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CKJt^^^ <fq<^ ,\^ 

^arfaarti Calltg 




Rumford Prof' 




Ti\i» book should be returne 
■ the Library on or before the last 
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A fin© of five cents a day is inci] 
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Please return promptly* 

tt*f^ /»* +- 

C^U^^^ <t^^ ,\^ 

f^arfaarti College Eibrars 



Rumford Professor and Lecturer on the Application 

of Science to the Useful Arts 








BY H. M. VERNON, M.A., M.D. 





^U^^ff, /^ 


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. 





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 



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 



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 




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 



/ 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 



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 




^ 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 



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 





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 



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. 


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 



Units of Biepsin washed out during 


at end of 



4 hout onwards. 

Kept 3 days at 14'' 
» 7 .. 12* 

Fresh . 
»f • • • 
i» • • • 

















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. 


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. 


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, 



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. 



Organic Substances 

Nitrogen (calculated as Protein) . 

Phosphoric Add 

Purin Bases 




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. 


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 

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, 

(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, 



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 

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. 


16 hours. 

22 hours. 

40 hoars. 

Spleen Juice alone . . . , 
+ .25% Acetic Acid 
+ .i%HCl . . . 
+ -37%Na2C03 . . 

Boiled Juice + .25% Acetic Acid . 












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. 



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 


(Total N=:66-8.) 

Nitrogen at or beyond Peptone Stage. 


2 days. 

5 days. 

1 month. 

Juice alone .... 
„ +.25% Acetic Add. 
„ +CaC08 





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. 


16 hours. 

8 days. 

16 days. 

Juice alone .... 
„ +.25% Acetic Acid. 
„ +CaC03 





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 



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- 

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. 


ment had destroyed an anti-ferment present in the spleen 

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, 


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 

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, 

3 Hofnieisteri ildd,y 6, p. 69 ; and Arch.f. exp, Patk.y 19, p. 8, 1885. 


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 

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. 



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 : ^ — 


Breptio Value. 


Ereptic Value. 

Duodenal muc. memb. . 


Submaxillaiy gland 
Thyroid gland . 


Jejunal „ 





Suprarenal gland , 


Large intest „ 


Cardiac muscle 


Gastric „ 


Brain . 


Kidney . . . 


Ovary . . . 




Skeletal muscle 


Lung . , , , 


Blood . . 




Serum . 


Liyer . . . • 


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. 



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 . 







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- 

The differences between endoenzymes and trypsin is well 
shown by their action upon polypeptides. Abderhalden, in 



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 


Ox or Dog's 
Liver Juice. 

Dog's Kidney 

Ox of Dog's 
Muscle Juice. 


Dl-leucyl-glvcyl-glydn . 
Dl-alanyl-g^(^l-giydn . 
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. 



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 : — 


10 dftys. 

20 days. 

80 days. 

40 days. 

50 days. 

Mono-amino adds . 







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. 



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, 


* Schiff, Ber,^ 29, p. 298, 1896 ; Ann, Chem, Pharm,^ 299, p. 236, 1897. 
' Kossel and Dakin, Zeitf.physioL Chem,y 41, p. 321, 1904. 



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. 


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; 


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. 



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 

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 : — 



Amide Nitrogen, directly separable . 
„ separable by adds . 

Total Amide Nitrogen 

Per cent. 

Per cent. 




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. 


These extractives were not ammonia, and so were presumably 

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- 

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. 


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 

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, — 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. 



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. 


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 

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. 


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. 



and carbon dioxide, whilst a stronger oxidising agent, such 
as cold nitric acid, converts it into alloxan and urea. By other 

N=C— NH, 




N— C N<^ 




CH C— NH. 


N— C N^ 


NH,— C C- 


11 II 
N— C- 











CO C— NH^ 

I II >co 

NH— C— NH/ 

Uric Acid. 

.NH— CH— NH. 

-NH— CO NH, 



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 


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. 


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. 


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 


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. 



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: — 





Liver of Ox 

„ Rabbit . 

» Dog . 

Spleen of Dog . 

" Pig . 

Pancreas of Pig . 











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. 


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. 


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 



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 

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 







Liver . . 
Kidney . 
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. 


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. 


different Or perhaps the allantoin formed first may have 
undergone a subsequent conversion into urea and other 

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. 



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 








Number of Observations 


8 to 10 






Cardiac muscle .... 
Skeletal muscle .... 




1 1.6 











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. 



8 days. 

81 days and 

Weight . 



81 gms. 

585 gms. 

Kidney . 


Cardiac muscle . 

Skeletal muscle . 









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 


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 

^ 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, 


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, 



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 





Kidney .... 
Pancreas .... 
Spleen .... 
Liver .... 
Cardiac muscle 
Skeletal muscle 
Brain .... 





2*1 : 1 




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. 



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, 


Normal Guineapigs 
(7a7 grms.). 

Wasted Ouineapigs 
(805 grms.). 

Died 8 days after 
injection (677 grms.). 

Kidney . 

Cardiac muscle 
Skeletal muscle 









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 

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 


Ereptic Valaes of 
Gat's Tissues. 

Per cent. Protein 

hydrolysed in Acid 

Solution (/3-protease). 

Spleen ^ . 
Liver . 
Cardiac muscle 
Skeletal muscle 
Submaxillary gland 
Blood . 
Serum . 




about 90 

about 45 


very slight 

very slight 


^ Hedin and Rowland, Zeit, f. physioL Chem.y 32, pp. 341 and 531, 1901. 


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 

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. 


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. 


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. 


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. 


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 

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. 


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 

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. 


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. 



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. 


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. 


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. 



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. 







Per cent. 

Per cent. 

Pancreas .... 



Liver .... 



Intestinal mucosa 



Stomach .... 



Muscle .... 



results, Nencki concluded that all tissues possess fat-splitting 

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 

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. 



minutes at 40°, the following amounts of butyric acid were 
liberated : 

Per cent. 

Per ceut. 

Liver of pig 

„ sheep . 

„ duck . . . 

„ ox . . . 




Liver of chicken 
Pancreas of pig . 
Kidney of pig . 
Submaxillary gland of pig . 




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. 


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 . 


Corpuscles .... 

Per cent. 


} ■" { 

Per cent. 
1 9*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 

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. 


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. 



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. 


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. 


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. 



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. 


Per cent, 
of Glycogen. 

Liver ..... r * 





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. 


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« 


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. 


•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. 


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- 

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. 



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, 

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. 


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. 


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 

GuO from 
Liver digest. 

CaO from 
Muscle digest. 






















Adult pig 


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. 


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. 


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 

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. 



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 : — 


Per cent. 




of Maltose 



Mucous membrane of 

Kidney ... 


small intestine . 


Gastric muc memb. . 




Pancreas . 


Lymphatic gland . 


Salivary gland . 


LiveTT . . . 


Skeletal muscle . 


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. 


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. 


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, 

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. 


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. 


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. 


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. 


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. 


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 

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. 


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. 


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. 



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. 


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>* 


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 



12-4 gm. of alcohol ; another, 8-9 gm. of CO2 and 8-9 gm. of 

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^ 


In pxeaenee of 
•2 C.C. of Toluol. 

Another Sample 

of Juice, 

without Antiseptic. 












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. 



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 :— 



Per cent. 









• 30 




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 



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 : — 




Fresh juice 

After I day at room temperature 

„ 6 days „ „ . . 





Per cent. 

Per cent. 




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. 


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- 

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 


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 


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. 



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. 







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 


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- 

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. 



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. 


20 cc Juice + 8 gm. Sugar 

20 C.C Juice + 8 gm. Sugar 
alone + 2 per cent As^Oj . 












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 









1 Abclcs, Ber.y 31, p. 2261, 1898. * Bokorny, PflUgef^s Arck.^ m, p. 37i» 



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. 



20 C.C Juice + 8 gra. 

Sugar + no Antiseptic, in 92 hours gave 




„ +.2 C.C. Toluol „ „ . 




i» T" ^ ^^ i» n f» • • 


I '69 


+ •2 gm. Thymol „ „ . 




„ +1 gm. „ „ „ 



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 


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. 


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 

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. 


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, 

2 Buchner and Klappe, Biochem, Zeit^ 8, p. 520, 1908. 


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, 

2 Meisenheimer, Zeit f. pkysiql Chem^^ 37, p. 526, 1903- 



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. 


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. 


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 : 


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. 


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. 


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. 


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. 


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. 


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. 



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 „ 


Pancreas „ 

u 36 

Pancreas + Muscle 

65 , 

Pancreas + Liver 


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. 


Hall's experiments, but no direct observations on the point were 

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 

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. 


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. 


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. 


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, 


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 

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. 


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 


Non- volatile 



Ox Liver, i day aseptic . 

„ 9 days „ . . . 
Dog's Liver, l day aseptic 

„ 6 days „ . . . 












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 : — 


Non-volatfle 1 Volatile 
Acids. Acids. 

Ox Liver, i day under aseptic conditions 
„ 2^ months with Chloroform 
„ 2^ „ with Toluol 
»i 6 i» »» ... 



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. 



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 : — 


Duration of 



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 . 








3-3 . 






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. 



that during liver autolysis the quantity of these acids remains 

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. 



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- 



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. 



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 : — 




Salivary glands 
Thymus , 
Spleen . 
Lungs . 
Brain . • 
Suprarenal glands 
Testis . 
Kidnev . 
Lymph glands 
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. 



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 : — 


No Antiseptic. 

1 per cent. NaF. 



Spleen .... 








Thyroid . 
Kidney . 



Th)rmus . 



Suprarenal gland 



Testis .... 



Abelous and Biam^, loc, cit 


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. 


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. 


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. 


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). 


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. 


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. 


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. 


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. 


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 

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. 



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 



















Rabbit . 









Dog. . 









Cat . 









Adder . 























Fish (^Leuciscus) 









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. 


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. 



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. 








5 gm. embyro • 






17 ., 








36 „ 








Foetus at term * 








2-day Guineapig . 








7-<iay „ 
Adult „ 















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. 


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. 


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. 


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. 


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, — 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. 


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 : — 


In Hydrogen Current 

In Air Current 

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. 


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. 




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 : — 

Heart muscle 
Skeletal muscle . 
Liver .... 

242 C.C. 
194 i^ 

184 » 
176 „ 

Brain .... 




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. 


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. 


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. 


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. 


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 

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 


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. 


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. 



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 


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. 


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. 


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 






CooliDg Juice to 0' (Schoumow-Simanowsky) 
Precipitating with (NH4)2S04 „ 
Dialysing Juice fNencki and Sieber) , 

„ „ (Pekelharing) 
Precipitating with (NH4)2S04 (Pekelharing) 











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^* 


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. 


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 

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 


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. 


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. 


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. 



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 


Precipitate dissolved 
in Water. 

•2 J 






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 . 





























a suitable method for the purification of these particular 

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. 


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. 


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. 


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, 


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. 


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. 


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. 


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. 



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 

Proportion of Dye 

Proportion of Dye 
in Paper. 


Per cent. 





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 






^ 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, 


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. 


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 

3 Frankel and Hamburg, Hofmeistet^s Beitr,^ 8, p. 389, 1906. 


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. 


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. 



(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. ^ 

inverted in 60 minutes. 

Inyerted in 60 minutes. 











•000$ I 

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. 








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. 


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. 



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 



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 



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 


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- 

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. 


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 : 




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. 



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 : — 


CoRMiponding Hydrolyto. 

BUbot of HexoM on rate of 




Lactase . 
Emulsin . 
Maltase . 

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 











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. 


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, 



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 

Molecules of 

Per cent, of Acid 











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 


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. 


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, 



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. 


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. 


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* 


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. 



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°). 


Percent, of Hydrolysb 
by means of 




per cent. 










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 


Free Add remaining 

in mixture of 

Acid + Alcohol. 

Free Add liberated 
flpom mixture of 
Bster+ Alcohol. 

Bquilibrlum Point. 





* Bodenstein and Dietz, Zeit / Electrochefn,^ 12, p. 605, 1906 ; Dietz, 
Zeitf.physioL Chetn,, 52, p. 279, 1907. 



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 

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 


Acid formed 

Equilibrium point with Hydrochloric Acid . 
„ „ Picric Add . 
„ „ Pancreatic Lipase . 

Per cent. 



about 75-0 




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 

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, 




Gnmines of Water present. 






12 hours . 

36 „ 

60 , . 

4 days . 
6 „ . 

5 „ 
10 „ 
14 » 















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. 


8 days. 

80 days. 

















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. 


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 , 

» »» 



= 77 

no , 

» n 



= 64 

100 , 

» »» 



= 51 


1 ti 



- 20 


) 11 



= 5 


) 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 

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 


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. 


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. 


Deutero-proteose .... 
Witte's Peptone .... 
Antipeptone (KUhne) 
Witte's Peptone, 81% hydrolysed . 














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. 


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. 


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 

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 


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. 


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. 


whilst 3 molecules of stearin and i of glycerin gave 8413 

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, 

2 Bach, Coniptes Rendus^ 116, p. 1145, 1^93. 

3 Euler, Ber., 37, p. 3415, 1904. 


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 


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. 


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. 



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 

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 



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. 



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 


Perfusion Liquid. 

SulMitances washed out per hour per 
1kg. of Kidney. 






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 









1 9^62 










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 


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. 


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 


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. 


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 

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. 


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 

In some cases it appears that enzymes act in the same way 


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. 



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 

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. 


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. 


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. 


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 

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 


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, 


7 Wttnon, Joum. Physiol, 3', P- 355, 1904. 


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, 


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. 


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 




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 

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. 


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 

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. 


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 

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. 


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 £ 


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. 


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. 


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. 



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. 



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* 





* 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. 


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. 


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. 


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. 


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 


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 

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. 


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. 


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 


Bach, 115, 120, 122, 134, 192, 193 

Baer, 10 

Baeyer, 122, 123 

Baker, H. B., 196 

Bang, 29, 30, 155 

Baranetsky, ^^ 

Barcroft, 139 


Barratt, 162, 163 

Bary, De, 76 

Battelli, 39, 103, 128, 129, 138, 139, 140 

Bayer, 189 

Bayliss, 160, 162, 163, 168, 186, 200, 

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, 

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 



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 


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 



Gulewitsch, 45 
Giimbel, 21 

JoUes, 130 

Jones, 27, 32, 33, 35, 39 


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 


Jacobson, 127 

Jacoby, 7, 11, 20, 22, 23, 35, 36, 45, 

Jackson, 113 
Jaquet, 117, 119 
Jelmek, loi, no 


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, 

Loewi, 0., 23 
Loisel, 48 



Lossnitzer, 153 
Lubarsch, 62 
Liidy, 53 
Lussana, 138 


Macallum, 212 

Macfadyen, 5, 82, 85, 86, 88 

Macleod, 29 

Madsen, 161, 209, 212 

Magnus, 206 

Magnus-Levy, iii, 112, 114, 143, 179, 

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 


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 



Rajewsky, io8 


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, 

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 


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 




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 


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, 



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 


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, 

Carbohydrate- splitting endoenzymes, 




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, 

Clupem, action of erepsm on, 13 
Coagulation, temperature of protein, 

Co-efficient, temperature, of enzymes, 

221 ; of various vital processes, 223 
Co-enzyme, of yeast, 96 ; of steapsin, 

Colloids, metallic, 132 ; and toxins, 

Creatin, action of endoenzymes on, 

Creatinin, action of endoenzymes on, 

Cytase, 78 


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, 

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 



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, 

Guiacum test, 116, 120, 121 


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 


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 


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, 

2 G 2 




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 


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 


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, 
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 




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, 

Respiration, of animal tissues, 135 ; of 
plants, 109, 136 ; of minced tissues, 

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, 

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, 

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 


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, 

Stereoisomers, action of proteolytic 

endoenzymes on, 16, 172 
Sublimate, corrosive, poisonous action 

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, 


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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" The book is thoroaghly sdentifie in its tnatmeiitof the nnmeroiu parts of the narvons system 
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THB BAOTBIRIOLOQT OF MILK. By Harold Switbinbanx, of the 
Bacteriolo^cal Research Laboratory, Durham, and Georgx Nswman, M.D., 
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Newman, MJ)., F.R.S.E., D.P.H. With numerous Illustrations. Medium 
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know, so fur as everyday problems of sanitation and preventive medidne demand. . . . Dr 
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By W. C. D. Whetham, M.A., F.R.S. Illustrated. Large Crown 8vo. 

Jstd fut, 
iMTBODuonon— Thi Philosophical Basis of Phtsioal BcnNOi— Ths LiQUirAonoH of 
Oask ahd TBS Absolutb Zmao of TsMPniATUBi— Fusion and Solidifioatioh— Ths Pboblbms 
OF Solution— Thx OoNDUonoH of Eubotbioitt thbouoh Oasis— Badio-Aotivitt— Atoms and 
jBtbbb^-Astbo-Phtsigs— Indnx. 


By Mrs WaTT-SmtTH. Large Crown 8vo. 6s nei, 

** It is to be questioned whether the Oommittee now inquiring into the allemd deterioration of 
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NOTBWORTHY FAWTTTiTHIS — (SOIBNOB). An Index to Kinships in 
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m 2 1 1921