses.
Lawrow and Salaskin^ obtained it from all of the proteose
fractions separated from Witte's peptone by Pick's method.
Kurajeff * obtained it by the action of gastric juice on secondary
1 Danilewsky, cited by Kurajeff, Hopneister^s Beitr,^ i, p. 121, 1901.
2 Sawjalow, Pftuget's Arch,, 85, p. 171, 1901.
« Lawrow and Salaskin, Zeitf.physioL Chem,, 36, p. 277, 1902.
* Kurajeff, HofnuisUt^i Beitr., I, p. 121, 1901 ; 2, p. 411, 1902.
PLASTEIN 189
proteoses, but not on primary proteoses, whilst he found that
papain gave a plastein precipitate with primary proteoses, but
not with secondary proteoses. Wait ^ fractionated the proteoses
of Witte's peptone with alcohol, and found that the secondary
proteoses gave a plastein precipitate with gastric juice, whilst
the primary ones did not. Bayer ^ found that no single
proteose gave any plastein, though a mixture of them did, and
Sawjalow^ confirms this conclusion, and says that a mixture
of all the primary and secondary proteoses is necessary. He
attributes the opposite results obtained by himself and other
observers to impurities in the proteose preparations.
A plastein precipitate is obtainable not only from Witte*s
peptone, but probably from every protein. Sawjalow prepared
it from egg albumin and globulin, serum albumin and globulin,
edestin, myosin, and casein, and he found that its percentage
composition was almost constant. It contained on an average
SS*3 per cent of carbon, 7-4 per cent, of hydrogen, 15-0 per cent,
of nitrogen, i-i6 per cent, of sulphur, and 21-2 per cent, of
oxygen, or had the composition of proteins. Wait and Lawrow
obtained similar figures for their analyses, but Kurajeff and
Rosenfeld* found their plastein preparations to contain about
59 per cent, of carbon. Sawjalow admits that the coniposition
is only constant if the plastein be obtained under certain
conditions, for he himself found that when casein was digested
two days it gave a plastein containing 55-7 per cent, of carbon,
and when digested four days, one containing 57-1 to 59-0 per
cent, of carbon. Bayer obtained a plastein containing 38-4
per cent, of carbon and 80 per cent of nitrogen, but his pro-
duct is so different from those obtained by all other investi-
gators that it must be an entirely distinct substance. Thus
it did not give the biuret, xantho-proteic, or lead sulphide
reactions.
Plastein is an acid body, insoluble in water, but it dissolves
in alkalis to form a soluble alkali salt, and from the amount of
* Wait, Diss, in Russian, cited by Sawjalow, Zeit f, physioL Ckem,^ 54,
p. 119,1907.
2 Bayer, Hofmeister^s Beitr,^ 4, p. 554, 1904.
3 Sawjalow, Zeitf, physioL Chem,^ 54, p. 1 19, 1904.
* Rosenfeld, cited by Sawjalow, loc, cit
190 REVERSIBLE ENZYME ACTION
alkali necessary to dissolve it, Sawjalow calculated its molecular
weight to be about 6ooa This is at least double the molecular
weight of proteoses. Sawjalow thinks that plastein is formed
by the reverse action of the pepsin of the gastric juice, as the
reaction goes on well only in concentrated proteose solutions
(30 to 40 per cent being best), whilst in dilute solutions the
plastein dissolves up again with formation of primary and
secondary proteoses and peptones.
A synthesis of similar character to that occurring in the
condensation of amino acids to polypeptides is found in the
dehydration of benzoic acid and glycin to hippuric acid. This
body is hydrolysed by the endoenzymes of minced kidney
substance, but Berninzone ^ states that if the kidney tissue is
allowed to act upon a mixture of glycin and benzoic acid in
presence of NaF, it is able to synthesise small quantities of
hippuric acid. Abelous and Ribaut^ confirm this result, but
the amount of synthetic product obtained by them was
extremely small For instance, a mixture of 425 gm. of
chopped horse's kidney, kept with 500 cc of horse's blood,
1-5 gm. of glycin, 3 cc of benzyl alcohol, and 2 per cent of NaF
for forty-two hours at 42° in a stream of air, yielded only -i i gm,
of hippuric acid.
Sufficient evidence has been adduced to prove that individual
members of all classes of enzymes are able, under suitable
conditions, to act synthetically, and hence we may assume with
a considerable degree of probability that all enzymes, endo-
enzymes no less than exoenzymes, can act in this w^y. It
seems highly probable, also, that enzymes synthesise the
substances which they hydrolyse, or that they are merely cata-
lytic agents which accelerate the velocity of two chemical
reactions, occurring in opposite directions, so as to hasten the
approach to an equilibrium point. They act in the same way
as inorganic catalysts, therefore, except than in virtue of their
asymmetric character they may act at unequal rates upon
opposite optical isomers. The catalytic agent of itself neither
adds energy to a reacting system or takes it away, but the
passage towards the equilibrium point, from whichever end of
^ Berninzone, AtL d. Sac, ligust d. Scienc, NaU^ 1 1, 1900.
2 Abelous and Ribaut, C. R. Soc, BioL^ 52, p. 543.
ENERGY RELATIONS 191
the reaction it occurs, is always accompanied by a liberation of
energy of some kind. It is this energy which forces on the
reaction towards the equilibrium point. Definite knowledge
of the energy relations concerned in the reversible actions above
described is wanting, but we know that the heat evolved or
absorbed during their progress is so small as to be practically
inappreciable. It cannot be measured directly, but some idea
of its magnitude can be obtained by comparing the heat of
combustion of a substance with that of its hydrolytic products.
For instance, it is found that in the hydrolysis of cane-sugar to
glucose and fructose only 31 rational calories are evolved, or
•23 per cent, on the heat of combustion of this sugar to water
and COg.^
C12H22O11 + H2O = QHiPe + CflHiPe + 31K
Cane-Sagar. Glucose. Fructose.
I3527K = 6737K + 67S9K + 31K
V. LengyeP digested egg albumin with pepsin for two to ten
days, and found that the heat of combustion of a definite quantity
of the mixture after digestion was 3736 calories on an average,
as compared with a value of 3739 calories before digestion.
Hari^ endeavoured to determine the energy loss in tryptic
digestion, but he could not find that there was any liberation
of energy whatever as the result of hydrolytic processes. That
such hydrolysis does occur is proved by the analyses of Mohlen-
feld,* Kossel,^ and Danilewsky. Hari found that the digested
protein showed a greater and greater increase in weight the more
prolonged the digestion, and after four to sixty-five days* tryptic
digestion, the increase amounted to i-i to 7-2 per cent. In the
case of fat hydrolysis, there is likewise no appreciable liberation
of energy. A gramme molecule of ethyl butyrate was found to
give 85 1 '3 calories on combustion, whilst gramme molecules of
ethyl alcohol and of butyric acid together gave 850-1 calories.
Again, a gramme molecule of stearin gave 8393 calories,
^ Quoted from article by B. Moore in Hill's Recent Advances in Physi-
ology ^ p. 40^ London, 1906.
* V. Lengyel, PflUger^s Arch.^ "5, p. 7, 1906.
3 Hari, ibid,^ 115, p. 52, 1906. * Mohlenfeld, ibid.y 5, p. 390, 1872.
^ Kossel, Zeitf, physioL Chem,^ 1879.
192 REVERSIBLE ENZYME ACTION
whilst 3 molecules of stearin and i of glycerin gave 8413
calories.^
It follows that though enzyme action is able to account for a
certain amount of synthesis under suitable conditions, it is unable,
as at present understood, to explain the storing up of energy.
Such storage of energy is constantly taking place in living cells,
and is most strikingly instanced in the formation of starch from
carbonic acid and water by chlorophyll-containing plant cells.
The energy stored up in the starch grains is in this case derived
from the sun's rays, or radiant energy from an external source
is by some mechanism unknown to us seized upon and trans-
formed into chemical energy. From ignorance of the true
explanation, such a transformation of energy is often attributed
to some " vital " property of the cell, though similar reactions
apart from living cells have long been known to chemists. For
instance, Berthelot found that acetylene was produced by passing
electric sparks between carbon points in an atmosphere of
hydrogen. In the presence of nitrogen, this acetylene formed
hydrocyanic acid. Sir Benjamin Brodie found that under the
influence of electric discharges carbon monoxide and hydrogen
unite to form methane and water. Again, hydrogen and iodine
combine together at a red heat to form hydriodic acid, with
considerable absorption of energy. It may be objected that
these syntheses only occur at very high temperatures, and can
have no bearing on any possible changes occurring in living
cells; but for aught we know to the contrary, some catalysts
may exist in the cells which are able to effect similar energy
tranformations at low temperatures. Bach ^ stated that if carbon
dioxide were passed through a 1-5 per cent, solution of uranium
acetate exposed to sunlight, a precipitate of uranium peroxide
and lower oxides was thrown down, whilst formaldehyde and
hydrogen peroxide were formed in solution. The uranium
oxides presumably acted as a catalytic agent, and enabled
the radiant energy of the sun to be transformed into the chemical
potential energy stored up in the formaldehyde. A repetition
of this experiment by Euler^ confirmed the synthesis, but the
^ Quoted from Leathes, Problems in Animal Metabolism^ p. 76, London,
1906.
2 Bach, Coniptes Rendus^ 116, p. 1145, 1^93.
3 Euler, Ber., 37, p. 3415, 1904.
SYNTHESIS IN PLANTS IM
product formed was found by him to be formic acid and not
formaldehyde. Usher arid Priestley ^ likewise found formic acid,
but they endeavoured to prove that formaldehyde is an inter-
mediate product. They found that the amount of decomposition
in three weeks of bright weather is extremely small, but that
the. reaction takes place more rapidly if the COj is under con-
siderable pressure. A 2 per cent solution of uranium sulphate
acted in the same way as the acetate, so the formic acid was not
derived from the acetic acid of the salt
Bach thought that the chemical change primarily eflfectied by
the sunlight and uranium salt was as follows :-—
CO2 + 3H2O = H.CHO + 2H2O2.
The hydrc^en peroxide, if actually formed in this way in the
green plant, would be at once split up into water and free oxygen
by the universally present catal^tse enzyme. Usher and
Priestley state that the catalase is strictly localised in the chbro-
plasts of the green leaf, and thoi^h this is improbable, we may
conclude that it is more concentrated in these structures than in
the rest of the leaf Formaldehyde is an extremely poisonous
body, so if produced in the green plant it doubtless undei^oes a
further transformation almost immediately. Loew ^ has shown
that certain chemical substances such as metallic oxides and
sulphites are able to condense formaldehyde to various carbo-
hydrates such as formose, a-acrose and methylenitan. Hence it
is possible that a similar condensation is effected in the plant
cell by the action of an enzyme. If such an enzyme exists, it
seems to be a very unstable body, intimately hound up with the
vitality of the cell, for Usher and Priestley found that exposure
of Ehdia to chloroform vapour for two hours destroyed its power
of condensing formaldehyde.
Usher and Priestley showed that if green sprigs of Elodta
were killed by dipping in boiling water for thirty seconds, and
were then placed in water saturated with COj and exposed to
sunlight, the green colour of the leaves disappeared in a few hours,
and the bleached leaves contained formaldehyde. They explain
1 Usher and Priestley, Proc. Ray, Soc, B. 77, p. 369, 1906 ; B. 78, p.
368, 1906.
2 Loew, Ber., 21, p. 271, 1888.
7 B
194 REVERSIBLE ENZYME ACTION
the course of events by supposing that hydrogen peroxide and
formaldehyde are at first produced from the COg and water
in the normal way, but that the peroxide, instead of being
decomposed by catalase as in living plants, oxidises the
chlorophyll to a colourless substance. The reaction thereby
comes to an end, for the chlorophyll is supposed to be the
catalytic agent which induces the reaction.
Some of Usher and Priestley's experiments have been
repeated by Ewart,^ and he finds that chlorophyll forms an
aldehyde when exposed to light, but he thinks that it is merely
a decomposition product of the chlorophyll, as it is formed even
if no carbon dioxide be present. He points out that chlorophyll
is bleached by sunlight in presence of air or oxygen containing
no CO2, and he says that there is no satisfactory proof of the
formation of hydrogen peroxide or of free oxygen by the
agency of chlorophyll, except inside the living cell. Hence we
must admit that for the present the synthesis of formaldehyde
or formic acid from carbon dioxide and water by an organic
catalyst has not been established.
Anabolic processes accompanied by a storage of chemical
energy are not limited to chlorophyll-containing cells, but
probably occur in every living cell. In the absence of
chlorophyll, the protoplasm must derive the energy necessary
for the synthesis from some source other than the sun's rays.
Usually it comes from the heat produced by the oxidation
processes occurring in the cell, or the energy set free by one
reaction is used to assist another reaction. Moore ^ points out
that it is this " linkage of one reaction with another, and the
using of the free energy of one to run another, which specially
characterises the cell and differentiates it from the enzyme,"
and that in the process " a set of manifestations peculiar to life
appear, which cannot be reproduced elsewhere than in living
cells." Somewhat inconsistently, Moore himself adduces an
example of linked reactions from inorganic chemistry, viz. that
of hydrogen peroxide upon certain metallic oxides, as those of
silver and gold. Hydrogen peroxide spontaneously undergoes
a slow conversion into water and oxygen, with evolution of
1 A. J. Ewart, Proc. Roy, Soc,^ B. 80, p. 30, 1908,
^ Moore, loc, ctt^ pp. 49 and 135 et seq.
SYNTHESIS IN ANIMALS 195
energy ; but in the presence of one of these oxides the velocity
of reaction is enormously inci:eased, though much of the energy
liberated is absorbed in order to induce another reaction, viz.
the reduction of the oxide to the free metal. As Moore points
out, " the induced reaction runs the inducing reaction backwards
away from its equilibrium point by means of the energy which
would otherwise be set free."
In living cells it is true that at present we know little or
nothing of the linkage of reactions, but their elucidation may be
only a question of time. In the plant cell we see that a
catalyst, perhaps chlorophyll, can transform solar energy into
chemical potential energy, and that possibly an enzyme may
then condense the synthetic formaldehyde into an actual
carbohydrate. In the animal cell, most of the syntheses such
as the formation of glycogen from glucose, of protein from
amino acids, and of neutral fat from glycerin and fatty acids,
are accompanied by an extremely small absorption of energy,
and, as Moore suggests, the variations of osmotic energy with
changes in concentration may account for the energy required,
and an enzyme which adds no energy to the reacting system
may effect the conversion. But other chemical changes occur
in animal cells, such as the transformation of carbohydrates into
fats, in which there is a very large absorption of energy. This
energy is presumably derived from the heat of combustion of
some of the food material stored up in the cell, by the same
kind of mechanism as that which in the plant converts solar
energy into chemical energy.
Hence it is of paramount importance for us to discover
catalytic agents which can effect the transformation of heat
energy into chemical energy, and investigate their action. As
far as I am aware, no instances of such energy transformation
are known to occur at temperatures compatible with the life of
a cell, but they are not unknown at higher temperatures. For
instance, in the vaporisation of ammonium chloride the
vaporised salt, under ordinary circumstances, is dissociated into
ammonia and chlorine with absorption of heat On cooling,
the gases combine together to form ammonium chloride, with
the evolution of a large amount of heat How much of this
heat is due to chemical combination, and how much to con-
\9t REVfeR^lBtE !BN%YMfe ACTIOK
densation from the gaseous to the solid state/ we do not know.
(L B. Baker ^ has shown that if the ammoniuni chhn'ide be
perfectly dry, it does not dissociate at all on va^risatton, and
so it seems to follow that the traces of water usually present act
as a catalytic e^ent, and bring about a transformation of beat
energy into chemical energy.
1 B^ktr^Jcum. Chem. Soc. Trans.^ 1894 and 1898, p. 482.
LECTURE VIII
ENDOENZYMES A19D PROTOPLASM
Comparison of living and dead tissues. Disintegration effected by chloro-
form and by lactic acid saline. Autodigestion of intact and of minced
tissues. Comparison of enzymes with agglutinins and lysins.
Zymoids. Antiferments. Constitution of biogens. Action of anti-
septics on living organisms and on enzymes. InHuence of tempera-
ture on enzymes, on metabolism of organisms, on heart beat and on
rate of propagation of nervous impulse. Optimum and maximum
temperatures.
We have seen in the preceding lectures th^t from dead
disintegrated tissues endoenzymes can be extracted which arc
capable of inducing most of the katabolic changes known to
occur in these tissues whilst still living. We have seen also
that enzymes possess synthetic powers, and so may be
responsible for some at least of the anabolic changes which
occur in living tissues. We therefore know a good deal about
many of the individual groups which, bound up together and
acting in harmony with one another, constitute living substance.
A far more difficult problem is to discover the manner in which
these constituent groups are united to one another, and can
exert their activity whenever it is required, independent of
what the other constituent groups may be doing. A few jrears
ago we were in total ignorance of this matter, but thanks to
the labours of Ehrlich and others, we are now making some
progress towards its solution. We regard the unit of living
substance, the biogen, as consisting of a nucleus with which
numerous side-chains are connected. These side-chains are in
most cases of a protein-like nature, and owe their different
capacities partly to differences of chemical composition, but
197
198 ENDOENZYMES AND PROTOPLASM
perhaps more especially to differences of structure and con-
figuration. At present we know very little about the chemical
differences of these side-chain groups. Still more ignorant are
we as to the nature of the linkages which bind them to the
bic^en nucleus. Any violent chemical or physical treatment of
living matter made with a view to determining its chemical
constitution, inevitably kills it, and so the evidence yielded by
such analysis pertains only to the dead and disintegrating
tissue, and does not necessarily hold in any degree whatever
for living protoplasm. In fact it is generally held that there
exists a fundamental difference between living and dead
substance, but extended research may prove that this view is
not justifiable, and that the difference is only one of degree,
not of kind Hence a study of the properties of dead and
dying tissues may prove of great value in assisting us to
understand and account for the seemingly inexplicable proper-
ties of living tissues.
A convenient method of studying the properties of dead
and dying tissues is that adopted by the writer,^ and already
referred to in a previous lecture. It consists in taking a fresh
and still living organ, such as the kidney or heart of a mammal,
and perfusing it with saline or some other liquid for several
days. The products of disintegration of the tissue, at the time
of death and subsequently, are carried away in the perfusion
liquid, and can be analysed qualitatively and quantitatively.
A kidney perfused continuously in this way at room tempera-
ture generally shows very little disintegration for several days
if putrefaction be prevented. Throughout the whole of this
time, however, the side-chains remain bound to the tissue
framework by very weak bonds, which are readily snapped by
almost any change in the conditions of perfusion. A temporary
stoppage of the flow of perfusion liquid or a change in its salinity,
may cause an immediate disintegration of the tissues, whereby
considerable quantities of proteins and other substances are
washed out of the organ. These other substances seem to
consist entirely of protein decomposition products, and contain
no appreciable quantity of fat or carbohydrate. As much as
74 per cent , of the total amount of protein present in the
* Vernon, Zeitf. allgem, PhysioL^ 6, p. 393, 1907.
TISSUE DISINTEGRATION
199
kidney tissues may be removed by perfusion : hence the
statement made above that the side-chains are of a protein-
like nature. The products removed by perfusion probably
include most of the endoenzymes. Thus the only one in-
vestigated by me, endoerepsin, might be almost completely
removed by a week's perfusion. The most effective method
of all for breaking the link between side-chain and tissue
framework is to perfuse with saline saturated with ether or
chloroform. In one experiment, a kidney was perfused with
oxygenated saline for the first two hours, and then saline
saturated with ether was substituted. The disruption was so
considerable that 19 per cent, of the total nitrogen and 16 per
cent, of the total erepsin in the kidney was washed out in the
first three hours of etherisation. In another experiment, a
kidney was perfused for 150 hours with 2 per cent, sodium
fluoride, and as can be seen from the data in the table, the
amounts of erepsin and of protein washed out during this time
were extremely small. Ringer's solution saturated with chloro-
Time of
Perftuioa of
Kidney.
Perfusion Liquid.
SulMitances washed out per hour per
1kg. of Kidney.
Brepein.
Protein.
Nitrogen
X6-26.
Nitrogen.
Hoora.
3 to 12
12 „ 48
48 „ 95
95 » 150
150 „ 151
151 » 156
156 „ 168
2% Sodium Fluoride
II »» •
II II •
II II •
r Ringer's Solution satura-\
\ ted with Chloroform j
»» II
II II
•003
.064
•245
38-46
1.36
.28
gm.
•022
•013
•029
•070
1 9^62
1.83
.40
gm.
}...3
•086
.106
19-98
1-82
•43
gm.i
.108
•057
•036
h -034
form was then substituted, and within the next hour 12 per
cent of the total protein contents of the kidney was washed
out, or more than three times as much as in the previous 147
houra The erepsin showed a still more remarkable degree of
disruption from the tissues, as 58 per cent of the total amount
present in the kidney broke away during the first hour of
chloroform perfusion, or the actual rate of disruption was
i3,(XX)times more rapid than during the 3rd to 12th hours of
200 ENDOENZYMES AND PROTOPLASM
perfusion. The only other agent found to be comparable to
these anaesthetics in disruptive power ^s free ammonia.^
Thus perfusion of a kidney for twelve hours with saline
containing -005 to -025 per cent of ammonia caused 29 to
35 per cent of the total protein contents of the tissues to be
washed out.
It has been pointed out to me by Dr W. M. Bayliss that
bodies like chloroform are known to increase the permeability of
the cell-limiting layer. Hence the substances escaping from the
cells may not be actually split off from combination with the
protoplasm, but merely be enabled to pass out owing to the cell
walls being rendered permeable to them. However, this criticism
would not apply, as far as we know, to the experiments in which
the kidney was perfused with dilute ammonia, or with saline
solutions of different strengths, and yet in their case the dis-
integration was almost as great, and as rapidly induced.
In these experiments the protein washed out from the
kidney was estimated by a colorimetric method dependent
on the biuret test, whilst the total nitrogen was estimated by
KjeldahPs method. The values so obtained, multiplied by
6-25 to bring them to terms of protein, are larger than the
biuret protein valuea That is to say, some of the nitrogen
was washed out of the kidney in a non-protein form. The
amounts so washed out in the above experiment are given in
the last column of the table, and it will be seen that they
are quite independent of the actual protein values. Though
between the 150th and i68th hours of perfusion the protein
broke away fifty times more rapidly on an average than between
the 3rd and 48th hours, the non-protein nitrc^en broke away
three times more slowly. It owes its origin to the autolytic
action of the intracellular proteolytic enzymes, and hence its
amount gradually diminishes during the course of a perfusion,
according as less and less of the enzymes and of proteins upon
which they can act are left in the tissues. If putrefaction were
prevented, it was found that whatever the conditions of per-
fusion the amount of this autolytic nitrogen remained fairly
constant, being on an average about -05 gm. per hour per
kilogram of kidney (expressed in terms of protein). If, how-
1 Vernon, Jaum. PhysioLy 35, p. 82, 1906.
RATE OF AUTOLYSIS 201
ever, the kidney were perfused with saline containing -oi. to
•2 per cent of lactic acid, this non-protein nitrogen was increased
two- to ten-fold, and in fact considerably more than half of the
total nitrogen came away from the kidney in a non-protein
form. This is an important point, as it shows that a large
fraction of the tissue protein is present, not as actual protein,
but as potential protein. Generally speaking, this unstable
potential protein breaks away as actual protein, and when
liberated it becomes quite stable, and is not hydrolysed by
dilute lactic acid saline even in the course of several months.
The fact that under the influence of dilute lactic acid it readily
breaks away as non-biuret-test^-yielding substances, shows that the
bonds uniting the numerous amino-acid groupings which together
constitute a potential protein molecule are very much weaker
than they are in free protein molecules. Such looseness of union
of the amino-acid groups, and ease of disruption, suggests a
corresponding ease in their synthetic combination in the tissues.
The average value given above for the protein autolysed
by the perfused kidney, viz. '05 gm. per hour per kilogram,
would come to 84 gm. per day per 70 kg. It is less, therefore,
than the protein metabolism of an average man, and much less
than that of a smaller animal. If the perfusions had been
carried out at 37° instead of at i S"* to 20°, the rate of autolysis
would have been at least four times greater, but making every
allowance for the temperature factor, it must be admitted that
in an intact perfused organ the autodigestion after death is not
extravagantly greater than that which occurs during life. As
long as the endoenzymes remain bound up in the tissues, even
if the tissue cells be dead, it is probable that they cannot exer-
cise their digestive powers to a much greater extent than in
the living cells. Presumably they can only act upon protein
groups which are anchored on to the biogens in their immediate
neighbourhood. Once they have broken free of their bonds,
however, they are able to attack any or all of the protein
groups present in the tissues. Evidence bearing upon this
hypothesis has been obtained by Miss Lane-Claypon and
Dr Schryver^ in their autolysis experiments. The liver or
* Lane-Claypon and Schryver, Journ, PhysioL^ 31, p. 169, 1904;
Schryver, ibid,^ 32, p. 159, 1905.
2 C
202 ENDOENZYMES AND PROTOPLASM
other organ examined by them was removed directly after
death, chopped up with a knife, and incubated with saline.
Two to twenty-four hours later samples were precipitated with
hot trichloracetic acid, and the estimations of the nitrogen in
the filtrates showed the amounts of tissue proteins which had
arrived at or beyond the peptone stage. It was found that
during the first two to four hours there was comparatively little
autolysis. For the next six or eight hours it became rapid, and
then gradually slowed dowa The actual change was consider-
able. In one liver autolysis, for instance, 13-2 per cent, of the
total nitrogen was initially present in the form of peptones :
I3«8 per cent, was so present after four hours* incubation : 427
per cent after eight hours, and 63*2 per cent after twenty-four
hours. The initial latent period must be taken to indicate that
at first most of the proteolytic endoenzymes were still bound up
in the tissues, and so were incapable of exercising more than a
very limited activity. Once they were free, however, they
digested the tissue proteins at many times their previous rate
Taking an average of the values obtained by Lane-Clay pon
and Schryver in the ten comparable experiments made upon
liver autolysis, I find that during the first two hours the
autolysis was -5 unit per hour; during the next two hours,
1-2 units; during the next six hours, 2-87 units; and during
the next fourteen hours, ^B/ unit
The protein side-chains are doubtless bound up to the
biogens in different ways and with different degrees of firmness.
I obtained^ a direct proof of this by comparing the rate at
which the endoerepsin breaks away from the tissues under
various experimental conditions with that at which the general
mass of protein groups breaks away. On changing the salinity
of the liquid with which a kidney was perfused from i per cent,
to 4 per cent or vice versa, I found that the rate of disruption
might be suddenly increased sixty-fold, and it increased to the
same extent for both the erepsin and the protein. On the
other hand, if already perfused saline were sent through the
kidney a second or third time, the protein disruption diminished
considerably (e.g, to a seventh its previous value), whilst the
ferment disruption increased as much as twenty-fold. That
* Vernon, Zeitf. allgem, PhysioL, 6, p. 393, 1907.
LINKAGE OF ENZYMES IN BIOGENS 203
is to say, one and the same change of condition caused dia-
metrically opposite results in different side-chains. Even the
different enzymes are bound up in the tissues with very
different degfrees of firmness. Thus I found ^ that on extract-
ing minced pancreas, the diastatic enzyme was removed far
more readily than the trypsinogen. In one experiment, in
which the gland substance was shaken up for two hours with
dilute alcohol, 55 per cent, of the total amount of diastase
present passed into solution, but only 14 per cent, of the total
trypsinogen.
It will be seen that the direct chemico-physical method of
studying the tissues during and after death is able to afford
some information as to the probable constitution of the biogens
during life, and hence it should be pursued simultaneously with
the indirect biological method which has yielded such remark-
able results during the last decade. It would be out of place
in this lecture for me to attempt even a brief summary of the
chief conclusions which have been deduced from the vast body
of experimental data we possess upon Immunity and allied
subjects, hence I will only refer to such portions as bear more
especially upon the question of endoenzymes.
The endoenzymes appear to be bound up in the tissues in
somewhat the same way as the side-chain receptors. As a rule
they are fixed with sufficient firmness to prevent them from
being cast off into the blood stream in other than small
amounts. But some of them such as maltase are liberated in
larger quantities, for the plasma is richer in this enzyme than
are any of the tissues. Exoenzymes, as we know, are liberated
in large amounts whenever they are required. Probably the
endoenzymes and the exoenzymes are formed and are bound
up in the tissues in a similar manner, only the linkage binding
the exoenzymes is more readily snapped under an appropriate
chemical or nervous stimulus than that binding the endoenzymes.
If minced pancreatic tissue be extracted with glycerin, the
endoenzymic erepsin seems to pass into solution as readily as
the exoenzymic trypsin and amylopsin.
We have seen that under conditions of greater functional
activity endoenzymes are elaborated and stored up in the
* Vernon, Jaum, Physiol,^ 28, p. 466, 1902.
204 ENDOENZYMES AND PROTOPLASM
tissues in increased quantity. That is to say, the tissues are
capable of over-regenerating their endoenzyme receptors, in
somewhat the same way as they r^enerate receptors to which
toxin molecules have become anchored Still they do not, as a
rule, over-r^enerate them to such an extent that they are cast
off in a free state into the blood, after the manner of antitoxin
molecules. Upon the mechanism of regeneration of receptors,
the side-chain theory tells us nothing. Presumably the tissues
build them up synthetically by means of their series of proteo-
lytic enzymes. The blood stream contains small quantities of
all the various amino acids of which a protein is constituted,
and the endoenzymes of the bic^ens must seize upon these amino
acid molecules one by one, as they are required, and build them
up tc^ether in accordance with some definite pattern which is
already laid down in the tissues. Failing such a pattern
protein molecule to model fresh receptors upon, it seems likely
that the synthesis of the particular kind of receptor in question
is impossible. Thus we saw in a previous lecture that certain
enzymes such as lactase and invertase were localised in a
definite region of the body, viz. the mucous membrane of the
alimentary canal, and that if the cells of this membrane lost
their power of secreting lactase, they were unable to recover it
when subsequently stimulated to do so by a milk diet
The action of enzymes may be regarded as similar to that
of Ehrlich's "receptors of the second order." Each of these
receptors has a haptophoric gfroup, which is supposed to anchor
on to a suitable receptor in the corpuscle or bacterium it is
going to agglutinate. The agglutinophoric group of the
receptor then acts upon the cell, and causes the cells to clump
together. Similarly, an enzyme attaches itself to a food
particle by means of what may be termed its haptophore group,
and acts upon it by what may be termed its zymophore group.
Endoenzymes are directly comparable to the agglutinating
receptors which are bound up in the tissue biogens, whilst
exoenzymes are comparable to the receptors which are over-
regenerated and cast off into the blood stream when the
blood of one animal is injected from day to day into another
animal.
In some cases it appears that enzymes act in the same way
MODE OF ACTION OF ENZYMES 205
as Ehrlich's receptors of the third order. That is to say, they
do not bind themselves directly to the food particle upon which
they are acting, but only through the intermediation of a third
substance or amboceptor. This amboceptor is comparable to
the thermostable " immune body " of Ehrlich, which is thrown
off by the tissue receptors into the blood of an animal on
repeated injection of a foreign blood, and which is able to bind
itself on the one hand to a suitable receptor of a blood
corpuscle, and on the other hand to some of the thermolabile
complement which is always present in the blood, and so enable
this complement to produce haemolysis of the corpuscle. The
researches of Fuld, Morawitz, and others ^ show that the con-
version of fibrinogen into fibrin is effected by a ferment produced
by the interaction of a thrombokinase derived from the
leucocytes and other cells with a thrombogen and calcium salts
present in the blood plasma. E. W. A. Walker^ found that
oxalate plasma which had been heated for two hours to 50° did
not coagulate on the addition of calcium chloride solution, but
did coagulate if fresh tissue extract were added as well. This
seems to indicate that a temperature of 50° slowly destroys a
thermolabile thrombokinase which is present in the oxalate
plasma, but does not affect a thermostable thrombogen.
Consequently the addition of calcium salts and of fresh throm-
bokinase (in the tissue extract), brings about coagulation.
Though there is no direct proof that thrombogen resembles
immune body in binding itself to the fibrinogen on the one
hand and to the thrombokinase on the other, yet the fact that
both it and immune body are thermostable, whilst both
thrombokinase and complement are thermolabile, suggests a
complete analogy.
In a similar manner Walker found that ptyalin solutions,
inactivated by heating to 50° to 53°, could be re-activated by the
addition of blood. This seems to show that ptyalin consists of
a specific thermostable substance, without independent activity,
and a non-specific kinase or complement, which is present in
blood and tissue extracts as well as in saliva. Again, he
found that rennet preparations could be inactivated by heating
* For literature, sec Buckmaster, Science Progress^ 2, p. 51, 1907.
2 E. W. Ainley V^dXVtr^ Joum. Physiol.^ 33, Proc. xxi., 1905.
V
206 ENDOENZYMES AND PROTOPLASM
to 55°, and re-activated by the addition of fresh liver extract
Donath^ worked with pancreatic steapsin, and he found that
the enzyme was inactivated by heating to 60** to 63", but could
be partially re-activated by the addition of normal horse serum.
If the steapsin were heated to 'J^'' to 80**, it could not be
re-activated.
However, the activity of steapsin appears to be controlled by
still a third factor. Magnus ^ found that liver extracts lost
their hydrolytic action upon amyl salicylate when they were
dialysed, but that they regained it on addition of boiled liver
extract. Hence he thought that the activity of the enzyme
depended on a diffusible " co-enzyme." Loevenhart * confirmed
this result, and he proved the co-enzyme to consist of bile salts.
Again, v. Fiirth and Schiitz * found that the action of steapsin
on olive oil might be increased fourteen-fold by the addition
of bile. Donath observed an even greater effect than this with
bile salts, and he concluded that the bile salts liberated free
steapsin from a zymogen precursor.
If further experiment confirms the results of Walker and
Donath, and the interpretation put upon them, it does not
necessarily follow that all ferments act in a similar manner
through the intermediation of a specific amboceptor or immune
body. Some may act directly and some indirectly in the same
way as Ehrlich's receptors appear to do.
The existence of separate haptophorous and zymophorous
groups in enzyme molecules is supported by other evidence.
Korschun ^ found that if rennin solutions were filtered through
a Berkefeld filter, the enzyme lost its milk-curdling power to a
much greater extent than its power of neutralising antirennin.
In fact the strength of the one property might be reduced ten
times more than that of the other. This seems to show that
the rennin solution consisted partly of ferment molecules with
both haptophorous and zymophorous groups, and partly of
molecules with haptophorous groups alone, and that the pores
* Donath, Hofmeistet^s Beitr,^ 10, p. 390, 1907.
2 Magnus, Zeitf.physiol. Chem.^ 42, p. 148, 1904.
3 Loevenhart, /^f^fiv. Biol. Chew.y 2, p. 391, 1907.
* V. Fiirth and Schiitz, Hofmeistet^s Beitr,^ 9, p. 28, 1906.
* Korschun, Zeit f. physioL Chetn.^ 37, p. 366, 1903.
ZYMOIDS 207
of the filter retained a larger proportion of the former molecules
than of the latter. Pollak ^ and Schwarz ^ obtained confirma-
tory evidence by another method. Pollak found that if trypsin
solution were inactivated by heating to 70°, it was still able to
retard the action of fresh trypsin upon gelatin, and to a much
smaller extent, its action upon serum proteins. Similarly,
Schwarz found that if pepsin solution were inactivated by
heating to 60', it had a paralytic effect upon the activity of
fresh pepsin solutions. Korschun's results indicate that the
inhibitory substances, the zymoids ^ or fermentoids as they have
been termed, are present, preformed in the original enzyme
solution. Hence the destruction of the trypsin or pepsin
molecules by heat serves merely to unmask these zymoids.
It was found by Donath* that pancreatic steapsin, if
inactivated by heating to 70° to 100°, exerted a paralytic effect
on active enzyme to which it was added. Again, Beam and
Cramer* found that solutions of rennin, taka-diastase, and
emulsin, inactivated by heating to 56° to 60° for about half an
hour, likewise exerted an inhibitory influence on the activity
of the corresponding enzymes, but it was necessary to add a
considerable amount of the inactivated enzyme to produce an
effect Different preparations of the same enzyme varied
considerably in their inhibitory power, and in some cases gave
absolutely negative results. But such failures do not disprove
the validity of the positive results. The zymoid molecules may
be thrown off by the cells in varying proportions as compared
with the enzyme molecules, or the heat inactivation may
have destroyed larger or smaller numbers of them in different
cases. Though Korschun's results indicate the presence of
preformed zymoids, they do not exclude the possibility of
conversion of enzyme into zymoid by heat inactivation, or
during the gradual deterioriation of activity which all enzyme
solutions undergo in course of time. Arguing from the analogy
of toxoid formation from toxins, such a conversion is highly
^ Pollak, Hofmeistet^s Beitr,^ 6, p. 95, 1904.
^ Schwarz, ibid*^ 6, p. 524, 1905.
3 Q» Bayliss, Arch, d. Set, bioLy 11, Suppl., p. 271.
* Donath, loc, cit
* Beam and Cramer, Biochem, Joum,^ 2, p. 174, 1907.
208 ENDOENZYMES AND PROTOPLASM
probable. Thus Ehrlich observed that the poisonous action
of toxin solutions might deteriorate rapidly, whilst their power
of binding antitoxin remained nearly constant For instance,
a diphtheria toxin, after being kept for nine months at room
temperature, was found to have lost two-thirds of its toxicity,
but to have retained its original capacity for binding antitoxin.
Ehrlich explained this result by supposing that the toxophorous
affinities of the toxin molecules had been destroyed, whilst the
haptophorous affinities remained intact
It seems probable, therefore, that enzyme and zymoid
combine with substrate in somewhat the same way that toxin
and toxoid combine with antitoxin. Proof of such combination
was obtained by Beam and Cramer in the case of rennin
zymoid. They found that if active rennin solution were added
to the diluted milk before the inactivated rennin, no retardation
whatever was produced; but if the inactivated rennin were
added before the active enzyme, the coagulation time was
nearly doubled. If the inactivated rennin were allowed to
stand with the milk at room temperature for five minutes
before the addition of the active rennin, the coagulation time
was twice as long as when the active rennin was added
immediately after the inactive rennin. These experiments
indicate that the rennin zymoid gradually combines with some
of the caseinogen of the milk and so prevents or delays the
action of the rennin enzyme upon it They require repetition
and confirmation, however, as Beam and Cramer state that
their results were irregular.
Antiferntents. — Enzymes closely resemble toxins in still
another respect, viz. in their power of stimulating the tissues
to form anti-bodies. In 1899 Morgenroth^ showed that
the subcutaneous injection of small doses of rennet ferment
immunised an animal against the ferment, and its blood serum
was found to contain an antirennin. Immune serum of
moderate strength was obtained. Thus in one experi-
ment the addition of 2 per cent of it to milk was
sufficient to necessitate the addition of i part of rennet
ferment in 15,000 before coagulation was induced. In the
absence of antiferment, only i part of rennet in 3,000,000
^ Morgenroth, Cent/. Bacty 26, p. 349, 1899 ; 27, p. 721, 1900.
ANTIFEEMENTS 209
was necessary, or 200 times less. The antirennin was specific
to the extent of being unable to neutralise vegetable rennet
ferment, and this ferment similarly formed an anti-body which
could not neutralise rennin of animal origin. Korschun ^ states
that the relation of rennin to antirennin closely obeys the laws
of toxin-antitoxin reaction, and he finds that at room tempera-
ture the union of rennin and antirennin is completed in fifteen
minutes. However, Fuld and Spiro^ state that ordinary blood
serum contains rennin in addition to the antirennin previously
shown to exist in it by Morgenroth, and that the ferment and
its anti-body can be separated by fractional precipitation with
ammonium sulphate. The addition of 28 to 33 per cent of this
salt to horse's serum throws down the rennin along with the
euglobulin, whilst 34 to 46 per cent of the salt throws down
antirennin along with pseudo-globulin. It seems difficult at
first sight to reconcile these statements with those of Korschun,
but perhaps ferment and antiferment react with one another
in the same way as a weak acid and weak base. As already
mentioned in a previous lecture, Arrhenius and Madsen*
showed that if ammonia and boric acid were allowed to interact,
the affinity of the acid and the base for one another is so small
that the solution contains, in addition to ammonium borate,
considerable quantities of free base and free acid. It seems
probable that in the same way mixtures of toxin and antitoxin
solutions contain free molecules both of toxin and antitoxin,
in addition to the loose toxin-antitoxin combinations. The
ratio of free molecules to combined molecules varies with
the affinity which the reacting bodies have for one another, and
if ferment and antiferment have but a weak affinity, a solution
of the two of them would contain sufficient free molecules
to render a partial separation possible by fractional salting
out
The attraction of ferment for antiferment and of toxin for
antitoxin is probably complicated by a physical factor, dependent
* Korschun, Zeit f. physioL Chint,^ 36, p. 141, 1902.
' Fuld and Spiro, ibid.y 31, p. 132, 1900.
^ Arrhenius and Madsen. See Arrhenius, ImmuiUhchemistry^ New
York, 1907, p. 174 ; also, article by J. Ritchie in Allbutt and Rollestotis
System of Medicine^ 2, Part I., p. 69, 1906.
^ 2D
210 ENDOENZYMES AND PROTOPLASM
on their colloidal or semi-colloidal nature. Craw^ found that
the reaction of the lysin and antilysin of Bacillus megatherium
does not obey the law of mass action, and so he concludes that
it is not a purely chemical change. It seems in many ways
analogous to the adsorption phenomena mentioned in a previous
lecture, but at present there is not sufficient evidence to enable
us to decide as to the relative degrees of importance to be attached
to the physical and the chemical factors of the interaction.
Rennin is by no means the only ferment for which an
antiferment has been obtained. Sachs ^ immunised geese
against pepsin, and the serum of these animals contained
so much antipepsin that in the presence of i c.c. of the serum
twenty times more pepsin was necessary to liquefy gelatin than
in the presence of i c.c of normal goose serum. Achalme^
injected sterile pancreatin preparations which had been filtered
through a clay filter into the peritoneal cavity of guineapigs,
and obtained a fairly active antitryptic serum. Camus and
Gley,* Landsteiner,^ and others have shown that normal blood
serum contains an antitrypsin. It is attached to the albumin
fraction of the serum proteins. Hedin® found that if trypsin
and serum albumin were mixed together before they were
added to the substrate (caseinogen), the neutralising effect
of the anti-body was much larger than if they were added
separately. If they were allowed to stand together at room
temperature for an hour or two before adding them to the
substrate, the neutralising effect was larger still. Hence the
ferment united or reacted with the anti-body rather slowly,
and, as far as one can judge from the data obtained, its affinity
for anti-body seemed to be no greater than its affinity for
caseinogen. Egg albumin has a much greater antitryptic
effect than serum albumin, for I found ^ that the addition of
^ Craw, Proc. Ray. Soc.y B. 76, p. I79i 1905 ; Joum, Hygiene^ 7, p. 501,
1907. See also, Arrhenius, ibid,^ 8, p. i, 1908.
* Sachs, Fortschr. d. Med,^ 20, p. 425, 1902.
3 Achalmc, Ann. de PlnsL Pcuteur^ 15, p. 737, 1901.
* Camus and Gley, C. R. Soc. BioL, 47, p. 825, 1897.
^ Landsteiner, Centralb. f. BacL, 27, p. 357, 1900.
^Hedin^/oum. Physiol., 32, p. 390, 1905; Biochem. Joum., i, p. 474,
1906.
7 Wttnon, Joum. Physiol, 3', P- 355, 1904.
ANTIFERMENTS 211
I part of egg albumin to 6000 of trypsin solution reduced the
digestive action of this ferment upon fibrin to about half the
normal value. Exposure of a solution of egg albumin to a
temperature of 60° did not diminish its antitryptic influence,
and even after keeping it for three hours at 100° C the coagu-
lated albumin still retained a good deal of inhibitory power.
It is therefore of quite a different nature from the antitrypsin
formed by injecting animals with trypsin, for this body is
weakened by heating to 56°, and destroyed by heating to 64°.
Antiferments against urease, tyrosinase, laccase, and fibrin
ferment have been obtained: also, though not with much
certainty, against diastase. Bertarelli^ immunised rabbits
with the lipase of castor-oil seeds, and found that the serum
contained an antilipase. This anti-body was active against
castor-oil seed lipase, but not against lipases of animal origin.
Bertarelli did not succeed in obtaining an antilipase on injecting
rabbits and dogs with animal lipases, but this negative result
may well have been due to technical difficulties. It seems
probable that every enzyme, if injected under suitable conditions
into a animal, is able to induce the formation of a specific anti-
body. This is another argument in support of the protein-like
nature of enzymes, for as far as we know proteins are the only
substances to the stimulus of which the cellular protoplasm
reacts in this way.
Constitution of Biogens, — Of the constitution of biogens or
biogen nuclei we know nothing. Indeed it is doubtful whether an
actual nucleus, in the ordinary acceptation of the term, exists
at all. All that we know of the constitution of protoplasm
concerns only the numerous intracellular enzyme groups
described in the previous lectures, and the still more numerous
toxin, antitoxin, lysin, agglutinin, precipitin, opsonin, and other
similar bodies which we know to exist in the protoplasm, or to be
capable of formation by it. Almost every essential constituent
of the protoplasm is probably, therefore, a body of protein-like
nature. These various protein molecules doubtless differ
considerably from one another in size. For instance, Arrhenius
and Madsen* found that diphtheria antitoxin diffused nearly
* Bertarelli, Centralb,/. Bact^ 40, p. 231,
2 Arrhenius and Madsen. See Arrhenius, ImmunO'Chemistryy p. 25,
212 ENDOENZYMES AND PROTOPLASM
ten times more slowly than diphtheria toxin, and Craw^
found that a lysin from B, megatlterium could pass
through a gelatin filter, whilst the antilysin could not. But
none of these protein bodies appear to be more complex than
the other proteins known to us, and hence their isolation in a
pure state, and the determination of their exact chemical com-
position and even of their chemical constitution, appears to be
only a question of time. What is the fundamental difference,
therefore, in the structure of a biogen, a unit of living protoplasm,
and of the protein molecules of which it consists ? We know
that it is in an extremely unstable condition, and is continually
undergoing decomposition changes and recomposition changes,
but does such instability and continual chemical change imply
the existence in the biogen of some central nucleus of an
entirely different chemical constitution from that possessed
by proteins ? It might be thought that a complex iron-contain-
ing nucleoprotein molecule forms the central nucleus to which
numerous protein side-chains are attached, and perhaps such
nuclei as this are actually present in the (morphological)
nucleus of the cell, which we know to be rich in iron and
phoq)horus compounds. But the cytoplasm of many cells is
extremely poor in organically bound phosphorus. By a
microchemical method Macallum ^ showed the presence of small
quantities of organic phosphorus in the cytoplasm, but Scott ^
states that the principle of the reaction used is wrong, and that
deductions from it £is to the distribution of organic phosphorus
compounds in cells are valueless. Burian and Walker Hall*
found that whilst lOO gm. of thymus contain -40 gm. of purin
nitrogen bound up as nucleoprotein, 100 gm. of muscle contain
only -015 gm. Most of this purin must be localised in the nuclei
of the muscle fibres, hence it is possible that the cytoplasm
of the muscle cells and likewise of many other cells contains no
organically bound phosphorus whatever.
* Craw, Proc. Roy, Soc,^ B. 76, p. 179, 1905.
2 Macallum, Proc. Roy, Soc.^ 63, p. 467, 1898.
5 Scoix^ Joum» PhysioLy 35, p. 119, 1906. See also Bensley, BioL Bulletin^
10, p. 49, 1906 ; Nasmith and Fidlar, yi7«r«. PhysioL^ 37, p. 278, 1908 ; and
Macallum, Ergebuisse der PhyHoLy vii., p. 637, 1908.
* Burian and Walker Hall, Zeit,f, PhysioL Chem^y 38, p. 336, 1903.
CONSTITUTION OF BIOGENS 213
In the absence of a nucleoprotein nucleus, it seems to me
that the biogens should be regarded rather as a congeries of
nunfierous protehi-like molecules, of different chemical constitu-
tion and functions, which are loosely bound together by weak
chemical bonds. The number of such protein groups in a
single biogen is so large that it is impossible for them to hold
together as a stable unit. Hence they are continually breaking
away and uniting to some neighbouring biogen, or uniting to-
gether in some other combination to form a new biogen. Or one
might even say that no really isolated biogen units exist at all,
but that the protoplasmic contents of a cell consist of a mass of
protein-like groupings, loosely bound together, but continually
breaking away from one another and uniting together again in
fresh combinations and arrangements. Every protein con-
stituent of a biogen is therefore a side-chain, and the central
nucleus of a biogen consists only of the general mass of side-
chains to which the particular side-chain under consideration is
attached. If the cytoplasm of most cells possess no fixed
structure or definite stable nuclei, it seems to follow that it
cannot perform synthetic functions, and elaborate toxin,
enzyme, and other groups from the individual amino acids
brought to it in the blood plasma. The evidence, so far as it
goes, seems to point to this being the case, for it is well
known that enucleated pieces of protoplasm of certain
protozoa are unable to assimilate food, or show other
synthetic powers, and so speedily die. If the synthetic pro-
perties of living tissues be confined to the nuclei of the cells,
therefore, it is probable that such synthesis can be induced only
by nucleoprotein-containing biogens of definite stable structure.
However, in the present state of our knowledge, such discussion
is of little value. I have brought forward these suggestions
chiefly to indicate that the biogen nucleus is only a theoretical
conception, which has at present very small experimental support.
If it be admitted that we have no grounds for assuming
that protoplasm contains chemical units of much greater
complexity than the protein and nucleoprotein substances
known to us, it follows that we must to some extent revise
our ideas concerning the anabolic and katabolic processes df
living tissues. It has frequently been assumed that protoplasm
n
I
214 ENDOENZYMES AND PROTOPLASM
IS of such enormous complexity that the ordinary protein
molecule, as known to us, is but a short stage in the chain of
synthetic processes required to elaborate actual living substance ;
and correspondingly, that the enzymes and other protein-like
bodies formed and secreted by living substance represent one
of the later stages of protoplasmic katabolism. On the view
above suggested, protein-like groups represent practically the
summit of synthetic processes. Once they are formed, they
loosely combine with other similar protein groups, but the
bonds so uniting them are probably of a weaker nature than
the bonds uniting the individual amino-acid molecules of
which they are composed. They still retain more or less of
their own individuality and constitution, therefore, and so
should be regarded as the actual working units of the living
substance.
It has already been pointed out that if an organ like the
kidney be perfused with saline for some days after death, the
endoenzymes and proteins of the tissues only very gradually
break away from the gland cells, and pass into solution. This
might be held to indicate that these products were formed by
a gradual breaking down of very complex precursors into
simpler and simpler substances, which finally arrived at the
stage in which they passed into solution.^ Though such an
explanation cannot be disproved, it is equally probable that the
gradual liberation of the enzyme and protein groups is dependent
partly on their colloidal nature, and partly on their being
bound up in the tissues with various degrees of firmness. We
have seen that in the pancreatic tissue the diastatic enzyme is
less firmly bound up than the tryptic-rennetic enzyme. Also
we know that colloidal bodies react very slowly in combining
with or in breaking free from one another, and that their inter-
actions are greatly influenced by physical considerations.
In the case of pepsin, trypsin, and some other ferments, we
know that the enzyme passes out from the cell in zymogen
form, and that only after such secretion does it become
converted into free enzyme. But even this conversion seems
to be merely some molecular transformation, and does not
involve a breaking down of larger molecules into smaller ones.
1 Cf. Langley,/^r». Physiol, 3, p. 290, 1882.
ACTION OF ANTISEPTICS 215
Thus I found ^ that the precipitability of trypsinogen from a
glycerin solution by various strengths of alcohol was practically
identical with the precipitability of the free trypsin.
Action of Antiseptics, — The argument that living tissues
differ from dead tissues and their disintegration products in
degree rather than in kind is supported by another entirely
different class of evidence, viz, by their reaction to anti-
septics. It is frequently supposed that antiseptics have little
or no influence upon the activity of enzymes, whilst very small
quantities of them are fatal to the vitality of living organisms.
Evidence controverting both suppositions has been adduced
incidentally in the course of previous lectures, but the subject
is one of such 'importance that it deserves to be treated more in
detail.
As already stated in an earlier lecture,^ Buchner and Rapp
divide antiseptics into two classes, viz, those which enter into
chemical combination with proteins (and presumably with the
protein constituents of living tissues, and with enzymes), and
those which do not. In the first class fall salts of the heavy
metals such as mercury, silver, and copper, and perhaps the
fluorides, arsenites, and cyanides. In the second class come
ether, chloroform, toluol, and other similar bodies, most of
which have a small solubility in water, and a great one in fats.
Of the heavy metal salts, corrosive sublimate is the best known
example. Its action upon protozoa is very variable. Bokorny^
states that a -005 per cent solution kills ParanuBctum and
Vorticella in six hours, whilst a 002 per cent solution kills in
two days. Davenport and Neal* found that a -001 per cent
solution killed Stentors in a minute or two, but in a -0001 per
cent, solution they not only lived, but became so acclimatised to
the poison that when subsequently placed in a -001 per cent
solution they survived two or three times as long as unac-
climatised Stentors. Algae are in some respects more sensitive
to corrosive sublimate than protozoa, for Bokorny ^ found that
1 Vemotkyjoum. Physiol,, 29, p. 318, 1903.
' See p. 91.
3 Bokorny, Pfiuger's Arch,, 85, p. 257, 1901.
* Davenport and Neal, Arch,f, Entwickelungsmechan,, 2, p. 564, 1896.
^ Bokorny, PftUgef^s Arch,, 108, p. 216, 1905.
216 ENDOENZYMES AND PROTOPLASM
Spirogyra was killed by a -00 1 per cent solution in a few hours,
whilst some of the cells were killed after twenty-four hours*
immersion in a -0001 per cent, solution. Cultures of the plant
in a -00001 per cent, solution, and in one ten times more dilute,
showed distinct changes, and after a few days in a solution ten
times more dilute still (i in 1000 million), the filaments con-
tained a much smaller store of glycogen than those kept in pure
water. Certain bacteria are much more resistant, for Koch
says that a -02 per cent, solution of sublimate does not disinfect
invariably.
Enzymes are in some instances just as sensitive to the action
of corrosive sublimate as living organisms. A -oi per cent
solution is said ^ to destroy malt diastase in twenty-four hours,
whilst a -02 per cent, solution destroys yeast zymase and maltase
in a similar period. On the other hand, • i per cent solution does
not affect rennet ferment, though -2 per cent delays its action.
This variation in sensitiveness seems very large, but it is due in
part at least to the different conditions under which the observa-
tions were made. The sublimate destroys an enzyme by com-
bining with it, and if the enzyme solution contains protein
impurity, it will combine with this instead, in proportion to the
amount of it present, and so the enzyme will be to a greater or
less degree protected. The ratio between total quantity of protein
and total quantity of sublimate present in a given solution is often,
therefore, of greater importance than the actual concentration of
the salt Arguing on this principle, first enunciated by Buchner,
Bokorny ^ calculated the lethal dose of various poisons for a
given weight of yeast cells and of Spirogyra. He found that if
ID gm. of pressed yeast (containing about 1-5 gm. of dry protein)
were mixed with 10 cc. of -05 per cent Hg^lg, the cells still
showed some reproductive activity after twenty-four hours, but
if mixed with 20 cc of the sublimate solution, they showed
none. Hence the lethal dose of sublimate for 10 gm. of yeast
is -005 to -OI gm. of HgClg. In a similar manner Bokorny
found the lethal dose of sublimate for 10 gm. of Spirogyra
(weighed in the moist condition), to be '0005 to '00005 gm.
This weight of alga contains '14 to -28 gm. of protein, or about
* Bokorny, loc. cit
2 Bokorny, Pfluget^s Arch.y iii, p. 348, 1906.
ACTION OF ANTISEPTICS 217
a tenth as much as lo gm^ of yeast, and hence the smaller
lethal dose roughly corresponds with the smaller protein content
of the alga.
To silver salts living organisms and enzymes are probably
about as sensitive as they are to mercury salts. A -02 per cent.
solution of silver nitrate kills most bacteria : a -oi per cent
solution destroys malt diastase, yeast maltase, and zymase in
twenty-four hours, whilst a -05 per cent, solution does not injure
rennet ferment (Bokorny).
Fluorides and arsenites are not, as a rule, nearly so
poisonous as the salts of the heavy metala A -i per cent
solution of sodium fluoride kills algae in twenty-four hours, and
according to Tappciner and to Loew, a -oi per cent: solution
prevents putrefaction. Bokorny found that if 10 gm. of yeast
were mixed with 50 c.a of -i per cent NaF, the cells still
retained their reproductive activity twenty-four hours later.
They were unable to resist twice this quantity of fluoride, how-
ever, so the lethal dose of the salt is -05 to -i gm., or ten times
greater than that of corrosive sublimate. Upon enzymes fluorides
are very much less poisonous than upon living organisms.
The most sensitive of them, zymase, has its action stopped by a
•55 per cent solution of ammonium fluoride, but most other
enzymes can act fairly well in the presence of i per cent or
more of NaF. Still they are retarded to some extent For
instance, I found ^ that intestinal erepsin took seventeen and a
half hours to split up half of the peptone in a given sample
when acting in presence of toluol: twenty-six hours when
in presence of chloroform, and forty-two hours when in
presence of i per cent NaF. Also it is to be remembered
that though living organisms cannot exist permanently in more
than very dilute fluoride solutions, yet their vitality is not
immediately destroyed by concentrated solutions. A i per cent
NaF solution takes two hours to kill frog's nerves,* and I found *
that on perfusion of a mammalian kidney with a similar solution,
the gaseous metabolism fell only to a half or third the normal
during the next three hours, and did not disappear entirely
^ Wttnon^ Jaum. Physiol,^ 30, p. 365, 1903.
2 Davenport, Experimental Marphologyy London, 1897, p. 22.
2 Vernon, ^«r». PhysioL^ 35, p. 77, 1906.
2 £
218 ENDOENZYMES AND PROTOPLASM
even after three days. Perfusion with i per cent, arsenious acid
solution gave a similar result.
Formaldehyde is sometimes quoted as a suitable poison for
the differentiation of enzymes and living organisms, but
probably it is no more efficient than those already quoted.
Bokorny states that a -i per cent solution acts fatally upon
bacteria and other organisms, whilst a '005 per cent, solution
kills Spirogyra in a few days. Also Borkorny found that 25 c.c
of a • I per cent, solution were insufficient to destroy the vitality
of 10 gm. of yeast in twenty-four hours, whilst twice this
quantity of the solution was more than sufficient. Hence the
lethal dose was '02S to '05 gm. Some enzymes are almost as
susceptible to the action of formaldehyde as living organisms,
for the activity of malt diastase is very greatly diminished by
exposure for twenty-four hours to -01 per cent, of it. A • i per
cent solution acts injuriously on yeast maltase in twenty-four
hours, whilst i per cent destroys it A -2 per cent solution
destroys zymase in twenty-four hours, whilst •$ per cent
hinders rennet ferment, though it does not entirely destroy it
Most resistant of all is the enzyme myrosin, which is not
affected by i per cent of formaldehyde, and is destroyed only
after twenty-four hours by 5 per cent of it^ Loew found that a
5 per cent solution rapidly destroyed catalase.
Antiseptics of the second class do not enter into chemical
combination with proteins, and their action perhaps depends
upon their solubility in the fatty constituents of the cell.
Hence, beyond a certain minimal limit, their action should vary
only with their concentration, and there should be no quanti-
tative relationship between the amount of protoplasm or
protein present and the amount of poison.
Antiseptics of this second class, such as ether, chloroform,
thymol, toluol, and phenol, are of much greater value than those
of the first class for differentiating between living organisms
and enzymes. No organism can live in their saturated aqueous
solutions, whilst most enzymes can still exert their activity,
often with extremely little retardation. The great sensitiveness
of living organisms probably depends upon the disintegrating
effect exerted by the antiseptic solution^ upon living tissues.
1 C/, Bokorny, Pfluget's Arch,^ 85, p. 257, 1901.
ACTION OF ANTISEPTICS 219
As already mentioned, I found that perfusion of a freshly
excised mammalian kidney with saturated solutions of chloro-
form or ether caused an immediate disintegration of the tissues,
and the passage outwards, in the perfusion liquid, of large
amounts of proteins and endoenzymes. It might be supposed
that the action of the antiseptic was first and foremost to kill
the cellular protoplasm, and that disruption of the biogens
ensued only subsequent to their death, and in consequence of
it. In that a similar disintegration of the tissues of a dead
kidney occurs immediately it is perfused with ether or chloro-
form saline, it looks rather as if the action of the antiseptic
were directly upon the chemical bonds which unite the protein
and enzyme groups to the biogens, and that death of the
protoplasm ensued subsequent to the breakage of these bonds,
or simultaneously with it
The number of instances in which the influence of these
antiseptics upon enzyme action has been investigated quanti-
tatively is comparatively small. As stated in a previous lecture,
Magnus-Levy ^ found that large pieces of liver and other organs
underwent more autolysis in a single day at 37° under aseptic
conditions than in several months if chloroform or toluol were
present In contrast to this, Lane-Claypon and Schryver^
found that minced liver, kept in saline at 37**, underwent
autolysis almost as rapidly in presence of toluol as in its
absence. Buchner found that toluol slightly retarded the
action of zymase upon sugar, whilst thymol reduced the COg
output by a half. Bokorny^ states that chloroform does not
influence the action of emulsin or of rennin, but that small
quantities of it destroy pepsin. On the other hand, a saturated
solution of thymol (i in iioo) destroys rennin. Kastle and
Loevenhart * found that '02 per cent solutions of toluol, chloro-
form, and phenol hardly affected the action of liver lipase upon
ethyl butyrate, whilst thymol and salicylic acid retarded it
slightly. In the case of phenol alone of the second class of
antiseptics is the action upon enzyme and living organisms
^ Magnus- Levy, HofmeisUf^s Beitr,^ 2, p. 261, 1902.
2 Lane-Claypon and Schryvcr, yi7«r«. PhysioLy 31, p. 169, 1904.
3 Bokomy, Pftiiget^s Arch,^ 85, p. 257, 1901.
* Kastle and Loevenhart, Amer, Chem,Joum,^ 24, p. 491, 1900.
220 ENDOENZYMES AND PROTOPLASM
closely comparable. A 5 per cent solution of it kills Anthrax
bacilli, but even a 5 per cent solution does not kill all spores.
A -I per cent solution kills yeast cells, but does not afiect
zymase and maltase. However, these two enzymes are
rendered inactive by a i per cent solution, though the invertase
which accompanies them does not seem to be affected (Bokorny).
With regard to antiseptics as a whole, therefore, we may say
that there is no sharply defined demarcation between the
reaction of living organisms and that of enzymes. Living
organisms differ more widely amongst themselves in their
sensitiveness to antiseptics than do the most resistant organisms
from enzymes. A striking instance of this is seen in the case of
Anthrax spores just recorded, for they can withstand a concen-
tration of phenol 100 times greater than that which kills most
other forms of life.
Influence of Temperature. — The essentially chemical nature
of enzyme action, and of the changes occurring in living tissues,
is indicated by the response of these processes to temperature
changes. Arrhenius^ and van*t Hoff* have shown that the
velocity of chemical reactions is roughly speaking doubled for
each rise of temperature of 10°. The observed quotients vary
between the limits of i-2 and 3-68, but the majority of them
vary only from 1-9 to 2-7. On the other hand, purely physical
processes such as the electrical conductivity of a wire, the
viscosity of a liquid, osmotic pressure and surface tension, are
not affected to anything like this extent by rise of temperature.
As can be seen from the data given in the table, the velocity
of enzyme action follows the law of chemical reactions, and not
that of physical processes. We see that in the case of various
proteolytic, milk-curdling, lipolytic, amylolytic, and catalytic
enzymes, quotients of about 2 were obtained. In sotne
cases more divergent results have been arrived at, but they
are omitted from the table as there seemed in their case good
reason to suspect secondary phenomena which modified the
primary temperature effects. It will be noted that the ijpper
temperature limit to which the quotient was estimated in no
^ Arrhenius, Zeit f, fhysik, Chem,^ 4, p. 226, 1899.
* Van't Hoff, Lectures .on Theoretical mnd Physical Chemisify^ LondoOi
1899, I., p. 228.
INFLUENCE OF TEMPERATURE
221
case exceeds 40°. It has in some instances been determined
up to 60° or 70"*, but it is usually found that at temperatures
above 40° the quotient gets smaller and smaller, and «ven
becomes negative. This is due entirely to the destructive
eflTect of high temperature upon the enzyme. The destruction
Enzyme and Sabstnte.
Tempenture
Range.
Quotient
ferlO'.
Action of Trypsin on Witte's Peptone * ,
„ Erepsin on Witte*s Peptone * . . .
„ Pancreatic Rennin on Milk f • . •
„ Gastric Rennin on Milk J . . . .
„ AmylopsinonStarch(in.2%NaCl)t .
„ Castor-oil Bean Lipase on Triacetin § .
„ Blood Catalase on Hydrogen Peroxide ||
15° to 25''
25: » 40:
20* „ 30"
18" „ 28'
0° „ 10*
2-3
2-6
2-1
3-1
2-0
2.6
i-S
* Vernon, Journ. PhyHoL, 80, p. 864, 1908. f Vernon, i&id., 27, p. 190, 1901.
t Fuld, HofmeisUr's Beitr., 2, p. 184, 1902. f Taylor, Jowm, BM, Chem., 2, p. 87, 1906.
II Senter, ZeU, /. physik. Chem., 44, p. 257, 1908.
becomes greater and greater the higher the temperature, till at
the so-called maximum temperature the enzyme is destroyed
almost immediately. The optimum temperature of enzyme
action is that temperature at which it acts most rapidly, or at
which the increased velocity due to high temperature is just
balanced by a diminution of effect resultant on destruction of
the enzyme. It is therefore a variable point, dependent on
the rate at which the reaction is progressing, and the presence
of other substances which may increase or decrease the stability
of the enzyme. For instance, Tammann^ found that when
a fixed amount of salicin was acted upon by one part of
emulsin, or sufficient to hydrolyse 34 per cent of it in twenty
hours, the optimum temperature was 26°. With two parts of
emulsin, the optimum was 35**, and 46*5 per cent was
hydrolysed in twenty hours. With four to sixty-four parts,
the optimum was 46'', and 64-0 to 94-5 per cent was hydrolysed ;
whilst with 128 parts, the optimum was 54°, and 94-5 per cent
was hydrolysed. Again, the writer ^ found that pancreatic
diastase, if allowed to act upon starch paste made up with tap
* Tammann, Zeit /, pAysioi. Chem,y 18, p. 436, 1894.
* Vernon, Jaurtk Physiol,,^ 27, p. 190, 1901.
222 ENDOENZYMES AND PROTOPLASM
water, attained its optimum at 35°; but if it were acting in
presence of *2 per cent NaCl, the conditions were so much
better adapted to its activity and stability that at 35° it
digested the starch four and a half times more rapidly, whilst
at its optimum temperature, viz. 50°, it digested it nine times
more rapidly. The maximum temperature in both cases lay
between 65° and 70°, whilst that of pancreatic rennet lay at
something above 70^ Tammann*s results show that the
maximum temperature for the action of emulsin on salicin is
about 75°, whilst that for yeast invertase on cane-sugar is about
62°. Brown and Heron ^ found that malt extract could be
heated to 76**, and the diastase would then act upon starch at
75°, so its maximum temperature must be a little above these
figures. Nicloux* found that the action of castor-oil seed
lipase upon olive oil is destroyed in ten minutes at 55^ hence
its maximum temperature is probably about 60°. Donath^
found that pancreatic steapsin was inactivated at 55° to 65°;
Schwarz,* that pepsin was inactivated by heating to 60° ;
Beam and Cramer,^ that rennin, pepsin, taka-diastase and
emulsin were inactivated at ^fi"* to 60°. Again, Mayer® states
that pepsin solutions lose their activity when warmed to 57°,
but Pekelharing ^ found that exposure of a pepsin solution for
two minutes to a temperature of 70° did not entirely destroy
the ferment.
We may conclude, therefore, that the great majority of
enzymes are destroyed at a temperature varying in different
cases from 60" to ^^'',
The metabolic changes of living organisms, being of an
essentially chemical nature, might be expected to comply
with the law observed for chemical reactions and enzyme
actions. This is actually the case unless disturbing factors
such as nervous control are introduced. As pointed out by
1 Brown and Heron, yt?«r«. Chem, Soc. Trans.^ 1879, P- 596.
2 Nicloux, C. R. Soc, BioL, 56, pp. 701^ 839, and 868, 1904.
3 Donath, Hofmeister^s Beitr,^ 10, p. 390, 1907.
* Schwarz, ifoV/., 6, p. 524, 1905.
6 Beam and Cramer, Biachem, Joum,^ 2, p. 174, 1907.
« Mayer, ZeiLf, BioLy 17, p. 351, 1881.
^ Pekelharing, Zeit f* physioL Chem.^ 22, p. 242, 1897.
INFLUENCE OF TEMPERATURE 223
van't HoflT/ the observations of Clausen 2 upon the carbonic acid
discharge of seedlings of wheat, lupins, and syringa flowers
show a temperature quotient of 2-5 between 0° and 25°. Miss
Matthaei ^ found that the assimilation of carbon dioxide by a
leaf of Prunus Laurocerasus had a temperature quotient of 2-4
between 0° and 10°; one of 2-i between 10° and 20°, and one
of 1-8 between 20° and 30°. Aberson * found that the fermenta-
tion of glucose by yeast was 29 times more rapid at 27° than
at 17-5°. The writer^ investigated the effect of temperature
upon the oxygen intake of various marine animals, and
between 10° and 20° obtained quotients of 1-9 to 3-7 for various
transparent pelagic Medusae, Ctenophores and Salpae, and
quotients of i-6 to 2-2 for various Mollusca and Fishes.
When we pass to higher forms of animal life, we find that
the temperature quotient does not by any means correspond
with the theoretical law* Not only does it vary considerably
in different animals, but in the same animal it shows great
differences at different temperature intervals. For instance,
in the frog (7?. temporaria) the writer found® it to be on an
average i«8 between lo"* and 20°, and 3'5 between zd" and 30°.
In the toad it was 1*3 and 4*1 for the same temperature inter-
vals respectively. Other cold-blooded animals showed similar
differences, and even the earthworm had quotients of 1-3 and
4- 1 for the respective temperature intervals mentioned.
Not only does the general metabolism of living organisms
follow the theoretical law, but many other vital processes not
necessarily dependent on chemical changes obey it also. Cohen ^
pointed out that the temperature effects observed by Hertwig^
upon the segmentation of developing frogs' eggs correspond
with it. The first stage of development gave a temperature
quotient of 2-2, and the later stages, one of 3-3. With Echino-
* Van*t Hoflf, Vorlesungen^ I., p. 224, 1901.
2 Clausen, Landwirtsch Jahrb,y 19^ p. 892, 1890.
3 Matthaei, Phil. Trans, Roy, Soc^ B. 197, p. 47, 1904,
^ Aberson, quoted from Arrhenius, ImmunO'Chemistry^ p. 139.
' Vernon, yi?«r«. Physiol,^ 19, p. 18, 1895.
* Vernon, ibid.y 21, p. 443, 1897.
^ Cohen, Varlesungen iiber pkysikaliscke Chemie^ p. 42, 1901.
^ Hertwig, Arch,f, mikrosk. Anal,, 51, p. 319, 1898.
224 ENDOENZYMES AND PROTOPLASM
devm eggs, Peter ^ obtained a quotient of 2*3 for the first stage,
and 2- 1 for the later stages, Loeb ^ found that the veloeity of
artificial maturation of Lottia eggs is more than doubled on
raising the temperature from 8° to 18°.
Upon the beating heart a number of observations has been
made. Snyder * observed that the isolated heart of the Pacific
terrapin {CUmys marmoratd) obeyed the law between the
temperature limits of 2-5° and 30°. Between lo"" and 20* the
quotient was 27, and between 20"* and 30*^, 2-2. At 35°, the
optimum temperature, the heart beat 48-6 times per minute,
but at 40"^ it sank to 43-3 per minute, so the high temperature
acted destructively in the same way as it is observed to do
upon enzymes. T. B. Robertson* determined the influence
of temperature upon the rate of heart beat in the transparent
fresh-water crustacean Ceriodaphnia, and between the limits of
1 1° and 29° he obtained an average quotient of 2-03. At 35'
the heart stopped permanently. Snyder^ investigated the
transparent nudibranch Phyllirrhat^ and he found that between
the limits of 16° and 29** the average temperature quotient for
the heart beating in situ worked out at 2*52. The isolated
heart of the crustacean Maia verrucosa had a co-efficient of
2-99 between the limits of 7** and 26^ Snyder also pointed
out that the observations of previous investigators upon the
mammalian heart likewise conform to the law of chemical
reaction velocities. Newell Martin^ experimented on the
isolated heart of the dog, and between the limits of 278"* and
42-5° he obtained quotients varying from i-8 to 3-3. Martin
and Applegarth^ obtained quotients of 1.3 to 3.2 for isolated
cats' hearts between the temperatures of 22-3** and 40-0°.
Langendorff ^ succeeded in maintaining the rhythm of isolated
cats' hearts between the temperature limits of 7* and 47°, and'
' Peter, Arch,f, Entwickelungsmechan,^ 20, p, 130, 1905.
* Loeb, " University of California Publications," Physiology^ 3, p. i-
^ Snyder, ibitL^ 2, p. 125, 1905.
* T. B. Robertson, Biol Bulletin^ 10, p. 242, 1906.
^ Snyder, Amtr.Joum. Physiol,^ 17, p. 350, 1906.
* N. Martin, Phil Trans, Roy, Soc,^ 174, p. 679, 18S3.
^ Martin and Applegarth, Studies from the Biol Laboratory^ Johns
Hopkins Univ,^ 4, p. 282, 1890.
« Langendorff, Pfliiger's Arch.y 66, p. 355, 1897.
PROPAGATION RATE IN NERVE 225
from his experimental data Snyder calculated that between the
temperatures of i6° and 39° the quotient varied only from i-8
to 3*7, and in most cases kept at about 27. At temperatures
above 39° it got less and less, but no actually negative quotient
was obtained. The human heart obeys the law no less than
that of other mammals, for Davy recorded his temperature
and pulse rate three times a day for eight consecutive months,
and found that when the mean temperature varied from 36-62**
to 37-07*', the mean pulse rate increased from 54-68 to 57-2,
Snyder calculated that these data give quotients of 2-3 to 3-1.
These numerous concordant observations upon the heart
beat appear to indicate that the beats are due to a constantly
repeated chemical reaction. The changes occurring in the
heart preparatory to a succeeding contraction are likewise of
a chemical nature, for, as Snyder points out, the refractory
period of the heart is affected by temperature in the same
way as the contraction rate. Burdon Sanderson and Page^
observed that between 12° and 27° the refractory period in the
frog's heart diminished from 2-0" to •S", or gave temperature
quotients of 1-8 to 2-5.
More interesting even than the effect of temperature upon
heart beat and gaseous metabolism is its influence upon the
propagation of the nervous impulse. The resistance of nerve
to fatigue, and the apparent absence of chemical changes on
stimulation, suggest that the transmission of the nervous
impulse is a physical rather than a chemical process. How-
ever, the results recently obtained show that temperature
influences the propagation rate in the same way that it affects
chemical reactions, or that the mechanism of propagation must
be of an essentially chemical nature. Nicolai ^ determined the
rate of propagation in the olfactory nerve of the pike, and found
that at temperatures ranging from 3-5' to 25* the rate varied
from 5-65 metres per second to 22-2 metres. From these data
Snyder^ calculated the temperature quotient to vary between
1-8 and 4-1, or to average 2-55. v. Miram* determined the
1 Sanderson and FaLgey/oum, Physiol.^ 2, p. 384, 1880.
2 Nicolai, PflUget^s Arch,^ 85, p. 65, 1901.
3 Snyder, Arch./, (Ana/, u,) PAysio/.^ 1907, p. 113.
* V. Miram, tdid.^ 1906, p. 533-
2 F
236 ENDOENZYMES AND PROTOPLASM
propagation rate in the motor nerves of the frog between 1 5*
and 35**, and his results give quotients varying from 1-4 to 2-8,
and averaging 2-i. MaxwelP experimented with the pedal
nerves of the giant slug, Ariolimax columManus. The mean
impulse rate is only -44 metre per second, and as it is possible
to use a nerve 10 cm. in length, the determination can be made
with considerable exactness. The temperature range was — i*
to 26^ and the quotients obtained varied from i-o8 to 3-15.
The majority of them varied only from 1-5 to 2-i, however,
and as they averaged 1-78, we may say that in all the experi-
ments recently recorded the nerve propagation rate was found
to be influenced by temperature in accordance with the chemical
law.
The fact that many of the vital processes of living cells are
of an essential chemical nature does not prove that they are
brought about by enzyme action. Indeed, a seeming proof
to the contrary is afforded by the fact that enzymes can
exert their action at temperatures of 60° to 76""^ whilst the
vitality of most living cells is destroyed at something under 5o^
In the case of the higher vertebrates death may be due to the
coagulation of the cell globulins, for extracts of most tissues
{e,g, liver, spleen, muscle, nerve) have been found * to contain a
globulin coagulating at 45** to 50°. But some organisms are
killed at temperatures considerably below that at which, as far
as we are aware, any protein coagulation occurs. I found,* for
instance, that the ova of the Echinoid Strongyhcentrotus lividus
were killed at 28* 5°. After fertilisation their death temperature
rose, during the next four hours, to 33'5'*, and during the next
twenty-four hours (by which time they had reached the pluteus
stage) to 39*5°. Hence the cause of death in these developing
organisms is absolutely obscure.
Under certain circumstances, however, living organisms can
withstand as high a temperature as enzymes. Dallinger*
gradually acclimatised certain Flagellata to increasing high
1 Maxwell, /<?«r«. BioL Chem,y 3, p. 359, 1907.
^ See article by Halliburton in Schafer's Text Book of Physiology^ i,
pp. 85, 87, 96, and 118.
' Vtxnon^ Joum. Physiol.^ 25, p. 135, 1899.
* Dallinger, /ii7i/r». Roy. Microsc. Soc^ 7, p. 191, 18^7.
DEATH TEMPERATURES 227
temperature, till after about six years they were able to live
and multiply at 70°. Various protophyta^ are known which
flourish in hot springs at temperatures as high as 93^ Some
spores {e,g. Anthrax) can be boiled in water for several minutes
without losing their vitality. It may be asked why such high
temperatures as these do not cause death by coagulating the
cell proteins. The explanation is probably to be found in the
fact that the temperature of coagulation of a protein varies
inversely with the aniount of water it contains. Lewith ^ states
that egg albumin coagulates at 74° to 80° when in presence of
25 per cent, of water; at 80° to 90° with 18 per cent of water;
at 14s'* with 6 percent, of water, and at 160° to 170° with no
water at all. So in all probability organisms capable of with-
standing high temperatures contain less water than usual.
This is almost certainly the case with spores. In this connection
it is interesting to recall the fact that dried enzyme preparations
can readily withstand a temperature of 100° or more.
Though the whole of the processes occurring in living cells
are certainly not due to the action of endoenzymes, we seem
justified in concluding that many or most of the chemical
processes are dependent upon them directly or indirectly.
Though our knowledge of the subject is at present very in-
complete, we have sufficient information to suggest that it is
only a question of time and extended research before we shall
be able to give final and conclusive answers to many of the
questions tentatively raised in the course of these lectures.
^ For literature see Davenport and Castle, Arch,f, Eniwickelungsmechan, ,
2, p. 227, 1895.
2 Lewith, Arch,f, exp, Path.^ 26, p. 341, 1884.
INDEX OF AUTHORS
Abderhalden, 13, 15, 16, 17, 18, 159,
172, 186, 187
Abeles, 92, 93
Abelous, 117, 118, 119, 128, 190
Aberson, 223
Achalme, 210
Acree, 181
AdamofT, 62
Albert, 94
Aldehoflf, 6i
Almagia, 35, 36, 37
Antoni, 95
Aptplegarth, 224
Arima, 114, 180
Arinkin, 10, 28
Annstrong, E. F., 168, 171, 173J 174,
178, 179
Armstrong, H. £., 59, 60
Amheim, 105, 106
Arrhenius, 161, 181, 209, 210, 211, 212,
220, 223
Arthus, 54, 108
Ascoli, 35, 69
Asher, 113
Astric^ 60
Aubert, 141
Austin, 35, 36
Austrian, 32, 33, 39
B
Bach, 115, 120, 122, 134, 192, 193
Baer, 10
Baeyer, 122, 123
Baker, H. B., 196
Bang, 29, 30, 155
Baranetsky, ^^
Barcroft, 139
229
Barratt, 162, 163
Bary, De, 76
Battelli, 39, 103, 128, 129, 138, 139, 140
Bayer, 189
Bayliss, 160, 162, 163, 168, 186, 200,
207
Beam, 207, 208, 222
B^champ, 78
Beijerinck, 24, 78
Bensley, 212
Bergell, 165
Bernard, 62, 63, 68, 72, 73, 107, 108
Bemeck, v., 132
Beminzone, 45, 190
Bertarelli, 210
Berthelot, 72, 78, 174, 192
Bertrand, 123, 125
Bial, 69
Biam^s, 117, 118, 119
Biedermann, 123
Biemacki, 152
Bierry, 72
Biffen, 59
Biondi, 11
Blank, 45
Blumenthal, loi, 103
Bodenstein, 182
Bohm, 65
Bokomy, 92, 215, 216, 217, 218, 219,
220
Bonfanti, 69
Boruttau, 65
Bourquelot, 78, 116, 124, 125
Brandt, 46
Bredig, 132, 133
Brodie, B., 192
Brodie, T. B., 135
Brown, A., 167, 168
Brown, H., 70, 71, 78, 167, 168, 222
Briicke, 146, 149
Brugsch, 43, 56
230
INDEX OF AUTHORS
Bruschi, 52
Buchner, E., 4, 82, 83, 84, 85, 88, 89,
90, 91, 93, 94, 95, 96, 97, 98, 99, 100,
loi, 126, 127,215, 216, 219
Buchner, H., 82
Buckmaster, 121, 205
Burian, 30, 33, 35, 37, 212
Buxton, 57, 131
Camus, 59, 210
Cathcart, 12, 17, 179
Castle, 227
Chapeaux, 48
Chassevant, 23, 35, 36
Chittenden, 152
Chocensky, no, 137
Chodat, 115, 120, 134
Chodschajew, 164
Cipollina, 36
Claus, 104
Clausen, 223
Cohen, 223
Cohnheim, 12, 45, 76, 103, 104, 105, 106
Connstein, 53, 59
Cotte, 57
Cramer, A., 62, 65
Cramer, W., 62, 207, 208, 222
Craw, 210, 212
Cremer, 180
Cuisinier, 78
Czemy, loi
Czyhlarz, 120, 121, 132
Edie, 162, 163
Edkins, 152
Edmunds, 156
Ehrlich, 154, 197, 204, 205, 206, 208
Embden, 104
Emmerling, 177, 178, 180, 181
Engelmann, 46
Erlenmeyer, 100
Ernest, no, 137
Euler, 60, 192
Eves, 63
Ewald, 132
Ewart, 194
Falk, 151
Feinschmidt, 107
Fermi, 158
Fidlar, 212
Fischer, E., 16, 74, 78, 127, 165, 169^
170, 171, 172, 173, 178, 179
Fletcher, 143
Ford, 107
Foster, 67
Frankel, 160, 164
Fr^d^ricq, 47
Friedenthal, 149
Fuld, 205, 209, 221
Fiirth, v., 47, 76, 120, 121, 123, 132,206
D
Dakin, n, 17, 18, 19, 21, 23, 44, 55,
16^, 166
Dalhnger, 226
Danilewsky, 153, 188, 191
Dastre, 153
Dauwe, 4, 161, 163
Davenport, 215, 217, 227
Davy, 225
Dietz, 182, 183
Dixon, 76
Doebner, 116
Donath, 206, 207, 222
Doyen, 108
Duclaux, 2, 99, 100
Durham, 124
Dzierzgowski, 21
Gaunt, 126, 127
Geduld, 78
Gerard, 59
Geret, 86
Gewin, 155
Gigon, 187
Glendinning, 167, 168
Gley, 210
Godlewski, 109
Gonnermann, 79, 80
Gorup-Besanez, 50, ^^
Gottlieb, 22, 44, 45
Gow, 73
Green, R., 50, 51, 58, 59, 60, 73, 76, 78,
79, 81, 125
Greenwood, 46, 76
Griitzner, 154
Guldberg, 182
INDEX OF AUTHORS
231
Gulewitsch, 45
Giimbel, 21
JoUes, 130
Jones, 27, 32, 33, 35, 39
H
Hahn, 82, 85, 86, 11 1
Hall, G. W., 105, 106, 107
Hall, W., 212
Halliburton, 66, 135, 226
Hamburg, 160, 164
Hamburger, 69
Hammarsten, 155, 164
Handel, 61
Hanriot, 54, 99, 181
Harden, 89, 95, 96
Hari, 191
Harris, 21, 73
Hartog, 76
Hausmann, 21, 22
Hedin, 4, 8, 9, 10, 42, 163, 164, 210
Henri, 168, 172
Heron, 70, 71, 222
Hertwig, 223
Herzog, 98, 10 1
Hildebrandt, 43
Hill, C, 176, 177, 178, 179
Hill, L., 29, 168, 191
Hinkins, 181
Hirsch, 105
Hirschler, 21
HofF, J. H. van't, 220, 223
Hofmeister, 2, 12, 149
Hopkins, 143
Hoppe-Seyler, 99, 164
Horbaczewski, 31
Hoyer, 59, 60
Hunger, 120
Hunter, 16, 172
Ikeda, 132
Inoko, 30
IwanofT, 27
J
Jacobson, 127
Jacoby, 7, 11, 20, 22, 23, 35, 36, 45,
119
Jackson, 113
Jaquet, 117, 119
Jelmek, loi, no
K
Kastle, 54, 116, 117, 120, 122, 123, 133,
142, 181, 219
Kikkoji, 28, 114, 180
Kirchoff, 76
Kisch, 66
Klappe, 96, 97
Kobert, 108, 127
Koch, 216
KoUiker, 79
Korscbun, 206, 207, 209
Kosmann, 77, 78
Kostytschew, 30, 109, 136, 137
Kossel, 18, 23, 191
Krassnosselsky, 136
Krauch, 77
Krukenberg, 47, 76
Kulz, 65
Kurajeff, 188, 189
Kiihne, 146
Kutscher, 13, 29
Landsteiner, 210
Lane-Claypon, 112, 201, 202, 219
Lang, 19, 23
LangendorfT, 224
Langley, 152, 214
Lapp«, 73
Latour, De, 82
Lawrow, 188, 189
Lea, S., 24
Leathes, 11, 19
Leavenworth, 40, 57, 62
Lengyel, v., 191
Lesser, 129
Leube, 24
Lewith, 227
Liebermann, 133
Liebig, 81
Lister, 76
Lochhead, 62
Loeb, 10, 30, 224
Loevenhart, 54, 120, 122, 123, 133,
142, 181, 206, 219
Loew, 120, 130, 131, 134, 157, 193,
217
Loewi, 0., 23
Loisel, 48
232
INDEX OF AUTHORS
Lossnitzer, 153
Lubarsch, 62
Liidy, 53
Lussana, 138
M
Macallum, 212
Macfadyen, 5, 82, 85, 86, 88
Macleod, 29
Madsen, 161, 209, 212
Magnus, 206
Magnus-Levy, iii, 112, 114, 143, 179,
219
Majendie, 68
Mann, G., 30, 162
Martin, C. J., 89, 95
Martin, N., 224
Matthaei, 223
Maximow, 136
Maxwell, 226
Mayer, 222
Maz^, 102
Meisenheimer, 97, 98, 99, 100, 10 1, 126
Meissncr, 76
Mellanby, 45
Mendel, 39, 40, 57, 62, 67, 72, 75, 76
Menschutkin, 175
Mering, v., 103
Mesnil, 48, 57, 76
Metschnikoff, 46
Meyer, De, 104
Miescher, 30
Milroy, 26
Minkowski, 103, 113
Miquel, 24
Miram, v., 225
Mitchell, 39, 72, 75, 76
Miura, 73
Mochizuki, 114, 180
Mohlenfeld, 191
Moll, 24
Moore, 168, 191, 194, 195
Morawitz, 205
Morel, 108
Morgenroth, 208, 209
Morris, 5, 78, 82, 85, 86, 88
Miiller, 79
Musculus, 23, 24
N
Nabokich, 109
Naegeli, 82
Nakayama, 28
Nasmith, 212
Nasse, 65
Neal, 215
Nef, 100
Nencki, 20, 45, 53, 99, '47, 148, 149,
154, 155, 164
Neumeister, 52, 113
Nicloux, 60, 222
Nicolai, 225
Niebel, 74
Oppenheim, 130
Oppenheimer, 108, 109
Orb^ 74
Osborne, 21, 163
Ostwald, 175
O'Sullivan, 151, 166
Page, 225
Palladin, 109, 136, 137
Pantanelli, 179
Parastschuk, 155
Partridge, 27
Pasteur, 81, 100, 109
Paton, 64
Pavy, 63, 64
Pawlow, 147, 155
Payen, 76
Pekelbaring, 147, 148, 149, 150, 164,
165, 222
Persoz, ^^
Peter, 224
Petruschewsky, 86
Pfliiger, 61, 62, 66, 141
Pick, 67, 69, 188
Pictet, 135
Plimmer, 74, 75
Pohl, 116, 119
Pollak, 159, 207
Polzeniusz, 109
Portier, 74, 103
Pottevin, 183, 184, 185
Pozzi-Escot, 134
Praussnitz, 65
Pregl, 74
Preti, 10
Priestley, 193, 194
Prym, 17, 18
Pugliese, 68
INP£X OF AUTHORS
288
Rajewsky, io8
Rapp,84,9i,94,2i5
Rcpiault, 139
Reinbold, 17
Reinders, 132
Reiset, 139
Rey-Pailharde, 125, 134
Ribaut, 190
Richardson, 135
Richet, 22, 23, 35, 36
Ritchie, 209
Roberts, 157
Robertson, 224
Rdhmann, 69, 73, 1 16
RoUo, 109
Rona, 16, 186
Rosell, 116, 119
Rosenbaum, 106, 128
Rosenfeld, 189
Rowland, 5, 8, 42, 82, 85, 86, 88
Seemann, 13, 19
Senter, 127, 130, 131, 221
Shaffer, 57, 131, i33
Shedd, 116, 117
Sieber, 99, 147, 148, 149, 154, i55,
164
Sicgert, 113
Sigmund, 58
Simdcek, 102, 103^
Sisto, 75
Snyder, 224, 225
Spallanzani, 141
Spiro, 209
Spitzer, 23, 31, 116, 127, 131
Stadehnann, 21
Stangassinger, 44, 45
Stauber, 68
Stem, 39, 128, 129, 138, 139, 140
Steudel, 30
Stoklasa, 101, 102, 103, no, 137
Stokvis, 35
Subkow, 36
Sachs, 27, 28, 82, 210
Saiki, 67
St Gilles, 174
Salaskin, 21, 188
Salkowski, 6, 119
Salomon, 7, 27
Sanderson, 225
Saunders, 46
Sawjalow, 188, 189, 190
Schafer, 226
Schiff, 18
Schittenhehn, 32, 33, 35, 36, 43
Schlesinger, 40, 43
Schmidt, 33
Schmiedeberg, 30, 45
Schneider, 123
Schonbein, 115, 127, 132
SchSndorff, 61
Schoumow-Simanowsky, 147, 148
Schryver, 112, 201, 202, 219
Schiitz, 206
Schiitzenberger, 58, 99
Schwann, 81
Schwarz, 22^ 207, 222
Schwarzschild, 45
Schwiening, 7
Scott, 212
Seegen, 65, 108
Takdcz, 65
Tammann, 172, 221, 222
Tappeiner, 217
Taylor, 59, 181, 185, 186, 221
Tebb, 64j 71
Teruuchi, 13, 16, 172
Thomson, 151, 166
Thunberg, 138, 141
Van Tieghem, 25
Tschemiajew, 136
U
Umber, 11, 26, 56, loi
Usher, 193, 194
Vassiliew, 41
Vcrwom, 141, 142
Villiger, 122, 123
Vines, 49, 50, Sh 52
Visser, 179
Vitek, loi, no
Vfigtlin, 17
2 G
234
INDEX OF AUTHORS
W
Waage, 182
Wait, 189
Walker, £. W. A., 205, 206
Wartenberg, 59
Weinland, 74, 75
Widdicombe, 73
Wicchowski, 5, 35, 36
Wiener, 10^ 35, 36
Wingler, 45
Winogradow, 154
Wintemitz, 27, 32
Winterstein, 141
Witt, De, 104, 105
Wittich, v., 63, 67, 160, 163, 164
Wohlgemuth, 57
WolfFhiigel, 164
Wroblewski, 88, 95, 164
Yamagiwa, 119
Yoshida, 125
Young, 89, 95, 96
Zaleski, 20
Zanichelli, 119
Zunz, 21
INDEX OF SUBJECTS
Acetic acid, formation of, by yeast juice,
loo ; in autolyses, iii ; formation of,
by alcohol-oxidase, 126
Acetone, sterilising action o^ on yeast,
94 ; action o^ on Bacillus Delbrucki^
98 ; as precipitant for glycolytic
enzymes, 106; for preparation of
alcohol-oxidase, 126
Action, products of enzyme^ 172
Adenase, 31; increase of, with growth.
Adsorption, of enzymes, 160 ; of dyes,
162 ; of lysins, 210
Agglutinins, 204
Alcohol, formation of, by zymase, 83 ;
formation of, by action of alkalis,
99; formation o^ from lactic acid,
100; presence o^ in blood, 107;
presence of, in various tissues, 108 ;
formation of, in plants, 109 ; oxida*
tion o( by alcohol-oxidase, 126
Alcoholic fermentation, 81 ; of various
sugars, 84 ; of animal tissues, 10 1 ;
of plants^ 109
Alcohol-oxidase, 126
Aldehydases, 115, 118
Algae, action of antiseptics on, 115
Alkalis, action of, on sugar, 99
Allantoin, constitution o^ 29 ; forma-
of, by enzymes, 36
Amboceptors and enzymes, 205
Amide nitrogen, 21
Amino acids, liberation of ammonia
from, by enzymes, 20
Ammonia, liberation of, by enzymes,
20 ; by acids, 21 ; disintegrative
action of, on living tissues, 200
Amygdalin, action of enzymes on, 79,
171
386
Amylases, in liver, 63 ; in muscle, 6$ ;
in other organs, 67 ; in blood, 68,
69 ; in invertebrates, 76 ; in plants, 76
Amylolysis in liver, 63
Anabolism in living cells, 194, 213
Antiferments^ 208
Antiseptics, influence of, on zymase,
91 J action of, on organisms, 215 ;
action of, on enzymes, 216
Antitrypsins, 210, 211
Arginase, 18 ; distribution of, 19
Asepsis, autolysis with, 1 1 1
Autodig;estion, 6
Autolysis, of intact tissues, 3; of
minced tissues, 6^ 202 ; of tissue
juices, 8 ; influence of acids on, 10 \
products formed by, 11, 17; am-
monia liberated by, 21 ; of embryos'
tissues, 40 ; of yeast juice, 86 ; aseptic
and antiseptic, iii ; in perfused
organs, 200 ; effect of antiseptics
on, 219
B
Bile, action of, on steapsin, 206
Biogens, constitution of, 142, 197, 211
Biuret test, disappearance o( in
autolyses, 6, 18 ; meaning o( 18
Blood, diastatic enzyme of, 68, 6p ;
alcohol in, 107; aldehydase m,
119 ; coagulation of, 205
Carbohydrates, synthesis of, in plants,
192
Carbohydrate- splitting endoenzymes,
60
2i6
iNDfiX OF SUBJECTS
Carbonase, 136
Castor-oil seed lipase, synthetic action
o^ 182
Catalase, variation of, with growth, ^g;
action of. 127 ; variation of, with
functional activity, 129 ; properties
o( 130 ; functions o( 134 ^ locaUsa-
tion of, in plants, 193
Catalysts, enzymic, 166 ; inorganic,
132, 175 ; organic and inorganic
compared, 182, 183, 190; energy
relations of, 191
Chloroform, action o( on living tissues,
199, 218 ; on enzymes, 219
Chloropliyll, synthetic action of, 192,
'95.
Clupem, action of erepsm on, 13
Coagulation, temperature of protein,
227
Co-efficient, temperature, of enzymes,
221 ; of various vital processes, 223
Co-enzyme, of yeast, 96 ; of steapsin,
206
Colloids, metallic, 132 ; and toxins,
210
Creatin, action of endoenzymes on,
44
Creatinin, action of endoenzymes on,
44
Cytase, 78
D
Death temperatures, of vertebrates,
226 ; of Flagellata, 227 ; of proto-
phyta, 227
Diastase, of liver, 63 ; of muscle, 65 ;
of other organs, 67 ; of embryos,
67 ; of invertebrates, 76 ; in plants,
76; precipitability of, 153; differ-
ences of, m different animals, 159;
dialysis of, 164 ; composite nature
of, 164
DifTusibility, of enzymes, 164 ; of anti-
toxin, 212
Disintegration of tissues, mechanical,
4
Dyes, adsorptionlo^ 162
Embryos, adenase of, 39; lipase of,
57 ; glycogen of, 62 ; amylase of,
67 ; maltase o^ 72 ; lactase of^ 75 ;
invertase o^ 76 ; catalase of^ 131
Emulsin, 79; action of, on stereo-
isomers, 170; retardation of, by
products of action, 172, 173 ; syn-
thetic action of, 179, 180 ; action of,
on salicin, 221
Endoenzymes and exoenzymes, i
Endotryptase, 85 ; action of, on
zymase, 87
Energy relations of enzyme action,
191, 195
Enzymes, composition of, 150; insta-
bility of, 151 ; effect of proteins on,
152 ; precipitability o^ 153 ; differ-
ences of, in different animals^ 159 ;
adsorption of, 160; diffusibility of,
164 ; optical activity of, 165 ; action
of, on racemic bodies, 165 ; combina-
tion of, with substrate, 166; velo-
city of action o^ 166, 184 ; in relation
to stereoisomerism, 169, 172 ; con-
figuration of, 188
Erepsin, 12 ; distribution of, 13, 38 ;
estimation of, 14; influence of
alkalis on, 15 ; variation of, with
functional capacity, 37 ; influence of
growth on, 38 ; of diet, 41 ; of
hibernation, 41 ; of disease, 42 ;
presence of, in invertebrates, 48 ;
presence of^ in plants, 49
Esterification, conditions o^ 175, 184
Esters, hydrolysis of, by endoenzymes,
53, 165 ; hydrolysis o^ by acids, 174 ;
synthesis of^ by enzymes, 181
Ethyl butyrate, hydrolysis of, by
enzymes, 54, 57 ; synthesis o^ 181
Extraction of endoenzymes from
tissues, 2 — 5
Fats, action of endoenzymes on, 56 ;
synthesis of, 184
Fermentation, alcoholic, 81 ; lactic,
81 ; of various sugars, 84 ; of animal
tissues, 10 1 ; of plants, 109 ; of
stereoisomeric sugars, 171
Fermentoids, 207
Ferments, inorganic, 132
Filtration, effect of, on yeast' juice, 89
Fluorides, poisonous action of, 217
Formaldehyde, synthesis of, 192 ;
condensation of, 193 ; poisonous
action of, 218
INDEX OF SUBJECTS
237
Formic acid, synthesis of^ 193
Fuagi, nuclease in, 27 ; lipase in, 59 ;
invertase in, 179
Germination, effect of, on plant en-
zymes, 51
Glucosides, action of enzymes on, 79 ;
sterecMsomeric, 170
Glutinase, 159
Glycogen, distribution of, 61 ; in
emlnryos, 62 ; disappearance of,
from muscle, 65 ; synthesis of, by
zymase, iSo
Glycolysis, in animal tissues, 102 ; in
blood, 107
Glycolytic enzyme, of yeast, 82 ; of
pancreas, loi ; of muscle, 103 ; of
liver, 105 ; c^ blood, 107
Growth, increase of enzymes with, 38,
51, 57 ; increase of glycogen with, 62
Guanase, 31 ; absence of, from em-
bryos, 39
Guiaconic acid, as test for oxidases,
116
Guiacum test, 116, 120, 121
H
Haemase, 127
Hsemalin, and peroxidase reaction, 121
Heart beat, influence oi temperature
on, 224
Hippuric acid, action of endoenzymes
on, 45 ; synthesis of, 190
Hydrogenase, 134
Hydrolysis, of proteins by acids, 17,
21, 22 ; energy changes in, 191
Hydrolyte, in relat]<»i to enzyme, 173
I
Immunity, work on, 203
Inactivation of enzymes, 207, 222
Indophenol reaction, 116
Inorganic ferments, 132
Intramolecular oxygen, 141
Inulase, 78
Invertase, in animals, 72 ; in embryos,
76 ; in plants, 78 ; in yeast juice, 83 ;
composition of^ 151 ; instability of,
151 ; velocity of siction of, 167 ; re-
tardation o^ by products of action,
173 ; synthethic action of, 179
Invertebrates, proteolytic endoen-
zymes o^ 46 ; lipolytic endoenzymes
ofy 57; diastatic enzymes of, 76;
tyrosinase of^ 123 ; influence of tem-
perature on heart beat o^ 226
Isolactose, synthetic formation of, 178
I sotnaltose, action of enzymes on, 171 ;
synthetic formation of, 177
K
Kalabolism, of protoplasm, 214
Kidney, autolytic products of, 1 1
Laccase, 125
Lactacidase, 98
Lactase, distribution o^ 74 ; adapta-
tion of, 75 ; in embryos, 75 ; in
plants, 7S ; retardation of, by pro-
ducts of action, 173 ; synthetic
action of, 180
Lactic acid, formatictt o( by yeast
juice, 97; by Bacillus Delbrueki^ 98 ;
by action of alkalis, 99 ; by animal
enzymes, 102 ; in autolyses, 1 1 1,
114; in rigor mortis, 143 ; action
of, on disintegrating tissues, 201
Lactic fermentation, 81 ; of animal
tissues, 102 ; of plants, no
Langerhans islands, glycolytic function
of, 104
Linked reactions, 194
Lipase, intracellular, 53, 56 ; in castor-
oil plant, 58 ; in moulds, 59 ; action
of, on racemic bodies, 165 ; synthetic
action o^ 181 ; antiferment of, 211
Lipolytic endoenzymes, of animals, 53 ;
of embryos, 57 ; of plants, 58 ; syn-
thetic action of, 181
Liver, autolysis o^ 6; influence of
acids on, 10 ; glycogen of, 61 ; amy-
lase of, 63, 68 ; aseptic autolysis of,
III; catalase of, 128
Living and dead substance, 198
Logarithmic course of enzyme action,
166
2 G 2
238
INDEX OF SUBJECTS
M
Maltase, of intestine, 70 ; of other tis-
sues, 71 ; of serum, 72 ; of embryos,
72 ; of plants, 78 ; in yeast juice, 83 ;
action o^ on stereoisomers, 170;
retardation o^ by products of action,
173 ; synthetic action o^ 176, 180
Mass action, law o^ 166, 168, 182, 183
Maximum temperatures of enzyme
action, 222
Melizitase, 78
Micro-organisms, extraction of en-
zymes mm, 4
Moulds, nuclease in, 27 ; lipase in, 59 ;
invertase in, 179
Muscle, glycogen in, 61 ; post-mortal
disappearance of glycogen from, 65;
respiration o^ i j8, 139, 140^ 143
Mycetozoa, digestion in, 46
Myrosin, 79
N
Nervous impulse, propagation rate of,
in relation to temperature, 225
Nitrogen, amide, 21
Nuclease, 27
Nucleic acid, action of nuclease on,
27 ; constitution of, 29
Nucleoproteins, acted on by pepsin,
trypsin, erep^sin, and intracellular
enzymes, 26 ; catalytic action of, 131
O
Olein, synthesis of, 184
Optical activity, of enzymes, 165 ; of
amino acids, 187
Optimum temperature of enzyme
action, 221
Oxidases, 32, 115, 118; non-catalytic
nature o^ 121 ; vegetable, 124 ; of
alcohol, 126
Oxygenases, 115, 118
Pepsin, ammonia-liberating power o^
21 ; preparation of, 146, 147 ; com-
position of, 148 ; relation of, to
rennin, 154; adsorption of, 160;
dialysis o/, 164 ; synthetic action o^
188 ; anti-body of, 210
Peptase of plants, 50
Peroxidases, 120; estimation of, 121
Peroxides, organic, 122
Phenol, poisonous action o^ 219
Phosphates, influence of, on zymase, 95
Plants, proteolytic enzymes o^ 49;
lipolytic enz^es o^ 58 ; amylolytic
enzymes o^ 76; sucroclastic enzymes
o^ 78 ; zymase o^ 107 ; tyrosinase
of^ 124 ; oxidases o^ 124 ; laccase
of, 12 J ; respiration o^ 136; action
of antiseptics on, 2 1 5
Plastein, 188
Polypeptides, action of endoenzymes
on, 16, 172 ; attempted synthesis of,
186
Precipitants of proteins, 8
Propagation rate in nerve, as influ-
enced by temperature, 22^
Protamines, action of erepsm on, 13 ;
action of arginase on, 19 ; syndesis
o^ by enzymes, 186
Proteases, 8, 10 ; action o^ on proteins,
II, 12 ; compared with erepsin, 42
Protein, potential, of living tissues, 201
Proteins, estimation o^ 8 ; protective
influence of, on zymase, 88, 92 ;
delicacy of tests for, 150 ; eflect o^
on stability of enzymes, 152
Proteolytic endoenzymes, 5; classifica-
tion o^ 7 ; action of, on polypep-
tides, 16; variation of, with func-
tional capacity, 37 ; in lower animals,
46 ; in plants, 49
Protoplasm^ constitution of, 197, 211
Protozoa, dis[estion in, 46 ; reaction of,
to antiseptics, 215
Pseudo-enzymes, oxidases as, 122
Ptyalin, composite nature of, 205
Purins, autolysis of, 7 ; chemical con-
stitution o4 29
Papain, enzymes in, 50, 51 ; formation
of plastein by, 189
Quotient, temperature, of enzymes,
221 ; of metabolism of organisms,
223 ; of heart beat, 224 ; of propa-
gation of nervous impulse, 225
INDEX OF SUBJECTS
239
R
Racemic bodies, action of enzymes on,
165, 172, 187
Raffinase, 78
Receptors and endoenzymes, 203
Rennin, precipitability of, 153; rela-
tion o^ to pepsin, 154; relation of,
to trypsin, 155 ; universal presence
o^ 156 ; synthetic action o^ 188 ;
composite nature of, 206 ; action of
inactivated, 208; antiferment of,
208
Respiration, of animal tissues, 135 ; of
plants, 109, 136 ; of minced tissues,
138
Respiratory enzymes, 135, 137
Retardation of ferment action, 172 ;
produced by products of action,
187 ; produced by inactivated
enzymes, 207
Reversible enzyme action, of maltase,
176 ; of other sucroclastic enzymes,
179 ; of lipolytic enzymes, 181 ; of
proteolytic enzymes, 186 ; of rennin,
188
Revertose, synthetic formation of, 177
Taka-diastase, synthetic action of, 178
Temperature, influence of, on velocity
of chemical reactions, 220; on
enzyme action, 220 ; on metabolism,
223 ; on rate of development, 223 ;
on heart beat, 224 ; on propagation
of nervous impulse, 225 ; optimum
and maximum of enzyme action,
221 ; of death in various organisms,
226
Thrombokinase, 205
Tissue respiration, 135 ; of plants, 136
Toxoids, 207
Trehalase, 78
Trypsin, action of, on polypeptides,
16 ; action of, on protems, 17 ;
ammonia-liberating power of, 21 ;
instability of, 152 ; precipitability
of, 153 ; relation of, to rennin, 155 ;
variable stability o^ 158 ; adsorp-
tion of, 163 ; dialysis of, 164 ; action
o^ on racemic bodies, 165 ; action
of, on polypepi* "
action of, 186 ; action of inactivated.
of, on polypeptides, 172 ; synthetic
207 ; antiferment o^ 210
Tyrosinase, 123 ; vegetable, 124
U
Side-chain theory, 203
Spleen, autolytic products of, 11
Steapsin, intraceUular, 53; synthetic
action of, 181 ; heat inactivation of,
206, 207
Stereoisomerism, in sugars, 169; in
polypeptides, 172 ; in amino acids,
187
Stereoisomers, action of proteolytic
endoenzymes on, 16, 172
Sublimate, corrosive, poisonous action
0^215
Substrate, combmation of, with
enzyme, 166, 168, 208; and related
enzyme, 173, 188
Sucroclastic enzymes, in animals, 70 ;
in plants, 78
Sugars, stereoisomeric, 169; fermenta-
bility of, 171
Synthesis, effected by enzymes, 176; in
plants, 192 ; in animals, 195
Urea, liberated by arginase, 18 ;
formed in autolysis, 22 ; hydro-
lysed by enzymes, 23 ; formed from
uric acid, 36
Urease, 23
Uric acid, formation of, by enzymes,
31 ; destruction of, by enzymes, 35
Uricolytic enzyme, 35
Ve|fetable enzymes, proteolytic, 49 ;
lipolytic, 58 ; amylolytic, 76 ; sucro-
clastic, 78 ; zymase, 107 ; tyro-
sinase, 124 ; laccase, 125
Velocity co-efficient, 167
Vital action of cells, 192, 194
Vital processes, chemical nature of,
226
240 INDEX OF SUBJECTS
Xamho-oxidase, 32 ; absence of, from ^5^^"*^' ^'^^^^^^'^ ^J' ^^' ^^^'°'^. °^' ^
lV«K«,^e ^^ *uj>^"^c *^t, **uiii various sugars, 84 ; destruction of,
emoryos, 39 ^^ endotryptase, 86 ; filtration of,
89 ; influence of desiccation on, 90 ;
influence of antiseptics on, 91, 217,
Y 218 ; influence of phosphates on,
95 ; in animal tissues, loi ; in blood,
108; in ova, 108; in plants, 109;
Yeast, extraction of zymase from, 82 ; action of^ on polypeptides, 187
influence of antiseptics on, 91, 93 ; Zymo^^en, of trypsin, 214
sterile, 94 ; formation of glycerin Zymoids, 207, 208
by, 100 ; action of antiseptics on, 216 Zymophore groups, 204
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SCIENCE PROGRESS
IN THE TWENTIETH CENTURY
A QUARTERLY JOURNAL
OP
SCIENTIFIC WORK AND THOUGHT
Published January i, April i, July i, October i
Alx)ut 192 Pages, containing some twelve to fifteen papers.
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ADVISORY COMMITTBB
H. B. ABHSTBONO. Ph.D., LL.D., F.B.S.
HOBACB T. BROWN, LL.D., P.B.S.
J. BBSTLAND FABHBR, H.A., D.Bc.. F.B.8.
B. T. OLAiSSBBOOK, M.A., So.D., F.B.8.
H. J. MAGKINDBB, M.A.
J. B. MABB, M.A., Bo.D., F.B.S.
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Sib WH. BAHSAT, K.C.B., Fs.D., So.D.,
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A. D. WALLBB, M.D., LL.D., F.B.S.
EDITORS
N. H, ALCOCK, M.D., and W. G. FREEMAN, B.Sc, F.L.S.
"Science Progress" owes its origin to an endeavour to found a Scientific
Journal containing original papers and summaries of the present state of
knowledge in all branches of Science. In the prospectus of the first
number which appeared in July 1906, it was pointed out that the necessity
for such a Journal was to be found in the fact that with the specialisation
which necessarily accompanies the modem development of scientific work
and thought, it is increasingly difficult for even the professional man of
science to keep in touch Mrith the progress achieved and the trend of
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This difficulty is often felt to a much greater degree by scientific workers in
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ture, by teachers and students in schools and colleges, and the general
educated public interested in scientific questions. " Science Progress," to
attain its objects, should therefore contain in conveniently accessible form
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should be of an authoritative character, preferably written by those actually
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That very tangible success has been attained in realising these ideals is
demonstrated by the papers which have appeared in the seven numbers
already issued.
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nvS AP!1 28 IS 16
m 2 1 1921
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