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A SYMPOSIUM ON RESPIRATORY ENZYMES

A Symposium on
RESPIRATORY ENZYMES

Contributors:
Otto Meyerhof
Eric G. Ball
Fritz Lipmann
Kurt G. Stern
Fritz Schlenk

T. R. HOGNESS

Elmer Stotz
Carl F. Cori
E. A. Evans, Jr.
Philip P. Cohen
K. A. C. Elliott
Dean Burk
C. J. Kensler

Erwin Haas
H. M. Kalckar
M. J. Johnson
Van R. Potter
H. G. Wood
R. H. BuRRis
C. H. Werkman
P. W. Wilson
F. F. NoRD
Ephraim Shorr
a. e. axelrod
Frederick Bernheim
E. S. G. Barron
Fredrick J. Stare

The University of Wisconsin Press

MADISON

UNIVERSITY OF WISCONSIN

PRINTED IN THE UNITED STATES OF AMERICA

Foreword

IN 1897 Buchner published his classical study on alcoholic fermen-
tation by cell-free yeast juice. In the same year Eijkman con-
cluded that beri-beri among the natives of the Dutch East Indies
was caused by a dietary deficiency arising from the use of polished
rice. These two discoveries may be said to have initiated the modern
investigations in two of the most important fields of biochemistry
and medicine: the nature of the respiratory enzymes and the function
of the vitamins in cellular metabolism.

For the next thirty years research workers in these two fields
pursued their investigation almost independently of one another and
more or less oblivious to the progress being made in the other's field.
Then in the early part of the last decade it was discovered that ribo-
flavin was the functional group in a respiratory enzyme and very
soon afterward this compound was shown to be vitamin Bg (G).
Further discoveries of a similar nature soon demonstrated that the
enzyme chemist and the nutritionist were to a great extent prospect-
ing the same territory. The time seemed to be ripe, therefore, for
the two groups to join in a discussion of the latest advances. Such
a meeting on the "Respiratory Enzymes and the Biological Action of
the Vitamins" was sponsored jointly by the Universities of Wisconsin
and Chicago, institutions that have long been leaders in these fields.

This book contains the lectures and discussions given at the Uni-
versity of Wisconsin. It deals with the fundamental nature of those
enzymes that are intimately connected with the functioning of the
vitamins. Informative presentation of the latest developments, in-
terpretation of past and present findings, and indication of some of
the problems still unsolved in respiratory enzyme research are given
by recognized international authorities in the field. Supplementing
these explanations of the fundamental nature of respiratory enzymes
are discussions applying the findings to specific problems.

The Program Committee wishes to thank the many members of
the faculty for their cooperation in arranging the meetings held at
the University of Wisconsin, the speakers for their papers and dis-
cussions, and the Wisconsin Alumni Research Foundation for the
grant which made these sessions and the publication of the present
volume possible.

Address of Welcome

C. A. DYKSTRA

President of The University of Wisconsin

THE University of Wisconsin is a happy host today. It welcomes
to its campus scientists from many laboratories who are drawn
together for the discussion of common problems and common aims.
It recognizes in this symposium the challenge that faces intelligent
men of good will everywhere— the great need there is in the con-
temporary world for sitting down and reasoning together. From such
a process comes progress.

We are concerned here with functions which operate in the
biological and chemical world. We seek these out and discover how
they work so that, knowing about them, we may cooperate with
nature for the good of man. This we do by observation, experimenta-
tion, analysis, and, finally, the objective setting down of results
that may yield a pattern or a principle. As we look about us and see
biological specimens called men reacting to special or group inter-
ests as passion and selfishness may happen to dictate, we ask our-
selves, a bit dismally perhaps, whether the statesmen and public
leaders of the world can ever be persuaded to try out the scientific
method as an approach to the problems of world organization. We
also need desperately a healthy society and a sound international
body.

Here, today, we pay tribute to the internationalism of science. As
we scan our program for the week we are struck by the fact that
men from different backgrounds and from many nationalities and
races can come together peacefully in a symposium to present the
results of long years of human effort in a field of science, check these
results, and try to establish what they mean or may mean to life on
this planet. Today and right here men labor together who, were they
still living in their family homelands, would be enemies, legally and
politically. This is the great modem paradox— that as the world of
communication has made the globe a unity and as the domains of
science, hterature, music, art, commerce, and industry have become
international, we have at the same time the phenomenon of a more
bitter nationahsm than ever before. Something is wrong that needs

vii

viii ADDRESS OF WELCOME

early correction, and intelligent men must give attention to the chal-
lenge.

We meet today to talk of many things in the wonderland of
science. We have the special vocabulary necessary for the accuracy
of our thinking and investigation. This vocabulary is a closed book
to the man in the street except for a few words, such as vitamin,
for instance. This man in the street, however, does get a partial
implication of your work as he hears or reads the advertiser who
expounds the merits of certain food products. He may even be led
to think that he can be a vigorous and whole man if only he has
a box of pills or capsules in his vest pocket. He may even be duped
or exploited because of this partial knowledge.

We therefore have the obligation in our special fields of science
which promise so much to all to attempt such simplification and
general statement that those things for which we can vouch will
become common knowledge at the earliest possible moment. Just
now there is a wide spread of interest in many areas which this
symposium deals with. It is a good time, therefore, to capitalize on
this popular interest, for we have a receptive pubhc. We of the
public are willing and anxious to learn from you.

We here at Wisconsin are glad you are with us. We are happy
too in the cooperation of our sister institution, the University of
Chicago, in the enterprise here represented. Our welcome is genuine,
and we wish for the conference unusual and distinguished success.

Contents

INTERMEDIATE CARBOHYDRATE METABOLISM
By Otto Meyerhof, University of Pennsylvania .

Introduction, 3

Inadequacy of older views of the
relation between aerobic and an-
aerobic carbohydrate breakdown, 4

Alternate pathways of oxidation, 4

The oxidation quotient, 5

Competition of oxygen and pyruvate

in oxidation of dihydrocozymase, 6

Aerobic phosphorylation, 7

Reversibility of the glycolysis re-
actions, 9

Formation of phosphopyruvate from
pyruvate through oxalacetate, 12

Inhibitors, 14

OXIDATIVE MECHANISMS IN ANIMAL TISSUES
By Eric G. Ball, Harvard Medical School ....

Introduction, 16

Oxygen activation and the cyto-
chrome system, 17

Substrate activation and the pyri-
dine nucleotide and flavoprotein
systems, 18

The energy relationships of these
systems and the transfer of elec-
trons between them, 21

The relative concentrations of cyto-
chrome c, diphosphopyridine nu-
cleotide, and flavoprotein in animal

tissues and their possible signifi-
cance, 25

The possibility of the existence of
pathways alternate to the cyto-
chrome system, 26

Cyanide poisoning of respiration and
a theory of its mechanism, 27

An interpretation of respiratory
mechanisms in tlie arbacia egg, 30

Azide poisoning of respiration and
a possible interpretation of its ac-
tion on muscle respiration, 30

DISCUSSION ON HYDROGEN TRANSPORT
Van R. Potter, chairman

The Possible Role of Intermedi-
ary Metabolites as Hydrogen
Carriers by K. A. C. Elliott, 33

The Role of the Carriers in Dis-

MUTATIONS AND CoUPLED OxiDO-
REDUCTIONS, WITH SPECIAL REFER-
ENCE TO THE Flavoproteins by

E. G. Ball and F. Lipmann, 38
The Physico-Chemical Mechanism
OF Hydrogen Transport by Kurt
Stem and Erwin Haas, 42
Possibility of a By-Pass around
the Cytochrome System by El-
mer Stotz, 46

PASTEUR EFFECT

By Fritz Lipmann, Massachusetts General Hospital

The efficiency of aerobic and an-
aerobic metabolism, 50

The metabolic structure of cells, 52
Bacteria, 53
Animal tissues, 56

Interpretation of the Pasteur effect,
59
Equilibrium schemes, 59

Inhibition of the Pasteur effect, 62
Reversible oxidative inhibition of
glycolysis in extracts, 65
Thiol influence on fermentation and
glycolysis in intact cells, 67
Pasteur effect with very low res-
piration, 69
Conclusion and outlook, 70

CTCf /s r^f**j

CONTENTS

OXIDASES, PEROXIDASES, AND CATALASE

By Kurt G. Stern, Yale University School of Medicine .... 74

Introduction, 74 On the mechanics of hemin catal-

The common denominator in hemin yses, 86

catalyses, 74 Autoxidizable iron compounds, 96

Enzyme-substrate intermediates, 84 Oxygen transfer in living cells, 99

NICOTINAMIDE NUCLEOTIDE ENZYMES

By Fritz Schlenk, School of Medicine, University of Texas

104

Historical introduction, 104
Codehydrogenase I and II, 104
Occurrence, 105
Preparation, 106
Properties, 108
Investigation of the structure of co-
dehydrogenase I and II, 109
Reversible reduction, 109
The "model compounds" of Kar-
rer, 111

Nicotinamide nucleoside, its prepa-
ration and properties, 113

Structure of codehydrogenase I and
II, 116
Methods of determination, 120
Apodehydrogenases dependent on
nicotinamide nucleotides, 122
Substrate and coenzyme specificity
of apodehydrogenases, 123
Mode of action of nicotinamide
nucleotide enzymes, 125
Spectrophotometric methods, 126
Biosynthesis of the codehydrogenases
I and II, 126

THE FLAVOPROTEINS

By T. R. Hogness, University of Chicago 134

Historical introduction, 134 Comparison of activities, 142
The general properties of die known Cytochrome a reductase: test, spec-
yellow enzymes, 137 troscopic demonstration, and prop-
Dissociation constants, 139 erties, 144

CYTOCHROMES

By Elmer Stotz, Harvard University

Properties of the cytochrome com-
ponents, 149

Cytochrome c: isolation, purifica-
tion, structure, and oxidation-
reduction potential, 149
Cytochrome b, 153
Cytochrome a, 154

Oxidation-reduction potential of the
cytochromes in yeast, 154

Cytochrome oxidase and cytochrome
03, 155

Identity with Warburg's enzyme,
157

Summary of absorption spectra of
the cytochromes, 158
Copper-containing oxidase, 159
Soluble cytochrome c peroxidase,
160

149

Physiological reduction of the cyto-
chromes, 161

By the succinate system-extra fac-
tor, 161

By flavoprotein (cytochrome re-
ductase), 163

Catalytic relations of the cyto-
chromes and oxidase: oxidation of
hydroquinone and p-phenylene dia-
mine, 164

Determination and distribution of
cytochrome c and cytochrome oxi-
dase, 166

Physiological functioning of the cy-
tochrome system, 168

Possibility of a by-pass of the cyto-
chrome system, 169

CONTENTS xi

PHOSPHORYLATION OF CARBOHYDRATES

By Carl F. Cori, Washington University School of Medicine, St. Louis 175

Introduction, 175 Transphosphorylation, 181

Uptake of inorganic phosphate, 175 Regeneration of inorganic phosphate.

Intramolecular migration of phos- 185

phate, 179 Summary, 188

DISCUSSION ON PHOSPHORYLATION
H. M. Kalckar, chairman

190

Myokinase; dephosphorylation

By H. M. Kalckar, 190
Dephosphorylating and transphos-

phorylating enzymes

By Otto Meyerhof, 192

Energy utilization mechanisms
By M. J. Johnson, 194

Evidence for acetylphosphate occur-
rence
By Fritz Lipmann, 195

METABOLIC CYCLES AND DECARBOXYLATION
By E. A, Evans, Jr., University of Chicago ....

Introduction: cycle reactions in bio-
logical systems, 197
Krebs' citric acid cycle, 198

Experimental basis of the citric

acid cycle, 198

The "Krebs reaction," 200

The eflFect of malonate on tissue

respiration, 201

Evaluation of the reactions of the

citric acid cycle, 201

Criticisms of the theory, 202

197

The citric acid cycle in pigeon liver,
203

The synthesis of a-ketoglutaric
acid, 203

The assimilation of CO2 in the syn-
thesis of a-ketoglutaric acid by
pigeon liver, 204

The mechanism of a-ketoglutaric
acid synthesis, 204
The nature of CO2 assimilation by
pigeon liver, 205

TRANSAMINATION

By Philip P. Cohen, University of Wisconsin

210

Types of transamination, 210
Discovery of transamination reac-
tion, 211
Substrates active in transamination,
211

Amino and keto acids, 211
Peptides, 212

"Primary" and "secondary" sub-
strates, 213

"Catalytic" transamination, 213
Preparation and properties of trans-
aminating enzymes, 214
Aminopherases, 214
Transaminase, 215
Substrate specificity, 215
Mechanism of transamination, 216
Transamination in diflFerent tissues:
Animal tissues, 218

Malignant and embryonic tissues,

219

Plant tissues, 220

Yeast and bacteria, 221

Transamination in vivo, 221

Influence of various substances on
transamination, 221
Inhibitors, 221
Hormones, 222
Carcinogens, 222
Vitamins, 222

Role of transamination in intermedi-
ary metabolism, 223
Protein and amino acid synthesis
and degradation: animal tissues,
plant tissues, transamination and
glycolysis, transamination and hy-
drogen transport, 223

CONTENTS

DISCUSSION ON TUMOR RESPIRATION
C. A. Baumann, chairman

229

Characteristics of Tumor Res-
piration by K. A. C. Elliott, 229

Phosphorylation Theories and
Tumor Metabolism by Van R.
Potter, 233

On the Specificity of Glycolysis
IN Malignant Liver Tumors as
Compared with Homologous

Adult or Growing Liver Tissues
by Dean Burk, 235
The Effects of Certain Diamines
on Enzyme Systems, Correlated
WITH THE Carcinogenicity of
THE Parent Azo Dyes by C. J.
Kensler, 246

DISCUSSION ON BACTERIAL RESPIRATION
W. H. Peterson, chairman

252

Criteria for Experiments with
Isotopes discussed by H. G. Wood
and R. H. Burris, 252

Mechanisms for the Complete
Oxidation of Carbohydrates by
Aerobic Bacteria discussed by

C. H. Werkman, E. S. Guzman
Barron, and P. W. Wilson, 258
Reactions in Cell-Free Enzyme
Systems Compared with Those
IN the Intact Cell discussed by
F. F. Nord and P. W. Wilson, 264

DISCUSSION ON ANIMAL TISSUE RESPIRATION
C. A. Elvehjem, chairman

268

Factors Affecting the Prepara-
tion of Tissue for Metabolic
Studies by Ephraim Shorr, 268

Comparison of Slices and Homo-
genized Suspensions of Brain
Tissue by K. A. C. Elliott, 271

The Homogenized Tissue Tech-
nique, THE Dilution Effect and
Ion Effects by Van R. Potter, 274

The Stimulatory Effect of Cal-
cium UPON the Succinoxidase Ac-

tivity OF Rat Tissues by A. E.
Axelrod, 275

Tissue Metabolism in Vitro and
in Vivo by Frederick Bemheim,
276

Pathways of Carbohydrate Me-
tabolism by E. S. Guzman Bar-
ron, 278

The Citric Acid Cycle in Tissue
Metabolism by Fredrick J. Stare,
280

A SYMPOSIUM ON RESPIRATORY ENZYMES

Addresses given at an Institute

Held at the University of Wisconsin

September 11-17, 1941

Intermediate Carbohydrate
Metabolism

OTTO MEYERHOF

University of Pennsylvania

THIS REVIEW OF THE intermediary carbohydrate metabolism must
necessarily be treated broadly and generally, for the subject
has many diflFerent aspects, and the detailed questions of hydro-
gen transport, Pasteur eflFect, pyridine nucleotides, cocarboxylase,
metabolic cycles, phosphorylations, indeed all the items which are
intrinsic elements of the present picture of carbohydrate breakdown,
will be dealt with by competent investigators of these subjects.
Moreover, I had the opportunity to discuss the special question of
oxidoreduction and dismutation in carbohydrate metabolism at the
Chicago congress some months ago.

If we take this occasion to look back fifty years and to compare
our present knowledge with that which existed at the end of the
last century we have reason to be very proud, for at that time this
whole field appeared nearly as tabula rasa. But two outstanding
achievements had already been accomplished: first, Claude Ber-
nard's work on the interconversion of glucose and glycogen in the
liver and on the role of blood sugar under normal and diabetic
conditions; second, the work of Pasteur on the different microbic
fermentations as manifestations of the anaerobic metabolism of
these organisms. Nothing was known about the oxidative break-
down of sugar. Although lactic acid formation in the blood and
especially in the muscles had been observed by Claude Bernard
and others, it was not known whether nor how this cleavage was
connected with respiration.

Since then the interconversion of glycogen and blood sugar have
continued to claim the attention of medical investigators, and re-
cently, as you know, a highly interesting development was reported:
Professor Cori's discovery of glucose-1-monophosphoric acid as inter-
mediary. The old problem of diabetes was shifted by the isolation
of insulin from the study of blood sugar regulation to the bio-
chemical task of studying tissue metabolism under the influence of
added hormones. The third old problem of the connection between

4 A SYMPOSIUM ON RESPIRATORY ENZYMES

fermentation and respiration remained for a long time a subject of
speculation, and even now many a question is unanswered. How-
ever, I should like to follow this latter trend of ideas a little more in
detail.

PfeflFer and Pfliiger, following Pasteur, held a rather simple view
of this relationship: the first step of respiration was assumed to be
always anaerobic. If no oxygen is present, the products of anaerobic
cleavage accumulate: alcohol in yeast and higher plants, lactic acid
in the tissues of higher animals and in some bacteria. But if oxygen
is present, these products are oxidized to carbon dioxide and water.

That this concept required modification became apparent twenty
years ago from studies of metabolism of muscle. In 1907 Fletcher and
Hopkins (1) showed that under anaerobic conditions frog muscles
formed lactic acid steadily during both activity and rest, and that
this lactic acid disappeared when oxygen was admitted. Parnas (2),
working some years later in the same Cambridge laboratory, claimed
to have found that this disappearance was a complete oxidation, thus
apparently confirming the views of Pfeffer and Pfliiger. In 1920,
because of the controversial state of this question, I repeated the
experiments of Parnas, avoiding especially all kinds of irritation or
injury of the muscles which would lead to extra-consumption of
oxygen (3). Under these conditions much more lactic acid disap-
peared in oxygen than could be accounted for by oxidation, and the
lactic acid unaccounted for was reconverted into carbohydrate. This
was true for the lactic acid formed during activity as well as for that
formed during rest. Similarly, it was shown that in equal periods of
rest much more lactic acid was formed anaerobically than could be
burnt aerobically by the resting respiration. Indeed, the amount of
oxygen which failed to be used in a period of anaerobiosis was about
the same as the excess consumed after that period. This oxygen was
suflBcient only to oxidize from a quarter to a sixth of the lactic acid
which disappeared.

These facts, which are independent of special interpretations, are
sufficient to invalidate the original theory of Pfeffer and Pfliiger in
that they show that the oxidative removal of fermentation products
is not necessarily identical with the oxidation of these products. But
we can pose the more limited question whether the oxidation on the
whole attacks the end products of anaerobic breakdown. With
respect to lactic acid formed in a preceding anaerobic period, we
must surely answer in the affirmative. We know that lactic acid is
easily oxidized by way of pyruvic acid. For example, Barron et al.

INTERMEDIATE CARBOHYDRATE METABOLISM 5

(4) showed that specially treated, washed bacteria may lose the
power to oxidize sugar and other substrates, but retain the power
to oxidize lactic to pyruvic acid. Experiments on muscle lead to the
same conclusion. After a muscle is poisoned with iodoacetic acid the
formation of lactic acid is blocked; at the same time the respiratory
quotient drops to 0.7, and is not changed by the addition of sugar,
but is brought to 0.95 by the addition of lactic acid. Respiration is
increased, and oxygen consumption is essentially equivalent to the
disappearance of lactic acid (5). Similar results were obtained by
Krebs with respiration of brain and testis after poisoning with
iodoacetic acid (6). Since oxidation of sugar is completely checked,
no interpretation is possible except that lactic acid is directly oxi-
dized.

But this is not necessarily the pathway of sugar oxidation in the
aerobic steady state. That independent ways of sugar oxidation
exist may be gathered from many observations, such as the rapid
oxidation of fructose in brain tissue, where, in contrast to glucose
(7), it does not give rise to anaerobic lactic acid. Furthermore,
Warburg and Christian showed that hexosemonophosphate can be
oxidized by the triphosphopyridine nucleotide in yeast extract to
phosphogluconic acid (8), and Lipmann demonstrated the complete
oxidation to carbon dioxide in this manner (9).

On the other hand, the oxidation of sugar by way of pyruvic acid
is also firmly established, and in this case the steps up to the forma-
tion of the acid are identical in respiration and in anaerobic gly-
colysis. As was discovered by Peters (10), pyruvic acid accumulates
during oxidation of carbohydrate by cells and tissues in cases of
vitamin B^ deficiency, which means that lack of cocarboxylase
blocks the oxidative decarboxylation of pyruvic acid. Many other
findings, such as the similarity of the oxidation of pyruvic acid to
that of sugar in tissue pulps and extracts, point in the same direction,
namely, that sugar is oxidized via pyruvic acid (11). Thus several
pathways of sugar oxidation exist, the choice of which may depend
upon the special set of enzymes in different tissues and also upon
hormonal and other controlling influences.

All this probably has some bearing on the relationship already
mentioned between oxidation and interference with the mechanism
of fermentation. I have mentioned before the two possible cases of
this relationship— the actual synthesis of split products to the initial
substance and the non-formation of the split products during the sta-
tionary state of respiration. Without fearing to be accused of a

e A SYMPOSIUM ON RESPIRATORY ENZYMES

biased judgment I dare say that both cases are characterized by the
same numerical relationship— the oxidation quotient, which expresses
the ratio of the aerobic disappearance of splitting metabolism in
moles sugar to the oxidized sugar equivalents (12). Critics have
objected that under extreme conditions this number may range
from zero to infinite, but it is equally true, and more important, I
think, that under physiological conditions living cells exhibit quo-
tients between 3 and 6— approaching 6 more and more as the con-
ditions of temperature, oxygen pressure, nutritional state, and milieu
become optimal for the cells in question. The same preference for
the quotient of 6 was demonstrated by O. Warburg for different
warm-blooded tissues where the anaerobic glycolysis is high enough
to allow the calculation of the quotient (13).

The original concept of a metabolic carbohydrate cycle involved
the assumption that in the stationary state the quotient results from
a continuous overlapping of anaerobic glycolysis and of oxidative
resynthesis of the cleavage products— the endothermic resynthesis
made possible by coupling with oxidation. Today it seems possible
to refine this scheme and to modify it somewhat without rejecting
the main argument. Indeed, in the past fifteen years a tremendous
amount of material has been collected to prove that the general
concept of these cycles in carbohydrate breakdown holds good,
that every oxidative step is coupled with an involuntary phosphoiy-
lation, and that the several intermediate stages of the anaerobic
breakdown can be reversed by means of the "energy-rich phosphate
bonds" (31) created in this way.

On the other hand, the original concept of a single complete
cycle passing through the stage of lactic acid cannot be exactly
true for a very simple reason, which has become clear since 1933;
namely, that pyruvic acid is the necessary precursor of lactic acid in
glycolysis and of alcohol in yeast fermentation (14). Under anaerobic
conditions the reduction of pyruvic to lactic acid is compensated
for by the oxidation of phosphoglyceraldehyde to phosphoglyceric
acid. The latter, in turn, is decomposed via two intermediaries to
pyruvic acid (15). The hydrogen transfer proceeds in both directions
by the way of cozymase, the diphosphopyridine nucleotide of War-
burg.

But if oxygen is present the dihydrocozymase can transfer its two
hydrogen atoms to oxygen instead of to pyruvic acid by a long chain
of oxidative catalysts : the pheohemin enzyme of Warburg, the three
cytochromes, and the flavinproteins; consequently the pyruvic acid

INTERMEDIATE CARBOHYDRATE METABOLISM 7

is not reduced. On the contrary, such an oxidation of dihydro-
cozymase shifts the equihbrium in the opposite direction, so that
lactic acid, if present, would be oxidized by cozymase to pyruvic acid,
whereas in the stationary state of sugar oxidation pyruvic acid would
be continuously formed by way of phosphoglyceric acid, without a
compensating reduction.

Therefore only pyruvic acid, and not lactic acid, is formed in the
stationary state of oxidation. This interpretation at the same time
gives a clue to the oxidation quotient, the numerical relationship
between the oxygen consumed and the lactic acid that is prevented
from being formed: if one atom of oxygen is required to oxidize the
two hydrogen atoms of dihydrocozymase, then this atom prevents
one niolecule of pyruvic acid from being reduced to lactic acid or in
yeast fermentation to alcohol. Therefore six atoms of oxygen (corre-
sponding to the complete oxidation of one molecule of lactic acid)
can prevent six molecules of lactic acid from being formed, and we
obtain the normal oxidation quotient of 6. Of course this refers only
to the principle. The cozymase reoxidized by oxidative catalysts
must dehydrogenate other intermediary stages besides triosephos-
phate, because every oxidative step in the breakdown of sugar acts
in the same way, preventing the formation of one molecule of lactic
acid per one atom of oxygen taken up.

And this is only one side of the picture. If the breakdown of sugar
in oxygen and in nitrogen proceeded with the same speed to the
stage of pyruvic acid, and the only difference consisted in the fate
of pyruvic acid to be reduced or further oxidized, then the oxidation
would not prevent, as it actually does, by this so-called "Pasteur
effect," the greater part of sugar from disappearing. But here the
concept of metabolic cycles has its place. Actually every oxidative
step is coupled with the phosphorylation of the adenylic system, and
by this means a corresponding phase of anaerobic breakdown is
reversed, so that for every oxygen atom consumed one three-carbon
molecule can return to its initial stage as sugar or glycogen. This
state of affairs is very neatly shown by the recent experiments of
Cori, Kalckar, and co-workers (16) with dialyzed extracts of kidney
and heart, and by experiments of Behtzer and Tzibakowa (17) with
washed pigeon muscle. Cori and his group found that in the pres-
ence of the complete glycolytic coenzyme system the organ extracts
oxidize glucose and phosphorylate an excess of it, so that for every
hexose molecule burned to carbon dioxide, ten molecules of phos-
phate are taken up to form five molecules hexosediphosphate; and

8 A SYMPOSIUM ON RESPIRATORY ENZYMES

since the oxidized molecule also had to be phosphorylated, altogether
twelve molecules of phosphates are taken up for one molecule glucose
or twelve oxygen atoms consumed. Therefore every step of glucose
oxidation consisting in an oxidoreduction between cozymase and an
oxidizable intermediary is coupled with phosphorylation. Not only is
this true for the two steps where it is already known, i.e., the oxida-
tion of phosphoglyceraldehyde and that of pyruvic acid, in which
Lipmann discovered acetylphosphate as the primary product of
oxidation (18), but for every such step an energy-rich phosphate
bond is created in adenosinetriphosphate, which enables a synthetic
step to take place.

The experiments of Belitzer and Tzibakowa are a little dijBFerent,
because they added creatine to cut muscle and obtained under these
conditions a synthesis of creatinephosphate when lactate, pyruvate,
or the four-carbon acids of the Szent-Gyorgy cycle were oxidized.
At the most two molecules of creatinephosphate were formed for
every oxygen atom taken up. Although the presence of creatine
diverts the pathway of synthesis from carbohydrate, the experiments
are important in that they demonstrate the uptake of two molecules
of phosphate by way of adenosinetriphosphate for one atom of
oxygen consumed; this relationship is comparable to the synthesis
of creatinephosphate in muscle extract, where two steps of glycolysis
are involved in the transfer of phosphate, namely, the oxidoreduction
and the dephosphorylation of phosphopyruvic acid (19).

Moreover, the reaction studied by Belitzer is closely analogous to
the recovery period of the living muscle, especially a muscle which is
only slightly fatigued. Here, during oxidative recovery, the oxidation
serves mostly for the resynthesis of creatinephosphate, and to a small
extent for that of glycogen. If two molecules of creatinephosphate are
synthesized for every atom of oxygen taken up, then about 40 per
cent of the combustion heat of sugar or lactate is consumed for the
endothennic synthesis, a result which comes very close to the
efficiency of the oxidative recovery in the living muscle.*

But to return from this digression to the significance, already men-
tioned, of the experiments for the theory of carbohydrate cycles.
One objection may be raised against this interpretation of the Pas-
teur effect. Many cases are known where the respiration remains
quantitatively the same, while the effect of the respiration on the

* Actually the same ratio of two molecules of creatinephosphate syntliesized
for one atom of oxygen taken up was found by O. Meyerhof and D. Nachman-
.sohn (Biochem. Z., 222, 1, 1930) during recovery of a partially fatigued muscle.

INTERMEDIATE CARBOHYDRATE METABOLISM 9

glycolysis is suppressed. In the picture outlined above the oxygen
used would automatically eliminate an equivalent lactic acid forma-
tion, in so far as the oxygen serves to reoxidize dihydrocozymase. But
we must have in mind that the oxygen intervenes only indirectly by
way of the oxidizing catalysts. Here the so-called "Pasteur enzyme"
assumed by Warburg (20) and demonstrated by Stern and Melnick
(21) plays its role in steering the oxidation. All oxidation not going
by the way of cozymase would be without "Pasteur effect"; it may
be oxidation of non-carbohydrate, which replaces sugar oxidation,
or it may be oxidation of sugar by way of triphosphopyridine nu-
cleotide.

Now we come to the second half of the problem, the actual con-
version of lactic acid to glycogen in the oxidative recovery of the

Glycogen (starch)
HaPO, If

Glucose-1-phosphate
(Cori-Ester)

Dihydroxyacetonephosphate
I- (a) -Glycerophosphate
(Glycerol -1- H3PO4)

Pyruvic acid + H3PO4

■J'
Phosphopyruvic acid (?) (hydrated)

H2 J,
Phosphoacetic acid -1- CO2

i
Acetic acid -f H3PO4

Acetaldehyde -|- CO2 <-
Ethyl alcohol

d-Glucose + H3PO4
-* Glucose-6-phosphate
Fructose-6-phosphate

H3P04|f

Fructose-l ,6-diphosphate

-» d-3-Phosphoglyceraldehyde

H3PO4JI
d-1 ,3-Diphosphoglyceraldehyde

d-l,3-Diphosphoglyceric acid
H3PO4II
<f-3-Phosphoglyceric acid

It
d-2-Phosphoglyceric acid

H2OII
enol-Phosphopyruvic acid

Pyruvic acid -1- H3PO4

HJt

Lactic acid

Figure 1. — Complete sequence of intermediaries in anaerobic breakdown

of carbohydrate
Insertion on the left: oxidative decomposition in lactic acid bacteria, accord-
ing to Lipmann ( 18 ) .

A SYMPOSIUM ON RESPIRATORY ENZYMES

isolated frog muscle or in the mammalian liver. Without going into
too much detail I will show you the table of consecutive inter-
mediary steps, neglecting the coenzymes concerned. As you see from
the double arrows in Figure 1, nearly all these reactions are re-
versible. The transformation of glucose-1-phosphate into glucose-6-
phosphate is also reversible, according to Cori's recent findings (22).
This reversibility is especially conspicuous for the oxidative step,

CMM CO

200

150

100

40 MIN

Figure 2. — Stoichiometric coupling reaction
Oxidation of phosphoglyceraldehyde is drawn upward. The points on the lines
were obtained by manometric measurement. The spectrographic measurements
were of dihydrocozymase (absorption maximum 340 mM-).

INTERMEDIATE CARBOHYDRATE METABOLISM

the oxidation of phosphoglyceraldehyde* to phosphogly eerie aeid.
Even in the presenee of a stoichiometric amount of cozymase, which
is simultaneously reduced, the reaction would proceed completely in
the direction of oxidation but for the coupling with phosphate up-
take by the adenylic system. Thereby a measurable equilibrium is
obtained, which was established in 1938 (23) in the Heidelberg Insti-
tute and which is shown in Figure 2. The kinetic nature of this
equilibrium can be neatly demonstrated by the use of radioactive
phosphorus. If radioactive inorganic phosphate is added to the
enzymatic mixture after the equilibrium has been established, the
adenosinetriphosphate in the solution rapidly takes up radioactive

loV

2.0

1.5

1.0

0.5

\
\
\
\
\

1 ■

-• Uptoke of lab

— ^ True rate of

fhe labile

9roups of t

cicd P by ATP
exchanpe of
phosphate
he ATP

to 2040

C O O

150

Figure 3. — Use of radioactive phosphate in the reversible coupling reaction
Left: Adjustment of the equilibrium of the coupling reaction; Right: Adjust-
ment of the equilibrium of the isotopes

phosphate, although no chemical change takes place (24), as shown
in Figure 3. This reversible coupling reaction was confirmed and ex-
plained by Warburg, Christian, and Negelein in the way shown in
Figure 4. They isolated the 1, 3-diphosphoglyceric acid as inter-

* This substance is identical with tlie d-component of the "Fischer-Baer
ester" (Chem. Ber. 65, 337, 1932).

12 A SYMPOSIUM ON RESPIRATORY ENZYMES

mediary, and consequently we have three consecutive steps (25).
The overall reaction goes from left to right or in the opposite direc-
tion, depending upon the concentration of the reactants (Figure 4).
If, for instance, adenosinetriphosphate is continuously resynthesized
by an independent reaction, so that its concentration remains high
and the concentration of the inorganic phosphate low, the reaction
is pushed in the direction of the reduction of phosphoglyceric acid.
This reaction is favored still further if the phosphoglyceraldehyde
is removed by isomerisation to dihydroxyacetonephosphate and by
condensation to hexosediphosphate and so on. In this way the syn-
thesis can actually take place.

There remains one reaction which in the light of experiments
with radioactive phosphorus, seems to be irreversible— the dephos-
phorylation of phosphopyruvic acid. Adenosinetriphosphate con-

I. Stoichiome trie-coupled reaction (Meyerhof, Ohlmeyer, Kiessling, 1937-38)

( Co=cozymase )

(i-3-Phosphoglyceraldehyde+Co-|-Adenosinediphosphate+H3P04?=^
d-3-Phosphoglyceric acid+CoH2+ Adenosinetriphosphate

II. Warburg and Christian's explanation of the coupled reaction ( 1939 )

A. d-3-Phosphoglyceraldehyde+H3P04?^l,3-Diphosphoglyceraldehyde (?)

B. l,3-Diphosphoglyceraldehyde+Co<^l,3-Diphosphoglyceric acid-j-CoHj

C. 1,3-Diphosphoglyceric acid+Adenosinediphosphate<^
3-Phosphoglyceric acid+ Adenosinetriphosphate

Figure 4. — Coupling of phosphorylation and oxidoreduction

taining radioactive phosphorus in its labile groups did not exchange
this radioactive phosphorus with phosphopyruvic acid. If the reac-
tion between phosphopyruvic and adenylic acid were reversible, an
exchange should have taken place. But we must concede that the
experimental basis for this negative result is not too large and there-
fore accept it with some reservation until it is more firmly estab-
lished. If for the moment we accept the result, it has important im-
plications. There is indeed reason to believe that the oxidation of
carbohydrate by way of the four-carbon acids intervenes to bring
pyruvic acid back to phosphopyruvic. Some years ago Kalckar ob-
served, during oxidation of fumarate by kidney extract, the formation
of an acid which seemed phosphopyruvic (26). The more recent
findings of several investigators (27, 28, 29, 30) on the assimilation
of isotopic carbon dioxide, which appears in oxalacetic and keto-
glutaric acid and in glycogen during synthesis from lactic acid, fit
very well into such a scheme. Now that the condensation of carbon

INTERMEDIATE CARBOHYDRATE METABOLISM

dioxide with pyruvic to oxalacetic acid has been proved by Evans
and by Wood and Werkman, we have only to assume that the car-
bonyl group of the latter is phosphorylated, forming a phospho-enol
oxalacetic acid; the phospho-enol group would then be equally
distributed between the alpha and beta position and the compound
again decarboxylated; so we obtain a phosphopyruvic acid, half of
which contains labeled carbon in the carboxyl group. Such a reaction
may well be coupled with oxidation of fumaric and malic acid to

CH,
I
o<C=0

I
COOH

Pyruvic Acid

C*OOH

CHp
I
a C=0 + H3PO4

COOH
Oxalacetic Acid

+ H,

-H;

Malic
Acid

COOH
I
CHp

I
CHOH

COOH

•HpO

+ HpO

Fumaric
Acid

COOH
I
CH

CH
I
COOH

I ^
+H2 '

-H=

Succinic
Acid

COOH
I

CHp
I

CH3
I '
COOH

C*OOH

II
ex C-O-HpPO,
I ^

COOH

Phosphoenoloxalacetic Acid

0*0 OH

C-O-HpPO,

II
a CH
J.
[C*00:H

Phospho-
pyruvic
Acid

C^OOH
I
C-O-HpPO,

II
CH,

(ctH.oOs),
Glycogen

Figure 5. — Possible mode of uptake of carbon dioxide containing radioactive
carbon (C*) during synthesis of glycogen from lactic acid

14 A SYMPOSIUM ON RESPIRATORY ENZYMES

oxalacetic acid. These hypothetical reactions are shown in Figure 5.
In this or in a similar manner, by the way of a phosphomalic acid,
as Lipmann (31) and Hastings (28) recently suggested, the gap
between pyruvic acid and enol-phosphopyruvic may be bridged with
simultaneous oxidation of four-carbon acids. Indeed, with the clos-
ing of this gap the whole chain of reactions leading from lactic acid
to glycogen will be completely understood. Furthermore, the real
point of attack of insulin, which has so far eluded all investigators,
may even be sought in the oxidative mechanism concerned with the
four-carbon and five-carbon acids, lying on the pathway of oxidative
sugar breakdown. Such an assumption, already proposed by Krebs
(32), has not been conclusively proved by experiments.

Other questions concerning the intermediate metabolism of carbo-
hydrate remain unsettled. One of these would seem to be relatively
easy to attack with our present facilities. Fermentation is inhibited
by not too high concentrations of cyanide, nitric oxide, hydrogen
sulfide, and o-phenanthroline, substances known to fonn complexes
with heavy metals. Such complex formation is responsible, as we
know from the work of Warburg and many others, for the inhibition
of respiration by these and similar substances. We cannot be sure
at present whether this explanation holds good also for fermentation
and glycolysis, since we do not know of any heavy metal indis-
pensable to fermentation. Lohmann (33) of Heidelberg discovered
that magnesium was essential for the phosphorylating enzyme sys-
tem and for the carboxylase (34, 34a), and magnesium can be re-
placed in many instances by still smaller concentrations of man-
ganese (35). But it is doubtful whether the latter is the metal re-
sponsible for the inhibitions, and we do not know which of the many
intermediary reactions these inhibitors attack. On the other hand, we
know that fluoride attacks mainly the enolase, which dehydrates the
2-phosphoglyceric acid to enol-phosphopyruvic acid, while oxalate
inhibits the dephosphorylation of the latter (36). But here too the
mechanisms are unknown. Only in the case of iodoacetic acid, which
affects the oxidoreduction steps in which cozymase takes part, does
the mechanism of inhibition seem to be explained, namely, by the
oxidation of the sulfhydryl groups of the dehydrogenase proteins
(37).

Therefore our pride in the progress achieved in the last decades
must be tempered by confession of ignorance regarding many cru-
cial points. There are still many problems for this generation of
research workers to solve.

INTERMEDIATE CARBOHYDRATE METABOLISM 15

REFERENCES

1. Fletcher, W. M., and Hopkins, F. C, J. Physiol., S5, 247 (1907).

2. Parnas, J., Centralbl. f. Physiol., 30, 1 ( 1915).

3. Meyerhof, O., Pfliigers Archiv., 182, 232, 284; 185, 11 (1920).

4. Barron, Guzman E. S., and Miller, C. P., J. Biol. Chem., 97, 691 (1932).

5. Meyerhof, O., and Boyland, E., Biochem. Z., 2S7, 406 (1931).

6. Krebs, H. a., Biochem. Z., 234, 278 (1931).

7. LoEBEL, R. O., Biochem. Z., 161, 219 (1925).

8. Warburg, O., and Christian, W., Biochem. Z., 254, 438 ( 1932); 287, 440
(1936).

9. LiPMANN, F., Nature, 138, 588 (1936).

10. Peters, R. A., Biochem. J., 31, 2240 (1937).

11. CoLOwicK, S. P., Welch, M. S., and Corn, C. F., J. Biol. Chem., 133, 359,
641 (1940).

12. Meyerhof, O., Chemische Vorgange im Muskel (Berlin, 1930).

13. Warburg, O., Posener, K., and Negelein, E., Biochem. Z., 152, 309
(1924).

14. Meyerhof, O., and Kiessling, W., Biochem. Z., 264, 40; 267, 313 ( 1933).

15. Meyerhof, O., Ergebnisse d. Physiol. ( Asher-Spiro ) , 39, 10 (1937).

16. CoLOwicK, S. P., Kalckar, H. M., and Com, C. F., J. Biol. Chem., 137,
343 (1940).

17. Belitzer, V. A., and Tzibakowa, E. T., Biokimia, 4, 516 (1939).

18. Lipmann, F., J. Biol. Chem., 134, 463 (1940).

19. Meyerhof, O., Schulz, W., and Schuster, P., Biochem. Z., 293, 309
(1937).

20. Warburg, O., Biochem. Z., 172, 432 (1926).

21. Stern, K. G., and Melnick, J. L., J. Biol. Chem., 139, 301 (1941).

22. Sutherland, E. W., Colowick, S. P., and Corn, C. F., J. Biol. Chem., 140,
309(1941).

23. Meyerhof, O., Ohlmeyer, P., and Mohle, W., Biochem. Z., 297, 90, 113
(1938).

24. Meyerhof, O., Ohlmeyer, P., Centner, W., and Meier-Leibnitz, H.,
Biochem. Z., 298, 396 (1938).

25. Warburg, O., and Christian, W., Biochem. Z., 303, 40 (1939).
Negelein, P., and Bromel, W., Biochem. Z., 303, 132 (1939).

26. Kalckar, H. M., Enzymologia, 2, 247 (1937); 5, 365 (1939); Biochem.
J., 33, 631 (1939).

27. Ruben, S., and Kamen, M. D., Proc. Nat. Acad. Sci. (U. S.), 26, 418
(1940).

28. Solomon, A. K., Vennesland, B., Klemperer, F. W., Buchanan, J. M.,
and Hasting, A. B., J. Biol. Chem., 140, 171 (1941).

29. Evans, E. A., and Slotin, L., J. Biol. Chem., 136, 301 ( 1940).

30. Wood, H. G., Werkman, C. H., Hemingway, A., and Nier, A. O., J. Biol.
Chem., 139, 365, 377, 483 (1941).

31. Lipmann, F., Advances in Enzymology, 1, 99 (New York, 1941).

32. Krebs, H. A., and Eggleston, L. V., Biochem. J., 32, 913 (1938).

33. LoHMAN, K., Biochem. Z., 237, 445 (1931).

34. LoHMAN, K., and Schuster, P., Biochem. Z., 294, 188 ( 1937).

34a. Green, D. E., Herbert, D., and Subrahmanyan, V., J. Biol. Chem., J35,
795 (1940); J 38, 327 (1941).

35. Ohlmeyer, P., and Ochoa, S., Biochem. Z., 293, 338 ( 1937).

36. Lohman, K., and Meyerhof, O., Biochem. Z., 273, 60 (1934).

37. Rapkine, L., Biochem. J., 32, 1729 (1938).

Oxidative Mechanisms in Animal Tissues

ERIC G. BALL

Harvard Medical School

LIFE REQUIRES energy, and the study of life processes has resolved
J itself largely into a study of various manifestations of the
utilization of energy by the living organism. The source of this
energy necessary for life was first indicated by the work of Lavoisier
in 1770. Since then it has become increasingly recognized, as F. G.
Hopkins has said, that "among the most fundamental of the dynamic
chemical events related to life are the oxidations which yield energy
to the cell."

Today we know a good deal about the oxidative processes taking
place within the living cell, and we know a little about the amount
of energy such processes may yield. We do not know, however,
whether all the energy released by oxidative processes is utilized by
the cell nor how it is utilized. Further knowledge concerning this
aspect of the subject must perforce await fuller understanding of the
mechanisms involved in the energy-yielding oxidative processes.
That we are, however, upon the threshold of the solution is wit-
nessed by the recent developments linking phosphorylation with
oxidative processes in the living cell. It may well be that this
symposium on respiratory enzymes and phosphorylation processes
will mark a milestone in our advance. Let me, therefore, as my part
in it, review briefly for you what we know today about the oxidative
mechanisms in animal tissues and the energy they may yield.

Any consideration of the oxidative mechanisms in animal tissues
has naturally centered about two points, oxygen and the organic
substance undergoing oxidation. Outside the living cell oxygen does
not react with the foodstuffs of the cell to any appreciable extent.
Within the cell reaction occurs readily. This fundamental fact early
suggested that within the cell either oxygen or the foodstuffs have
become activated in some way that permits their interaction.

During the decade 1920-30 a controversy raged between two
schools. One, championed by Warburg, claimed that oxygen activa-
tion was the all-essential mainspring. Once oxygen was activated,
its direct attack upon the substrate was thought possible. The other

OXIDATIVE MECHANISMS IN ANIMAL TISSUES

school, headed by Wieland, claimed that activation of the substrate
was most important. In particular, activation of the hydrogen of the
substrate was stressed. Such activated hydrogen was believed
capable of reacting with atmospheric oxygen to form water. Both
schools seemed to agree that direct reaction between molecular
oxygen and the substrate could occur. A release of energy in one
tremendous burst was thus implied. As more data became available
it became evident that both schools of thought were right and that
biological oxidations took place only after both oxygen and the food-
stuff to be burned were acted upon by intracellular enzymes. Within
the last ten years we have also learned that it is doubtful whether
any direct reaction occurs between oxygen and the substrate to be
burned. Interposed between oxygen and the substrate are a series
of so-called carriers through which electron exchange occurs, and
energy is released in a series of successive steps.

Beginning with the oxygen end, let us examine the chain of
events more closely. Evidence for the activation of oxygen has de-
pended largely upon the use of so-called respiratory poisons. As a
result of Warburg's earlier belief that iron in some form or other
was the activator of oxygen, cyanide and carbon monoxide have
become classical tools for the study of respiratory mechanisms.
With the aid of these tools it has been proved, thanks to the labora-
tories of Warburg and Keilin, that at least four iron porphyrin com-
pounds participate in biological oxidations. One of these, now
commonly called cytochrome oxidase, is known largely as the result
of its shadow-boxing with carbon monoxide. The other three, known

CO dark . Fe^^CO - Cytochronne Oxidase

+ re**Cytochrome Oxidase

-» Fe ^O, ■ Cytochrome Oxidase

■H*

Fe^^^CN-Cytochrome Oxidose , ' CN' t Fe***Cytochrome Oxidase + H^O

Fe*t!^^^^ Fe*t* Fe^t^. Fe^*-^

Cylochrome Oxidase~---,^Cy+ochrome 1 g ^\^tochrome | c ^\^ytochrome b
Fe** ^"~~^ Fe** ^^ Fe** ^^ Fe**

Figure 1. — The cytochrome system

18 A SYMPOSIUM ON RESPIRATORY ENZYMES

as cytochromes a, b, and c, are spectroscopically visible in their
reduced forms. Their role in biological oxidations so far as we know

it is shown in Figure 1. As depicted here, oxygen reacts with ferrous
cytochrome oxidase, presumably to form an oxygenated compound
similar to oxyhemoglobin, as evidenced by the competitive aflBnity
shown by carbon monoxide. In the case of cytochrome oxidase, how-
ever, the oxygen is able to strike in and oxidize the ferrous iron to
ferric. The role of oxygen in biological processes is now ended.
Combining with hydrogen ions withdrawn from the acid-base con-
tinuum of the cell, it forms water. The ferric cytochrome oxidase thus
formed can now bring about the oxidation of the cytochromes a, b,
and c. In the presence of cyanide their oxidation is somehow pre-
vented. Whether the c)'tochromes react as a chain or individually
with ferric cytochrome oxidase we cannot say with certainty. If they
react as a chain, we may align them as shown here in view of their
relative oxidation-reduction potentials. The reaction involves the
transfer of an electron from one iron compound to another without
involving oxygen or hydrogen ions in the oxidation. Thus the oxi-
dizing agent in the cell that we now have to deal with is ferric
iron in organic combination. With what does it react? If we could
answer that question, one of the largest gaps in our knowledge of
the mechanisms of biological oxidations would be filled.

Since we can follow the pathway from the oxygen side no further,
let us turn our attention to the substrate side, to the studies made
upon its activation. The chief tools employed in these studies have
been certain dyestuffs capable of undergoing reversible oxidation
and reduction. Methylene blue in particular has been widely used;
as we now know, its choice was a most fortunate one in view of the
relative oxidation-reduction potentials of the systems concerned. By
using methylene blue as the oxidizing agent in place of oxygen it
was possible to show that the reducing action of various substrates
can be elicited only when certain tissue constituents are also present.
This was the most striking evidence that had been mustered for the
view that substrate activation must take place in biological oxida-
tions. In the hands of Thunberg and his co-workers this technique
proved most useful in demonstrating the existence of a group of
enzymes which were called dehydrogenases or dehydrases because
their function appeared to be the activation of the hydrogen of the
substrate in preparation for its removal to a suitable acceptor.

Each substrate or class of substrates, it was demonstrated, pos-
sesses its own specific dehydrogenase. Now since leuco-methylene

OXIDATIVE MECHANISMS IN ANIMAL TISSUES 19

blue is autoxidizable, it was possible in some cases to carry out the
air oxidation of a substrate by the addition of its specific dehydro-
genase and methylene blue. Such an oxidation was not, however,
affected by cyanide or carbon monoxide and in this respect did not
resemble the oxidation of the substrate by the living cell. As knowl-
edge concerning the cytochrome system increased, it was soon
realized that these iron porphyrin compounds played the role of
methylene blue within the cells. Thus it was generally agreed about
ten years ago that activation of the substrate was brought about by
a specific dehydrogenase and that then the substrate reacted with
oxygen through the cytochrome chain.

This, then, was the state of affairs in 1930 when Professor War-
burg came to this country to deliver lectures on his work on what
we now call cytochrome oxidase. Barron and Harrop (9) had shortly
before published experiments showing that the addition of methyl-
ene blue to non-nucleated red blood cells brought about an oxygen
consumption if glucose was present as a substrate. While Professor
Warburg was at Johns Hopkins, Dr. Barron obligingly repeated his
experiments at the request of his distinguished visitor, who watched
the proceedings carefully. Upon his return to Germany, Warburg
himself repeated the experiments and with his collaborators began
the isolation of the red blood cell constituents responsible for this
effect. Thus was begun a series of studies which brought forth some
of the most noteworthy advances ever made in this field. As you
know, these experiments led to the discovery of the vitamin-
containing coenzymes essential to the functioning of most dehydro-
genase systems. They showed that the activated hydrogen of the
substrate did not react directly with the cytochrome system, but
that at least two reversible oxidation-reduction systems were inter-
posed.

So we have today the following general picture of the pathway
of oxidations from the substrate side. In the presence of a specific
protein and of a particular organic compound of low molecular
weight, often called a coenzyme, the substrate loses two electrons
and two hydrogen ions. In the majority of cases so far studied this
coenzyme is one of the pyridine nucleotides. In these cases two elec-
trons and one hydrogen ion are accepted by the pyridine nucleotide,
the other hydrogen ion being released to the environment. We do
not know whether both the pyridine nucleotide and the protein are
concerned in the activation of the substrate molecule. If the protein
alone is responsible for this activation, the pyridine nucleotide may

20 A SYMPOSIUM ON RESPIRATORY ENZYMES

be looked upon as a highly specific electron acceptor. Dixon and
Zervas (11) favor this view and have presented evidence that several
compounds can substitute for the pyridine nucleotide as electron
acceptors. They have shown, for example, that alloxan can be re-
duced by malate or alcohol in the absence of the so-called pyridine
nucleotide coenzyme, the specific protein alone being present.
Whether alloxan plays such a role as an electron acceptor in living
tissues is an open question. The observation of Jacobs (15) that the
injection of alloxan into rabbits produced hypoglycemic convulsions
is suggestive in view of the role of the pyridine nucleotides in sugar
metabolism. Further evidence that the pyridine nucleotides are
highly specialized electron acceptors is the fact that they participate
in reactions in which the substrates possess markedly different
chemical properties. Moreover, they show no great affinity for the
specific protein also concerned in the reaction. Whatever their role,
we know that the result of the reaction is the reduction of the
pyridine nucleotide and the formation of the oxidized product of the
substrate. The latter may in turn act as the substrate in another oxi-
dation in which the same pyridine nucleotide is involved but with
another specific protein.

Now in order that the pyridine nucleotide may act as a catalyst
the reduced form must be reoxidized. The reduced pyridine nucleo-
tides are, however, not unlike the substrates themselves in that they
react sluggishly with most oxidizing agents. In the living cell they
appear to be oxidized readily by only one specific class of sub-
stances, the flavoproteins. In this reaction two electrons and one
hydrogen ion from the reduced pyridine nucleotide are transferred,
along with a hydrogen ion from the environment, to the flavin por-
tion of the flavoprotein. Whether the protein part of the flavoprotein
functions by activating the sluggish reduced pyridine nucleotide is
not known, though these flavoproteins might well be classified as
reduced pyridine nucleotide dehydrogenases. It is worth noting that
the electron acceptor is now firmly attached to a protein molecule
as in the case of the cytochromes. The pyridine nucleotides thus
occupy a unique position as electron acceptors in that they exist
largely in the free state. This fact undoubtedly enables them to play
their important role in anaerobic oxidation-reduction reactions.

The flavoproteins reduced by the pyridine nucleotide must now
in turn be oxidized. This can be accomplished by methylene blue
in the case of the isolated systems. In the intact cell, however, this
cannot be the pathway. The direct oxidation of flavoproteins by

OXIDATIVE MECHANISMS IN ANIMAL TISSUES

molecular oxygen is too slow to be of physiological significance;
moreover, none of them except one recently isolated from yeast by
Haas, Horecker, and Hogness (14) reacts rapidly with cytochrome
c. How then are they Hnked to oxygen in the living cell? This ques-
tion we cannot at present answer. We are thus left with a gap be-
tween the flavoproteins on the one hand and the cytochromes on
the other.

How large is this gap? Let us attempt to answer this question by
considering the various known systems in our chain in relation to

Volts af
pH 70

0.8.

06.
04.

02J

+
0.0

Q2.

0.4.

OXYGEN

Cytochrome Oxidase

Cytochrome g

Cytochrome c

Succinate
Fumarate

Cytochrome b

Methylene
Blue

Flavoproteins

Pyridine Nucleotides

Substrates

HYDROGEN

Acceptor

le ("2)

le ("2)
le 0<2)

le (x2)

2e+2H'

2e+H*

2e+2H^

Environ-
ment

2, H^"

>70%

>30%

Figure 2. — Oxidation-reduction systems concerned in biological oxidations

The source of the potential values used is given in reference 2 except for the

diphosphopyridine nucleotide system, which is taken from reference 5.

their oxidation-reduction potentials. As shown in Figure 2, we are
able to plot fairly accurately according to their potentials all the
systems discussed above. Cytochrome oxidase is the chief exception,
but presumably we may place it between cytochrome a and oxygen.
We thus have interposed between the substrate and oxygen, reading
in the order of the potential of their systems, pyridine nucleotides,
flavoproteins, cytochrome h, cytochrome c, cytochrome a, and finally
cytochrome oxidase. Now if cytochrome h functions in this chain,
and it must be remembered that we are not certain that it does,
the possibility that another system lies between it and the flavo-

22 A SYMPOSIUM ON RESPIRATORY ENZYMES

proteins seems rather small. At any rate, the gap between these two
systems in terms of the energy that would be released upon their
interaction is only a small fraction of the overall. There seems little
doubt, however, that cytochrome c and the flavoprotein systems are
necessary links in the chain. No flavoprotein has yet been obtained
from animal tissues that will react directly with cytochrome c. This
implies that there is some link in the chain between these two con-
stituents. Until cytochrome h can be isolated its claim to this posi-
tion must remain in dispute. Another system that we have not yet
mentioned must also be considered in this connection. Szent-
Gyorgyi has suggested that the succinate-fumarate system links the
flavoprotein systems to the cytochromes. It will be seen that the
potential of the succinate-fumarate system is such that it could play
this role. Here again, however, clean-cut proof is lacking, for suc-
cinic dehydrogenase and the cytochrome system appear to be
intimately tied together and have so far defied separation. Pennit
me in passing to call your attention to the position of the methylene
blue system. Situated as it is at this crossroad, it is well adapted to
react with the flavoproteins on the one hand and on the other to
bypass the cytochrome system in reacting with oxygen.

The chief pathway, then, by which energy is released in the living
cell, so far as we can tell today, appears to be that shown here. The
energy liberated when substrates undergo air oxidation is not
liberated in one large burst, as was once thought, but is released in
stepwise fashion. At least six separate steps seem to be involved.
The process is not unlike that of locks in a canal. As each lock is
passed in the ascent from a lower to a higher level a certain amount
of energy is expended. Similarly, the total energy resulting from the
oxidation of foodstuffs is released in small units or parcels, step by
step. The amount of free energy released at each step is propor-
tional to the difference in potential of the systems comprising the
several steps. As indicated in this diagram, the steps involving the
cytochromes account for more than two-thirds of the total energy
released by this chain.

Now also, just as each lock in a canal must be passed in sequence,
so here each link in the chain appears to be indispensable. Each
component of the chain seems to react readily only with that com-
ponent lying immediately above or below it. This marked specificity
of interaction is most extraordinary in view of the fact that these
substances may react with oxidizing and reducing agents foreign to
the living cell. Methylene blue has already been given as an example

OXIDATIVE MECHANISMS IN ANIMAL TISSUES 23

of such a reaction occurring with the flavoproteins. Another example
is the catalysis by the cytochrome system of the oxidation of certain
organic substances, such as p-phenylenediamine. It should be noted,
however, that such extraneous substances or their oxidized products
may by their lack of specificity react in a way that is harmful to the
cell mechanism. Methylene blue, for example, though not reduced
by the d-amino acid oxidase system, gradually inactivates it in the
presence of light (6). Mr. Kerr, working in my laboratory, has
recently been investigating the mode of action by which butter
yellow produces liver tumors. He has found that the oxidation
catalyzed by a heart muscle preparation of extremely small quanti-
ties of p-phenylenediamine (an apparent breakdown product of
butter yellow) completely inactivates the succinic oxidase activity
of such a preparation. Thus the marked specificity of interaction of
these compounds may also serve to prevent unwanted and harmful
reactions from occurring within the cell.

Next arises the question whether the energy released at each step
in this chain is utilized by the living organism and if so, how. At
present direct evidence for the utilization of energy furnished by
individual oxidative processes such as these is limited to the demon-
stration of coupled phosphorylation reactions. Such demonstrations
have thus far been largely confined to that portion of the chain in-
volving the pyridine nucleotides. Whether the large bulk of energy
release that occurs through the cytochromes is useful for phosphor-
ylations or for energy-utilizing mechanisms other than phosphoryla-
tion has not yet been definitely ascertained. It should be noted,
however, that Korr (16) has pointed out that in the fertilized arbacia
egg respiratory and functional activity are both inhibited by
cyanide. Restoration of the respiratory rate by the addition of a
substance such as methylene blue to replace the inactivated cyto-
chrome system does not, however, restore functional activity.

A point that should perhaps be mentioned in connection with
this pathway is the use of the term "hydrogen transport" to describe
biological oxidations. E' values of the cytochrome c system exhibit
a zero slope in the neutral pH region. Preliminary experiments of
the author indicate the same to be true for the cytochrome a and
h systems. This indicates that only electron transfer occurs with
these systems and that hydrogen is not concerned in the reaction.
If the cytochrome oxidase system behaves similarly, only a fraction
of the total energy released in biological oxidations involves hydro-
gen transport. Thus only the electrons of the substrate can be con-

0 "50

Cyfochrome _c_

Flavin-Adenine

3
if)

"e 100

Dinucleo+ide f|j|jjjj]

Di phosphopyridine
Nucleotide | |

o
o-

-5 50

nil

HEART

BRAIN

KIDNEY

LIVER

Figure 3. — The cytochrome c, flavin-adenine dinucleotide, and diphospho-
pyridine nucleotide content of four rat tissues
Values for the cytochrome c content are recalculated from those given by Stotz
(24) by assuming a molecular weight of 13,000. Flavin-adenine dinucleotide
values are calculated from the data of Ochoa and Rossiter (22) and Warbrug
and Christian (25). Diphosphopyridine nucleotide values are calculated from
tlie data of Axelrod and Elvehjem ( 1 ) with the exception of that for heart
muscle, which is from a value given by von Euler et al. ( 12 ) . The values given
by von Euler et al. are much lower than those reported by otlier workers.

OXIDATIVE MECHANISMS IN ANIMAL TISSUES 25

sidered to be transferred to oxygen in an unbroken chain by the
various acceptors. Hydrogen as hydrogen ion may enter or be
withdrawn from the acid-base continuum at several places in the
chain. Also, the various components of the chain may be classified
in two groups according to their ability to transport electrons. The
cytochromes can transport only one electron for each cycle of
oxidation and reduction of their prosthetic groups. The functional
groups of the flavoproteins and the pyridine nucleotides are capable,
however, of transporting two electrons for each cycle. Thus the
possible interaction of the cytochrome system with a flavoprotein
would be one where a two-step, one-electron transfer, with the
formation of a semiquinone flavoprotein intermediate, might play
an important biological role. The ability of the free flavins to under-
go such a stepwise oxidation has been amply demonstrated by the
work of Michaelis and Schwarzenbach (21).

Now a consideration of the pathway just outlined might suggest
that tissues contain each of these constituents in somewhat similar
amounts. That such is not the case can be seen from Figure 3.

The cytochrome c, flavin-adenine dinucleotide, and diphospho-
pyridine nucleotide content of four tissues from the rat are here
plotted in terms of millimoles per gram of wet tissue. The concentra-
tion of diphosphopyridine nucleotide in all four tissues is far
greater than that of the other two constituents. Cytochrome c is
present in lowest concentration in all these tissues. In liver, for
example, the concentration of the pyridine compound, expressed on
a millimolar basis, is 340 times that of cytochrome c. Since cyto-
chrome c transports only one electron per mole, this ratio becomes
680:1 when expressed in terms of equivalents. In the other tissues
the ratio is lower. From such relationships one might conclude that
the cytochrome system is far more efficient in the transport of elec-
trons than the other systems. Such indeed may be the case. A dif-
ferent explanation, however, is supported by more experimental
proof, namely, that the pyridine nucleotides and the flavoprotein
systems are involved in reactions other than those concerned in the
main oxidative pathway. The known role of the pyridine nucleo-
tides in certain anaerobic cycles is discussed elsewhere. With re-
spect to the flavoproteins, recent studies have indicated that some of
them are concerned in oxidative reactions which do not require the
cytochrome system. Substrates such as the d-ammo acids, hypo-
xanthine, xanthine, and certain aldehydes are so oxidized. The flavo-
proteins concerned in these reactions are unusual in that their

26 A SYMPOSIUM ON RESPIRATORY ENZYMES

reduced forms react directly with oxygen at a rapid rate. Thus
they differ in this respect from the flavoproteins responsible for the
oxidation of the reduced pyridine nucleotides. The reduced form
of the fZ-amino acid oxidase flavoprotein also differs from all other
flavoproteins in that it does not react with methylene blue or with
dyestuffs of even higher oxidation-reduction potential (6). This varia-
tion in the behavior of different flavoproteins containing the same
prosthetic group resembles the variation in the behavior of the
various iron porphyrin protein compounds. Obviously the protein
partner exerts a marked influence on the behavior of the prosthetic
group.

Now such flavin systems as those just mentioned are probably
of minor importance in furnishing the energy required by the cell;
it can be shown that they are not affected by cyanide, which blocks
the bulk of the oxygen consumption of the cell. In fact, these
systems might be looked upon as incinerators for disposing quickly
of unwanted products. Franke and Hasse (13) have termed them
"rudimentary." It may be that they represent the earliest types of
mechanism to emerge for the furtherance of biological oxidations
and thus might be classed as primitive. They differ from
cytochrome-linked systems in that hydrogen peroxide appears as a
by-product of their reaction with oxygen. Thus there arises the
question of the relationship of catalase and peroxidase to such
systems. It is interesting to note that, of the tissues examined, the
liver, among the richest in catalase, has the highest flavin and the
lowest cytochrome c content.

Finally, one other interpretation of the relatively low cytochrome
c content of tissues must be considered. This is the possible exist-
ence of pathways as yet unidentified which parallel the cytochrome
system or supplement it. The existence of such unknown pathways
has already been postulated as a result of certain experimental data
obtained from a study of the action of inhibitors of respiration that
are believed to poison cytochrome oxidase. Two of the most com-
monly employed inhibitors of this type are cyanide and azide. Let
us consider, therefore, two examples of experiments involving the
use of these respiratory poisons and examine the validity of the
conclusions that may be drawn from such experiments.

The respiration of the unfertilized arbacia egg is insensitive to
cyanide. Upon fertilization the egg consumes oxygen at a markedly
increased rate, and the additional oxygen consumed is found to be
cyanide-sensitive. These facts have been interpreted to indicate

OXIDATIVE MECHANISMS IN ANIMAL TISSUES 27

that in fertilized and unfertilized eggs the pathways for the oxida-
tion of substrates are different. It should be noted, however, that at
best the evidence merely indicates the possible existence in un-
fertilized eggs of a system alternate to the cytochrome system.
Cyanide and azide do not effect the reduction of methylene blue by
substrates acting through the pyridine nucleotide and flavoprotein
systems. Hence these systems may be functioning in the unfertilized
egg. Supporting evidence is furnished by the findings of Krahl
et al. (18) that the unfertihzed egg contains flavin-adenine di-
nucleotide and is also rich in diphosphopyridine nucleotide (17).
Now does the cyanide insensitivity of the unfertilized egg indicate
that an iron porphyrin system is not functioning in its respiratory
mechanisms? Such an interpretation is indeed possible. One may,
for example, postulate that the respiration is of a primitive type and
passes directly through a flavoprotein to oxygen. Such a contention
is supported by the fact that the presence of cytochrome a, b, or
c cannot be demonstrated in the unfertilized arbacia egg. It is
possible, however, to demonstrate in the egg the presence of hemin
substances (4). Moreover, Krahl and his co-workers (19) have re-
cently shown that the eggs contain a substance resembling cyto-
chrome oxidase, in amounts equal in activity to the cytochrome
oxidase of mammalian tissues. This substance was found to be
cyanide-sensitive if it was functioning in an oxidation requiring the
addition of cytochrome c. Are we to conclude, then, that this egg
"cytochrome oxidase" plays no role in the respiration of the un-
fertilized egg because such respiration is cyanide-insensitive? Is it
not possible that a reaction can occur directly between "cytochrome
oxidase" and flavoprotein in the arbacia egg? In view of the wide
variety of properties exhibited by flavoproteins such a reaction
might well occur. But it appears that such a postulate is contradicted
by the evidence cited above that this egg "cytochrome oxidase" can
be inhibited by cyanide.

Before we decide what is the correct interpretation of these data,
let us review our knowledge about the mechanism of cyanide poison-
ing. Cyanide apparently inhibits respiration by reacting with cyto-
chrome oxidase, since it prevents the air oxidation of the three re-
duced cytochromes. At low concentrations it apparently does not
combine with the cytochromes. Presumably it combines with the
ferric form of cytochrome oxidase, since it is methemoglobin and not
hemoglobin that reacts with cyanide. We may conclude, then, that
somehow cyanide prevents the reduction of ferric cytochrome oxi-

~9 cr

^UJ

Unfertil
Arbacia

fvl

-XL

lAI

a) ''

/ o

/ l_
/ -C

/ -2

L4_

/ '>^

^2
'S

/ ^

•w

' 1 [

4-»

bCrt

-ro

S'^

c /

/ /

C '3
o o

/ ^/

CP '

/ O/

'o "rt

J$ \

/ ^

/ 7

'c
o

a, o

°\

1
- in

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a
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O u

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2

d) 0)

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O U

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1 1

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o a

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o

' ^

OXIDATIVE MECHANISMS IN ANIMAL TISSUES 29

dase by the cytochromes. Now Barron (8) has presented ample
evidence that cyanide hemochromogens are able to act as reversible
oxidation-reduction catalysts. If the iron in such cyanide complexes
can be reduced, why is the ferric iron of the cyanide complex of
cytochrome oxidase not reduced?

As one answer to this question I suggested several years ago (3)
that the oxidation-reduction potential of the cytochrome oxidase
system is lowered in the presence of cyanide below that of cyto-
chrome a or c. The reduction of cytochrome oxidase by these com-
pounds could then not occur and the respiratory chain as a whole
might thus be blocked. The experiments of Barron (8), which showed
that the cyanide hemochromogen systems were the most negative
of the hemochromogen systems investigated, were cited in support
of this idea. Since then Clark and his associates (10) have presented
their thorough analysis of such hemochromogen systems. As a result
of this study it may be said that the potentials of iron porphyrin
systems vary according to the type of nitrogen compound associated
with them. Also, if the aflBnity of the nitrogenous compound is great-
est for the ferric form, the potential of the system will decrease
progressively as the concentration of the coordinating compound is
increased. The reverse holds true if the feiTous form displays the
greatest affinity. Now assuming that the cytochrome oxidase system
behaves in a similar manner, we may picture the effect of cyanide
on the potential of the cytochrome oxidase system as shown in
Figure 4. Here the cytochrome oxidase system is arbitrarily assigned
a potential of 0.5 volts at pH 7.0. The potential of this system is
plotted against the log of the concentration of the coordinating com-
pound, as is done by Clark et al. (10) for the hemochromogen
systems. It is assumed that only the ferric form of cytochrome
oxidase reacts with cyanide and that since the first noticeable effects
of cyanide poisoning result at concentrations of 10~^ to lO*' molar, it
is within this range that a potential shift will first occur.* The
potential is assumed to change according to a 0.12 slope, since this
value has been found to hold for certain hemochromogen sys-
tems (10). It will be seen that, according to such a scheme, when the
concentration of cyanide reaches 10'^ molar, the potential of the

* As shown by Clark et al. (10) for hemochromogen systems, this point de-
pends also upon tlie concentration of the hemochromogen system. The con-
centration of the cytochrome oxidase system is of course not known. It is
probably of the order of magnitude of the systems studied by Clark et al. (10)
if tlie concentration of cytochrome c in the tissues can be used as an index.

30 A SYMPOSIUM ON RESPIRATORY ENZYMES

cytochrome oxidase will be depressed below that of the cytochrome
a and c systems. In such a tissue as muscle, then, we might expect
100 per cent inhibition of the cytochrome-mediated respiration. It
is at such a concentration that cyanide exerts its maximum effect
upon respiration.

Let us now return to the case of the unfertilized arbacia egg,
where the cytochromes appear to be absent. Here cytochrome
oxidase may be considered to react directly with a flavoprotein. The
addition of cyanide may be pictured as depressing the potential
of the cytochrome oxidase in the same manner. In this case, how-
ever, the potential of the cytochrome oxidase still remains above that
of the flavoprotein with which it normally reacts. Oxidations can
therefore still proceed, and no cyanide inhibition is observed. In
fact, a stimulation of respiration by cyanide such as was observed
by Marsh and Goddard (20) in the fully mature leaf might be
encountered if such a set of conditions exists. Barron (7) has shown
that the rate of reaction of oxygen with reduced dyestuffs increases
as the oxidation-reduction potential of the system decreases. A
similar change may occur in the case of the cytochrome oxidase
system as its potential is lowered by cyanide.

Fertilization of the arbacia egg may then be looked upon as the
gearing of the cytochrome oxidase system to another system of
higher potential than the flavoprotein, which, though serving to in-
crease the respiratory rate, also causes the system to become
cyanide-sensitive. The fact that arbacia sperms are rich in cyto-
chromes a, b, and c is of interest in this connection. For a further dis-
cussion of this aspect see Krahl et al. (19).

One other example of an inhibitor study which may be employed
is furnished by the work of Stannard (23). This investigator has re-
cently presented data to show that resting and stimulated frog
muscle respond differently to cyanide and azide. Whereas cyanide
inhibits the oxygen consumption of both resting and stimulated
muscle, azide inhibits only the oxygen consumption of stimulated
muscle. The results have been interpreted to indicate that the path-
ways of oxidation are different in resting and in stimulated muscle.
As Stannard points out, the cyanide sensitivity of the resting respira-
tion seems to preclude the possibility that it represents an independ-
ent functioning of a flavoprotein system.

Now azide is believed to inhibit respiration also by reacting with
cytochrome oxidase. Quantitatively, however, its action is different
from that of cyanide. Inhibition of respiration by azide appears to

OXIDATIVE MECHANISMS IN ANIMAL TISSUES 31

begin at concentrations higher than those required for cyanide
poisoning. As the azide concentration is increased above this initial
value, inhibition progresses as rapidly as with cyanide until a
concentration of about 10"^ molar is reached. Further increase in
azide concentration produces no further effect, and inhibition of
respiration remains incomplete, never reaching the maximum value
obtained with cyanide. How then can this diflPerence in the be-
havior of cyanide and azide be explained if they both act on cyto-
chrome oxidase? Is it necessary to postulate that azide and cyanide
inactivate separate systems?

An explanation of the difference in the behavior of cyanide and
azide may be given in terms of their diflFerent effects upon the poten-
tial of the cytochrome oxidase system. Azide, like cyanide, is as-
sumed to combine with the ferric form of cytochrome oxidase. Un-
like cyanide, it is also assumed to combine with the ferrous form.
In Figure 4 the effect of azide upon the potential of the cytochrome
oxidase system is plotted on the basis of these assumptions. Since
azide inhibition first manifests itself at concentrations higher than
those for cyanide, its affinity for the ferric form of the oxidase is
assumed to be less than that of cyanide. Depression of the potential
of the oxidase system is therefore portrayed as starting when the
concentration of azide reaches a value between 10 * and 10 "' molar.
As the concentration of azide is increased above this value, the
potential is assumed to be lowered along the same slope as for the
cyanide system. Now since inhibition with azide reaches a maximum
at a concentration of 10"^ molar, it is assumed that at this concentra-
tion the azide begins to combine with the ferrous form of cyto-
chrome oxidase. According to Clark et al. (10), the effect of such a
combination on tlie potential of the system will be to alter the
slope to a 0.0 value, and thus no further change in potential occurs
as more azide is added. On the basis of this assumption it can be
seen that, as depicted in Figure 4 the potential of the cytochrome
oxidase system in the presence of azide can be depressed only to a
level corresponding to that for the cytochrome a system. At this level
the cytochrome oxidase may still function to oxidize the cytochromes,
but its efficiency will be at least 50 per cent impaired. It is thus
possible to conceive of the respiration of resting muscle as being
unimpaired by azide, since even at a lower level of efficiency the
cytochrome oxidase may still be capable of supplying the oxygen
needs of the resting muscle. If, however, increased demands for
oxygen are made upon the muscle by stimulation, the impaired

32 A SYMPOSIUM ON RESPIRATORY ENZYMES

eflBciency of the cytochrome oxidase system becomes a limiting
factor.

By this time I can hear you muttering, "Sheer speculation," and I
agree with you. I have, however, labored my point concerning the
action of such inhibitors because I believe that until we know more
about their mode of action any hypotheses of the existence of other
oxidative pathways must also be labeled speculation. I do not wish
to imply that other unknown pathways do not exist. I heartily agree
that they may. I only beg that, in these days of ever-widening use
of such inhibitors, more fundamental investigations be made into
their mode of action before we becloud the issue with false inter-
pretations.

REFERENCES

1. AxELROD, A. E., and Elvehjem, C. A., J. Biol. Chem., 131, 77 (1939).

2. Ball, E. G., Symposia on Quantitative Biology, Cold Spring Harbor, 7, 100
(1939).

3. Ball, E. G., Discussion published as part of reference 8.

4. Ball, E. C, and Meyerhof, B., J. Biol. Chem., 134, 483 (1940).

5. Ball, E. C, and Ramsdell, P. A., J. Biol. Chem., 131, 767 (1939).

6. Ball, E. C, and Ramsdell, P. A., unpublished experiments.

7. Barron, E. S. G., J. Biol. Chem., 97, 287 (1932).

8. Barron, E. S. G., Symposia on Quantitative Biology, Cold Spring Harbor,
7, 154 (1939).

9. Barron, E. S. G., and Harrop, G. A., Jr., J. Biol. Chem., 79, 65 (1928).

10. Clark, W. M., Taylor, J. F., Davies, T. H., and Vestling, C. S., J. Biol.
Chem., 135, 543 (1940).

11. Dixon, M., and Zervas, L. G., Biochem. J., 34, 371 (1940).

12. von Euler, H., Schlenk, F., Heiwinkel, H., and Hogberg, B., Z. physiol.
Chem., 256, 208 (1938).

13. Franke, W., and Hasse, K., Z. physiol. Chem., 249, 231 (1937).

14. Haas, E., Horecker, B. L., and Hogness, T. R., J. Biol. Chem., 136, 747
(1940).

15. Jacobs, H. R., Proc. Soc. Exp. Biol. Med., 37, 407 (1937).

16. KoRR, I. M., Symposia on Quantitative Biology, Cold Spring Harbor, 7,
120 (1939).

17. Krahl, M. E., Verbal communication at a seminar on August 26, 1941, at
Marine Biological Laboratory, Woods Hole, Mass.

18. Krahl, M. E., Keltch, A. K., and Clowes, G. H. A., Proc. Soc. Exp. Biol.
Med., 45, 719 (1940).

19. Krahl, M. E., Keltch, A. K., Neubeck, C. E., and Clowes, G. H. A., J.
Gen. Physiol., 24, 597 (1941).

20. Marsh, P. B., and Goddard, D. R., Am. J. Botany, 26, 724 (1939).

21. Michaelis, L., and Schwarzenbach, G. J., J. Biol. Chem., 123, 527
(1938).

22. Ochoa, S., and Rossiter, R. J., Biochem. J., 33, 2008 (1939).

23. Stannard, J. N., Symposia on Quantitative Biology, Cold Spring Harbor,
7, 394 (1939).

24. Stotz, E., J. Biol. Chem., 131, 555 (1939).

25. Warburg, O., and Christian, W., Biochem. Z., 298, 150 (1938).

Ball and Stotz
From flavoprotein to cytochrome

Meyerhof and Group
At the Fountainhead

XoKD, Neuberg, axu Kletzien'
Was it the weather or the argument?

BETWEEN THE SCIENTIFIC SESSIONS

Discussion on Hydrogen Transport

VAN R. POTTER
University of Wisconsin, Chairman

Dr. Potter:

This afternoon's discussion has been organized with the idea of
studying some of the problems which may be considered as open
questions at this time. The first speaker will be Dr. Elliott.

THE POSSIBLE ROLE OF INTERMEDIARY METABOLITES

AS HYDROGEN CARRIERS

K. A. C. ELLIOTT

Institute of the Pennsylvania Hospital

This subject has not been reviewed at length during this sym-
posium, and it is impossible to cover it fully in a brief discussion.
Some famiharity with the subject will therefore be assumed, and
only certain outstanding problems will be discussed. (For details and
bibliographies see references 1-3.)

The most important theory of intermediary metabolites as hydro-
gen carriers is that of Szent-Gyorgyi. It may be represented by the
following highly simplified diagram, in which heavy arrows indicate
transfers of hydrogen atoms (or electrons) from one substance to the
next.

OONATORS OXALACETATE FUMARATE REDUCED CYTOCHROME

I .2H^ il Jil^ It .2H^ n -^^OXYGEN

^^ ^^, ., MALATE SUCCINATE OXIDIZED CYTOCHROME

OXIDIZED DONATORS

According to this theory, hydrogen from tissue donators reduces
oxalacetate to malate; the malate is reoxidized to oxalacetate and the
hydrogen is transferred to fumarate, reducing it to succinate; and
the succinate is then reoxidized to fumarate by the cytochrome-
cytochrome oxidase system. Malate-oxalacetate and succinate-
fumarate thus perform functions similar to that of reduced cyto-
chrome-oxidized cytochrome. Known dehydrogenases and cozymase
are concerned in the catalysis of the various steps; also, a flavo-
protein was believed by the Szent-Gyorgyi school to mediate in the
reduction of fumarate by malate.

Considerable evidence that this mechanism can function in
pigeon breast muscle brei has been adduced by the Szent-Gyorgyi

34 A SYMPOSIUM ON RESPIRATORY ENZYMES

school, by Stare and Baumann, and, in connection with work on the
citric acid cycle, by Krebs and coworkers, and also by various other
workers. Krebs has also shown that at least the succinate-fumarate
mediation can occur in B. coli (Escherichia coli). If we assume that
the evidence for the Szent-Gyorgyi mechanism actually proves that
it does operate in pigeon breast muscle (see, however, addendum
below), the following questions arise:

1. In what tissues may the mechanism he important? Tissues
other than pigeon muscle have not been studied exhaustively from
this point of view. One of the main tests for the system consists in
finding increased or better maintained respiration when small
amounts of fumarate or malate, which are interconvertible by tissue
fumarase, are added to the tissue brei. Our experience with this
test and that of other workers suggests that the system may be im-
portant for the respiration of liver, testis, and possibly kidney, but
is not very active in brain or skeletal muscle. However, individual
reactions of the system, namely, succinate oxidation and oxalacetate
reduction, occur rapidly in all these tissues, and Banga, Cori, and
coworkers have shown that the presence of fumarate is necessary
for the oxidation of pyruvate by kidney and brain dispersions.
Banga showed that it was not easy to remove all the four-carbon
acids from tissue. It is therefore possible that when added four-
carbon dicarboxylic acids have little effect on the respiration of
tissues, these substances may already be present in the tissues in
such amounts that their concentration is not a limiting factor of the
respiration rate.

The volume of respiration passing through the system would be
limited by the activity of the relevant enzymes. Dr. Greig and I
found that there was sufficient cytochrome-cytochrome oxidase and
succinic dehydrogenase activity in many normal tissues to account
for all the respiration through succinate-fumarate, but the succinic
dehydrogenase activity was quite low in chick embryo, rat thymus,
spleen, pancreas, and some tumors. Breusch found that the rates of
oxalacetate reduction were high in muscle, liver, and kidney and
moderate in brain and pancreas, but negligible in spleen, lung,
placenta, peripheral nerves, and certain rat tumors; the rate in
embryo was found to be low by Blaszo.

2. Does the four-carbon dicarboxylic acid system always operate
in the same waif? Szent-Gyorgyi himself pointed out that not all
substances should be expected to utilize the whole system. Lactate,
for instance, might be oxidized through fumarate-succinate but

DISCUSSION ON HYDROGEN TRANSPORT 35

probably not through oxalacetate-malate, since the redox potential
of the latter system is close to that of pyruvate— lactate.* According
to the complete theory, fumarate and malate should behave alike,
an equilibrium mixture of the two being rapidly produced by the
action of fumarase when either is added. But Dr. Libet and I have
found that added malate and fumarate (and citrate) have different
effects on the repiration of brain suspensions. Greig and Munro
found that fumarate, but not malate, caused a lowered respiratory
quotient with ox retina and chick embryo.

3. What metabolites are oxidized through the system? The Szent-
Gyorgyi school indicated that carbohydrate derivatives— triose-
phosphate, alpha-glycerophosphate, and pyruvate— were oxidized
through the four-carbon dicarboxylic acid system by tissue sus-
pensions. But Greville reported that not more than 70 per cent of
the respiration brought about by fumarate catalysis in muscle was
due to carbohydrate oxidation. With suspensions of liver from
fasted rats, my wife and I found low respiratory quotient values
for the extra respiration caused by adding malate. Leloir and Munoz
found that added four-carbon dicarboxylic acids increased the rate
of butyric acid oxidation by liver suspension. Dewan and Green,
with isolated enzyme preparations, showed the oxidation of beta-
hydroxybutyrate by fumarate. Annau, and my wife and I, noted

* Dr. Eric Ball, Harvard University: My chief objection to the Szent-
Gyorgyi theory is that the inclusion of the malate-oxalacetate system in the
chain of reactions as it is written seems to be pointless. Attention has been called
to tliis fact previously ( 4-6 ) . If we break the scheme into the separate reactions,
this becomes evident. Assuming the substrate to be oxidized by means of
diphosphopyridine nucleotide (Py(P04)2), we may write the first reaction as fol-
lows:

( 1 ) Substrate + Py(P04 ) 2 -> H^PyCPO^ ) 2 + Oxidized Substrate

If the pyridine nucleotide is to act as a cyclic catalyst for this reaction, it must
be oxidized. According to the Szent-Gyorgyi scheme, tliis reoxidation is brought
about by oxalacetate. The reaction may be written:

( 2 ) Oxalacetate -|- H2Py(P04 ) 2 -> Py(P04 ) 2 -f Malate

Now if the oxalacetate in turn is to function as a catalyst, it must be regen-
erated. This requires that malate be oxidized. The oxidation of malate in the
body, however, is known to proceed only through the diphosphopyridine nucleo-
tide:

( 3 ) M alate -f Py (PO4 ) 2 ^ H2Py(P04 ) 2 + Oxalacetate

This equation is, however, the reverse of equation 2. Thus what is produced is
reduced pyridine nucleotide, and we are right where we started when we wrote
equation 1. The introduction of the malate-oxalacetate system into diis cycle
merely leads us into a blind alley.

36 A SYMPOSIUM ON RESPIRATORY ENZYMES

that added four-carbon substances removed, or prevented the for-
mation of, acetoacetic acid in liver and kidney brei. Krebs, working
vi^ith B. coli [Escherichia coli] indicated that fumarate-succinate
mediated the oxidation of glucose, malate, lactate, acetate, glycerol,
glyceraldehyde, butyrate, pyruvate, acetoacetate, Z( + )-glutamate
and molecular hydrogen. Apparently the oxidation of many types
of metabolite can be mediated by four-carbon dicarboxylic acids.

Szent-Gyorgyi considered that the four-carbon dicarboxylic acids
were quite analogous to coenzymes in their catalytic role. But it
must be remembered that they behave also as combustible sub-
stances, particularly with kidney cortex slices and also to some ex-
tent with brei of various tissues, when they are added in excess.
The four-carbon dicarboxyhc acids are not the only substances that
can act both as catalysts and as substrates for respiration. Carrier
possibihties have been shown for pyruvate-lactate, as well as other
alpha-keto— alpha-hydroxy acids, adrenochrome, and transamination
reactions. It has been suggested that in B. coli formate-bicarbonate
plays a similar role, and in plants catechol derivatives and dihydroxy-
maleic acid may be important carriers. Perhaps many other sub-
stances may act in this way. In fact, carrier functions could be postu-
lated for all reversible oxidation-reduction systems, and we should
perhaps think of all oxidizable metabolites as capable of acting as
both substrates and carriers in a dynamic oxidation-reduction con-
tinuum.

Addendum.'* —The Szent-Gyorgyi theoiy was first advanced at a
time when the succinic dehydrogenase system was the only one
definitely known to reduce cytochrome. Mediation by succinate-
fumarate of hydrogen transport between the majority of metabolites
and the cytochrome system thus naturally suggested itself. However,
the discovery of flavoproteins, which mediate oxidation of the re-
duced coenzymes, suggests that a flavoprotein catalyst may bring
about more direct oxidation of those substrates that are oxidized
through the action of coenzyme-deteiTnined dehydrogenases. Dr.
Hogness has described cytochrome c reductase, a flavoprotein from
yeast that causes cytochrome c reduction by dihydrocoenzyme II. It
seems likely that similar catalysts for the oxidation of both dihydro-
coenzymes I and II may occur in animal tissues. In that case there
would seem to be no necessity for mediation by fumarate-succinate.
(Another flavoprotein in yeast, fumarate reductase, has been de-
scribed by Fischer and coworkers. This enzyme causes the reduction

" In the liglit of private discussions during the symposium. Dr. EUiott has
written an addendum to his remarks. — Ed,

DISCUSSION ON HYDROGEN TRANSPORT 37

of fumarate to succinate by certain leuco dyes; it is distinct from
succinic dehydrogenase but could perhaps replace the latter enzyme
in the Szent-Gyorgyi scheme).

While it has been proved that the four-carbon dicarboxylic acids
and other metabolites may act as carriers, the previous paragraph
indicates that we cannot conclude that fumarate-succinate actually
does so. Since malonate inhibits succinate oxidation, the observed
inhibition of respiration by malonate has been taken as evidence of
the carrier function of succinate-fumarate. But the inhibition of
respiration by malonate and the promoting effect of added fumarate
may be explained as inhibition and promotion of oxidative metabol-
ism through Krebs' cycle.

Dr. Potter has emphasized the above points (6), and he points out
that carrier functions for four-carbon dicarboxylic acids and other
substances have been assumed on the basis of the following four
criteria: 1. The compound is a natural constituent of tissues. 2. It
can be reduced by tissues at rates compatible with the actual rate
of oxidation of the substrate whose oxidation it is presumed to cata-
lyze. 3. The reduced compound can be oxidized by the tissue prepa-
ration at an adequate rate. 4. The compound is able to stimulate
catalytically the rate of hydrogen transport in the system under
investigation. But it appears that a fifth requirement is necessary to
prove carrier function, namely, the compound must be directly re-
duced by one system and directly oxidized by a second system which
is not identical with the first. As Dr. Ball and Dr. Potter have pointed
out, oxalacetate is reduced by dihydrocoenzyme I and malate is
oxidized by coenzyme I, yielding tlie dihydrocoenzyme. That is to
say, hydrogen from the donator metabolite is passed to the co-
enzyme, producing dihydrocoenzyme, but subsequent reduction of
oxalacetate to malate and reoxidation of the latter merely results
in producing the dihydrocoenzyme again. Thus no effective trans-
port of hydrogen has occurred and, unless another biological mecha-
nism for the oxidation of malate is discovered, it seems unnecessary
to postulate a carrier function for malate-oxalacetate. It is conceiv-
able, however, that the structural relations of enzymes in tissue may
render the passage of hydrogen from the donators to fumarate or
flavoprotein easier via coenzyme-oxalacetate-malate-coenzyme than
directly via coenzyme in one step.

It seems likely that in tissues there is a dynamic equilibrium be-
tween the oxidized and reduced forms of numerous metabolites
and that flavoprotein-cytochrome-cytochrome oxidase mechanisms
continually abstract hydrogen (or electrons) from the system, while

38 A SYMPOSIUM ON RESPIRATORY ENZYMES

few, if any, of the metabolites can be singled out as specially con-
cerned in the hydrogen transport. (This view of the mechanism was
suggested to me by Dr. Potter.)

REFERENCES

1. Szent-Gyorgyi, a., Studies in Biological Oxidation (Leipzig, 1937).

2. Stare, F. J., and Baumann, C. A., Cold Spring Harbor Symposia on Quan-
titative Biology, 7, 277 ( 1939).

3. Elliott, K. A. C, Physiol. Rev., 21, 267 ( 1941).

4. Ball, E. G., Cold Spring Harbor Symposia on Quantitative Biology, 7,
100 (1939).

5. Martius, C, Ergebnisse Enzymforschung, 8, 247 (1939).

6. Potter, Van R., Medicine, W, 441 (1940).

Dr. Potter:

The second question to be considered is the role of the carriers in
dismutations and coupled oxidoreductions.* Since it is agreed
that the various metaboHtes may take part in coupled oxido-
reductions, it becomes of interest to determine how these reactions
may be brought about, and what features these "fermentation" re-
actions have in common with the oxidative mechanisms. Dr. Ball and
Dr. Lipmann will open the discussion.

THE ROLE OF THE CARRIERS IN DISMUTATIONS AND

COUPLED OXIDOREDUCTIONS

With Special Reference to the Flavoproteins

Eric Ball, Harvard University:

The chief carriers that play a role in dismutations or coupled
oxidoreductions are the pyridine nucleotides and flavoproteins.
Since the participation of the pyridine nucleotides in such reactions
is more common, examples of dismutations and of coupled oxido-
reductions involving diphosphopyridine nucleotide may be given
first.

The classical example of a dismutation is the so-called Cannizzaro
reaction, which may be represented by the following equations:

(1) R-CHO + 2e + 2H^->R-CH20H

(2) R-CHO-f H2O — 2e-2H*^R-COOH
(1) + (2) 2RCHO + H20->R-CH20H-f R-COOH

" This term was not included in the original statement of the question. Dr.
Barron rose to point out that the term "dismutation" has a very narrow applica-
tion and that "coupled oxidoreduction" is the more general expression.

DISCUSSION ON HYDROGEN TRANSPORT 39

One molecule of aldehyde undergoes an oxidation to the corre-
sponding acid at the expense of another molecule of aldehyde which
is reduced to alcohol. An enzyme which catalyzes this type of re-
action, called aldehyde mutase, has been found in hver by Dixon
and Lutwak-Mann (1). It requires as a coenzyme diphosphopyridine
nucleotide, which thus appears to function in the role of carrier of
electrons and hydrogen ions from one aldehyde molecule to another.
Another example that might be given is the dismutation of triose-
phosphate.

The role of a carrier in a so-called coupled oxidoreduction diflFers
from that in a dismutation reaction only in that electrons and
hydrogen ions are transferred between molecules of two different
substances. A well-known example of such a reaction (alcohoHc
fermentation) may be written as follows if we omit the coupled
phosphorylation steps that accompany it:

( 3 ) CHs • CHO -H 2e + 2H" -> CH3CH2OH

(4) CHO COOH

I I

CHOH -1- H2O - 2e - 2H^ -> CHOH

HaC-OPOaH, H2COPO3H2

The carrier of the electrons and hydrogen ions between these two
aldehyde molecules is also diphosphopyridine nucleotide. The reac-
tion is not classed as a dismutation simply because the aldehyde mole-
cules involved are not identical. One could give other examples of
coupled oxidoreductions in which the reacting molecules are more
dissimilar and involve diphosphopyridine nucleotide as a carrier.

Such reactions constitute the main type of energy exchange in
anaerobic processes. Under aerobic conditions that half of the re-
action which involves the loss of electrons and hydrogen ions to the
pyridine nucleotide may still occur. The reduced pyridine nucleo-
tide, however, under aerobic conditions, loses its electrons and
hydrogen ions to a flavoprotein rather than to another substrate
molecule. This type of reaction, then, might be hsted as still another
class of coupled oxidoreductions.

As compared with the pyridine nucleotides, the flavoproteins par-
ticipate in only a few direct reactions with substrate molecules.
Examples of dismutations or coupled oxidoreduction reactions such
as have been given for the pyridine nucleotides are less plentiful
for the flavoproteins. One example of a dismutation reaction which
may be classed as involving a flavoprotein is that for the substrate

40 A SYMPOSIUM ON RESPIRATORY ENZYMES

xanthine. Green (2) has shown that, in the presence of xanthine
oxidase, xanthine undergoes the following reactions:

(5) Xanthine + 2e + 2H* —> Hypoxanthine

( 6 ) Xanthine — 2e — 2H^ -» Uric Acid

2 Xanthine — > Hypoxanthine + Uric Acid

The carrier of the electrons and hydrogen ions between two mole-
cules of xanthine may in this case be due to the flavin portion of
xanthine oxidase. Examples of oxidoreduction reactions between
two dijfferent substrates involving flavoproteins are not known to me.
It may be that no such reactions exist, since each flavoprotein appears
to be substrate specific. An indirect reaction of a flavoprotein as a
carrier between two different substrates is, however, conceivable.
This would be the case with two different substrates whose direct
reaction occurred only with each of the pyridine nucleotides, as
shown by the following equations:

Oxidized Substrate A + HjPyCPOi) 2 -^ Py(P04)2 + Substrate A

H2Py(P04)2 - 2e - 2H^ -^ Py(P04)2
PyCPOJs + 2e + 2H^ -^ H2Py(P04)3

Substrate B + Py(P04)3 -^ H2Py(P04)3 + Oxidized Substrate B

Here in order for oxidized substrate A to react with substrate B, the
reduced triphosphopyridine nucleotide must react with diphospho-
pyridine nucleotide. Whether such a direct reaction is possible is, I
believe, unknown. It is possible, however, that a flavoprotein might
act as a carrier of electrons and hydrogen ions between the two
pyridine nucleotides. Such a reaction could thus be classed as a
coupled oxidoreduction involving a flavoprotein.

Fritz Lipmann, Massachusetts General Hospital:

Flavoproteins are at present known to react in two ways: (1) with
oxygen directly, i.e., as mediators between substrates and oxygen;
(2) as mediators between pyridine enzymes and other catalysts or
oxygen.

The second function has been thoroughly studied in reactions
representing anaerobic parts of essentially aerobic reaction chains:
the bridging between pyridine nucleotide and methylene blue (Haas,
Straub, Green) and between pyridine nucleotide and cytochrome
(Haas, Horecker, and Hogness). Although it would be suspected
that a flavin mediator is needed in a great variety of purely an-
aerobic reactions, especially in those between a pyridine and a non-

DISCUSSION ON HYDROGEN TRANSPORT 41

pyridine enzyme, not much is definitely known about such action
of flavoproteins. One example, however, is the dismutation of pyruvic
acid (Lipmann), in which a flavin component was shown to par-
ticipate in a coupled oxidoreduction. Here the flavin mediates
between two enzyme systems of different types, i.e., between lactic
and pyruvic dehydrogenase:

( 1 ) pyruvate — > lactate
2HT (flavin)

pyruvate phosphate — > acetylphosphate + CO2

The presence of a flavoprotein in yeast, which catalyzes the reduc-
tion of fumarate (Fischer), suggests the mediator function of such a
flavoprotein between pyridine enzymes and fumarate. For example,
in the dismutation of fumaric acid (Green) electron transfer pre-
sumably occurs between malic dehydrogenase, a pyridine enzyme,
and succinic dehydrogenase, a non-pyridine enzyme:

( 2 ) fumarate — » succinate
2H T (flavin)

fumarate «^ malate -^ oxalacetate

It is probably accidental that in both examples for anaerobic
flavin mediation, the coupled oxidoreduction is a so-called dismu-
tation. This might serve to show that in most enzymatic dismutations
the underlying reaction is, in fact, an oxidoreduction between two
enzyme systems catalyzing two fundamentally different reactions.
But in dismutation the metabolic substrates for both enzyme sys-
tems derive from the same compound. In the cases discussed, the
oxidant in the oxidation-reduction reaction is the compound proper,
pyruvate or fumarate, and the reductant is a transformation product
of the added compound. Oxidant and reductant belong, respectively,
to two different oxidation-reduction systems with widely different
oxidation-reduction potentials. In dismutation (reaction 1) the lac-
tate-pyruvate system of EJ —0.18 volts reacts with the pyruvate...
HX —acetate +CO2 system of Eo' below —0.4 volts. In the sec-
ond reaction the succinate-fumarate system of Eq' 0.0 volts reacts
with the malate-oxalacetate system of Eo' —0.17 volts. These large
energy differences between the reacting oxidation-reduction sys-
tems explain why dismutation occurs.

REFERENCES

1. Dixon, Malcolm, and Lutwak-Mann, Cecilia, Biochem. J., 31, 1347
(1937).

2. Green, Davto Ezra, Biochem. J., 28, 1559 (1934).

42 A SYMPOSIUM ON RESPIRATORY ENZYMES

THE PHYSICO-CHEMICAL MECHANISM OF
HYDROGEN TRANSPORT

Kurt Stern, Yale University:

Dr. Ball, on Thursday morning, spoke of a gap between the cyto-
chrome oxidase-cytochrome system and the substrate-dehydrogenase
systems. It appears that this gap may now be considered filled by
several flavoproteins, e.g., Hogness and Haas's cytochrome reductase,
Euler and Green's diaphorase (coenzyme factor), or Szent-Gyorgyi's
dicarboxyhc acid system, etc.

It is incorrect to label the entire process of cell respiration one
of hydrogen transport, since the first stages, from oxygen through
the four iron atoms of the respiratory ferment and the three cyto-
chromes, are concerned exclusively with electron transfers. This
brings up a difficulty in the formulation of the elementary steps that
connect the iron systems with the dehydrogenase systems. The
oxido-reductive changes taking place in the former are one-electron
transfers; the dehydrogenation reactions, on the other hand, are
formulated as bivalent processes, involving the loss and uptake of
two hydrogen atoms per molecule. The most satisfactory way of re-
solving this dilemma without invoking the existence of highly
problematical trimolecular reactions is to assume, with Michaelis,
that the apparently bivalent dehydrogenations are actually two-step
processes, involving the transfer of one hydrogen atom or its equiva-
lent at a time, with the intermediate formation of semiquinoid radi-
cals.

It seems to be no mere coincidence that the macromolecular prepa-
rations, called cytochrome oxidase by Keilin, contain a number of
components: the oxidase, cytochromes a and b, succinic dehydro-
genase, etc., which are all concerned with what Oppenheimer calls
the "terminal oxidation" of metabolites. It is reasonable to assume
that these particles represent functional units which contain these
catalysts in a spatial arrangement which facilitates the progress of
this important phase of cell respiration in a constant pattern and at
a constant and high rate.

Dr. Potter:

The dilemma of which Dr. Stern has spoken, that is, the mech-
anism for getting a bivalent dehydrogenation system to react with
a one-electron system, may possibly be resolved by the formation
of a complex made up of the proper components of the hydrogen

DISCUSSION ON HYDROGEN TRANSPORT

(electron) transport system. I believe Mr. Haas has some experi-
mental data which have considerable bearing on this question.

Erwin Haas, University of Chicago:

For a long time the mechanism of respiration was studied only
by considering intermediary metabolites. A more direct approach
to that problem is now possible, since some of the respiratory
enzymes have become available in isolated form and since their
functional groups are known.

cytochrome c

cytochrome
reductase

HEMIN PROTEIN^

JiL

ALLO'XAZINE MONO- PROTEIN

NUCLEOTIDE ^ ' -9
K = 10

TRIPHOSPHOPYRIDINE PROTEIN

NUCLEOTIDE ' -5
K =10

GLUCOSE -6 -PHOSPHATE
FIGURE 1

K = 10

' K 10

Figure 1 illustrates a part of the respiratory system which brings
about the reduction of cytochrome c by glucose-6-phosphate. The
components of the system are arranged in the order in which they
react; the details concerning their chemical structure are omitted.
Hydrogen or electrons from the glucose are passed on to the pyridine
nucleotide, thence to the alloxazine mononucleotide, and then to the
iron atom in cytochrome c. In each step a specific protein must be
present which, together with the prosthetic group, forms the active

44 A SYMPOSIUM ON RESPIRATORY ENZYMES

enzyme. By selecting proper concentrations any reaction in this
scheme can be made the hmiting factor. Thus by measuring the rate
with which cytochrome c is reduced, it is possible to determine rates
of reaction and to demonstrate formation of enzyme complexes for
any of the reactions involved. Negelein and Haas have shown by
ultraviolet spectroscopy the formation of a complex between protein
I, triphosphopyridine nucleotide, and glucose-6-phosphate. With the
method indicated above the dissociation of the cytochrome reductase
into alloxazine mononucleotide and protein II can be demonstrated
if to a small but constant amount of protein II increasing amounts of
alloxazine mononucleotide are added.

The dissociation constant of cytochrome reductase is small
(K = 1 X 10"^ M), and under physiological conditions this enzyme
will therefore be present as the practically undissociated complex.
The value of the spectrophotometric method for the study of enzyme
reactions is well demonstrated here, since 10"'' mg. of flavin are
suflBcient for accurate determinations. Not only the relation between
the prosthetic group and protein of one enzyme may be studied
with this system, but also the interaction of two different enzymes.
For example, we can measure the rate of reaction when increasing
amounts of dihydrotriphosphopyridine nucleotide are added to a
constant amount of cytochrome reductase. The velocity of the reduc-
tion of the enzyme is given by

(1) d(CR)_ kr(T) (CR)

dt ~ {T)+Kd

in which ( T) and ( CR) are the concentrations of triphosphopyridine
nucleotide and cytochrome reductase, respectively, K is the first-
order velocity constant, and Kd the dissociation constant of the
pyridine-alloxazine complex. Rate constants and dissociation con-
stants have been determined at different temperatures, and by apply-
ing the Arrhenius equation the energy of activation is found to be
about 10 kg. cal., and the heat of dissociation about 2 kg. cal., for
the reaction in which cytochrome reductase is reduced by dihydro-
triphosphopyridine nucleotide.

Similar experiments have been made for the purpose of studying
the oxidation of cytochrome reductase by cytochrome, and again
the formation of a complex between the two reaction partners could
be established. Furthermore, from the results of the kinetic deter-
minations it can be concluded that in the course of the oxidation of
alloxazine free radicals are involved. This could almost be antici-

DISCUSSION ON HYDROGEN TRANSPORT 45

pated, for the alloxazine undergoes a valence change of two,
whereas cytochrome undergoes a valence change of one, as Dr.
Stern has just pointed out.

To summarize, the facts concerning this part of the respiratory
system are, then, as follows: The protein of the cytochrome reductase
is bound simultaneously to alloxazine mononucleotide, to triphos-
phopyridine nucleotide, and to cytochrome. Approximate values for
the different dissociation constants are given in Figure 1. The energy
of activation is low, about 10 kg. cal. One may venture to say that
the binding forces are of the Van der Waal type rather than ordinary
bond forces, which would involve much higher bond energies.

The task of the protein may be (a) to estabhsh the proper geo-
metrical configuration between the different prosthetic groups; and
(b) to aid in the formation and stabihzation of free radicals. The
first point may be ofiFered as a working hypothesis to explain the
specificity of the proteins, and the second point may explain the
tremendous activity of this catalyst. These experiments were done
in Professor Hogness' laboratory, with Drs. B. L. Horecker and
C. J. Harrer.

Mr. Haas:*

The formation of complexes in the course of these enzymatic
reactions can be demonstrated in two independent ways.

1. Kinetic Measurements.— In agreement with equation 1, the
velocity of the reaction is proportional not to (T) or to (CR), as one
would expect from an ordinary bimolecular reaction, but to the
amount of enzyme present in the form of the complex.

2. Spectroscopic Measurements.— Alloxazine mononucleotide has
its maximum absorption at wave length 445 mix; the addition of
the protein of the old yellow enzyme causes the maximum absorp-
tion to migrate to wave length 465 m[i, and the further addition of
triphosphopyridine nucleotide to wave length 475 mpi. Thus the
free flavin, the flavin-protein complex, and the flavin-protein-pyridine
complex can easily be distinguished by their color.

Dr. Potter:

Thus far the emphasis has been on the cytochrome system as one
of the hnks in the hydrogen transport system. Yet there is an accu-
mulating body of evidence which indicates that an alternate path

* This statement of Mr. Haas' was made in reply to a question from tfie floor
regarding tlie proof of formation of an enzyme-substrate complex.— Ed.

46 A SYMPOSIUM ON RESPIRATORY ENZYMES

of hydrogen transport may exist. Dr. Stotz has agreed to open the
discussion of this interesting possibiHty.

POSSIBILITY OF A BY-PASS AROUND THE

CYTOCHROME SYSTEM

ELMER STOTZ
Harvard University

The possibihty of a 'Tsy-pass" around the cytochrome system in
certain phases of tissue respiration has eUcited considerable discus-
sion of late. The discovery of the flavoproteins, and their function
in isolated systems as autoxidizable substances, had raised a doubt
as to the exclusive role of the iron system in "oxygen activation."
However, this threat to the autonomy of the iron compounds was
dispelled by the correction of certain technical points and by the
work of Theorell and of Barron. The most recent threats have come
from a study of the effects of cyanide and azide ( as oxidase inhibit-
ors) on the respiration of "resting" and "stimulated" tissues. The work
of Stannard and of Korr on this topic is reviewed in my paper on
page 169. Their results may be summarized briefly by the state-
ment that the respiration of "resting" tissue is insensitive to azide,
whereas that of the stimulated tissue becomes azide-sensitive. Tissues
in either phase may, however, be cyanide-sensitive. The 'Iby-pass"
theory therefore implies that in the resting tissue an oxidation path-
way other than the cytochrome system is functioning. In Korr's
terminology, tissues that have been stimulated may liberate sub-
stances that "link" or "gear" the reducing systems to the cytochrome
system.

The by-pass theory, although possibly correct, is in my judgment
a rather sweeping conclusion to make from evidence based chiefly
on the use of these inhibitors, and without knowledge of the nature
of an alternative pathway. Even if we assume an identical action
of the inhibitors in isolated systems, as in the muscle and tissue
slices, we are far from understanding the mechanism of their action.
Stannard believes that cyanide affects not only cytochrome oxidase
but also other enzymes essential in respiration, and, to be sure, he
offers some evidence for this belief. On the other hand, this does
not imply that azide is any more specific than cyanide and that only
the effects of the former need be considered.

It would seem more conservative at the moment to consider how
the differences between cyanide and azide might be explained on
the basis of the existing knowledge of the cytochrome-cytochrome

DISCUSSION ON HYDROGEN TRANSPORT 47

oxidase system and of the action of nitrogen compounds on iron
systems.

The relations of cytochrome oxidase and cytochrome c are such
in the oxidation of hydroquinone, for instance, that a decrease of
oxidase, which would ordinarily cause a decreased hydroquinone
oxidation, could be compensated for by an increase in reduced
cytochrome. It has been shown that cyanide does in effect "remove"
a certain portion of the oxidase. Hence in a resting tissue, where
most of the cytochrome is in the oxidized state, a decreased oxidase
could be compensated for by increased reduction of the cytochrome.
In the more active state of metabohsm, where there is a small
reserve of oxidized cytochrome, such compensation is less possible.
The more undissociated the oxidase-inhibitor complex the less eflB-
cient would be the compensation. Since azide is a "less powerful"
oxidase inhibitor than cyanide, and since the greatest sensitivity to
azide is found in the "active" state, these factors may be operative.

Dr. Ball has considered at some length, in this symposium, the
possible differences between cyanide and azide as nitrogenous com-
pounds uniting with the oxidase ( Fe) and the lowering of potential
caused thereby. He has pictured how a difference in the abihty
of the two compounds to unite with the oxidized and reduced forms
of the oxidase could lead to differences in the effective potential
of the complex formed. Thus the union with cyanide could lead to
an oxidase complex with a potential lower than that of cytochrome
c, hence possessing a low catalytic power. As a result of combina-
tion of higher concentrations of azide with both oxidized and

reduced oxidase, an effective potential might be reached which could
be somewhat higher than that of cytochrome c. Although such a
complex would be less eflBcient than the original oxidase, it might
nevertheless be suflBcient for the low metabolism that exists in the
resting state, although insuflBcient for the metabolism of the "active"
tissue. This theory was not advanced to overthrow the "by-pass"
theory, but only to call attention to other possible explanations
for the differences between azide and cyanide. Perhaps these con-
siderations should be exploited before postulating, through the
mechanism of as yet unknown enzymes, a "by-pass" around the cyto-
chrome system in respiration.

Pasteur Effect

FRITZ LIPMANN

Massachusetts General Hospital

WITH RESPECT TO their dependence on oxygen supply, organisms
may be classified into (1) strict aerobes, equipped only with
respiratory metabolic systems, (2) strict anaerobes, equipped only
with anaerobic fermentative metabolic systems, and (3) facultative
organisms, equipped with both respiratory and fermentative systems.
This commonly used classification should not be followed too rigidly,
however, for intermediate states between the main classes are com-
mon in nature, and adaptive interconversion has been widely ob-
served.

The organisms in each of the first two groups rely exclusively on
one form of energy supply, respiratory or fermentative, respectively.
The third group, however, has developed the two mechanisms side
by side. It is with this latter group that we shall deal, and more
specifically with the interrelation between their respiratory and
fermentative mechanisms.

Most doubly equipped organisms possess in the Pasteur effect
a regulatory device that enables them to use, as occasion demands,
either their aerobic or their anaerobic systems. By the operation
of this effect their fermentative apparatus is blocked in the presence
of sufficient oxygen, and energy is furnished almost exclusively by
the far more efficient and powerful respiratory apparatus. When
oxygen is lacking, however, the fermentation system is brought into
operation.

The following example may serve to illustrate the energetic struc-
ture of a facultative anaerobic organism. A power plant uses as a
source of energy cheap water power; this may be compared to the
"cheap" respiratory energy. But because of seasonal variations of
flow the water power may not be entirely reliable and hence as a
safeguard against a deficiency in the supply of power a more ex-
pensively operating steam engine is built into the plant; this may
be compared to "expensive" fermentation. For obvious reasons the
plant will be equipped with a switch mechanism— its "Pasteur effect"
—which keeps the steam engine from functioning so long as the
water flow supplies suflBcient energy but throws it into operation
when water power is lacking.

PASTEUR EFFECT

An impressive physiological example of a mechanism utilizing
both respiratory and fermentative energy supply is the muscle.
Figures 1 and 2, after experiments by Bang (1), reproduce measure-
ments of oxygen consumption and lactic acid formation (blood
lactate) on human beings during physical work. During a prolonged
period of not too hard work (Figure 1) blood lactate at first increases
moderately but returns, during the first quarter of the period, almost
to the resting level. An anaerobic energy supply is observed only at
the beginning, when an adequate oxygen supply is lacking, and until
respiration climbs to the equilibrium level. For excessive short-term
work the picture is different, as shown in Figure 2. An excessive and
long-continued increase of blood lactate signifies a large expenditure
of anaerobic energy. In such a situation the adaptation is much too

WORK

BLOOD

LACTIC ACID

millimolcs per liter

I ••

60 Minutes

OXYGEN
liters per minute

Figure 1. — Oxygen consumption and blood lactate with moderate work. The
slight initial rise of lactate output coincides with the period of adaptation, before
the oxygen consumption rises to the equilibriimi level. (After O. Bang, ref. 1.)

A SYMPOSIUM ON RESPIRATORY ENZYMES

slow to supply oxygen in time, and the muscle has to rely almost
entirely on the anaerobic energy of glycolysis.

The Efficiency of Aerobic and Anaerobic Metabolism

Fermentations are energy-yielding rearrangements of the atoms
constituting the glucose molecule. These are oxidation-reduction
reactions in which, after cleavage, one part of the molecule is

Figure 2.— Oxygen consumption and blood lactate with strenuous work. The
excessive lactate formation signifies predominantly anaerobic energy supply.
A moderate rise of oxygen consumption occurs first after completion of the
work in tlie period of restitution. The slow fall of lactate in tlie blood indicates
a relatively slow removal of lactate by resynthesis or oxidation. (After O. Bang,
ref. I.)

PASTEUR EFFECT 51

oxidized at the expense of the other part, which accordingly is re-
duced. Energetically probably the most eflBcient reaction is one such
as the propionic acid fermentation: 3 CgHigOg = 4 CoHjCOOH +
2 CH3COOH + 2 CO2 + 2 H2O. The energetic yield is 61 kg.-cal.
of heat per mole of glucose. According to Burk (2), the change in
free energy is approximately 18 kg.-cal. higher per mole of glucose
than the heat exchange. The probable maximum for a fermentative
breakdown of carbohydrate thus amounts to 79 kg.-cal. per mole.
This is 11.5 per cent of the 686 kg.-cal. to be obtained by respira-
tory breakdown. The more common lactic acid and alcoholic fer-
mentations do not reach this maximum but yield only 54 kg.-cal.
(36 kg.-cal. plus 18 kg.-cal. entropy change), or 7.9 per cent of the
heat of combustion.

These values represent the theoretical maximum for fermentation
and respiration. To compare the eflBciencies of the two reactions in
the cell we must know how much of the energy of each reaction is
actually available to the cell. In a recent paper (3) the author has
pointed out that from 40 to 70 per cent of the theoretical fermenta-
tion energy is utilizable. This is deduced from the fact that in the
muscle up to 40 of the 54 kg.-cal. derived from glycolysis can be
stored as four energy-rich phosphate bonds in phosphagen, the
energy of which is utilizable for muscular work and other purposes.

Until fairly recently the view was favored that respiration energy
was much less utilizable than fermentation energy— in other words,
that fermentation energy was relatively more valuable than would
be indicated by a comparison of theoretical caloric yields. Recent
results, however, for the conversion of oxidation into phosphate
bond-energy strongly indicate that such is not the case. With
oxidation of pyruvic acid in the brain, Ochoa (4) found that for each
molecule of oxygen consumed four energy-rich phosphate bonds
were generated. With carbohydrate oxidation in heart muscle, ac-
cording to Belitzer and Tzibakova (5) as many as seven energy-rich
phosphate bonds (for nomenclature cf. ref. 3) might be formed per
molecule of oxygen. One energy-rich phosphate bond represents
from 10 to 12 kg.-cal. of utilizable energy. The six moles of oxy-
gen oxidizing one mole of carbohydrate could therefore generate
from 11 X (4 to 7) X 6 = 260 to 460 kg.-cal. of utilizable energy,
or 40 to 68 per cent of the theoretical.

The unexpectedly high yield obtained in these recent experiments
shows that there is probably no great diflFerence in utilizability be-
tween fermentation and respiration. Therefore it seems permissible

52 A SYMPOSIUM ON RESPIRATORY ENZYMES

to take the theoretical caloric value of 54 and 686 kg.-cal., respec-
tively, as a basis for comparing their efficiencies, and to conclude
that only one-twelfth to one-ninth of the total possible energy is
made available to the cell by the anaerobic fermentation of the
glucose molecule.

From this calculation the superior economy of respiratory me-
tabolism becomes evident. To draw the same amount of energy
from fermentation as from respiration the cell must use from nine
to twelve times as much substrate. In reahty the anaerobic energy
is rarely equal to the aerobic. As a rule a fully developed facultative
anaerobe uses anaerobically only four to eight times as much sub-
strate as aerobically, thus reaching on the average half the energy
level of the aerobic state. From these considerations the economical
and regulatory aspect of the Pasteur effect becomes evident.
Through its operation the voluminous fermentative metabolism is
allowed to proceed only in anaerobiosis, as is indicated in the fol-
lowing scheme, which represents the increase in glucose utilization
following change from aerobic to anaerobic conditions:

OXYGEN NITROGEN

(Qo, = 6; Q^^ = 0) (Q^^=10)

1. Glucose + 6 Oj -> 6 CO2 + 6 H2O 1. Glucose -> 2 Lactate

2. Glucose — > 2 Lactate

3. Glucose — > 2 Lactate

4. Glucose — > 2 Lactate

5. Glucose — > 2 Lactate

Some examples of "ideal" facultative anaerobic cells are given in
Table 1. From the Q values the corresponding glucose consumption
and the caloric yields are calculated. One cubic millimeter of respira-
tion oxygen corresponds to the utilization of 1.34 micrograms of
glucose and the yield of 5.2 X 10^^ cal.; one cubic millimeter of
fermentation carbon dioxide corresponds to the utilization of 4.03
micrograms of glucose and the yield of 1.2 X 10^^ cal.

In the cases cited the large fermentative metabolism disappears
completely in aerobiosis. In the experiment with the fish retina
almost three-fourths of the aerobic energy is made available through
anaerobic metabolism and correspondingly a three- to sevenfold an-
aerobic increase of glucose consumption occurs.

The Metabolic Structure of Cells

In the middle of the metabolic type scale are placed the organ-
isms that alternate between anaerobic and aerobic metabolism of

PASTEUR EFFECT

similar eflBciency. In the upper part are predominantly aerobic
types, e.g., kidney and liver and many plant cells with relatively
small fermentative capacity, and at the top of the scale is a strict
aerobe, azotobacter, with Qog of 2000 and no trace of fermentation.

Table 1.— Effect of oxygen on glycolysis

Organism or
tissue

Qo, Qo^ QN=F

Substrate
consumption

Caloric
yield

Refer-

^ anaerobic . , anaerobic ence
rate* — — ratej

aerobic

Torula

anaerobic . . . — 260 1.04

aerobic ... .-180 18 — 0.31

Embryonic heart

anaerobic . . . — 28 0.11

aerobic .... -13.6 0 — 0.018

Pigeon brain

anaerobic . . . — 28 0.11

aerobic .... -16 0 — 0.022

Fish retina (30° C.)

anaerobic . . . — 29 0.12

aerobic .... - 9.6 1 — 0.017

3.4

6.0

5.0

7.0

0.31

0.94

0.33

0.034

0.071

0.48

0.034

0.083

0.41

0.035

0.050

0.70

* mg. glucose per mg. dry weight per hour. f calories per mg. dry weight per hour.

On the anaerobic side, below the middle, are a variety of types
representing gradations down to exclusively anaerobic life. Here
fermentation is partly or wholly persistent in the presence of oxy-
gen, and respiration becomes a more or less residual function,

BACTERIA

The anaerobic type of life is most common among the bacteria,
but it occurs frequently in the animal kingdom, esoecially among
invertebrates. In all stages of phylogenetic development, life either
chooses or is forced to adapt to anaerobic conditions, and similar
metabolic arrangements correspond to similar environmental condi-
tions. Transition from alternative to exclusive anaerobiosis is well
illustrated by a tabulation of the metabohsm of the common yeasts
(Table 2), taken from Meyerhof s classical paper (6).

The almost exclusively anaerobic cultured yeasts used in the
manufacture of alcoholic beverages presumably developed from the

54 A SYMPOSIUM ON RESPIRATORY ENZYMES

alternating torula or wild yeast type. Baker's yeast is intermediate,
having a fair respiration and partially persistent aerobic fermenta-
tion. The metabolic type is not rigidly fixed. With aeration there is
adaptation to the respiratory type, and with the exclusion of air the
reverse is easily achieved (Table 2). It may be noted that the re-
appearance of respiration is accompanied by aerobic repression of
fermentation. The history of the manufacture of baker's yeast is an
impressive illustration of the economic superiority of aerobic me-
taboHsm (10). The earlier "Vienna" procedure of growing yeast

Table 2.— Yeast metabolism

Type Qo, Q^'f Q^

(Q^'f-QQ'f)X3 Inhibition
Q02 per cent

Wild yeast -180 18 260 4 93

Baker's yeast -87 95 274 6.2 65

Brewer's yeast -8 213 233 7.5 8

Same after 15 hours aeration -73 113 193 3.3 42

without agitation has now been almost entirely replaced by the
aeration procedure, for it has been found that through aeration the
yield can be greatly increased with the same amount of culture
fluid. In metabolic terms, the same amount of metabolized substrate
yields a larger amount of yeast material with economical respiration
than with uneconomical fermentation.

As a measure of the Pasteur effect two differently derived units
are recorded in the last two columns of Table 2. In the first of the
two the Meyerhof Oxidation Quotient is calculated. This relates the
disappearance of fermentation to the magnitude of respiration.
When three times the difference between fermentation in nitrogen
and fermentation in oxygen is divided by the respiration in oxygen,
the quotient represents the relation between the glucose equivalent
of fermentation and that of respiration. Disregarding underlying
theoretical implications, it states how much fermentation glucose
is replaced by oxidized glucose when respiration is allowed to occur.
Stressing the economical significance of the quotient, we have pro-
posed to call it a replacement quotient (3). In the next column the
percentage of inhibition is calculated. From the recorded figures one
would suspect a relationship of some kind between the magnitude
of respiration and the Pasteur effect. This observation originally led
Meyerhof to a universal application of his resynthesis theory as an
explanation of the Pasteur effect.

PASTEUR EFFECT 55

Although a broader discussion of the theory of the Pasteur effect
is reserved for a later paragraph, some interesting experiments re-
lating to the constancy of the Meyerhof Quotient may be mentioned
here. These experiments on the effect on the oxidation quotient of
the Z-factor of v. Euler, a factor stimulating only fermentation, are
taken from a paper by Meyerhof and Iwasaki (11). See Table 3.

For the same yeast, Q values, the oxidation quotient, and the
percentage of inhibition are hsted with and without the stimulating

Table 3.— Influence of medium on fermentation and oxidation

quotient
(Reference 11)

Inhibition

Yeast

Medium

Qo,

QO'F

Q^'F

O.Q.*

per cent

Baker's yeast I

glucose in phosphate

-37.5

44.5

119

plus wort

-37

212

9.4

Baker's yeast II

glucose in phosphate

-40.5

105

199

plus molasses

-45.6

228

374

9.6

Strain XII

sugar in phosphate

-10

2.3

plus yeast extract

-10.5

137

167

8.5

* Meyerhof oxidation quotient.

factor. Here with the same respiration but variations in anaerobic
and aerobic fermentation, the relative influence of respiration on
the disappearance of fermentation increases, whereas the per-
centage of inhibition remains constant. These experiments are more
consistent with an explanation of the Pasteur effect as an inhibition
of some kind resulting from the presence of oxygen but independent
of the magnitude of respiration. Similar results were recently re-
ported by Burk, Winzler, and du Vigneaud (12), who studied tlie
metabolism of a biotin-deficient yeast. They found oxidation quo-
tients up to 20, whereas 12 is the theoretical maximum for a re-
synthesis theory.

The results of these experiments are discussed in some detail in
order to draw attention to two lines of approach to an explanation
of the phenomenon. The first set of data, showing parallel rates of
respiration and fermentation disappearance suggests an interaction
between the two lines of reaction which leads to the establishment
of a dynamic equilibrium of some kind. The second set of data
shows conditions where the aerobic inhibition of increased fermenta-
tion is independent of the rate of respiration, which remains prac-
tically constant. This suggests a direct inhibition through oxygen.

56 A SYMPOSIUM ON RESPIRATORY ENZYMES

Table 4 surveys lactic and propionic acid bacteria and chlorophyll-
bearing plant tissue. The three types of lactic acid bacteria are
representative examples which duplicate in every respect the
metabolic types of the yeasts shown in Table 2. Lactobacillus del-
bruckW* deserves special comment. It shows a well-developed

Table 4.— Bacterial and plant metaboHsm in glucose

Aerobic

Material

Organism

Q05

QO'F

QN'F

O.Q.

inhibi-
tion
per cent

Lactic acid

Bacterium cereale

-189

305

3.9

bacteria (13)

Bacterium DelbrUckii
(hemin-free)

-109

188

Lactobacillus casei

287

316

-3

255

277

Propionic acid

Propionibacterium

bacteria (14)

pentosaceum

-15

Lathyrus plant

Sprout

-3.5

(15)

Leaf

-1.4

0.8

Algae (15)

Chlorella pyrenoidea

-5

1.6

Coelastrum proboscideum

-12.7

2.7

Pasteur eflFect without possessing any catalysts of the hemin type. The
oxygen-producing plant tissues show only a small fermentative
capacity.

ANIMAL TISSUES

The fairly frequent occurrence among invertebrates of organisms
adapted to anaerobic life has been mentioned. Particularly among
the worms is found a great variety of partly or wholly anaerobic
forms. Here, on a higher phylogenetic level, appears a metabolic
stratification similar to that in the yeasts and bacteria. There seems
to be a gradual transition from the facultative anaerobic free-living
worms to the obligate anaerobic parasitic forms (16). As early as
1909 Lesser (16a) made important quantitative experiments on the
interrelation between respiration and fermentation in earthwonn
metabolism. He found that fennentation resulted in the con-
sumption of from four to six times as much glycogen. These same
ratios were later found by Meyerhof to hold likewise for frog

* This organism has been referred to by a variety of names in the biochemi-
cal literature, including: Bacterium DelbrUckii, Lactobacillus Delbriikii, Bac-
terium cereale. Bacillus acidificans longissimus, etc.

PASTEUR EFFECT

muscle (17). As end products in worm fermentation Lesser found,
in addition to lactic acid, large amounts of higher fatty acids,
especially valeric acid (cf. also 18 and 19). An increase of glycogen
utilization in anaerobiosis is described for many other types of
worms (19). The parasitic worms living in the practically oxygen-
free intestinal fluids show predominantly anaerobic metabolism. Al-
though they are able to respire aerobically, their fermentation does
not seem to be inhibited nor their glycogen consumption dimin-
ished in oxygen (19).

The higher vertebrates and especially the warm-blooded animals
must be considered as essentially aerobic organisms. This is not true
for all their parts, however, nor under all conditions. The experiment
shown in Figure 2 is an example of partial anaerobiosis: the muscle
suddenly put under high strain must rely predominantly on a supply
of anaerobic energy. In Table 5 metabolic figures for representative

Table 5.— Metabolism of animal tissues

Refer-

Type

Tissue

Qo,

QO'Q

QN'G

U.Q.

ence

Largely aerobic

Kidney

0.8

-20

0-8

Liver

0.6-0.9

-12

0-2

0-12

Facultative

Brain

-16

4.9

anaerobic

Pituitary

-12

3.3

Testis

0.7-1

-14

1.3

Medulla of kidneys

0.97

- 9

3.3

Mucosa jejuni

0.86

-16

Largely anaerobic

Sperm, human

- 1

6.5

tissues are assembled. During recent years interesting examples of
adult tissues with predominantly anaerobic metabolism have been
described. Relatively large and aerobically persistent glycolysis has
been found, for example, in the medulla of the kidney and in
cartilage (20, 21). Dickens correlates the metabolic pattern with
the relatively poor blood supply of these tissues. Large and aerobi-
cally persistent glycolysis in the intestinal mucosa recently reported
by Dickens and Weil-Malherbe (22) might likewise be correlated
with the previously mentioned lack of oxygen in the intestinal fluid
surrounding it.

These observations show that the pronounced anaerobic me-
tabolism of embryonic tissue and of malignant growth (Table 6) is
not an isolated phenomenon. Here are tissues, as has been pointed

58 A SYMPOSIUM ON RESPIRATORY ENZYMES

out in an earlier paper (23), which through environmental conditions,
such as insuflBcient circulation, are forced to rely partly on a supply
of anaerobic energy (cf. 20). Recent measurements by Philip (24)
on respiration of the early developmental stages of the chick embryo
give evidence that this must be the case. This corroborates our earlier
findings based on less conclusive experimental data. Philip's remarks
may be quoted here: "The study of the early blastomere," he says.

Table 6.— Metabolism of tumor tissue

^°^'^'' Refer-

Tumor Qo, Q^'g Q^'g CQ. tion "®^^^

^ , ence

per cent

Flexner-Jobling carcinoma,

rat -15 23 23 0 0 54

- 7 16 24 3 33
Adenocarcinoma, human

male - 9 16 29 4.2 42 75

- 1.2 5 12 15 58
0 9 22 M 59

Jensen sarcoma -14 16 34 3.9 53 39

Walker sarcoma 256 ... -22 25 46 2.9 46 39

The average R.Q. for all the tissues was 0.85.

"has revealed that oxygen diffusion limits the oxygen consumption
in oxygen tensions of the air. This indicates that the early blastomere
may actually be in a state of partial anaerobiosis." And later in the
same discussion: "The considerations presented suggest that some
of the energy used during early periods of growth can be pro-
vided to the embryo by anaerobic processes. This condition is prob-
ably associated with the rapidly increasing size of the embryo
during early periods before the circulation system can function as
an adequate oxygenating mechanism." The relatively poor vascu-
larization of most tumors is evidence that the last statement holds
likewise for malignant growth. We are led then to the conclusion
that the high capacity of the anaerobic metabolism present in normal
and in malignant growing tissues should be attributed to their partly
anaerobic state of life rather than to an unlikely special growth
function of glycolysis.

A curious phenomenon of hyperfunction of the Pasteur effect is
observed in human beings at high altitudes (25). The relatively high
lactic acid level of the blood which would be expected at low
oxygen pressure is observed only before adaptation occurs (25).

PASTEUR EFFECT 59

After adaptation to the new environment the lactate level of the
blood becomes normal. Even with exhausting work the lactic acid
concentration remains very low, 2 to 3 millimolar, as compared with
a blood level of 13 millimolar reached with exhausting work at sea
level (Figure 2). Apparently a special mechanism prevents the
muscle from utilizing too much of the anaerobic energy supply even
at low oxygen pressure. Dill in his book on life at high altitudes (26)
makes interesting comments on this phenomenon: "It is as though
the body, realizing the delicacy of its situation with regard to oxygen
supply, sets up an automatic control over anaerobic work which
renders impossible the severe acid-base disturbances which can be
voluntarily induced at sea level."

Interpretation of the Pasteur Effect

During recent years discussion has centered more or less around
the question whether the effect depends upon respiratory activity
as such, or upon an inhibition produced by the action of oxygen.
In the first case the rate of respiration with its output of energy
would be a determining factor and a state of dynamic equilibrium
would result; part or even all of the respiration energy would be
spent or fixed to revert or repress glycolytic breakdown. If, however,
the effect is brought about through oxygen, or more specifically by
oxidative inhibition of an essential part of the glycolytic enzyme
system, then the reaction may be independent of the rate of respira-
tion and involve no transfer of energy.

equilibrium schemes

A fuller understanding of the partial reactions involved in fer-
mentation and respiration has given new impulse to the discussion
of their interrelationship as manifested in the Pasteur effect. The
fact that cozymase and adenylic acid, the two transmitter substances
in fermentation, are likewise participants in respiration has given
rise to some interesting suggestions.

Ball (27) has pointed out that, aerobically, respiratory oxidants
such as flavin may compete with pyruvic acid for the reduced
cozymase. Pyruvic acid would disappear largely through oxidation
rather than through fermentative reduction. Adler and Calvet (28),
however, comparing the ratio of oxidized to reduced cozymase in
aerobic and in anaerobic baker's yeast found no significant differ-
ence, but a ratio of nearly one to one in both cases.

Adenylic acid, the other common transfer system, was first linked

60 A SYMPOSIUM ON RESPIRATORY ENZYMES

with the Pasteur effect through work done by Ostern and Mann (29).
They found that the addition of adenosine triphosphate (ATP) to
mashed muscle depressed aerobic glycolysis and raised the Meyer-
hof Quotient from 2.2 to 4. Later Lennerstrand (30) discussed the
possibility that with aerobic over-phosphorylation of adenylic acid
(Ad), the ratio ATP: Ad might become too high to permit adenylic
acid to function effectively as a transmitter in fermentation.

A scheme based on the recent development of the biochemistry of
phosphate turnover has been presented and discussed in detail else-
where (3). Figure 3 is taken from this paper. The upper cycle, re-
volving clockwise, represents anaerobic glycolysis; the lower cycle,
revolving counter-clockwise, represents aerobic resynthesis. It ap-
pears that the clockwise run of the glycolytic cycle depends on the
outflow of the energy-rich phosphate created in the reaction. An
actual reversal of the cycle back to the aldehyde stage may occur
when through the aerobic influx of new energy-rich phosphate
the carboxyl-bound phosphate in 1,3-diphosphoglyceric acid (Ph-
glyceryl-Ph) cannot be removed.

In the Meyerhof-Warburg equilibrium reaction, as the diagram
shows, inorganic phosphate is bound when the reaction proceeds to
the right and is set free when it proceeds to the left. Therefore in-
organic phosphate concentration can become a rate-determining
factor. Meyerhof et al. (31) and Belitzer (32) have pointed out that
in muscle the increased concentration of inorganic phosphate
through creatinephosphate breakdown should be regarded as the
cause of the release of metabolic activity due to stimulation. Along
similar lines, Johnson (33) recently suggested that the lowering of
inorganic phosphate concentration might be a possible cause of
aerobic inhibition of glycolysis. Inorganic phosphate concentration
seems, however, to be high in most cells except in resting muscle,
where most of the phosphate is bound to creatine. Phosphate is
generally considered to be the intracellular anion. How much of
this is really free phosphate and how much is labile phosphate
broken down by chemical manipulations remains to be determined
(34).

In our opinion, the value of such schemes is limited because they
disregard controlling factors in the cell which undoubtedly must
regulate the routes of phosphate turnover— that is, the synthesis as
well as the breakdown of intermediates. The Pasteur effect cannot
be due merely to an "open" equilibrium; it must be due to specific
transmitter systems. Evidence for this is the fact that the 'linkage"

PH-T R I 0 S E 4- PH

+ 2 H

^GLUCOSE
< + PH

H.DPN

PH-G L Y C E R Y L^^PH

/ t

PH-G LYCERATE + AD'^PH
/
PYRUVIC

E N 0 L~PH

FUMARATE + PH

rH-O L Y c t-

/^ /a>-PH— ►

2PYRUV1C /

E 0 0 L-~PM

2 PYRUVIC AC.

Figure 3. — Schemes for anaerobic breakdown (upper cycle) and aerobic re-
syntliesis (lower cycle) of carbohydrate. (From F. Lipmann, Advances in
Enzymology, vol. 1, 1941.)

A SYMPOSIUM ON RESPIRATORY ENZYMES

between respiration and glycolysis may be interrupted without im-
pairing the reactions proper.

INHIBITION OF THE PASTEUR EFFECT

A considerable variety of compounds are able to interrupt the
Pasteur eflFect, or the Pasteur reaction, as Warburg (35), after dis-
covering the specific action of ethyl carbylamine, first called the
phenomenon. Table 7 presents a survey of the agents which have
been given the most study and which have proved most effective.
Similar effects were found with phenylhydrazine by Dickens (36),
with dinitrocresol and dinitrophenol by Dodds and Greville (41),
and with HCN on certain plant cells by Genevois (42).

Table 7.— Aerobic release of glycolysis

, , ., .^ Concentra-
Inhibitor

tion

Qo,

QOg

R.Q.

Tissue

Refer-
ence

Ethyl carbylamine

0
10-3 M

- 13

- 14

19
32

Jensen sarcoma

Carbon monoxide, light

88%

- 11

0.72

Allantois

Carbon monoxide, dark

88%

— 11

Oxygen pressure

95%

- 25.5

1.05

Chorion

- 22

0.7

Phenosafranine

- 13

1.03

Brain

10-6 M

- 13

0.98

Glutathione

-230

Yeast

.5X10-3 M

-205

255

The common effect is the release of aerobic glycolytic action up
to an anaerobic level, while respiration remains quantitatively un-
changed. In Laser's experiments (37, 38) with carbon monoxide and
low oxygen pressure the respiratory quotient was lowered, indicating
qualitative changes of respiration. With phenosafranine, however,
Dickens (39) found that the respiratory quotient of the brain re-
mained unity and the manometric and chemical determinations of
lactic acid were in excellent agreement. In this case, at least, it seems
very probable that the interruption of the Pasteur reaction occurred
without a qualitative change of respiration.

The action of metal-specific inhibitors has been of great interest.
Work in this field has revived discussion of the question whether
the effect is dependent on, or independent of, respiratory activity.
The old observation that in most tissues cyanide released aerobic

PASTEUR EFFECT 63

fermentation by inhibiting respiration was taken as indisputable
proof of the dependence of the Pasteur eflFect on the intactness of
respiration. Consequently ethyl carbylamine action, affecting only
the Pasteur effect, but leaving primary respiration intact, was in-
terpreted as inhibition of a reaction linking respiration to glycolysis.
A differential inhibition of respiration and Pasteur reaction by
carbon monoxide was observed by Warburg (43a) in yeast experi-
ments. Mainly interested in the respiratory effect of carbon
monoxide, he remarked only incidentally upon the relatively higher
sensitivity of the Pasteur reaction. Later, Laser (38) showed that
in animal tissues the differences in sensitivity were pronounced. Fre-
quently, he found, carbon monoxide had little or no effect on respira-
tion but did cause aerobic glycolysis to appear. The release of
aerobic glycolysis in animal tissues had been observed by Warburg
and Negelein (43), but had been considered as a secondary effect
due to inhibited respiration. From some preliminary measurements
of the effect of light on aerobic glycolysis in retina in the presence of
carbon monoxide, the spectrum of the Atmungsferment was charted.
Since Laser (38) had found respiration in retina to be uninfluenced
by carbon monoxide, these measurements, as Stern and Melnick (44)
recognized, had to be reinterpreted as preliminary measurements of
the spectrum of the Pasteur agent-carbon monoxide compound.
Stern and Melnick then measured carefully with the Warburg il-
lumination technique the relative absorption spectrum of the Pasteur
agent-carbon monoxide compound. The decrease in aerobic fer-
mentation on irradiation was plotted against wave length. This de-
crease may be assumed to be due to the decomposition of the
Pasteur agent-carbon monoxide complex. The resulting spectrum
was very similar to that of the respiratory enzyme. Such measure-
ments were made on retina (44) and yeast (45). Recently Melnick
(45a) charted the spectrum of the respiratory enzyme of animal
tissue by using heart muscle extracts in which, in contrast to the
intact tissue, respiration is sensitive to carbon monoxide (46). The
bands developed from these measurements are reproduced in
Figure 4. The spectra of the respiratory enzymes in yeast and in
animal tissue, respectively, differ greatly, as do those of the Pasteur
enzymes. In each case, however, the spectrum of the Pasteur enzyme
follows closely that of the respiratory enzyme, deviating only in the
absorption at longer wave lengths. The consistent, although small,
differences are considered as evidence of the existence of two

A SYMPOSIUM ON RESPIRATORY ENZYMES

definitely diflFerent enzymes, one catalyzing the final step in the
oxidation of metabolites, the other catalyzing the oxidative inactiva-
tion of a part of the glycolytic system.

This analysis seems a very promising approach at least to an
elucidation of the events taking place on the oxygen side. The simi-
larity between the respiratory and Pasteur enzymes suggests a direct

400

450

50C

■

) 550

600

650

RAT TISSUE

CYTOCHROME
OXIDASE

PASTEUR
ENZYME

YEAST

PASTEUR
ENZYME

RESPIR^T0RY
ENZYME

1— , 1

400 450 500 550 600 m/

Figure 4. — Spectra of the respiratory and Pasteur enzymes
in animal tissue and in yeast

reaction between oxygen and the transmitter. This is further in-
dicated by the difference in the affinity of the two enzymes for
oxygen (37, 47, 76). The peculiarities of the carbon monoxide effect
on respiration and a change of respiratory quotient at low oxygen
tension (37) and with carbon monoxide (38), however, seem to in-
dicate that the present interpretation may not represent the final
solution.

In spite of the interest that attaches to the metal-specific anti-
catalysts, it should not be overlooked that such inhibitors as phe-
nosafranine, dinitrocresol (41), and glutathione can hardly be con-
sidered metal-specific. In an extensive study of the action of pheno-

PASTEUR EFFECT 65

safranine and other phenazine derivatives, Dickens (39, 48) presents
evidence which suggests that a flavin enzyme may participate in
the transmission of the aerobic inhibition. The relationship between
flavin and the Pasteur eflFect is indicated also by its occurrence in
the hemin-free Lactobacillus delbriickii (13), where flavin is the
only respiratory catalyst.

The disturbance of the Pasteur effect in brain that attends a lack
of ionic balance represents a phenomenon of great complexity. Ash-
ford and Dixon (49) observed a profound metabolic change in brain
slices suspended in tenth molar potassium chloride. Aerobically they
found increased respiration and appearance of glycolysis; and
anaerobically, gradual and irreversible disappearance of glycolysis.
They correlated the metabolic changes with the well-known increase
in cell permeability through potassium ion (50). Dickens and
Greville (51) showed subsequently that the potassium effect is spe-
cific for brain and is not found in other tissues, and that omission
of calcium had a similar effect. Continuing on similar hues, Weil-
Malherbe (52) observed definite effects of potassium and also am-
monium ions at much lower concentrations than those used by
Ashford and Dixon.

This effect of electrolyte on brain metabolism signifies a great
lability of the Pasteur mechanism. Warburg (54) has emphasized
that the Pasteur mechanism is universally very sensitive to un-
physiological surroundings. For example, in rat embryo aerobic
glycolysis is high in Ringer solution but low or absent in serum or
amniotic fluid. Effects of this type must be taken as an indication
that aerobic disappearance of glycolysis is the result of an easily
disturbed balance of reactions.

REVERSIBLE OXIDATIVE INHIBITION OF GLYCOLYSIS IN EXTRACTS

In order to approach experimentally the possibility that oxidative
inhibition might be the cause of aerobic disappearance of fermenta-
tion, I studied some time ago the effect of oxidizing agents on
glycolysis and fermentation in extracts (55, 56). It was shown that
the fermenting system was inactivated by small amounts of iodine
and quinone. By adding indophenols as oxidants inhibition in oxygen
was provoked, which disappeared in its absence, when the oxidizing
dye was reduced by constituents of the extract and through its
enzymatic activity. Two experiments of this type with muscle extract
and yeast juice, respectively, are summarized in Table 8. It appears
that addition of the dye reproduces a Pasteur effect, which occurs,

66 A SYMPOSIUM ON RESPIRATORY ENZYMES

however, with neghgible respiratory activity, demonstrating the
possibihty of a reversible oxidative inactivation.

The inhibition of glycolysis with iodine was subsequently studied
more carefully by Gemmill and Hellerman (57). With concentra-
tions just high enough to obtain fairly complete inhibition they were
able to recover the activity by adding glutathione or cysteine. This
suggested strongly that the oxidative inhibition was due to reversible
oxidation of enzyme SH-groups. Rapkine (58, 59) then showed that
the oxidoreduction between phosphoglyceraldehyde and pyruvic
acid was at least one of the partial reactions being blocked by
oxidation, presumably of enzyme-SH. This reaction system could be
inactivated by S-S-glutathione and reactivated by SH-glutathione.
More recently Rapkine found the same reaction reversibly inacti-

Table 8.— Induced Pasteur eflFect in cell extracts

Extract

Addition

COsin
O2 O2

Cubic millimeters

C02in

per hour

O.Q.

Aerobic
inhibi-
tion
per cent

Ref.

Yeast

6X10-3Mnaphthol-
sulfonate indo-
phenol

None

-49 106
— 1140

710
1120

85
0

Muscle

10-3 M dichloro-

phenol indophenol
None

- 4 20
— 440

380
425

270

95
0

vated by dichlorophenolindophenol (personal communication), which
might explain my earlier results with the complete glycolytic sys-
tem. With these experiments the possibihty of a reversible oxidative
inactivation has become firmly established. It is therefore of little
significance for the question at issue that, as was shown by Michaelis
and Smythe (60), many dyes, irrespective of oxidation-reduction
potential, inhibit irreversibly by various mechanisms, or that naphthol-
sulfonate indophenol with different yeast preparations leads earlier
to irreversible inactivation than in our experience.

Resides the system studied by Rapkine, a number of partial
enzymes of glycolysis were found to undergo oxidative inactivation
followed by reactivation with glutathione. These reactions are as
follows:

PASTEUR EFFECT 67

phosphoglyceraldehyde + pyruvate -^ phosphoglycerate -|- lac-
tate (58)

glycogen + phosphate ±5 glucose-1-phosphate (61, 62)

glucose-1-phosphate ?± glucose-6-phosphate (62)

adenosinediphosphate + glucose -^ adenyhc acid + glucose-6-
phosphate (63)

The activation by thiol compounds of glycolysis in extracts, de-
scribed by Geiger and Magnes (64) and Michaelis and Runnstrom
(65) thus becomes easily understandable.

THIOL INFLUENCE ON FERMENTATION AND GLYCOLYSIS
IN INTACT CELLS

The function of glutathione is not yet well understood. It is
present in practically every cell in fairly large amounts. Frequently
it has been suggested that it performs the role of an oxidation-
reduction buflFer. The very complexity of intracellular metabolism
prevents us from making more than vague statements of that type.
The protection against oxygen injury which thiol compounds give
to strict anaerobes, first observed by Quastel and Stephenson (53),
lends support to the assumption that their function is one of
stabilization.

Observations on intact cells as well as on cell-free enzyme systems
suggest a regulatory effect of thiol compounds on glycolysis and
fermentation. As yet it is impossible to correlate definitely the action
on intact fermenting cells and on fermenting enzyme extracts or
partial systems, but a promising approach seems to be opened which
is worth very careful consideration.

Release of aerobic glycolysis with glutathione was first observed
by Bumm and Appel (66) with sliced animal tissues. Soon after-
ward Quastel and Wheatley (40) made an interesting study of the
effects of glutathione and cysteine on the metabolism of baker's
yeast. One of their experiments is included in Table 7 above. Gluta-
thione interrupts the Pasteur effect without affecting respiration.
They noted that an extract of brewer's yeast had much the same effect
as glutathione, which they ascribe to the large content of thiol com-
pounds in brewer's yeast. With cysteine the effect on aerobic glycol-
ysis was the same, but respiration was markedly inhibited. The
respiratory inhibition was specific for glucose and absent when
glycerol was used as substrate. More recently Runnstrom and
Sperber (67) undertook a study of the cysteine effect. Accompanying

68 A SYMPOSIUM ON RESPIRATORY ENZYMES

the release of aerobic fermentation they found an inhibition of the
synthesis of higher carbohydrates from glucose. This interesting
observation would be still more significant if respiration were not
inhibited at the same time. The alternative between glucose fer-
mentation and synthesis to glycogen suggests that thiol compounds
are able to upset the normal aerobic reaction course from glucose-6-
phosphate over Cori-ester to glycogen, forcing the glucose mono-
ester into the fermentation cycle.

With Propionibacterium pentosaceum a number of interesting
observations were made by Fromageot and Chaix (14). Dilute sus-
pensions of repeatedly washed bacteria did not ferment in the pres-
ence of minute amounts of oxygen. This inhibition was counteracted
by very small concentrations of cysteine or hydrogen sulfide. With
unwashed and concentrated suspensions small amounts of oxygen
did not affect the fermentation, but aerobically fennentation dis-
appeared (normal Pasteur effect, Table 4) and was released by
thiol compounds. They concluded that normally a substance is
present in bacteria which protects the fermentation system against
the action of small amounts of oxygen. Since with impoverished or-
ganisms protection can be restored with cysteine, they assumed the
protecting substance to be a thiol compound. With high oxygen
pressure the physiological concentration of the protective system is
not high enough to counteract the oxidative inhibition and aerobic
disappearance of fermentation; that is, the Pasteur effect occurs.
When the concentration of thiol compound is increased, the oxidative
inhibition is blocked again, and aerobic fermentation appears. In
other words, the occurrence of fermentation depends on the relative
concentrations of SH-compound and oxygen, respectively.

An observation reported by Dickens (68) with pyocyanine should
be mentioned here. In the presence of this dye "anaerobic" glycolysis
of sarcoma was inhibited when measured in unpurified nitrogen
containing 0.3 per cent oxygen. At the same time a slight color
remained, indicating slight reoxidation of the dye. The color and
the inhibition disappeared when chromous chloride was used to ab-
sorb the traces of oxygen. The parallel between this phenomenon
and our dye-induced Pasteur effect in extracts, as well as Froma-
geot's effect of low oxygen pressure on propionic acid bacteria, is
obvious.

Despite the complexity of dye effects on living cells (68, 69),
Dickens came to the conclusion that in general there is a tendency
for dyes with high oxidation-reduction potential to increase the

PASTEUR EFFECT

Pasteur eflFect. In harmony with this generahzation is the increase
of the Pasteur effect in tumors by ferricyanide (70) and in yeast by
indophenols (71). The complexity of the dye effects, however, is il-
lustrated by the action of methylene blue, which, according to early
observations by Gerard (72), releases aerobic glycolysis in muscle,
while it was found by Barron (73) to inhibit aerobic glycolysis in
erythrocytes. Nevertheless there seems to be a parallelism between
dye action in extracts and in cells and a correlation between thiol
and dye effects.

PASTEUR EFFECT WITH VERY LOW RESPIRATION

That aerobic inhibition of fermentative metabolism is inde-
pendent of respiration can be most clearly demonstrated through
the occurrence of the Pasteur effect with very low respiration.
Aside from the dye-induced Pasteur effect in extracts, some examples
of such phenomena in living cells have already been discussed, such
as inhibition by traces of oxygen or inhibition in the presence of
relatively low respiration in yeast (11, 12). As a rule parallelism
between the appearance of anaerobic metabolism and the dis-
appearance of respiration is to be expected by the very nature of
the phenomenon. The fact that the most-used inhibitors of respira-
tion are metal-specific and are likewise more or less pronounced
inhibitors of the Pasteur reaction, has greatly complicated the
analysis. Inhibitors of respiration which interrupt the chain, not at

Table 9.— Pronounced Pasteur effect with very low respiration

Aerobic

inhibi-

Refer-

Tissue

Addition

Q02

Q°^G

Q^^G

O.Q.

tion
per cent

ence

Adenocarcinoma,

none

-1.2

human male

none

Brain of rat

10-2 M male-

Ihr.

-10

ate

2hr.

-5

0.5

3hr.

-2

19.5

Embryonic

2.5X10-2 M

(1)

-1

chicken heart

malonate

(2)

-4

(3)

-11

6.8

(4)

Ihr.

-11

6.5

2hr.

-6

in serum-

malonate

(5)

-9.5

3.5

70 A SYMPOSIUM ON RESPIRATORY ENZYMES

the end where the iron catalysts are operating but at an earher
stage, might be expected under favorable conditions to interrupt
respiration without afiFecting the Pasteur reaction. Malonate and
maleate, which block the Szent-Gyorgy cycle, might react in this
way. Weil-Malherbe (52) has indeed found with maleate poisoning
that there is no appreciable aerobic glycolysis in brain when respira-
tion has already declined to very low levels. I found, with malonate,
similar effects on embryonic heart (74). In Table 9 a survey is given
of these and other experiments, where with animal cells a Pasteur
effect was found with low respiration. This was observed by Rosen-
thal and Lasnitzki (75) with some human cancer without inhibitors
and by Kempner and Gaffron (76) with myeloblasts at 6 per cent
oxygen pressure. It should be remembered that in Kempner's experi-
ments with myeloblasts, while the Pasteur reaction was unaffected,
respiration declined with falling oxygen pressure: the Qo^ in 95 per
cent oxygen was 8; in 6 per cent oxygen, 3.2. Laser (37) found the
reverse with chorion, retina, and mouse liver; that is, little influence
of low oxygen tension on respiration but inhibition of the Pasteur
reaction.

Malonate does not have the effect described above on all tissues.
I found with pigeon brain a decrease of respiration accompanied
by a large increase of aerobic glycolysis (74). Similar results were
reported by Kutscher and Sarreither (77) with skeletal muscle.

Conclusion and Outlook

It has not been my purpose to give a complete survey of the work
in this field. The recent reviews by Burk (83, 84) constitute a com-
petent discussion of the problem as a whole, especially with regard
to earlier work and thoughts. Our purpose here has been to sum-
marize mainly the facts that indicate the occurrence of an oxidative
inhibition. In general the evidence may be considered indicative
but not conclusive, except in a few instances. The cell may choose
to eliminate unneeded anaerobic metabolism by an inhibitory
mechanism rather than by a counterforce, but there are indications
that such inhibition acts upon reactions directing the internal flow
of energy. Substances interrupting the Pasteur linkage likewise in-
terrupt synthetic reactions, as has been shown in the case of cysteine
(67) and especially dinitrophenol (41). Clifton (78), while in
Kluyver's laboratory, made the discovery that dinitrophenol inhibits
completely the synthetic processes in microorganisms. His study was
based on the work of Barker (85), who demonstrated that with

PASTEUR EFFECT 71

"resting" organisms only part of the disappearing non-nitrogenous
metabolite could be accounted for by oxidation, while a large part
was converted into cell material, presumably carbohydrate. This
conversion was completely interrupted in the presence of dinitro-
phenol, in which case catabolic breakdown continued until all ma-
terial was oxidized (78, 79).

Dinitrophenol has therefore become an important tool for the
study of the relation between anabolic and catabolic processes,
which must be determined by the flow of energy-carrying reactions.
In a recent paper (3) where I have discussed the generation and
transfer of energy-rich phosphate bonds it is stated that a major
part of metabolically yielded energy is converted primarily into
phosphate bond energy. An understanding of the means by which
the cell directs the flow of energy-rich phosphate bonds into pre-
determined reactions should lead to a more precise understanding
of the mechanism of regulative cell reactions such as the Pasteur
effect and the probable related action of the hormones.

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72 A SYMPOSIUM ON RESPIRATORY ENZYMES

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40. Quastel, J. H., and Wheatley, A. H. M., Biochem. J., 26, 2169 (1932).

41. DoDDS, E. C, and Greville, G. D., Lancet, 112: I, 398 ( 1934).

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PASTEUR EFFECT 73

75. Rosenthal, O., and Lasnitzki, A., Biochem. J., 196, 340 (1928).

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Oxidases, Peroxidases, and Catalase

KURT G. STERN

Yale University School of Medicine

IN THIS PAPER no attempt will be made to treat the subject in a
comprehensive or systematic manner. A number of review ar-
ticles and monographs (4, 49, 50, 51, 62, 84, 85) have been written
on hemin catalysts and respiratory enzymes, to which the reader
is referred for information on historical developments, basic facts,
and details. By way of introduction some of the available data on
hemin catalyses and on hemin-containing enzymes are given in
Tables 1 to 5, which are documented by references to experimental
studies.

The chief aims of this presentation are to bring out certain funda-
mental features shared by all the catalysts under discussion; to
analyze critically some of the controversial issues in the field; and to
trace some of the more recent developments. To conserve space
and to avoid overlappings with other papers, enzymes such as poly-
phenoloxidases, which contain copper rather than iron in their
prosthetic group, and the cytochromes, which cannot be regarded
as independent enzymes, will be considered only in so far as their
relationship to the hemin enzymes may require it.

The Common Denominator in Hemin Catalyses

All reactions and catalyses in which hemins participate are either
of the oxidative type or at least involve oxygen as a reactant. Under
this heading are grouped a variety of processes, ranging from the
transport of molecular oxygen by the respiratory pigments to the
activation of oxygen or the transfer of electrons from ferrous to ferric
iron. The few reports which have claimed that hemins or porphyrins
have promoted hydrolytic reactions remain unconfirmed. The pri-
mary step in many hemin catalyses appears to be an interaction be-
tween coordinatively linked, porphyrin-bound iron and the bond
between two oxygen atoms as it exists either in molecular oxygen
or in the forai of a peroxide bridge. This elementary process pre-
cedes or, indeed, represents what is mysteriously called the phe-
nomenon of "oxygen activation." All that we know about it is that
oxygen atoms thus captured acquire a state of high reactivity. The

OXIDASES, PEROXIDASES, AND CATALASE 75

explanation that this activation is caused by a "deformation of the
electron shells of the oxygen atom" is little more than a clever means
of hiding our ignorance about a process which in biological import
may be likened to the primary reaction of carbon dioxide with
chlorophyll during photosynthesis. It is much easier to comprehend
the type of interaction represented by the oxidation of ferrocyto-
chrome by the ferri form of the respiratory ferment of Warburg.
This reaction must be determined, at least partly, by a difiFerence in
oxidation-reduction potential between the two iron compounds, al-
though it is not absolutely necessary that the normal potential of the
Warburg enzyme be considerably more positive than that of the
cytochrome. We know of instances where the reduced form of a
reversible system is oxidized partly by the oxidized form of a more
negative system, such as the methemoglobin formation by methylene
blue. The extent of such an interaction is governed by rigid thermo-
dynamic principles only in isolated and homogeneous systems where
no side reactions take place. In a living cell, where the ferrous
form of the respiratory enzyme is rapidly reoxidized by molecular
oxygen and the ferri form of cytochrome is rapidly reduced by
Hogness and Haas's "cytochrome reductase," the ratio of the various
forms, in the steady state of respiration, may differ considerably from
that in the isolated system.

The catalytic power of hemins is, in the last analysis, a function
of the catalytic power of the central iron atom. The schema, traced
in Figure 1, illustrates how the highly complex and specialized
hemin protein enzymes stem from iron in its simplest form. In the
lowly iron sulfate we already encounter, in a rudimentary form, some
of the features which distinguish the oxidases, peroxidases, and
catalase. It is fascinating to watch the increase in catalytic activity
and the increasing degree of specialization that takes place as the
iron atom is riveted into compounds of increasingly complex struc-
ture. In many respects hemin occupies a central position in the
scheme. In its linkage to the porphyrin skeleton the iron atom
reaches a new level of catalytic activity. Under conditions where
simple iron salts decompose 10"^ moles of hydrogen peroxide, hemin
will split 10"^ moles, representing a thousandfold gain. That the
essential feature here is the iron-nitrogen-carbon bridge is indicated
by Warburg's classical experiments with charcoal prepared from
hemin, where this bridge apparently remains standing among the
ruins of the iron-porphyrin ring system and imparts a power to
oxidize cysteine and amino acids which is vastly superior to that

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OXIDASES, PEROXIDASES, AND CATALASE 79

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IRON CATALYSTS

Homogeneous Sysfems
Inorganic Fe- Salts

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IRON OXIDES (MITTASCH)

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Figure 1. — A family tree of iron catalysts

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84 A SYMPOSIUM ON RESPIRATORY ENZYMES

peroxidatic, and catalatic activity. But the enzyme peroxidase has
lost the power of splitting hydrogen peroxide without the presence
of an oxygen acceptor, and, conversely, the enzyme catalase is ap-
parently devoid of any peroxidatic activity. The faculty of reacting
with molecular oxygen, finally, is reserved to a small number of
autoxidizable hemin proteins, i.e., the respiratory fennents and the
Pasteur enzymes. The ultimate in specialization is reached with
hemoglobin, which alone of all iron derivatives and hemochromo-
gens will combine with but will not be oxidized by molecular oxy-
gen. The formally analogous reaction with carbon monoxide is
shared by most ferrous complexes, such as ferrous cysteine and
ferrohemochromogens of all types. As may be seen from Tables 4
and 5, at least two diflFerent types of hemins occur in hemin enzymes,
namely, the red protohemin and mixed-colored pheohemins.

Enzyme-Substrate Intermediates

It is diflBcult to understand why there should be so much con-
troversy over the existence of well-defined intermediates in the
course of enzymatic catalyses in general and hemin catalysis in
particular. Once it is admitted that enzymes, like all other catalysts,
exert their function not by mystical, long-range forces but by actu-
ally combining, at some stage of the process, with their substrates
and thus creating a new pathway which, although more complex,
yields a higher overall rate of reaction, it will depend solely on the
lifetime of these intermediates and on their spectroscopic or other
properties whether their existence can be detected by experimental
means. There are now on record several perfectly clear-cut cases
where such enzyme-substrate compounds have been demonstrated
by the spectroscope: the intermediate formed in the catalase-ethyl
hydrogen peroxide reaction (60), the complexes foiTiied between
peroxidase and various proportions of hydrogen peroxide (28), and
the less clear-cut observations on complexes between catalase,
hydrogen peroxide, and certain inhibitors, such as hydrazine and
hydroxy lamine (26). The first-named complex exhibits precisely the
behavior that the kinetic theories of Michaelis and Henri postulate
for an intermediate enzyme-substrate compound: it is unstable and
disappears at the rate at which the end products of the reaction are
formed. The peroxidase-hydrogen peroxide complexes are also un-
stable, but their decomposition cannot be due to a true peroxidatic
reaction, since it occurs also in the absence of oxygen acceptors.
However, the recent work by Karush (25) and Chance (12) on

OXIDASES, PEROXIDASES, AND CATALASE 85

rapid reactions affords further proof of the existence of enzyme-
substrate complexes as intermediates in the peroxidate catalysis.
The nature of the ternary complexes formed by catalase, hydrogen
peroxide, and some inhibitors is not yet clear. They may represent
ternary complexes between enzyme, substrate, and inhibitor, but
they may also be binary enzyme-substrate compounds the lifetime
of which is prolonged by the presence of the inhibitors. The inter-
pretation of the phenomena observed in the foregoing enzyme
reactions is reinforced considerably by observations made on such
closely related, non-enzymatic models as the methemoglobin-
hydrogen peroxide (21) and methemoglobin-ethyl hydrogen per-
oxide (58) complexes and the hemin-hydrogen peroxide intermedi-
ate (22). If we go back a little, we find that in 1905 Spitalsky
observed analogous phenomena in the chromic acid-hydrogen per-
oxide catalysis. It is safe to conclude that similar inteimediates arise
in all hemin-catalyzed reactions even if they cannot yet be demon-
strated experimentally. The writer has just been informed by
Professor Hogness that cytochrome peroxidase, too, forms a typical
enzyme-substrate complex with hydrogen peroxide.

The oxidation of ferrous iron to ferric iron by oxygen is usually
regarded as a process fundamentally different from the oxygenation
of hemoglobin. However, it would simplify matters a good deal if
both types of reactions could be brought to a common denominator.
In agreement with Haber* and Warburg the reviewer believes that
this common link is that in the ferrous-ferric transformation by
molecular oxygen an oxygenated intermediate of a structure analo-
gous to oxyhemoglobin is interposed (see Oppenheimer and Stern
(49), pp. 14ff.): Fe^+ + O, -» Fe^O^ -» Fe^^\

The reasons for this hypothesis are, first, that molecular oxygen,
despite its high potential ( + 0.8 volt), appears to be too sluggish an
oxidant to react rapidly with ferrous iron without prior activation.
It is logical to assume that the hydrogen peroxide, formed as a result
of the oxidation of the ferrous iron, arises by interaction of the
ferrous-oxygen intermediate with water molecules or hydrogen ions.
Secondly, the well-known competition of molecular oxygen and
carbon monoxide for the ferrous fomi of the respiratory ferment,
which is governed by Warburg's distribution equations, would be

* This concept appears applicable even to the simplest reactions in the gas
phase. Thus Haber and Sachsse (16), on the basis of kinetics experiments,
conclude tliat during the reaction of sodium vapor with oxygen one sodium
atom combines with one oxygen molecule.

86 A SYMPOSIUM ON RESPIRATORY ENZYMES

diflBcult to understand if the primary reaction of the enzyme with
oxygen were not a reversible equihbrium reaction. This is particu-
larly true for the photochemical dissociation of the iron-carbonyl
complex, where it is assumed that, under the influence of light,
carbon monoxide is reversibly exchanged with oxygen. All phe-
nomena may be satisfactorily explained on the basis of the assump-
tion that the primary step consists in the formation of a ferrous-
oxygen intermediate and that the further course of events is gov-
erned by the lifetime and reactivity of this complex. In the instance
of oxyhemoglobin the intermediate stage is fixed in a unique manner
under the influence of the globin component. In that of the oxidases,
the intermediate is very short-lived, with the possible exception of
the respiratory ferment in baker's yeast (75), where Warburg has
observed an absorption band which he tentatively attributes to an
oxygen addition compound of the enzyme. A unified theory of
oxidase, peroxidase, and catalase action could, then, be based on
postulating, with a fair degree of probability, the formation of
"moloxides" and "molperoxides," respectively, as the primary process
in the catalysis. The mechanism of the oxidation of hemoglobin to
methemoglobin by oxygen deviates from this schema because of
the high stability of oxyhemoglobin. The well-defined maximum
of reaction velocity at low oxygen pressures is in this instance
(10, 48) largely due to a decrease in the concentration of the free,
autoxidizable ferroform as the partial pressure of oxygen in the
system is increased. The rate of oxidation is proportional to the
concentration of reduced hemoglobin and to a function of the
oxygen pressure. In this case oxygen represents both a reactant and
an inhibitor, not only because of oxyhemoglobin formation but also,
perhaps, through breaking of chain reactions.

On the Mechanics of Hemin Catalyses

When considering reactions promoted by hemins, one almost in-
variably encounters the notion that they must all conform to the
pattern of the ferri-ferro cycle. There is no question that this is the
most handy explanation: everybody knows that the hemin iron may
exist in the reduced (Fe+^) and in the oxidized (Fe+++) state and that
this transformation may be accomplished in a reversible manner
even if no bases are linked to the heme. But like many generaliza-
tions, this concept as the only explanation is not only hazardous
but definitely too narrow.

Let us go back for a moment to the theories that have been ad-

OXIDASES, PEROXIDASES, AND CATALASE 87

vanced to explain the catalytic or semi-catalytic effects of iron and
other metal ions (37). As in the case of the hemins, we find a
widespread inclination to explain everything by a valency change
of the metal during catalysis: in the first stage the substrate reduces
the trivalent iron (or the bivalent copper) to bivalent iron (or mono-
valent copper); in the second stage the oxidizing agent, e.g., oxygen
or hydrogen peroxide, regenerates the ferric iron or cupric copper,
respectively. There are unquestionably instances where this simple
hypothesis will serve to explain all the facts, e.g., in the metal-
catalyzed oxidation of phenols by oxygen. But in many other in-
stances the hypothesis proves inadequate. Two of the main obstacles
are, first, the fact that bivalent iron salts and complexes are so
often superior in catalytic activity to the corresponding trivalent
compounds and, second, the phenomenon of the "primaerstoss"
(alpha-activity). By this we mean the frequent observation that
oxidation catalyses, in the presence of ferrous ions, exhibit an initial
phase of high velocity which is followed by a steady state of a
much lower reaction rate (beta-activity). The transition of ferrous
into ferric iron in the course of this process is accompanied by a
turnover of many more substrate molecules than would correspond
to the number of iron equivalents.

Three different theories have been proposed to explain the facts.
All postulate the fonnation of labile and highly reactive intermedi-
ates which are formulated as peroxides by Manchot, as complex
compounds by Wieland, and as free radicals by Christiansen, Baeck-
stroem, and Haber. Manchot assumes that only the bivalent iron
is capable of forming peroxides of the type Fe^Og or, more recently,
of molperoxides of the type Fe'^^HoOg. Such peroxides could oxidize
two substrate equivalents and, subsequently, react with hydrogen
peroxide present in excess to yield inactive ferric iron, or they could
interact with excess hydrogen peroxide to form oxygen while the
ferrous iron is regenerated. Wieland, on the other hand, believes
that the ferrous iron forms a complex with the substrate or with
other substances present in the system with the possible inclusion
of the oxidizing agent. By the complex formation tlie substrate
hydrogen is "activated" and is thus made accessible to the attack
by the oxidant. The alpha-activity ("primaerstoss") is attributed to a
temporary protection of the active ferrous iron contained in the
complex against the transition into inactive ferric iron. In this way
one ferrous ion is enabled to oxidize a larger number of substrate
molecules, the oxidized molecules being released from the complex

88 A SYMPOSIUM ON RESPIRATORY ENZYMES

and replaced by fresh substrate molecules. In the well-known chain
reaction schemas, as developed by a number of authors, the metal
is assumed to initiate the reaction by reacting with a substrate
molecule and producing a molecule of high energy content ("energy
chains" of Christiansen and of Baeckstroem) or monovalent radicals
of high reactivity ("radical chains" of Haber and Franck), where-
upon the chain is propagated by such intermediary radicals or
"hot" molecules without the further participation of the metal.
The chain length, i.e., the yield in product molecules per elementary
act initiating the process, is determined by the probability with
which two of the intermediary radicals or energy-rich molecules
will collide and inactivate each other, and by the presence or ab-
sence of specific inhibitors, so-called "chain-breakers." It is im-
portant to note that in the metal catalysis of sulfite oxidation, cupric
copper and ferrous iron but not ferric iron are able to initiate
reaction chains (14). In this connection, the work of Haber and
Weiss (17) on the decomposition of hydrogen peroxide by ferrous
salt and the nature of the alpha -activity is of particular interest.
As is well known, a small amount of ferrous salt, when brought
together with a large excess of hydrogen peroxide, is oxidized to
ferric salt with the simultaneous liberation of oxygen. The yield
depends upon the rate at which the two reactants mix; the ratio
AHgOa/AFe'^'^ may reach values as high as 15.6. The underlying chain
reaction is formulated by the authors as follows:

FV" + H2O2 = Fe^^^OH + OH
OH + H2O2 = H2O + OH
OH + H2O2 = 02 + H2O + OH
Fe^^ + OH = Fe^^^OH

The last equation depicts a chain-breaking reaction. If all the
Fe'^^ has been oxidized, the reaction stops.* It is perfectly true that
this process, like many reactions studied by Wieland and his stu-
dents, is not a true catalysis but an induced reaction. But it is like
the true catalyses in that small amounts of a promoter or inductor
bring about the reaction of a disproportionately large number of
substrate molecules. On the other hand, we know that all enzymes
are slowly but irreversibly "consumed" during the reactions which
they catalyze. Only a finite, although large, amount of protein or
hydrogen peroxide can be split by a given quantity of proteinase

" For an analysis of the catalytic decomposition of hydrogen peroxide by
ferric salts in acid solution the reader is referred to the papers by Haber and
Weiss (18), and Kuhn and Wassermann (36).

OXIDASES, PEROXIDASES, AND CATALASE 89

or catalase, respectively. This observation is usually interpreted in
terms of an irreversible destruction of the active protein component
of the enzyme in some side reaction, e.g., by denaturation or as a
result of attack by other enzymes present as impurities. While this
may be so, it is not always easy to distinguish clearly between a
true catalysis, where the catalyst is progressively eliminated by side
reactions, and an induced reaction, where the inductor is slowly
converted into an inactive form. This is particularly true of experi-
ments in biological systems. According to all three theories outlined
above, the reaction comes to a standstill once all ferrous iron has
been converted into ferric iron. A continuation of the process is
obviously possible only if the substrate or some other component
in the system is able to reduce Fe^^^ back to Fe^^.* One could
visualize "hybrid" processes where the ferrous iron, in the main
reaction, acts as an inductor and where the inactive ferric iron
is slowly reduced to the active ferrous form by some "outsider"
such as a thiol and is thereby enabled to start the induced reaction
all over again. Such a situation, if encountered in living cells, would
probably defy any attempt to distinguish between true and apparent
catalysis, especially if the reducer is constantly replenished from
suitable precursors, e.g., glutathione from protein breakdown.

We go one step further. If iron can break down the potential
barrier, shielding stable substrate molecules, either by the ferri-
ferro cycle or by the induced reaction mechanisms just mentioned,
is it not possible that iron, when linked up in suitable complexes,
could bring about changes in certain substrate molecules or initiate
chain reactions without itself suffering a change in valency? It is
on this possibility that the issue of the mechanism of catalase action
largely hinges. It is well established that the iron in catalase exists
in a remarkably stable ferri state. The enzyme is invariably isolated
from all sources as the ferri foiTn and it defies reduction with
activated hydrogen or with hydrosulfite. This property is unique
among hemin proteins; it is not shared even by peroxidase, which
may be readily reduced to the ferro form by agents such as sodium
hydrosulfite. Moreover, it can be shown that the spectroscopically
well-defined intermediate arising in the catalase-ethyl hydrogen

* Theoretically, ferri ion could act catalytically by being reversibly oxidized
to a higher stage of valency. Supporting such a view are the spectroscopic
observations of Bohnson and Robertson (J. Amer. Chem. Soc, 45, 2493 (1923),
on weak acid solutions of ferric salts in the presence of hydrogen peroxide. The
color changes occurring in this system were interpreted by these workers to
be due to the formation of ferric acid. See, however, Haber and Weiss ( 18 ) .

90 A SYMPOSIUM ON RESPIRATORY ENZYMES

peroxide reaction contains the enzyme in the ferric state (61).
Carbon monoxide, which will inhibit many hemin catalyses where
ferrous iron is involved, has little or no specific effect on the catalase-
hydrogen peroxide reaction (61), other reports to the contrary
notwithstanding. Some years ago Haber and Willstaetter (19) pro-
posed a chain reaction schema for this catalysis which embodied,
as the initial step, a reduction of the enzyme iron to the ferrous
form. A little later the present writer modified this schema some-
what (55) with a view to avoiding the necessity of assuming a
ferri-ferro cycle. The initial step in that schema consisted in the
interaction of two adjacent porphyrin-bound ferri atoms with one
hydrogen peroxide atom to yield two monovalent OH-radicals which
could then propagate the chain. The iron was assumed to remain in the
trivalent form throughout. More recently Keilin and Hartree have
advanced a different hypothesis, incorporating the idea of the ferri-
ferro cycle (27):

Step I 4 Fe"*^ + 2 H^O^ = 4 Fe"^ + 4 H" + 2 O,

Step II 4 Fe^" + 4 H^ + O^ = 4 Fe^^^ + 2 H2O

2 H2O2 = 2 H2O + O2

The authors state that, in accordance with this schema, the
catalysis is greatly inhibited or even suspended in the absence of
free oxygen. It will be noted that the hydrogen peroxide is here
assigned the role of a specific reducer of the ferri form of catalase,
whereas molecular oxygen is considered as the oxidizing agent in
the regeneration of the ferri form. The concept of hydrogen peroxide
as a reducing agent in itself, although somewhat startling, is not
new and finds support in the earlier finding of Kuhn and Wasser-
mann (36) that ferric salts, in the presence of such complex formers
as a, a'-dipyridyl or o-phenanthroline, are quantitatively reduced by
hydrogen peroxide. But the schema is open to other objections, both
on theoretical and on experimental grounds. Thus Johnson and
van Schouwenburg (24), Weiss and Weil-Malherbe (80), Sumner and
Dounce (cf. 68), and the writer (61) failed to confirm the observation
of Keilin and Hartree that catalase is inactive under strictly an-
aerobic conditions. Furthermore, it seems somewhat strange that,
according to the schema of these workers, molecular oxygen should
be required for the reoxidation of the ferrous form of catalase in a
system containing hydrogen peroxide, which is generally considered
a more active oxidizing agent than oxygen, as Dr. M. Gorin has
pointed out, in a private communication to the writer. According

OXIDASES, PEROXIDASES, AND CATALASE 91

to Dr. Gorin, one would expect, if Keilin's theory were correct, that
the reduced form of catalase would accumulate in contact with
hydrogen peroxide and in the absence of air. The spectrum of the
ferro form of the enzyme, as obtained by treatment with sodium
hydrosulfite in the presence of hydrogen sulfide and subsequent
removal of the latter, has recently been described by Zeile et al.
(87). No spectral change, on the other hand, has as yet been reported
for a mixture of catalase and hydrogen peroxide under nitrogen.
Attempts to "catch" those ferro-catalase molecules, which might
possibly be formed as intermediate products during the catalase-
hydrogen peroxide reaction, with the aid of carbon monoxide have
been unsuccessful (61). The story of the eflFect of carbon monoxide
on catalase under various conditions is just as controversial as the
subject of its reaction mechanism (cf. 11, 61, 27). It may suffice
here to mention Keihn and Hartree's observations (27) that purified
carbon monoxide, in the absence of oxygen, exerts an inhibiting
eflFect on the enzyme which is not reheved by light, whereas certain
catalase preparations may be made sensitive to carbon monoxide
inhibition in the presence of oxygen by adding traces of azide,
cysteine, and glutathione. This eflFect of carbon monoxide is stated
to be completely relieved in a reversible manner by light. The
mechanism of this "sensitization" and the reason why crude enzyme
preparations are more readily inhibited by carbon monoxide than
chemically purified fractions are still obscure. It would seem that, for
the present at least, the observations made with the use of carbon
monoxide aflFord no basis for supporting or rejecting Keilin and
Hartree's reaction schema. The same is true, in the writer's opinion,
of the spectroscopic observations made by Keihn and Hartree (26)
on catalase solutions containing sodium azide or hydroxylamine in
addition to hydrogen peroxide.

Let us now turn to the theoretical objections raised against Keilin
and Hartree's schema. As Weiss and Weil-Malherbe (80) point out,
an exclusive reoxidation of ferro-catalase by oxygen would in eflFect
prevent a decomposition of the hydrogen peroxide. For unless a
radical chain mechanism is postulated, the oxygen formed by the
reduction of the ferri form of the enzyme by hydrogen peroxide
(Step I, p. 90) is quantitatively used up again for the reoxidation
of a stoichiometric amount of the ferrous form with a simultaneous
reduction of the oxygen to hydrogen peroxide. To avoid this diffi-
culty, Keilin and Hartree formulate the oxidation reaction (Step II,
p. 90) in such a way that the reduction of oxygen to water does

92 A SYMPOSIUM ON RESPIRATORY ENZYMES

not involve the intermediary formation of hydrogen peroxide. This
is in opposition to the general views on the mechanism of autoxi-
dation and would appear highly improbable, since it postulates a
reaction of a very high order. Furthermore, on the basis of Keilin
and Hartree's hypothesis, the reaction, when proceeding in nitrogen,
should exhibit the characteristics of an autocatalytic reaction be-
cause of the production of increasing amounts of oxygen during the
process. This, however, is not indicated in the data of these authors.
Sumner and Dounce (cf. 68), who also were unable to confirm Keilin
and Hartree's observations with respect to the importance of oxygen
for catalase action, prefer the following schema, which is based on
earlier ideas of Haber, Euler, and Liebermann:

Step I Fe-OH + H^O^ = Fe-OOH + H2O

Step II Fe-OOH + H2O2 = Fe-OH + H2O + O2

In this schema, catalase in its ferric form is represented by the
symbol Fe-OH, which is often employed for methemoglobin, a
molecule very similar to catalase. The symbol Fe-OOH represents
an intermediary catalase peroxide which is assumed to react with
a fresh substrate molecule to yield oxygen, water, and the free ferri
form of the enzyme. It will be noted that this hypothetical schema,
in contrast to that of Keilin and Hartree, does not involve the ferri-
ferro cycle and is in agreement with the spectroscopic observation
of a catalase-peroxide complex in the enzyme-ethyl hydrogen per-
oxide reaction (58, 60). But the writer doubts very much whether
so simple a schema is adequate to explain all the features of the
enzymatic catalysis. We must not forget that the decomposition of
hydrogen peroxide can be catalyzed by a variety of agencies besides
the enzyme, such as ultraviolet light, dust, metallic and non-metallic
surfaces, colloidal platinum, inorganic ferri and ferro salts, cobalti
salts, etc. We are therefore confronted with the necessity of finding
an explanation for the reaction mechanism which will be equally
applicable to these various catalysts. The central theme is, of course,
the mode in which hydrogen peroxide is transfonned into water
and oxygen. The most cogent formulation for this central process
has been given by Haber and Weiss (18):

( 1 ) OH -f H2O2 = H2O + HOl

(2) Ha + HaOj = O2 -(- H2O + OH

In this schema OH and HO. are monovalent radicals. As Haber em-
phasizes, in a paper published posthumously (18), the progress of

OXIDASES, PEROXIDASES, AND CATALASE 93

the reaction through radicals is the main point of the concept, while
the propagation of reaction chains through such radicals, although
an interesting phenomenon, is of secondary importance. In fact, the
two features are not necessarily associated with each other. The
detailed study of the kinetics of the decomposition of hydrogen
peroxide by ferrous and ferric salts suggests strongly that under
certain conditions the radicals may give rise directly to the formation
of the end products, water and oxygen, without initiating a chain
by further reacting with hydrogen peroxide and thereby repro-
ducing themselves. Thus the catalytic breakdown of hydrogen per-
oxide by ferri ions in acid medium does not, in general, represent
a chain reaction; the oxygen is released here by the process

(3) Fe^^^ + Ha = Fe*" + H^ + O2

Upon slightly changing the experimental conditions, e.g., by
increasing the hydrogen peroxide concentration or decreasing the
Fe'^^'^, reaction 2 is favored, with the result that chains appear (18).

The second question relates to the way in which the central
process, consisting of steps 1 and 2, is initiated by the various
catalytic agents. The radicals OH and/or HO, can be created only
by a monovalent attack on hydrogen peroxide. It is the monovalent
character of the primary reaction of the catalyst with the substrate
which, according to Haber and Weiss (18, 78), is the common feature
of the chemical, photochemical, and electrochemical primary proc-
esses. Thus the radicals may arise by the transformation of a metal
ion into the one of next higher valency, as in the instance of ferrous
ions,

(4) Fe"" + n^O. = Fe"^^ + OH- + OH

or by the reduction of a higher valent state, as in the case of ferric
ions:

(5) Fe^*^ + HO=- = Fe^" + Ma

Hydrogen peroxide may also be split into two OH radicals by
ultraviolet light:

(6) H202 + hv = 2 OH

In heterogeneous systems, involving metal surfaces, the radical OH
is beUeved to result from a simple electron transfer,

(7) H,02 + electron („,etai) = OH- + OH

While reaction 7 is an expression of the oxidizing action of H2O2,
the reducing properties of the molecule are attributed to its anion
(HO2"), in accordance with the equation

94 A SYMPOSIUM ON RESPIRATORY ENZYMES

( 8 ) HO2- = Ha + electron

Both these functions are related by the dissociation equihbrium

(9) H2O2 ^11"^ + HOr (dissociation constant, K)

It follows that there is no fundamental difference between the
homogeneous and heterogeneous reactions with regard to the ele-
mentary process. Weiss (79) has recently discussed the mechanism
of catalase and peroxidase action from the same point of view. In
his presentation of the central reaction, step 2 is replaced by the
equation

(2a ) O2- + U2O2 = OH- + OH + O2

where O2- has been substituted for HO^ on the basis of new evidence.
These two radicals are related to each other by the electrolytic dis-
sociation equilibrium

(2b) O2- + H* <^ HO2 (dissociation constant, KHO2).

Weiss treats the catalase-hydrogen peroxide reaction as a hetero-
geneous catalysis and as an analogon to the catalytic decomposition
of the same substrate by colloidal platinum, which has been so
carefully studied by Bredig. The similarity is accentuated by the
fact that neither catalase nor colloidal platinum shows peroxidase
activity toward acceptors of the type of iodide ion or pyrogallol. In
both instances the primary process is formulated as a surface reac-
tion involving the radicals OH and HO,. The author assumes that
the iron atom of the enzyme is alternatively reduced to the ferrous
and reoxidized to the ferric form by the hydrogen peroxide during
the course of the catalysis. The role of the metal atom in the
porphyrin skeleton is regarded as that of facilitating rapid electron
transfers, since the valency change of the iron in the porphyrin ring
system takes place without appreciable dislocation of heavy par-
ticles, in contrast to the situation in the instance of free ferrous and
ferric ions, where the water dipoles in the hydration shell must
undergo rearrangements upon a change in the charge of the central
atom. Furthermore, he believes that the system of conjugated double
bonds surrounding the iron in the heme group makes for a rapid
"conduction" of the inner electron by virtue of their loosely held
n-electrons.

In analogy to the action of colloidal platinum on hydrogen per-
oxide, as formulated by the same author, the mechanism of catalase
action is depicted as follows:

OXIDASES, PEROXIDASES, AND CATALASE 95

(10) Fe^^^ + HOr ^ Fe^- + HO2

(11) Fe^^ + H2O2 = Fe^^^ + OH- + OH

( 12 ) Fe+^ + OH = Fe*^^ + OH"

The production of molecular oxygen is attributed to reactions 1
and 2a.

It will be noted that the process, although involving free radicals,
is not a conventional chain reaction, since the enzyme iron plays
an active role in three stages: 10, 11, and 12. This is partly based on
the finding that in the model system platinum-hydrogen peroxide
an average chain length of only about five links may be assumed.
It is therefore considered probable that in the enzymatic catalysis,
under the usual experimental conditions, the chain reaction prac-
tically degenerates to a simple radical reaction. It will be recalled
that one of the criticisms directed against the chain reaction theory
of Haber and Willstaetter by Haldane was that one would expect
the rate to be proportional to the square root of the enzyme con-
centration rather than to the enzyme concentration itself, as is
actually the case. By postulating very short chains, as Weiss does,
the feature of the proportionality between reaction velocity, enzyme
concentration, and substrate concentration is retained without sacri-
ficing the essential concept of Haber, i.e., the postulate of inter-
mediate, monovalent radical formation. The further objection of
Haldane that the assumption of the same type of radicals (OH and
HO2) in various kinds of enzyme reactions, as was done by Haber
and Willstaetter, was in conflict with the well-known specificity
of the oxidizing enzymes, is also met if very short chains are as-
sumed. In this case the radicals are present only in so low a con-
centration that their oxidizing action on acceptors, e.g., iodide ion
or oxyphenols, remains below the threshold of sensitivity of the
analytical methods. All these considerations refer to the "normal"
course of the catalase reaction, i.e., to conditions where the enzyme
and the substrate concentration are within the range usually em-
ployed in kinetic studies and activity determinations. There can be
little doubt that the explosive type of hydrogen peroxide decompo-
sition, such as is produced in concentrated peroxide solutions when
a relatively large amount of enzyme is added, represents a chain
process with a long chain length. There may even be branched
chains, such as are assumed to occur during "knocking" in internal
combustion engines, when the relatively slow combustion along a
flame front of regular rate of progression changes over into detona-
tion (cf. Lewis and van Elbe, 40).

96 A SYMPOSIUM ON RESPIRATORY ENZYMES

It is not feasible, within the space allotted to this discussion, to
cite all the experimental evidence and theoretical arguments in
favor of the intermediary formation of free radicals or of the chain
reaction character of the hydrogen peroxide catalysis or of oxidative
enzyme action in general. It must suffice, therefore, to refer to the
original publications where the existence of OH radicals during
hydrogen peroxide photolysis (73a) or the formation of the HO2
in reactions with molecular oxygen (78a) have been observed. The
experiments of Schwab et al. (53a) on the effect of typical chain-
breakers on the catalase reaction did not yield conclusive results.
But the experiments of Barron (5) on the effect of antioxidants
on the rate of oxidation of unsaturated fatty acids by hemochromo-
gens are very suggestive. The pioneer work of Michaelis on the
radical nature of the semiquinones, which arise through monovalent
reduction or oxidation of reversible dyestuffs makes it all but im-
perative to admit the intermediary existence of monovalent radicals
(monohydropyridine, monohydroalloxazine, monohydrothiamine,
etc.) during cellular respiration, unless reactions of high order are
postulated.

Those who are interested in the manner in which the action of
other oxidative enzymes, e.g., peroxidase or dehydrogenases, may be
interpreted in terms of radical chain mechanisms are referred to the
papers by Haber and Willstaetter (19), Weiss (79), and the review
article by Moelwyn-Hughes (45).

AUTOXIDIZABLE IrON COMPOUNDS

We return to the starting point of the discussion: What makes
the iron atom and the oxygen molecule "click" during the primary
process? Why are some iron compounds autoxidizable and others
not? No satisfactory answer may at present be given to this ques-
tion, which certainly attracts the attention of many workers in the
field of biological oxidation. Only partial solutions have been of-
fered to this key problem.

Ferrous iron is relatively stable in acid solution and is rapidly
oxidized to ferric iron by molecular oxygen in alkaline solution.
The state of the metal under these two conditions obviously differs
in one respect. In the acid medium the iron is present as ferrous ion,
whereas in alkaline solution it exists as non-ionized ferrous hydrox-
ide. Smythe (54), in an interesting paper, points out that the oxi-
dation of ferrous ion involves the separation of a negative charge

OXIDASES, PEROXIDASES, AND CATALASE 97

(electron) from a nucleus which already carries two positive charges.
Non-ionized ferrous hydroxide, on the other hand, should be more
easily oxidized, since this process represents only the separation of
the electron from an electroneutral substance. If this reasoning is
sound, one would expect that ferrous iron, if built into a non-ionized
compound, should be readily autoxidizable regardless of the acidity
of the medium. The rate of autoxidation of ferrous complexes would
then be expected to be a function of the pH only in so far as the
stability of the complex is affected by changes in hydrion concen-
tration. In other words, under any given condition the velocity
of the reaction with oxygen should be proportional to the concen-
tration of the ferrous complex, which in turn is a function of the
concentrations of the ferrous ions and the complex-forming anions.
Since the latter, within a given pH range, depends on the hydrion
concentration, one would expect that within this range the rate of
oxidation would vary with the pH. Smythe proceeded to test this
working hypothesis on a series of inorganic and organic iron com-
plexes of relatively simple configuration. His manometric study of
the rate of autoxidation of ferrous pyrophosphate and ferrous meta-
phosphate, which are stable over a wide pH range, bore out the
prediction. The reaction rate varied strongly with the hydrion con-
centration, decreasing markedly in both instances with an increase
in acidity. The case of ferrocyanide is of special interest because its
structure bears a certain resemblance to the core of iron porphyrin
complexes. In both instances the iron atom is surrounded by four
groups, containing nitrogen linked to carbon atoms. In the case
of the ferrocyanide these groups carry negative charges. To escape
from the metal atom, an electron must pierce this "negative atmos-
phere," which is obviously difficult. We are not surprised, therefore,
to find that the rate of autoxidation of ferrocyanide is extremely small
in spite of the fact that the iron is present in covalent rather than in
ionic linkage. If it were possible to reduce the negative charge on
the groups encircling the iron atom, its reaction with oxygen should
be facilitated. Indeed we find that if the charge is reduced by re-
placing one of the CN~ groups by ammonia, a compound, penta-
cyanoammine-ferroate, is formed which is attacked by oxygen at
an appreciable rate. The striking observation of Baudisch and
Davidsohn (7) that the oxygen uptake is more rapid at pH 2 than
at 7 or 12, in contrast to the findings obtained with iron pyro- or
metaphosphate, is explained by Smythe in terms of a suppression of
the ionization under the influence of the increased hydrion concen-

98 A SYMPOSIUM ON RESPIRATORY ENZYMES

tration and a consequent decrease of the negative charges around
the iron atom.

However, Smythe's views are not shared by Weiss (78). This
author beheves, in direct contradiction to the hypothesis that only
coordinatively bound ferrous iron is autoxidizable, that only "free"
ferrous ions react, in general, with molecular oxygen. By "free" ions
Weiss means iron with incompletely filled electron orbits. The
autoxidation of ferrous sulfate is formulated in accordance with
Weiss's theory of simple electron transfers as follows:

O2- + H* ?^ HOT

(13)

Fe^^ + O2 -> Fe^^^ + O^-,

(14)

Fe^^^ + O2- -> Fe^* + O2

(15)

Fe^^ + HO2 -^ Fe^^^ +HO2-,

HO2- + H^ = H2O2
Equation 15 explains the formation of hydrogen peroxide as an
end product of autoxidation processes. If, however, it is not caught
in statu nascendi, e.g., by cerium hydroxide, the hydrogen peroxide
is relatively rapidly decomposed by reaction with ferrous iron-
ferric ions and hydroxyl ions or water being formed as final prod-
ucts. During the entire process the oxygen molecule is stepwise
reduced by four electrons with the formation of four hydroxyl ions.

The sequence given above, according to Weiss, provides a logical
explanation for the slow rate at which an acidified ferrous sulfate
solution is autoxidized. The latter does not indicate that the inter-
action between the ferrous ions and oxygen (reaction 13) is slow
but, on the contrary, that the rate of the back reaction 14, which
involves a reduction of ferric ions, is quantitatively significant.
Under stationary conditions and for a given partial tension of oxy-
gen, the rate of reaction 15 is defined by the ratio of the velocities
of the partial reactions 13 and 14; in other words, by the ratio
[Fe^*] to [Fe^^*]. The greater this ratio the more positive will be the
oxidation-reduction potential and the higher will be the rate of
ferrous salt oxidation. The essential feature in reactions of this type,
according to Weiss, is not the formation of stable ferrous iron com-
plexes but the fact that the ferric ions are protected against reduc-
tion by the formation of still more stable ferric iron complexes. Thus,
in instances where the ferrous complex is more stable than the corre-
sponding ferric complex, as in the case of ferrous tridipyridyl sulfate,
no autoxidation takes place.

A decidedly more mechanistic explanation is offered by Theorell
(73) for the failure of cytochrome c to react with molecular oxygen
in the physiological pH range: "The heme of the cytochrome is
. . . built into the protein component in a manifold way: by means

OXIDASES, PEROXIDASES, AND CATALASE 99

of thioether bindings from the side chains of the porphyrin to the
protein, and by means of two histidine-imidazole groups strongly
bound to Fe on each side of the flat heme disc. Thus the heme group
appears to be built into a crevice in the protein molecule. This ex-
plains why cytochrome c is not autoxidizable, since oxygen can never
approach the iron atom, and why no CO-compounds or cyanide
compounds are formed at physiological pH values."

Oxygen Transfer in Living Cells

In every aerobically living cell we find a number of hemin pro-
teins, e.g., a respiratory ferment, three diflFerent cytochromes, cata-
lase and/or peroxidase. There is also frequently present what Keilin
calls the "unspecific cell hematin." More recently another functional
type of hemin enzymes has been found in such cells, which cata-

CHAIN OF RESPIRATORY CATALYSTS

Op-

RESPIRATORY CYTOCHROMES ^CYTOCHROME ^PYRIDINE.

■ ENZYME * A-»C^B REDUCTASE ENZYMES

SEQUENCE DURING PASTEUR REACTION

PASTEUR
ENZYME

FERROUS

IRON
CATALYST

FERI^ENTATION
ENZYME
SYSTEM

SUBSTRATES

STARCH

GLYCOGEN

GLUCOSE

FRUCTOSE

Figure 2. — Function of molecular oxygen in respiration and Pasteur reaction

lyzes the inhibiting eflFect of oxygen on fermentation and glycolysis
(Pasteur reaction). Some data pertaining to these various iron por-
phyrin proteins are given in Tables 4 and 5.

Two groups of these intracellular hemin proteins are endowed
with the power to react directly with molecular oxygen: the oxygen-
transferring enzymes of respiration (for short, "Warburg enzymes")
and the aerobic fermentation-inhibiting catalysts (for short, "Pasteur
enzymes"). Inasmuch as the Pasteur reaction is the subject of an-
other paper, it must suffice here to present only the particular
working hypothesis which the writer is advancing on the basis
of recent work in this laboratory. See Figure 2.

Photochemical work (44, 61) has revealed the pheohemin nature
of the prosthetic group of the Pasteur enzymes in rat retina and in

100 A SYMPOSIUM ON RESPIRATORY ENZYMES

baker's yeast. It is thus seen that catalysts of the same type, al-
though differing in details, have been developed by nature to keep
in check both alcoholic and lactic acid fermentation in the presence
of oxygen. As Figure 2 indicates, oxygen is "mobilized" for its two
chief tasks in aerobic cell life by two autoxidizable hemin proteins
(Warburg and Pasteur enzymes) acting in analogous manner but
independently of each other. The effect of a variation of the oxygen
tension (and, for that matter, of carbon monoxide concentration in
inhibitor experiments) on the overall phenomena of cellular respira-
tion and aerobic fermentation will depend on the affinity which the
iron contained in the two types of enzymes has for these gases.
Just as is the case with the respiratory transport protein hemoglobin
in various vertebrate species, there will be variations in the gas-
dissociation curves for these enzymes from cell to cell. Depending
on whether a particular Pasteur catalyst has a higher or a lower
aflBnity for oxygen or carbon monoxide than the Warburg ferment
in the same cell, we will expect to find respiration more or less
readily afiFected by a lowering of oxygen tension or a given ratio
of carbon monoxide to oxygen than the Pasteur reaction in that cell.
If these gases do not vary in their effect on the two processes, as
Warren (unpublished observations) found to be true in the case of
bone marrow, it must be because the affinity of the enzymes for
the gases is equal. In general it would appear that in higher animal
tissues the Warburg ferment has a greater affinity for oxygen and a
lesser affinity for carbon monoxide than the Pasteur enzyme in the
same tissue (Laser effect, 39); in unicellular systems (certain bac-
teria, human myeolocytes) the reverse seems to be true (Kempner
effect, 29). It would appear that there are as many (slightly) different
Warburg and Pasteur enzymes as there are living forms. Probably
the difference resides in the protein rather than in the hemin group
of the molecules, except in the case of Azotobacter, which seems to
have a Warburg enzyme with a green hemin rather than a mixed-
colored or pheohemin in the prosthetic group (47). The reasons for
interposing a hypothetical ferrous iron catalyst between the Pasteur
enzyme and the fermentation system, as indicated in the figure,
cannot be explained here because of lack of space.

In closing, some model experiments may be mentioned which are
being carried out at present in this laboratory. Some years ago, Lip-
mann (41) showed that a Pasteur effect in cell-free systems (yeast
and muscle fermentation extracts) may be produced by the addition
of suitable reversible dyestuffs of suflBciently high potential (see

OXIDASES, PEROXIDASES, AND CATALASE

101

Figure 3). If our concept of the mechanism of the Pasteur reaction
is correct, it should be possible to replace the dyestuffs in Lipmann's
experiments by an autoxidizable hemochromogen of suitable poten-
tial. Pyridine, histidine, and picoline hemochromogen have been
tried without positive results. The experiments with the very posi-
tive systems nicotine and nicotinic acid amide hemochromogen, on

PASTEUR
ENZYME

FERMENTATION
ENZYME SYSTEM

SUBSTRATE

2.6-Dichlorophenol

GLYCOLYZING

O2 * fndophenol

MUSCLE * STARCH

Eo=-0.l89(pH74)

EXTRACT

Naphthol-Sulfonafe-

FERMENTING fCLUCOSE

O2 — » mdophenol

YEAST MACERATION — > < ♦Hexose Diphosphafe

Eo=*Ol47v.(pH6.6)

EXTRACT [sucrose

Nicotine Fern-

Hemochromogen

Eo=>*0.200v.(pH6.0)

FERMENTING
YEAST MACERATION
EXTRACT

["glucose

<^fructose

[sucrose

Figure 3. — Attempts at reconstruction of Pasteur reaction in cell-free systems

the other hand, have yielded some encouraging results which, un-
fortunately, did not prove to be reproducible at will. One has the
impression that the underlying idea is correct but that some of the
factors involved in the complex reaction have thus far evaded
adequate control.

The problem of the Pasteur reaction, as the writer sees it, is now
shifting from the nature of the catalyst to the nature of its substrate.

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1939).

50. Reid, a., Ergebnisse d. Enzymforschung, J, 325 (1932).

51. Reid, A., Z. Angew. Chem., 47, 515 (1934).

OXIDASES, PEROXIDASES, AND CATALASE 103

52. Reuter, F., Willstaedt, H., and Zirm, K. L., Biochem. Z., 261, 353
(1933).

53. Robinson, M. E., Biochem. J., 18, 255 (1934).

53a. Schwab, G. M., Rosenfeld, B., and Rudolph, L., Ber. Deutsch. Chem.
Ges., 66, 661 (1933).

54. Smythe, C. v., J. Biol. Chem., 90, 251 (1931).

55. Stern, K. G., Z. physiol. Chem., 209, 176 (1932).

56. Stern, K. G., Z. physiol. Chem., 215, 35 (1933).

57. Stern, K. G., Z. physiol. Chem., 219, 105 (1933).

58. Stern, K. G., Nature, 136, 335 (1935).

59. Stern, K. G., J. Biol. Chem., 112, 661 (1936).

60. Stern, K. G., J. Biol. Chem., 114, 473 ( 1936); Enzymologia, 4, 145 ( 1937).

61. Stern, K. G., J. Gen. Physiol., 20, 631 (1937).

62. Stern, K. G., Yale J. Biol, and Med., 10, 161 ( 1937).

63. Stern, K. G., Cold Spring Harbor Symposia on Quantitative Biology, 7,
312 (1939).

64. Stern, K. G., and Gordon, W., unpublished observations.

65. Stern, K. G., and Kegeles, G., unpublished.

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67. Stern, K. G., and Wyckoff, R. W. G., J. Biol. Chem., 124, 573 (1938).

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71. SvEDBERG, Tn., J. Biol. Chem., 103, 311 (1933).

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Taylor, H. S., and Gould, A., J. Am. Chem. Soc, 55, 859 (1933).

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Nicotinamide Nucleotide Enzymes

FRITZ SCHLENK

School of Medicine, University of Texas

REPORTS OF INVESTIGATIONS in the field of the nicotinamide nu-
^ cleotide enzymes are now so numerous as to make impossible
a complete review of the subject here; and in any case it would seem
to be unnecessary in view of the comprehensive articles that have
been published in recent years (la-k). Emphasis will therefore be
laid upon those details that have not been extensively discussed in
previous articles and to report some recent advances.

CODEHYDROGENASE I AND II

Of the two coenzymes in this group, codehydrogenase I (cozymase,
diphosphopyridine nucleotide, Co I), was detected in 1906 by
Harden as a coenzyme of alcoholic fermentation (2). The other,
codehydrogenase II (triphosphopyridine nucleotide, Co II), was dis-
covered in 1932 by Warburg (3). In a series of investigations by the
von Euler school (1921-34) methods for the purification and deter-
mination of cozymase were elaborated. Its classification as a nu-
cleotide was ascertained, its codehydrogenase nature was pro-
pounded, and the numerous reports denying its existence were
refuted (le, f). In 1934 the most noteworthy discovery in the field
was made: Warburg and Christian isolated nicotinamide from co-
dehydrogenase II (4) and demonstrated its function as part of a
hydrogen-transporting coenzyme (5). In 1935 nicotinamide was
isolated also from cozymase (6), and soon the very close relationship
between these two coenzymes was established by the work of the
institutes in Berlin and Stockholm.

Both coenzymes are nicotinamide-adenine dinucleotides, the only
difference between them being that codehydrogenase II contains
three molecules, and codehydrogenase I two molecules, of phosphoric
acid. Later the transformation of one coenzyme into the other was
achieved by enzymatic dephosphorylation of codehydrogenase II
and enzymatic as well as chemical phosphorylation of codehydro-
genase I (7). The yield of codehydrogenase II by this reaction is,
however, rather low; the isolation from red blood cells is still the

104

NICOTINAMIDE NUCLEOTIDE ENZYMES 105

only method that has been described for preparing it in the pure
state.

Codehydrogenase II occurs in much lower concentration than
codehydrogenase I. In Table 1 are given some typical examples
of the occurrence of the two coenzymes. The values are only ap-
proximate because crude extracts were used for the determinations.
With improved methods of determination it probably will be neces-
sary to revise these values considerably, especially those for co-
dehydrogenase II.

Table 1.— Some examples of the occurrence of the codehydrogenases

I and II

Codehydrogenase content in micrograms

_, . , . , per gram of fresh material
Material exammed

Co I Co II

Bottom yeast >500 <10

Top yeast >500 5-10

Erythrocytes (horse) 100 >12

Liver (rat) >200 30

Muscle (rat) 200 50

Kidney (rat) 160 40

It is remarkable that despite the low content of codehydrogenase
II in the source material and the difficulty and lengthiness of the
isolation procedure, the preparation of codehydrogenase II in a
pure state, the isolation of nicotinamide therefrom, and the demon-
stration of its mode of action were carried out by Warburg and
Christian in a remarkably short time. This work greatly facilitated
the isolation of nicotinamide from cozymase and the subsequent
work on this coenzyme in the Stockholm institute.

In Tables 2 and 3 the methods of isolating the two compounds are
shown schematically. The procedures are diflFerent in the two cases,
but typical steps of purification known from earUer work in nucleo-
tide chemistry are involved in both instances. Significant steps,
some of them new, in the preparation of codehydrogenase II are the
following: removal of proteins by acetone; fractionation of the crude
mixture of nucleotides as barium salts, yielding adenosine polyphos-
phate and coenzyme I as by-products; solution of the coenzyme in
methanol-hydrochloric acid and reprecipitation by ethylacetate (5, 8).

/ '-4 AT-^^-i^ ^^\ C

106

A SYMPOSIUM ON RESPIRATORY ENZYMES

The method that led to the isolation of codehydrogenase I in a pure
state is based in the initial stage on the purification steps recom-
mended by Myrback (le). A definite improvement is the precipita-
tion of cozymase by cuprous chloride dissolved in a concentrated

Table 2.— Preparation of codehydrogenase II from horse erythrocytes
WASHED ERYTHROCYTES

hemolysed by water;
precipitated with acetone

SOLUTION

acetone removed by
vacuum distillation;
fractional precipitation by Hg(0Ac)2

PRECIPITATE (protein)
(discarded)

FRACTIONS 2 and 3 (contain Co II)

+H2S (HgS i )
+acetone

FRACTIONS 1 and 4
(discarded)

PRECIPITATE (contains Co II; purity about 15%)
dissolved in H2O;

fractional precipitation by Ba(0H)2
-f alcohol (separation from Co I and cophosphorylase)
-|-Hg(0Ac)2; precipitate +H2S;
solution -{-acetone

PRECIPITATE (contains Co II; purity about 30%)
dissolved in HCI-CH3OH;
precipitation by ethylacetate

CODEHYDROGENASE II (purity about 50%)

fractional precipitation by lead acetate-}- alcohol

FRACTION 1 (about 60%) FRACTION 2 (about 60%)

FRACTIONS 3
and 4 (about 100%)

solution of potassium chloride (9). This procedure has been em-
ployed in all preparative methods subsequently recommended
(10-13). Since pure cozymase does not give a stable precipitate with
the cuprous chloride reagent, such precipitation cannot be repeated.
Apparently some impurity in the crude cozymase solutions plays an
important role in producing a stable precipitate. Decomposition of
the precipitate by hydrogen sulfide involves a considerable loss of

Table 3— Preparation of codehydrogenase I from yeast

YEAST

Extracted with HjO at 80-100° C.

|+Pb(Ac)2

SOLUTION

+Ba(Ac)2+NaOH (pH 8)

PRECIPITATE (discarded)

SOLUTION

+phosphotungstic acid

PRECIPITATE (discarded)

SOLUTION (discarded)

PRECIPITATE

suspended in dilute H2SO4
+amyl alcohol-ether

H2O-H2SO4 PHASE

+Ba(0H)2 (BaS04 i )
+AgN03+NH40H (pH 7.5)

AMYL ALCOHOL-ETHER PHASE
(discarded)

PRECIPITATE

|H2S(Ag2S i )

SOLUTION (discarded)

SOLUTION

l+CuCl in concentrated KCl+HCl

SOLUTION (discarded) PRECIPITATE

+H2S (CU2S i )
concentration in vacuo
alcohol precipitation

CODEHYDROGENASE I (60-80 per cent purity)
IHjO, Ba(0H)2 (pH 8)

SOLUTION

+H2SO4 (BaS04 i )

+Pb(Ac)2

PRECIPITATE (discarded)

PRECIPITATE

(adenylic acid and
impurities)

SOLUTION

fractional precipitation by alcohol

FRACTION 1 FRACTION 2 ^^^F.SS:^ a

IHoS (PbS i ) IH2S (PbS 1 ) H2S (PbS i )

Ualcohol l+alcohol +alcohol

i i i T

CODEHYDROGENASE I CODEHYDROGENASE I CODEHYDROGENASE I

(80-90 per cent) (90-100 per cent) (90-100 per cent)

107

108

A SYMPOSIUM ON RESPIRATORY ENZYMES

the coenzyme by adsorption on the copper sulfide. For desorption
the sulfide precipitate is aerated until slight oxidation of copper
sulfide is attained. This method of desorption, the details of which
were described some years ago (9, 14), has also proved advantageous
in preventing losses by adsorption on silver sulfide. The final puri-
fication is brought about by fractional barium and lead precipitation.
Both codehydrogenases prepared according to the methods given
in Tables 2 and 3 contain traces of impurities which complicate
their use in the spectrographic methods employed in dehydrogenase
investigations. The great stability toward oxidizing agents (le) per-
mits the destruction of impurities by treatment with bromine water

Table 4.— Properties of codehydrogenases I and II

Property

Codehydrogenase I

Ref.

Codehydrogenase II

Ref.

Empirical formula

C21H27O14N7P2

CaiHasOnNyPs

Molecular weight

663

743

Structural units

1 Mol. nicotinamide
1 Mol. adenine

2 Mol. pentose

2 Mol. phosphoric acid

3 Mol. phosphoric acid

Base equivalent

3-4 (?)

Stability:

J. £7

Oxidized form

In 0.1 N HCl at

50% destroyed after

100° C.

8 min.

7.3 min.

In 0.1 N NaOH

50% destroyed after
17 min. (20°)

50% destroyed after
12 min. (23°)

Reduced form

In 0.1 N HCl at 20° C.

activity disappears

immediately

In 0.1 N NaOH at

slight decrease in activ-

100° C.

ity after 10 min.

In 0.1 N NaOH at

stable

20° C.

Absorption spectrum:

Oxidized form

Maximum at 260mjit

cm^

E = 3.8X107 ^

|_Mol.

li
5

8
10

E-3.5X10' ""'
[_Mol._

Reduced form

fcm""

, r cm 2

Maximum at 260m/u

E = 3.3X107 z-z-r
Mol.

^ = ^-^X^«' [mo1.J

340m/i

E = 1.1X10^ ^
[_Mol.

E- 1.0X10' ^'f^
_Mol.

NICOTINAMIDE NUCLEOTIDE ENZYMES 109

(8). Filtration through a column of activated alumina has also been
employed successfully (15).

Recently some modifications in the preparation of cozymase as
given in Table 3 have been described (10-13), The improvements
consist mainly in omitting some of the purification steps, which can
be done without complications if a good quality of yeast is used
as the source material. B. J. Jandorf has introduced the adsorption
of cozymase on charcoal in his method of preparation (13). S. Ochoa
has developed a method in which muscle tissue is used as source
material (11), and P. Ohlmeyer has described a method for the
preparation of dihydrocozymase (10). From 10 kilograms of yeast
one gram of almost pure, or 0.5 gram of pure codehydrogenase I is
obtained. The yield of codehydrogenase II from 1000 liters of
erythrocytes is about 2.5 grams of almost pure, or 1.0 gram of abso-
lutely pure preparation. The coenzymes precipitated from aqueous
solution by organic solvents are not crystalline. Table 4 shows the
composition and properties of codehydrogenase I and II.

The most important part of the work on the structure of the co-
enzymes has been concerned with the nicotinamide moiety, its mode
of action, and the linkage between nicotinamide and the rest of
the molecule. This work was begun by Warburg (8) and continued
by Karrer and his co-workers (23a-f). Warburg showed first that in
codehydrogenase II the nicotinamide reacts with two atoms of
hydrogen in the presence of substrate and apoenzyme, forming a
dihydro compound. This compound can also be obtained by reduc-
tion with hydrosulfite in a shghtly alkaline medium. The reduced
coenzyme has an absorption maximum at 340 n\\i, whereas the maxi-
mum at 260 miJ, has lost some of its intensity by the reduction (see
Figure 1). By reoxidation the original state is restored.

Catalytic hydrogenation yields an uptake of six hydrogen atoms
by the oxidized coenzymes and of four hydrogen atoms by the
biologically reduced coenzymes. Experiments with adenine and its
derivatives showed that under the same conditions these compounds
are very slowly reduced by catalytic hydrogenation, whereas free
nicotinamide exhibits the same properties as the coenzymes upon
catalytic reduction (5). It should be remembered that the catalytic
reduction which leads to the hexahydro compounds is irreversible,
and the products obtained are inactive as coenzymes. These ex-
periments demonstrated that the place of the reversible (biological)
reduction— i.e., the center of the coenzyme activity— is the nicotin-
amide nucleus.

/
/
/

\
\

<r^

^^— ^— — — ^^^

■zr^ — -^

^^^

■^Cn---

— .^_^

-/-

E E

3. °

•

■0 XI

V «)

■5-5

O a

1 /

^^ ^^

_ 1 ,- ^ '

^— — "^

^ "

c — :

^^ ^ ■*"

^^^

^-^

^=^:=:^—

H =

t— 1

fVJ

-13

■<r

c
o

<X)

c«

1—!

^
n

si)

110

NICOTINAMIDE NUCLEOTIDE ENZYMES

111

The next problem was to determine what type of linkage existed
between nicotinamide and the rest of the molecule and what
changes were brought about in the ultraviolet absorption by reduc-
tion of the coenzymes to the dihydro form. This question was an-
swered by the extensive experiments of Karrer and his co-workers.
In the first place, the three functional centers of the nicotinamide
molecule had to be taken into consideration. "Model compounds"
were therefore prepared which were substituted in these positions
with simple organic groups. Among these compounds may be men-
tioned the following typical representatives: nicotinamide iodo-
methylate (I), nicotinic acid ethylimido ether (II), and monomethyl
nicotinamide (III).

FORMULA 1

CONH2

OC2H5

■CONH-CH,

(I)

(11)

(HI)

Of these compounds only the nicotinamide iodomethylate exhib-
ited properties similar to the coenzymes. Like the coenzymes it is
reducible by hydrosulfite (see formula 2), and the absorption maxi-
mum of its dihydro product is 360 m[jL (340 m[x is the typical ab-
sorption maximum of the dihydrocoenzymes), which disappears
when the solution is acidified, as does that of the dihydrocoenzymes.

FORMULA 2

CONH;

+ 2Y^p

CONH.

+ 2NaHS03
+ HI

These results indicated strongly that in the codehydrogenases the
nicotinamide is bound as a quaternary pyridinium base. The ex-
perimental data accumulated in testing this working hypothesis soon
established its validity. It was already known that quaternary pyri-

112 A SYMPOSIUM ON RESPIRATORY ENZYMES

dinium bases are readily subject to reduction processes. Concerning
the position where the reduction takes place, the following possi-
bilities had to be considered.

FORMULA 3
H H Hg

HC'^^'^C-CONH^ HC^^VCONH^ '-'9j^^^^"^^^^^2

I ' k

R R R

(a) (b) (c)

A comparison of the earlier known compounds of this group
showed noteworthy diflFerences between the p-dihydro compounds
and the reduced coenzymes, but very good agreement between the
latter and the model o-dihydro compounds. Whether the reduction
takes place according to formula 3a or 3b could not be decided
(23a, c).

The next step was the preparation of nicotinamide derivatives
which are substituted by carbohydrate radicals on the ring nitrogen
atom. The properties of these compounds showed a still better
correspondence with those of the coenzymes. The best representa-
tive of these compounds which can be prepared in a pure state
was found to be tetra-acetyl-glucosido-nicotinamide bromide. The
absorption maximum of its dihydro derivative is very similar to that
of the reduced codehydrogenases. In addition to similarity in optical
properties the model nucleosides share with the coenzymes the
sensitivity of the glycosidic linkage toward alkali when in the oxi-
dized form, and stability toward alkali when reduced; also, the
action of strong acid on the dihydro compounds yields, according
to Karrer, products which no longer have the absorption band at
340 m[x. Furthermore, both the model compounds and the co-
enzymes are very sensitive toward hypoiodite, which destroys the
pyridine ring (24).

When the pyridinium model compounds are reduced to the di-
hydro compounds, the ring nitrogen of the reaction products is
trivalent, and they do not contain the acid group. Corresponding to
this acid group in the model compounds is the phosphoric acid in
the coenzymes. Upon reduction an acid group is liberated according
to the following scheme (8, 21):

NICOTINAMIDE NUCLEOTIDE ENZYMES
FORMULA 4

113

CONH.

I O^ pR'
R-0-P=0

■^

CONH.

N' ,

I HO, PP'
R-0-P = 0

According to Haas this reaction can be controlled manometrically
if bicarbonate is present in the medium (25). The attempts of Karrer
to synthesize nicotinamide derivatives with pentose as a substituent
were not successful. Attempts with arabinose and xylose gave oily
products which could not be obtained in a pure nor in a crystalline
form. The closest link between the model compounds and the co-
enzymes, therefore, was missing. The compound consisting of nico-
tinamide and pentose has been obtained from cozymase (26). It
possesses all the expected properties and has some biological in-
terest. Since no extensive publication on this subject has yet ap-
peared, the preparation and properties of the compound are here
discussed in somewhat more detail.

As has been pointed out, the splitting of the linkage between
nicotinamide and pentose is the first result of acid as well as of
alkaline hydrolysis of cozymase. Therefore only enzymatic splitting
could yield this very labile nucleoside. For this purpose the nucleo-
tidase discovered by Bredereck in sweet almond press-cake was
chosen (27). After purification this enzyme has the following prop-
erties, which make it suitable for the preparation of the nucleoside:
The pH for optimum activity is about the same as that for the
optimum stability of cozymase. The preparations are free from

Table 5.— Enzymatic splitting of cozymase

Spectro-

Time of

Fermentation

Percentage

photometric

hydrolysis,

test, per-

phosphorus

nicotinamide

determination;

in horn's

centage Co I
found

spht off

spHt off

percentage
"pyridinium
compound"

100

96*

120

100

<10

A fresh enzyme was added after 96 hours.

114

A SYMPOSIUM ON RESPIRATORY ENZYMES

nucleosidase, but contain some nuclease, which causes splitting into
the mononucleotides, and a nucleotidase, which removes the phos-
phoric acid. Table 5 illustrates the course of the enzymatic hydrolysis
of cozymase.

The isolation of the nicotinamide nucleoside is complicated by
the fact that thus far only one reagent has been found which pre-
cipitates the compound, namely, phosphotungstic acid. The isolation
and purification, therefore, as given in Table 6, consists mainly in
removing the other compounds and impurities from the mixture.

The nicotinamide nucleoside gives no characteristic precipitates

Table 6.— Preparation of nicotinamide nucleoside

COZYMASE

enzymatic hydrolysis at pH 4.5

(Nicotinamide nucleoside
UYDROLYS ATE\ Adenosine

I Phosphoric acid
[Protein
dialysis; Ba(OH)2(P04i)

SOLUTION (nicotinamide nucleoside, adenosine)

+ Ag2S04

SOLUTION (nicotinamide nucleoside)
+H2S (Ag2S i )
-f-phosphotungstic acid

PRECIPITATE SOLUTION

suspended (discarded)

in dil. H2SO4,

phosphotungstic acid removed
by amyl alcohol-ether
extraction

SOLUTION

+picric acid (impurities [ )

ether extraction;

precipitation by acetone -|- ether

NICOTINAMIDE NUCLEOSIDE (crude product)
filtration through
aluminum oxide;
fractional precipitation

NICOTINAMIDE NUCLEOSIDE

PRECIPITATE (adenosine)

-f-HsS (Ag2S i )
-|-picric acid

ADENOSINE PICRATE

ether extraction;

repeated

recrystallization

ADENOSINE

NICOTINAMIDE NUCLEOTIDE ENZYMES 115

with any of the typical reagents used in nucleotide chemistry, such
as picric acid, picrolonic acid, Reinecke salt, chloroplatinate, and
aurichloride. It is difficult, therefore, to remove the last traces of
impurities from the nucleoside preparations. The elementary analysis
and the quantitative determination of nicotinamide and pentose,
however, rule out every other composition except that of a pentose
nucleoside. It remains to be determined what pentose we are deal-
ing with; for this purpose relatively large amounts of the nucleoside
must be prepared, a task which is in progress at present.

It was of great interest to compare this split product with the
model substances of P. Karrer. It was found to be strikingly similar
to the synthetic compounds in all respects. In the first place, the
reduction with sodium hydrosulfite should be mentioned (see for-
mula 5).

FORMULA 5

fT^

CONH

KJ ^

CONH

I A I

Pentose Pentose

Like the codehydrogenases and the synthetic pyridine derivatives
with pentavalent ring nitrogen, the nicotinamide nucleoside yields
an o-dihydro compound which has the same characteristic absorp-
tion maximum at 340 m[x (see Figure 2). In other respects also the
nucleoside has the expected properties. It has in the oxidized form
a stability optimum of about pH 3-4, and is extremely labile in
alkali, whereas the dihydro derivative is stable in alkali and is
sensitive toward acids, as are the dihydro coenzymes. Alkaline as
well as acid hydrolysis separates the carbohydrate from the nico-
tinamide.

The nucleoside cannot replace either of the coenzymes in the
dehydrogenase systems. The phosphoric acid and adenylic acid
which are present in the codehydrogenases are necessary for the
combination of the pyridinium compound with the apoenzymes.

The investigations of Warburg and Karrer, described above, on
the linkage between nicotinamide and the rest of the molecule,
which were completed by the isolation of the natural nicotinamide
nucleoside, justify the claim of a quaternary pyridinium linkage.
Beyond this, our knowledge on the combination of the structural

116

A SYMPOSIUM ON RESPIRATORY ENZYMES

units is more complete for cozymase than for codehydrogenase II;
investigations on the latter have been less numerous, since it is
difficult to obtain in quantity.

MOLE

1.5

Oxidiz ed

form

duced

form

1.0
0.5

>4
S

^._

300

320

340

7V<

360

Figure 2. — Absorption spectnim of nicotinamide nucleoside

Our knowledge of the structure of cozymase is based on the
following findings: Besides the isolation of nicotinamide and adenine
(6, 9), the isolation of pentosephosphoric acid was attained by acid
hydrolysis (17). By means of the periodate method it was showni
that the phosphoric acid is linked to the fifth carbon atom of the
pentose molecules, as is indicated in formula 6.

CONH

2 Mol.

FORMULA 6

I
HC

II II
N C

C-NH,
I 2

C — N

OH OH OH

III /

OHC-C-C-C-CH 0-P=0
III 2 \

H H H OH

NICOTINAMIDE NUCLEOTIDE ENZYMES

117

Depending on the experimental conditions, the product from
alkaline hydrolysis was nicotinamide (28) or adenosine diphosphoric
acid (29), the structure of which is well established by the work of
Lohmann (30), Embden (31), and Levene (32). Alkaline hydrolysis
in the cold gives, besides nicotinamide, a product of adenosine
diphosphate plus pentose (formula 7). This degradation product
of cozymase is inactive as coenzyme (33).

N = C-NH

I I

HC C— N\

II II ^CH
N — C — N

FORMULA 7

CONH.

OH OH H OH OH

II 11/

-C— C — C — C — c-o-p-o-p=o

I I I I I II \

H H H H H O OH

OH OH

I I
Adeni ne — pentose — P-0-P-peniose -f-

II II
O O -N

The enzymatic hydrolysis gave the two nucleosides:

FORMULA 8

■CONH.

X-

C-

I
N

■CONH-

■N
II
■C-NH.

I
HCOH

I
HC

^C^

HC-

I
HCOH

CH^OH

118

A SYMPOSIUM ON RESPIRATORY ENZYMES

In view of these results and the fact that cozymase is a monobasic
acid composed of its structural units minus five molecules of water,
the structural formula published in 1936 as a working hypothesis
(18) (see formula 9) seems well established. It remains, however,
to determine whether or not the pentose of the nicotinamide part is
identical with cZ-ribose.

FORMULA 9

■C-

CONH.

.<^

HC-

C = C-
I I

N N

^C^

•N
II
C
I
NH.

HC-

HCOH

I O

HCOH

I
HC

HCOH

I
HCOH

I
O HC

CH^O — P — O

— P-O-CH.
I
OH

Concerning the structure of codehydrogenase II the following
results may be mentioned: Warburg and his co-workers stated that
the molecule consists of nicotinamide, adenine, two molecules of
pentose, and three molecules of phosphoric acid. These compounds
minus six molecules of water form the coenzyme. The experiments
on the linkage of the nicotinamide have already been mentioned.
In a very early stage of the work, Theorell, on the basis of cata-
phoretic experiments, claimed that the coenzyme is a tetrabasic acid
and that the amino group of the adenine is not free (19). Warburg,
Christian, and Griese (5) later revised this claim; their titration ex-
periments indicate that the coenzyme is a tribasic acid, and they
showed that the amino group is free. The finding of Adler and
Euler (7) that the enzymatic reaction Co I ^ Co II can take place
narrowed considerably the possibilities respecting the structure of
codehydrogenase IL The location of the third phosphoric acid group
now seems to be the only question concerning the structure that has
not been settled. The fact that cozymase contains adenosine diphos-
phate as an essential constituent suggested (15) that codehydrogenase

NICOTINAMIDE NUCLEOTIDE ENZYMES 119

II might contain adenosine triphosphate as a part of its molecule
(formula 10, 1).

FORMULA 10

O OH OH

I Nicofin amide -pentose — P— O— P— O— P — pentose- adenine

11 II II

0 0 0

®0 OH

® I I

2. Nicotinamide- pentose — P — O — P — pentose- adenine

o o 'p^hI

This hypothesis, however, was not substantiated by experimental
results. Experiments carried out with rather limited amounts of pure
coenzyme II showed that it contains no readily hydrolyzable phos-
phate, and yields no alkaline degradation product active as a co-
phosphorylase (34). Therefore it seems probable that the third phos-
phoric acid group is linked to the adenylic acid part of the molecule,
perhaps in much the same way as in yeast adenylic acid (formula
10, 2). This phosphoric acid group should block the coenzyme prop-
erties of the adenylic acid part when liberated by alkaline hydrolysis.
Further experimental work is needed to decide this question.

The small amounts of adenosine-5' -phosphoric acid and its homo-
logues which occur as contaminants of impure codehydrogenase
preparations and which are formed from alkaline hydrolysis of
cozymase but not from codehydrogenase II can be traced by a test

FORMULA 11

GOGH GOOH GOOH

1 , I /OH _x I /OH

GHOH > GH-0-P=0 7 G-0-P=0

I /OH < I ^oH <— N. OH

GH 0-P=0 GHOH GH^

GOOH GOOH

2 C-P^o'~' 4- ADENYLIC \ p C-OH + ADENOSINE-

II Vu ACID II TRIPHOSPHATE

GH^^ GH^

ADENOSINE- TRIPHOSPHATE — > ADENYLIC ACID
+ 2 PHOSPHORIC ACID

120 A SYMPOSIUM ON RESPIRATORY ENZYMES

based on the coenzyme properties of adenylic acid and its homo-
logues (cophosphorylase) in the enzymatic spHtting of phospho-
pyruvic acid (see formula 11). In the absence of a suitable acceptor
the phosphoric acid appears in a free state in an amount propor-
tional to the amount of cophosphorylase present in the system; by
this method 10 to 100 micrograms of adenylic acid can be deter-
mined (35).

Methods of Determination

Only the methods based on the coenzyme properties will be dis-
cussed here. For determining the presence of cozymase Harden (2)
originally used a press extract of yeast as a source of apoenzymes
and a boiled yeast extract as a source of coenzyme. A great im-
provement over this method was made when Euler and Myrback
found that dried brewer's yeast loses its cozymase by repeated ex-
traction with cold water (le, 36). The remaining product was found
to contain all the apoenzymes, activators, and coenzymes necessary
for fermentation except cozymase. Therefore it was called apo-
zymase. The use of this preparation is still the simplest and perhaps
the most accurate method of determination, its accuracy being Hm-
ited only by the errors of the manometric measurements. Another
test system recently elaborated by Jandorf , Klemperer, and Hastings
uses, instead of the function of cozymase in fermentation, the co-
enzyme properties of cozymase in glycolysis, according to the
following scheme (37):

Hexose diphosphate -^ phosphoglyceraldehyde + dihydroxy-
acetone phosphate

Phosphoglyceraldehyde + H3ASO4 -^ arsenophosphoglyceral-
dehyde

Arsenophosphoglyceraldehyde + DPN -> arsenophospho-
glyceric acid + DPN-Hg

Arsenophosphoglyceric acid -^ 3-phosphoglyceric acid +
H3ASO,

Phosphoglyceraldehyde + DPN-Ho -> glycerophosphoric
acid + DPN
6) Hexose diphosphate -^ 3-phosphoglyceric acid + glycero-
phosphoric acid

The amount of phosphoglyceric acid produced in a given time in
the presence of bicarbonate buffer can be measured manometrically
with the Warburg apparatus. The test system for codehydrogenase
II as given by Warburg uses the dehydrogenation of Robison ester

NICOTINAMIDE NUCLEOTIDE ENZYMES 121

by codehydrogenase II, Robison ester apodehydrogenase (Zwischen-
ferment), and riboflavin enzyme. If the codehydrogenase II is the
speed-hmiting factor in this system, the oxygen uptake is a measure
of its concentration (5). It is probable that the old yellow enzyme
used by Warburg and Christian is an artifact. The cytochrome c
reductase of Haas, Horecker, and Hogness (38) seems to be the
natural acceptor for the hydrogen of the reduced codehydrogen-
ase II.

These methods of determination have been used not only to
elaborate the purification of the codehydrogenases but also to study
their distribution from the standpoint of vitamin research. The
codehydrogenases occur in an equilibrium: coenzyme ^ dihydroco-
enzyme. Since the stabilities of the oxidized and the reduced forms
are different (see Table 4), the proportions of coenzyme and dihy-
drocoenzyme can be determined by heat extraction with acid or
with alkali, according to Adler and Calvett (39). If the extraction is
made with boiling water, the reduced coenzyme is oxidized by air.
Therefore no loss occurs if some of the subsequent steps of the
preparation are carried out in acid medium.

In most tissues somewhat more of the oxidized than of the reduced
form of cozymase was found, but in the Jensen rat sarcoma, Euler
and his co-workers found a large excess of dihydrocozymase (20, 40).
Recent experiments confirm this finding, but apparently it is not
true for all cancerous tissues. An excess of dihydrocozymase was
repeatedly found in methyl-cholanthrene rat tumor, but not regularly
in benzpyrene tumors of mice and in Brown-Pearce rabbit carci-
noma (33). The data on human carcinomata are still too limited to
permit any conclusion.

It must be remembered that all methods of determination in tis-
sues involve complications: incompleteness of extraction, limited
stability of the codehydrogenases in oxidized and reduced form at
high temperature, rapid changes in the equilibrium Co I <=s Co II,
enzymatic destruction upon disruption of the cells, and finally
errors in the methods of determination. The claims for accuracy
which are made by many publications dealing with coenzyme con-
tent of tissues seem far too optimistic.

The growth-promoting properties of the codehydrogenases for
certain microorganisms (Hemophilus influenzae and Hemophilus
para-influenzae) can be used for detennining small quantities of
these compounds (41). Whereas the number of nicotinamide-
requiring microorganisms is rather high, only H. influenzae and

122 A SYMPOSIUM ON RESPIRATORY ENZYMES

H. para- influenzae have been found to require cozymase or code-
hydrogenase II. According to LwoflF only 0.004 microgram per milli-
liter of peptone solution is necessary to produce visible growth under
standard conditions. The method does not distinguish between co-
dehydrogenases I and II. Dihydrocozymase was found to be in-
ferior as a nutrilite to an equivalent amount of cozymase (42). This
may be due to a difference in the permeability of the cells to the two
compounds.

For the examination of pure or almost pure coenzyme prepara-
tions the spectrophotometric determination of the dihydro com-
pounds is a very accurate method. The solutions must, however, be
free from impurities that absorb in the ultraviolet region in which
the dihydrocoenzymes exhibit their characteristic absorption. The
chemical methods of determination cannot be discussed here in
detail.

The Apodehydrogenases

Since the history of the apodehydrogenases has been reviewed in
several comprehensive articles (la, c, g, 43a-d), they will simply be
listed here, and our present knowledge about their purification, na-
ture, and function will be summarized and their coenzyme speci-
ficity discussed.

As can be seen from Table 7, some of the apodehydrogenases have
been prepared in a pure state and obtained in a crystalline form.
The main progress in the field was made by Warburg and his co-
workers, especially Christian, Negelein, Gerischer, Haas, Wulff,
and Kubowitz. Warburg's methods for purification consist mainly
in precipitations of the apoenzymes at the isoelectric point, their
fractional precipitation by ammonium sulfate or organic solvents,
and removal and inactivation of other enzymes by heat denatura-
tion at a temperature of about 50° C. Of special interest is the pre-
cipitation of the diphosphoglyceraldehyde apodehydrogenase in a
step of its purification by addition of nucleic acid (44),

The isolation of the apodehydrogenases in a pure state was im-
portant for a detailed study of their relation to coenzymes and sub-
strate by the spectrophotometric technique. Whereas the use of
crude apoenzymes does not exclude the possibility of more com-
plex reactions, a definite conclusion can be drawn respecting the
mechanism of a reaction if pure apoenzyme, coenzyme, and sub-
strate are used. Our previous conception of the function of cozymase
in the dehydrogenation of triosephosphate was greatly revised in

Table 7.— The apodehydrogenases dependent on nicotinamide

nucleotides

Substrate and dehydrogenation Co-
product enzyme

Note on the apodehydrogenase Ref.

l,3-diphosphoglyceraldehydei=^
1,3-diphosphogly eerie aeid

Triosephosphatei^phospho-
glyceric acid

Lactic acid<=^pyruvic acid

Alcohol<=^acetaIdehyde

Methyll

Propyl plcohoI<=^
Amyl J
Form- )

Propion- [aldehyde
Valer- J

2 R • CHO+HjO^R COOH
+RCn20H

Malic acid<=^oxalacetic acid

a-glycerophosphate<=^phospho-
glyceraldehyde

/3-hydroxybutyric acid^aceto-
acetic acid

Formic acid<=^C02

Glucose-6-monophosphate
(Robison ester)— >phospho-
hexonic acid

Phosphohexonic acid

Decarboxylation and dehydro-
genation

Isocitric acid<^a-keto-/3-carboxy-
glutaric acid

Glucose^gluconic acid
Glutamic acid

Iminoglutaric acid

Co I Obtained by Warburg and Christian 4 1
in crystalline state from yeast

Co I Crude preparations from animal tis- 70
sues. Identical with 1,3-diphospho- 71
glyceraldehyde apodehydrogenase?

Co I Prepared by Straub in a crystalline 72
form from heart

Co I Obtained by Negelein and Wulff in 51
crystalline form from yeast

Co I Crude preparations from animal tis- 46
sues. Identical with ethyl alcohol
apodehydrogenase ?

Co I Aldehyde mutase; crude prepara- 73
tions from liver

Co I Crude preparations from animal tis- 74
sues

Co I Crude preparations from yeast and 75
tissues

Co I Crude preparations from animal tis- 76
sues

Co I Crude preparations from seeds and 49
B. coli

Co II Obtained from yeast in a highly 77
purified state by Negelein and
Gerischer

Co II Purified preparation obtained from 78
yeast by Warburg and Christian

Co II Crude preparations from seeds, ani- 79
mal tissues, and yeast

Co I or Crude preparations from liver and 45
Co II yeast

Co II For apoenzyme from yeast and B. 80

coli;
Co I or For apoenzyme from animal tissues;
Coll

Co I For apoenzyme from plants

123

124 A SYMPOSIUM ON RESPIRATORY ENZYMES

this way by Warburg and Christian. They showed that diphospho-
glyceraldehyde rather than triosephosphate is the substrate of
cozymase in fermentation and perhaps in glycolysis (44). The main
purpose of giving, in Table 7, the degree of purity of the apoenzymes
obtained thus far is to afford some idea of the reliability of our
knowledge respecting the reactions in question.

The most noteworthy fact regarding the apoenzymes is that they
are responsible for the specificity of the dehydrogenase, whereas
codehydrogenases I and II combined with different proteins form
a relatively large number of dehydrogenases. The protein moiety
is therefore more specific than the prosthetic group.

It was found that both codehydrogenase I and codehydrogenase
II can act as coenzymes for glucose apodehydrogenase (45). This
apoenzyme, however, has not yet been sufficiently purified, and
therefore it is not impossible that a transformation of codehydro-
genase I into codehydrogenase II or vice versa may occur in this
system.

In connection with alcohol apodehydrogenase it should be men-
tioned that methyl, propyl, and amyl alcohol can also serve as sub-
strate (46). This seems to indicate that the specificity of the
apodehydrogenases even toward the substrate is not always an
absolute one.

Warburg has claimed that the protein part of a given dehydro-
genase probably differs for each type of cells. Robison ester apode-
hydrogenase from yeast and from rat blood were found to be
different, their isoelectric points being at pH 4.8 and 5.8 respectively
(lb). In some cases the difference is so great as to suggest that the
specificity for the codehydrogenases depends on the source of the
apoenzyme. Thus glutamic acid apodehydrogenase from liver was
found by Adler to require codehydrogenase II; the apoenzyme
from plants uses codehydrogenase I as a prosthetic group (47).
Besides the cozymase-dependent glycerophosphate apodehydro-
genase, a glycerophosphate dehydrogenase was found to occur in
muscle tissue which does not require a coenzyme (48). Similar
findings have been reported for formic dehydrogenase. Whereas the
dehydrogenase preparations from seeds consist of cozymase plus
apoenzyme (49), the corresponding enzyme from Bacterium coli
does not need cozymase (50).

If there are actually as many variations of apodehydrogenases as
there are different types of cells, a further important specificity may
be based on this difference. To obtain more evidence, the apode-

NICOTINAMIDE NUCLEOTIDE ENZYMES 125

hydrogenases must be prepared from different sources and the re-
sulting products compared with one another. It is probable that the
differences will consist in a slight variation in the pH for optimum
activity or a change in the equilibrium between protein, substrate,
and coenzyme rather than in a completely different mode of action.

The preparation of some apodehydrogenases in crystalline form
has permitted an estimation of the amount present in the cell. They
are found in much smaller concentration than the codehydrogenases.
O. Meyerhof (Ic) has pointed out that if each cozymase molecule
in rabbit muscle or yeast were accompanied by one molecule of
each of the apodehydrogenases, the protein of the cells would con-
sist exclusively of the apodehydrogenases.

The apodehydrogenases combine with the nicotinamide nucleo-
tides, the reduced nucleotides, the substrate, and the reaction
product. The extent to which the complexes thus formed are dis-
sociated detennines the direction and the rate and equilibrium of the
reaction. As an example may be mentioned the following system:
alcohol, acetaldehyde, cozymase, dihydrocozymase, and apodehy-
drogenase. This system was investigated by Negelein and Wulff,
who used the pure components (51). They found that under their
experimental conditions the concentration of each substance at
which the apodehydrogenase is half saturated with it is as follows:
cozymase, 0.0001 M.; dihydrocozymase, 0.00003 M.; acetaldehyde,
0.0001 M.; and ethyl alcohol, 0.024 M. The reduced coenzyme and
acetaldehyde are bound to a much greater extent than cozymase and
alcohol, and under normal conditions the reversible reaction acetal-
dehyde + dihydrocozymase ^ alcohol + cozymase proceeds in the
direction of alcohol formation. Experiments carried out on other
pyridine dehydrogenase systems have yielded similar results. The
dehydrogenases requiring codehydrogenase II, however, seem to be
less dissociated.

The high degree of dissociation between the pyridine nucleotides
and their protein parts corresponds to that of the cophosphorylases
and their specific proteins. M. Dixon and L. G. Zerfas have pointed
out the differences between this group of enzymes and the other
type, in which the prosthetic group is in a relatively stable linkage
to the protein and the ratio between prosthetic group and protein
is 1:1 (52). On the basis of interesting experimental results with
artificial hydrogen acceptors they state in their discussion that the
use of the term "pyridine-proteid" is misleading and should be dis-
continued. It would seem, however, that this suggestion is too radi-

126 A SYMPOSIUM ON RESPIRATORY ENZYMES

cal. The work that has been done in this field leaves no doubt that
we are dealing with real enzymes, which, after bringing about the
dehydrogenation of substrate under biological conditions, are un-
changed. Furthermore, at the moment when they exhibit their
activity, the nicotinamide nucleotide is combined with its specific
protein just as the riboflavin nucleotide is combined with its specific
protein. That we deal in the one case with a relatively stable linkage
and in the other with a very unstable one is only a difference in
degree. Hence a change of the whole nomenclature would seem to
be unnecessary and would probably increase the confusion in this
field.

The physiological reoxidation of the reduced coenzymes is
brought about by the oxidized form of the metabolites listed in
Table 7, as, for example, acetaldehyde in fermentation or pyruvic
acid in glycolysis. In other instances the dihydrocoenzymes are re-
oxidized by alloxazine proteids such as diaphorase (84), coenzyme
factor (85), cytochrome c reductase (38), or the enzyme recently
detected by Altschul, Persky, and Hogness (86).

Spectrophometric Methods

In studying the function of the nicotinamide nucleotide enzymes
we have been greatly aided by the spectrophometric methods de-
veloped by Warburg. The basis is the appearance of an absorption
band at 340 m\}. when the codehydrogenases are reduced. This
absorption band disappears upon reoxidation. Under proper ex-
perimental conditions the concentration of coenzyme or apoenzyme,
the substrate and acceptor specificity of the dehydrogenases, and
the speed of reactions can be studied, as has been done in some
laboratories, notably those of Warburg, Euler, and Meyerhof (lb,
i; 53).

As an example the action of the reducing and the oxidizing
fermentation system may be given (44). See Figure 3.

Biosynthesis of the Codehydrogenases

In view of the relatively complicated configuration of the code-
hydrogenases and of their nucleotide character, it is improbable that
a satisfactory synthesis will be obtained by present-day methods of
organic chemistry. Therefore experiments designed to carry out a
biosynthesis from the structural units are very important from a
practical point of view.

Mention may be made first of the experiments dealing with the

NICOTINAMIDE NUCLEOTIDE ENZYMES

127

formation of the codehydrogenases from nicotinamide in the or-
ganism. The biological function of the nicotinamide-containing
coenzymes and their wide distribution in nature (If) were known
before their vitamin properties. There was little doubt that the
nicotinamide ingested as a vitamin is used to form the coenzymes.
Nevertheless an exact demonstration had to be obtained.

1.6

1.2 -

0.8

0.4

Protein II added

Mi N.

Figure 3. — Action of oxidizing and reducing fermentation enzymes. Spectro-
photometric experiment, absorption at 340 m\i ( dihydrocozymase ) . d = 0.557
cm. Protein I: diphosphoglyceraldehyde apodehydrogenase. Protein II: acetal-
dehyde reductase (alcohol apodehydrogenase). The concentrations are as
follows: cozymase, 0.183 mg. per ml.; (i-phosphoglyceraldehyde, 0.733 mg. per
ml.; acetaldehyde, 1.47 mg. per ml.; apoenzymes (protein I and II), 0.0008 mg.
per ml.; orthophosphate, 3.3 X 10 '^ mole per ml.; pyrophosphate, 3.3 X 10"'
mole per ml.

Experiments on animal tissues were performed by Axelrod and
Elvehjem. The cozymase level of pigs and dogs living on a diet low
in nicotinamide was found to be decreased in muscle and liver.
Upon administration of nicotinamide the normal cozymase content
was rapidly restored (56). Later the same effect was observed in
pellagrins by Axelrod, Spies, and Elvehjem (58). Experiments on
rats also seemed to indicate the same result (57), but according

128 A SYMPOSIUM ON RESPIRATORY ENZYMES

to more recent findings the vitamin nature of nicotinamide for the
rat is doubtful (81, 82). Dann and Handler recently have shown
that nicotinic acid is formed by the chick embryo (83).

An important observation made in these experiments was that the
organism does not synthesize coenzyme beyond the normal level of
coenzyme content under favorable dietary conditions. Even an ad-
ministration of nicotinamide far in excess of the normal requirement
results in no significant synthesis beyond the normal level. In
erythrocytes, however, an increase in coenzyme content upon ad-
ministration of an excess of nicotinamide was observed by Kohn and
Klein, Vilter and Spies, Axelrod and others (59, 60).

Numerous experiments with isolated enzyme preparations from
yeast, liver, and muscle have thus far failed to give a noteworthy
synthesis of the coenzymes from the structural units. The destruc-
tive tendency of these preparations has always been found to be
remarkable. It was observed by Euler and co-workers (64—66) that in
tissues the coenzyme content after death, and especially upon
destruction of the cell structure, decreases rapidly. The significance
of this finding for the methods of determination and the precautions
necessary have been pointed out repeatedly.

Mann and Quastel have recently confirmed the findings of Euler.
Of special interest is their finding that free nicotinamide in great
excess prevents the postmortem decomposition of cozymase (67), a
result which the authors explain by assuming that nicotinamide and
cozymase compete for the active center of the nucleotidase which
destroys the coenzyme.

Lennerstrand found some years ago (68, 69) that a destruction
of cozymase by washed dried yeast (apozymase) takes place, but is
inhibited by phosphate and hexosediphosphate. Apparently the
substrate protects the coenzyme from destruction. It is not yet pos-
sible to say what products are formed by the inactivation of cozy-
mase by apozymase. If glucose and phosphate are added to the
inactivated cozymase plus apozymase, a resynthesis takes place
after several hours, and as much as 50 per cent of the cozymase
originally present is restored.

A similar effect was observed recently with cocarboxylase. When
cocarboxylase is incubated with aetiozymase, it is destroyed. In the
presence of pyruvate a much slower inactivation takes place (33).
It is possible that underlying these findings is an important principle
of regulation of the coenzyme level.

It seemed possible that a regeneration of cozymase from the nico-

NICOTINAMIDE NUCLEOTIDE ENZYMES 129

tinamide nucleoside might take place in a fermentation system,
similar to the formation of cocarboxylase from thiamine. Experi-
ments designed to carry out such a biosynthesis of cozymase by
incubation with yeast preparations in the presence of phosphate
and adenosine phosphoric acids have not yet been successful. Never-
theless it can be assumed that the nicotinamide nucleoside is an
intermediate in the course of biosynthesis of the codehydrogenases.

Among the lower organisms of the plant kingdom— for example,
Bacterium aerogenes, Torula, and other yeasts— we have examples
of biosynthesis of cozymase from very simple nitrogen and carbon
sources. Hutchens, Jandorf, and Hastings have shown recently that
the protozoon Chilomonas paramecium is capable of synthesizing
cozymase in a synthetic medium containing ammonia as the only
source of nitrogen and acetate as the sole source of carbon (62).
Other microorganisms, such as Staphylococcus aureus (54), Proteus
vulgaris (55), and Shigella paradysenteriae (63), require nicotinamide
to complete the synthesis of cozymase, a fact which permits these
organisms to be used for the bio-assay of nicotinamide. It has been
suggested that these bacteria require nicotinamide for growth as a
result of their parasitic existence (55). Most striking in this connec-
tion is the finding of Lwoff and Lwoff that Hemophilus influenzae
and para-influenzae require codehydrogenase I and II (41). This
specificity surpasses even that of man and other mammals.

Gingrich (42) has shown that the "V" requirement of hemophihc
bacteria can be satisfied not only by the oxidized codehydrogenases
but also by dihydrocozymase, acid-treated dihydrocozymase (com-
pletely inactive as codehydrogenase), and desamino cozymase, an
artificial derivative of cozymase (61) in which the adenylic acid is
replaced by inosinic acid. Furthermore, the fact that the nicotinam-
ide nucleoside has been found (42) to promote the growth of these
organisms is highly interesting in that it demonstrates that the only
special requirement is the preformed linkage of nicotinamide to the
pentose. It is obvious, then, that the nutrihtes required for the
biosynthesis of cozymase are related chiefly to the nicotinamide
moiety and its linkage to the rest of the molecule. The steps in
biosynthesis between simple nitrogen and carbon compounds and
the pyridine ring remain a promising field of investigation.

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130 A SYMPOSIUM ON RESPIRATORY ENZYMES

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NICOTINAMIDE NUCLEOTIDE ENZYMES 131

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25. Haas, E., Biochem. Z., 285, 368 (1936).

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27. Bredereck, H., Beuchelt, H., and Richter, C, Z. physiol. Chem.,
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28. Schlenk, F., v. Euler, H., Heiwinkel, H., Gleim, W., and Nystrom,
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29. Vestin, R., Schlenk, F., and v. Euler, H., Ber. Chem. Ges., 70, 1369
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30. Lohmann, K., Biochem. Z., 282, 120 (1935).

31. Embden, G., and Zimmermann, M., Z. physiol. Chem., 167, 114, 137
(1927).

32. Levene, p. a., and Bass, L. W., Nucleic Acids (New York, 1931), pp.
187-192.

33. Schlenk, F., unpublished experiments.

34. Schlenk, F., Hogberg, B., and Tingstam, S., Svenska Vet. Akad. Arkiv
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35. Schlenk, F., and Schlenk, T., J. Biol. Chem., 141, 311 (1941).

36. Myrback, K., Z. physiol. Chem. 177, 158 (1928).

Schlenk, F., and Vowles, R. B., Svenska Vet. Akad. Arkiv f. Kemi,
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37. Jandorf, B. J., Klemperer, F. W., and Hastings, A. B., J. Biol. Chem.,
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38. Haas, E., Horecker, B. L., and Hogness, T. R., J. Biol. Chem., 136,
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39. Adler, E., and Calvett, F., Svenska Vet. Akad. Arkiv f. Kemi, 12 B,
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40. V. Euler, H., Malmberg, M., and Gunther, G., Z. f. Krebsforschung,
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41. LwoFF, A., and Lwoff, M., Comptes rendus Acad. Sci., 203, 896
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b) Thunberg, T., Ergebnisse d. Physiologic, 39, 76 ( 1937).

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44. Warburg, O., and Christian, W., Biochem. Z., 303, 40 ( 1939).

45. Das, N. B., Z. physiol. Chem., 238, 269 (1936).
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46. Lutwak-Mann, C, Biochem. J., 32, 1364 (1938).

47. V. Euler, H., Adler, E., Gltnther, G., and Das, N. B., Z. physiol.
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48. Green, D. E., Biochem. J., 30, 629 (1936).

49. Adler, E., and Sreenivasaya, M., Z. physiol. Chem., 249, 24 (1937).

50. Gale, E. F., Biochem. J., 33, 1012 (1939).

51. Negelein, E., and Wulff, H. J., Biochem. Z., 293, 351 (1937).

52. Dixon, M., and Zerfas, L. G., Biochem. J., 34, 371 ( 1940).

53. Meyerhof, O., Ohlmeyer, P., and Mohle, W., Biochem. Z. 297, 90
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54. Knight, B. C. J. G., Biochem. J., 31, 731, 966 (1937).

55. Fildes, p., Proc. Roy. Soc. (London), B 124, 4 (1937); Brit. J. of Exp.
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132 A SYMPOSIUM ON RESPIRATORY ENZYMES

56. AxELROD, A. E., Madden, R. J., and Elvehjem, C. A., J. Biol. Chem.,
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Dann, W. J., and Handler, P., J. Nutrition, 22, 409 (1941).

57. V. Euler, H., Schlenk, P., Melzer, L., and Hogberg, B., Z. physiol.
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Frost, D. V., and Elvehjem, C. A., J. Biol. Chem., 121, 255 ( 1937).

58. AxELROD, A. E., Spies, T. D., and Elvehjem, C. A., J. Biol. Chem., 138,
667 (1941).

59. Kohn, H. I., and Klein, J. R., J. Biol. Chem., 130, 1 ( 1939); 135, 685
(1940).

60. ViLTER, R. W., ViLTER, S. P., and Spies, T. D., J. Am. Med. Assoc,
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62. HuTCHENs, J. O., Jandorf, B. J., and Hastings, A. B., J. Biol. Chem.,
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d) Bass, A., Berkman, S., Saunders, F., and Koser, S. A., J. Inf. Diseases,
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65. V. Euler, H., and Gunther, G., Z. physiol. Chem., 243, 1 (1936).

66. V. Euler, H., HEivvntNKEL, H., and Schlenk, F., Z. physiol. Chem., 247,
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67. Mann, P. J. G., and Quastel, J. H., Biochem. J., 35, 502 (1941).

68. Lennerstrand, A., Biochem. Z., 287, 172 (1936).

69. Lennerstrand, A., Svenska Vet. Akad. Arkiv f. Kemi, 14 B, 1 (1940);
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70. Adler, E., and Gunther, G., Z. physiol. Chem., 253, 143 (1938).

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NICOTINAMIDE NUCLEOTIDE ENZYMES 133

80. V. EuLER, H., Adler, E., Gunther, C, and Elliot, L., Enzymologia, 6,
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(1941).

The Flavoproteins

T. R. HOGNESS

University of Chicago

r-|-iHE flavoproteins constitute a relatively large class of the
_L respiratory enzymes. As we know them today they are charac-
terized by having as prosthetic groups either alloxazine mono-
nucleotide, i.e., riboflavin phosphate, or alloxazine adenine
dinucleotide, which is composed of both riboflavin phosphate and
adenylic acid. They are further characterized by their reactivity
toward oxygen— some to a very limited extent— when in the re-
duced form. Many of them are oxidized by methylene blue; one is
oxidized by fumaric acid; and one is specifically oxidized by a
known member of the hydrogen transport system: the flavoprotein
cytochrome c reductase, in its reduced state, is oxidized by cyto-
chrome c.

In the oxidized state the flavoproteins as a group can be reduced
by a variety of substrates— dihydrotriphosphopyridine nucleotide
(cozymase II), dihydrodiphosphopyridine nucleotide (cozymase I),
the d!-amino acids, xanthine and other purines, and some of the
aldehydes. Each particular flavoprotein, however, is specific toward
one (or one class) of the above substrates. The chemical structures
of both alloxazine mononucleotide and alloxazine adenine dinucleo-
tide are depicted in Figures 1 and 2.

Stern and Holiday (1), by spectrographic methods, first found
that the prosthetic group of Warburg's old yellow enzyme (2),
which will be considered in more detail below, was a derivative
of alloxazine, and the structure of the riboflavin phosphate was
finally deteiTnined independently by Kuhn (3), Karrer (4), and their
collaborators. Theorell (5) demonstrated that the enzyme contained
one molecule of phosphate; and later Kuhn, Rudy, and Weygand (6),
by synthesizing riboflavin-5-phosphoric acid, demonstrated the posi-
tion occupied by the phosphate group.

In Figure 1 the hydrogenation of the alloxazine mononucleotide is
also indicated. The process of hydrogenation undoubtedly takes
place in such a way that one hydrogen atom at a time is transferred
from the substrate molecule to the riboflavin phosphate. The forma-
tion of a red-colored intermediate in the reduction process consti-
tutes much of the evidence in favor of this view. Kuhn and Wagner-

134

THE FLAVOPROTEINS

135

HOCH
HOtH

pO-PO,H;

'jng

H3C

HX-

:-i^^VV%o

HOCH
HOCH
^ H

^fS^y^^r^^

ALLOXAZINE
MONONUCLEOTIDE

1^ 6

DIHYDROALLOXAZINE
MONONUCLEOTIDE

FIGURE 1

Jauregg (7) made the observation that when riboflavin and also
alloxazine are reduced in acid solution red-colored intermediates
with absorption bands having maxima at 490 m[jL were formed.
Haas (8) was able to demonstrate this intermediate formation with
the old yellow enzyme and it has also been found in the case of

H H
H2^0-§-0-P-0-CH2

HOCH 0 0 HO9H
HOCH

ho6h

H3C

CH2

HOCH
HOCH
CH2

c^Vh

"%y^^^^^

^N'

-C>. ^NH
X'

ALLOXAZINE ADENINE
DINUCLEOTIDE

FIGURE 2

a -=i

C/-V

0^-x

a
o

4J !«!

2 ttJ O 4)

•r 3.& 3
i3 fl-d 0

e 4-1

m CI.

S c

.2
'J

.'"

f-,

'G '3a

CU •

.a o
= a

2 6cg

O 4)
O D

tT g i::^

O 4)

-G-d

.2 o

05 3
^1

& ~

'<^

I ^

-g-d^T3
o <= S o

is c-d q

«-^
9 o

CS U

"3 "O

. en O <u
n 0; _c "d

•- >s 55 o
S-t3 ay

-d

= 1

"cS -d

w> 9

J3 03

o >jS

g
■3

-2.9

13 "d

-d
a

-a

1-^

-g

.25 >>.22

.2 o

3-S

P^-d

^
^

O O

-d

"3^

fl2

o c«

-d ti
o 3

-d

•^

_ft

4-1

rS-^ '

""^

ffi

tn
tn

tn
03

r^^ 1

C 1

136

THE FLAVOPROTEINS 137

cytochrome c reductase (9). In Table I the characteristics of the
known and identified flavoproteins are presented in outhne form.

Inasmuch as three excellent reviews of the respiratory enzymes
by Kalckar (20), Green (21), and Oppenheimer and Stern (22) have
recently appeared which collectively deal at length with the flavo-
proteins, I shall consider only the highlights that characterize them
and treat them by comparison with one another. Later I shall con-
sider in some detail that particular flavoprotein with which I am
most familiar and which was isolated by my two collaborators, Haas
and Horecker— cytochrome c reductase. I should like to add that
Mr. Haas is responsible for much of the experimental work reported
by me here.

The Old Yellow Enzyme— Kher Barron and Harrop (23) found
that methylene blue could bring about the respiration of erythro-
cytes, Warburg and Christian repeated these experiments, using an
extract of horse erythrocytes, with hexose monophosphate as the
substrate. They were able to separate from this extract three factors
necessary in the respiration process. From these three separate com-
ponents, the old yellow enzyme, triphosphopyridine nucleotide, and
Zwischenferment were later isolated. The old yellow enzyme was
thus the first flavoprotein to be discovered. The prosthetic group of
the old yellow enzyme is alloxazine mononucleotide or riboflavin
phosphate.

An interesting observation that has never been explained came
out of these first experiments of Warburg and Christian. Whereas it
was necessary to add methylene blue to a fresh extract of horse
erythrocytes to bring about respiration, the addition of the dye was
not necessary when the extract had been dried and subsequently
dissolved. In the latter case the old yellow enzyme presumably
reacted directly with oxygen. If so, then why did not the same
reaction also take place with the fresh extract?

The d- Amino Acid Oxidase— In 1934 Krebs (10) identified both
the d- and the Z-amino acid oxidases in an extract obtained from
kidney cortex. He found that whereas the Z-amino acid oxidase is
inhibited by cyanide, the d-amino acid oxidase is not. Furthermore,
when the extract is dried, the Z-amino acid oxidase is destroyed,
whereas the cZ-amino acid oxidase remains active.

Beginning with Krebs' findings, Warburg and Christian (11)
isolated the cZ-amino acid oxidase and found that its prosthetic
group was a dinucleotide made up of riboflavin phosphate and
adenylic acid. The cZ-amino acid oxidase reacts with most of the

138 A SYMPOSIUM ON RESPIRATORY ENZYMES

ff-amino acids, oxidizing them to the alpha-keto acids and ammonia;
d-glutamic acid is a notable exception. Its reactivity with oxygen is
greater than that of the other flavoproteins.

The New Yellow Enzyme.— The new yellow enzyme, isolated by
Haas (12) immediately after the first isolation of the c?-amino acid
oxidase, was found to have associated with it, as its prosthetic group,
alloxazine adenine dinucleotide. It reacts with diphosphopyridine
nucleotide as does the old yellow enzyme; its reactivity toward
oxygen is considerably less than that of the old yellow enzyme, but
it is very active toward methylene blue.

The Straub Yelloio Efizyme.—This enzyme (13) was isolated from
heart muscle and in all probability is the same enzyme that Haas
isolated from yeast.

The Crossed Yellow Enzy7ne— Warburg and Christian (14) "syn-
thesized" a new enzyme by having the protein moiety of the old
yellow enzyme combine with alloxazine adenine dinucleotide rather
than with the mononucleotide in combination with which it was
isolated from yeast. The properties of this crossed enzyme are
similar to those of the old yellow enzyme.

Xanthine Oxidase.— This enzyme, isolated in a purified state by
Ball (15), catalyzes the oxidation of the purines, particularly
xanthine, the aldehydes, and diphosphopyridine nucleotide (24), by
oxygen. Its prosthetic group is alloxazine adenine dinucleotide. Since
its exact nature has not yet been elucidated, it is possible that this
enzyme contains components not yet accounted for. As defined by
its activity toward aldehydes, it was once known as "Schardinger's
Enzyme." It was first identified in milk by Morgan, Stewart, and
Hopkins (25).

Fumaric Dehydrogenase.— This enzyme catalyzes the reduction of
fumaric acid by one of several leuco dyes, the products of the
reaction being succinic acid and the oxidized or colored dye. The
activity of the enzyme is measured by the rate at which the color
appears. It was discovered by Fischer and Eysenbach (16) in 1937,
and in 1939 Fischer, Roedig, and Ranch (17) purified it further by
electrophoresis. No physiological reducing agent has been found
with which this enzyme is active.

Aldehyde Oxidase.— hike xanthine oxidase, this enzyme catalyzes
the oxidation of aldehydes but diflFers from the xanthine oxidase in
that it does not catalyze the oxidation of xanthine. It was isolated
in 1939 from liver by Gordon, Green, and Subrahmanyan (18).

Cytochrome c Reductase— This flavoprotein acts as the inter-

THE FLAVOPROTEINS 139

mediary link between cytochrome c and triphosphopyridine nucleo-
tide. It will later receive special attention.

A glance at Table 1 shows clearly that many of these flavopro-
teins are closely associated with either disphospho- or triphosphopyr-
idine nucleotide. They therefore constitute a link in the hydrogen
transport system. Nor is it surprising that so many of them react
with molecular oxygen. In fact, we should expect all of them to be
autoxidizable, at least to a small extent, for all of them are pre-
sumably dissociable into the prosthetic group and a protein, and
the prosthetic group itself is autoxidizable. In some cases, however,
the prosthetic group, when attached to the protein, is very probably
also autoxidizable. If this were not true, we might expect that those
flavoproteins which dissociate to the greatest extent, i.e., those
having the largest dissociation constants, would have the greatest
rate of autoxidation. There is evidence that those flavoproteins with
the greater dissociation constants are more autoxidizable, but it
is not conclusive. In making such a comparison we must compare
with each other only those flavoproteins having the same prosthetic
group and under such conditions that the oxidation of the flavo-
protein is the rate-determining step.

Each of the flavoproteins dissociates to a difi^erent extent into its
prosthetic group and its protein moiety. The degree of dissociation
of any flavin nucleotide— protein complex is determined by a pro-
cedure which involves first splitting the complex into its two con-
stituent parts and separating them. By adding increasing amounts of
the prosthetic group to a fixed amount of protein and choosing
conditions such that the reaction velocity is proportional to the
amount of complex formed, it is then possible to determine the dis-
sociation constant from the well-known Michaelis-Menten equation,
or some modification of it. This equation states that

(1) ^ '^'

Vm Kt+(S)

in which V is the velocity, Vm the maximum velocity (when all pro-
tein is in the form of a complex), (S) is the total concentration of the
prosthetic group, and K^ is the dissociation constant. When
y/Vm = 0.5, then K^ is equal to (S). This equation is valid only
when the dissociation constant is so high or the concentration of the
protein so low that the amount of the prosthetic group bound to the
protein is small as compared with the total amount in the solution.
Under these conditions the concentration of the uncombined pros-

140 A SYMPOSIUM ON RESPIRATORY ENZYMES

thetic group and the total concentration (combined and uncombined)
may be regarded as the same. If this assumption cannot be made,
then a somewhat more comphcated equation must be used (see ref.
19).

The spHtting of the flavoprotein has been accomphshed in two
ways. Theorell (26) separated the riboflavin phosphate and the
protein by a 72-hour dialysis against a dilute acid solution. By
adding the riboflavin phosphate to the protein remaining after the
dialysis, the original activity was restored. Warburg and Christian
(27) later developed a simpler and more rapid method in which
the solution containing the flavoprotein is acidified in the presence
of large concentrations of ammonium sulfate. In this solution the
flavoprotein is split and the protein is precipitated. The ammonium
sulfate protects the protein moiety against denaturation.

The known dissociation constants, determined in the manner
previously outlined, are presented in Table 2.

Table 2.— Dissociation constants

Enzyme Dissociation constant Reference

Amino acid oxidase 250X10-9 28

Old yellow enzyme 60X10-» 2

New yellow enzyme 27X10-9 12

Cytochrome c reductase 1 X 10"' 19

In comparing the activities of various enzymes, several factors
must be taken into account. These are (1) the concentrations of the
reacting substances, (2) the aflBnity of the enzyme for the reacting
substrate molecules, and (3) the absolute reaction velocity of the
reactants when in the form of the protein-substrate complex. The
last two of these factors determine the intrinsic activity of the
enzyme, but in determining how great a role any enzyme plays in a
given cell, the concentrations are of prime importance.

All the evidence indicates that in every case substrate and enzyme
form a complex before reaction sets in. As an example we may
consider the reaction between reduced triphosphopyridine nucleo-
tide (TPNH2) and oxidized cytochrome c reductase (CR).

( 2 ) TPNH, -f CR = TPN -f CRH2

A complex is first formed and the velocity of the reaction is pro-
portional to the concentration of this complex.

THE FLAVOPROTEINS

141

The equations representing the dissociation and the equiUbrium
expression or dissociation constant for the formation of the complex
are as follows:

( 3 ) Complex = CR + TPNHj

_ (CR) (TPNH=)
( Complex )

The velocity of the reaction is proportional to the concentration
of the complex.

( 5 ) v = Kr i Complex )

Since ( 6 ) ( CR ) totai = ( CR ) + ( Complex )

Kr(TPNH2)(CR) total

(7) V = — ^^

Ki+ (TPNHO

This equation is essentially the Michaelis-Menten equation. I am
considering it in some detail only because I wish to use it in refer-
ence to the determination and definition of enzyme activity.

In the above derivation I have assumed that the products of the
reaction have no inhibitory influence, or that the conditions are such
that the concentrations of the products are negligibly small. The
velocity plotted against the concentration of dihydrotriphosphopyr-
idine nucleotide is given in Figure 3. This curve is strictly in accord
with equation 7.

2.0 X 10

FIGURE 3

142

A SYMPOSIUM ON RESPIRATORY ENZYMES

The magnitude of Kr denotes the velocity of the reaction within
the complex, and the value of K^ gives a measure of the concentra-
tion of the complex. Thus in more dilute solutions of the substrate,
when the enzyme is not saturated, both Kr and K^ determine the
activity— the larger Kr and the smaller K^, the more active is the
enzyme. Unfortunately not enough data are present to permit of so
specific a comparison of enzymatic activities. An approximation,
however, can be made. If the concentration of the substrate, TPNHj
in this case, is very small as compared with K^, then equation 7
reduces to:

2^
(8) t; = —^(TPNHO(CR) total

or u = K'(TPNHO(CR) total

Under these conditions the velocity is proportional to the concen-
tration of the TPNH2 (lower left part of curve), and K\ which is
approximately equal to Kr/K^ is a measure of the activity— the
larger the velocity constant Kr and the smaller the dissociation con-
stant Ki, the more active is the enzyme. Neither Kr nor K^ are
aflFected by the concentration of the substrate or enzyme, whereas
the turnover number may be.

In more concentrated solution, i.e., when the enzyme is saturated
with substrate, only Kr determines the activity (u = Kr(CR)totai)-
Thus the significance of the dissociation constant is apparent only
when the substrate is present in low concentration— a low dissocia-
tion constant enhances the activity. On the basis of this criterion of
activity, a comparison between the activities of the various flavo-
proteins for low concentrations of substrate is given in Table 3.

Table 3.— Specific reaction velocities at 25° C,

Reaction with

dihydrotri-

Reaction

Enzyme

Prosthetic
group

phospho-

pyridine

nucleotide

with
oxygen

with cyto-
chrome c

Old yellow enzyme

alloxazine mono-

nucleotide

6X10«

10.0X10*

0.3X105 (?)

New yellow enzyme

alloxazine adenine

dinucleotide

22X109

1.4X10*

Cytochrome c reduc-

alloxazine mono-

tase

nucleotide

170X108

0.8X10*

53,000X10«

THE FLAVOPROTEINS 143

Theorell (29) found that reduced old yellow enzyme reacted to a
very small extent with cytochrome c. In view of the fact that the
activity of cytochrome c reductase toward cytochrome c is more
than 100,000 times greater than that of the old yellow enzyme in low
concentrations, it is conceivable that Theorell's sample of the old
yellow enzyme contained a trace of the reductase.

My two collaborators, Haas and Harrer (9), have found that when
the cytochrome c reductase is kept at 0° C. for four weeks the
enzyme becomes partially denatured. Its activity, as measured by
the second-order velocity constant, with respect to triphospho-
pyridine nucleotide and cytochrome c, decreases 91 per cent, and
its activity toward triphosphopyridine nucleotide and oxygen de-
creases 36 per cent. These experiments indicate that Warburg's
old yellow enzyme is not denatured cytochrome c reductase, for
if it were, one would expect the activity of the reductase toward
oxygen to increase with this specific deactivation, since the activity
of Warburg's old yellow enzyme toward oxygen is greater than that
of cytochrome c reductase.

Only two of the flavoproteins are oxidized by physiological sub-
strates other than oxygen, namely, fumaric dehydrogenase and
cytochrome c reductase. But since it has not been demonstrated that
fumaric dehydrogenase is reduced by physiological substrates, cyto-
chrome c reductase constitutes the only known link between the
pyridine protein system (specifically triphosphopyridine nucleotide)
and the cytochromes in the hydrogen transport system. Szent-
Gyorgyi (30), on the basis of the catalytic efiPect of small amounts
of dicarboxylic acids, succinic-fumaric and malic-oxalacetic, on tissue
respiration, and on the basis of the finding that dihydro-alloxazine
can be oxidized enzymatically by fumaric acid, postulated that the
succinate-fumarate couple served as the missing link between old
yellow enzyme and cytochrome c. However, no direct experimental
evidence has been produced to substantiate this viewpoint. The
fact that cytochrome c reductase, a very active flavoprotein itself,
acts in this capacity has obviated the necessity for a link between
the old or new yellow enzymes and the cytochrome system.

The hydrogen transport system (or one branch of it), as we now
definitely know it, consists of the following series of reactions:

Hexose- 2H Triphospho- 2H H

mono- >- pyridine ->. Cytochrome c > Cytochrome c

phosphate Zwischen- nucleotide reductase
ferment

144 A SYMPOSIUM ON RESPIRATORY ENZYMES

The cytochrome c is oxidized by oxygen through the intermediation
of an enzyme or enzymes known as cytochrome oxidase, and by
hydrogen peroxide with cytochrome c peroxidase as the catalyst
(31). The cytochrome c oxidase, or one component of it, is probably
Warburg's oxygen-carrying ferment, but it has not yet been defi-
nitely identified. Nor do we yet understand the whole mechanism
involved in the oxidation of cytochrome c.

The test for determining the relative concentration of the cyto-
chrome c reductase involves all these components. If oxidized cyto-
chrome c is placed in a solution containing all the other components
and if the concentration of the hexose monophosphate is in excess,
the cytochrome c will be reduced, and the rate at which it is re-
duced will depend upon the concentrations of the other components :
Zwischenferment, triphosphopyridine nucleotide and the cytochrome
c reductase. The concentrations of all substances can be so adjusted
that the logarithmic rate of reduction of the cytochrome c will be pro-
portional to the concentration of the reductase.

(9) -^ = K(CR)

In this equation (CyFe+'^+) represents the concentration of the oxi-
dized cytochrome c and (CR) the concentration of the cytochrome
c reductase. The equation is an empirical one.

It is easy to demonstrate that all components of the system must
be present before the cytochrome c will be reduced. In the reduced
state cytochrome c displays three bands with maxima at about 410,
520, and 550 m^., and any one of these bands may be used to deter-
mine the rate of reduction of the cytochrome. For most purposes the
band at 550 m[j. is most convenient for analytical purposes, although
for very dilute solutions of cytochrome c (10^ M) a wave length
of 418 is used. As the cytochrome c is reduced, these bands appear.
In the following spectroscopic demonstration, the general method
employed in this test is illustrated. If all components except the
reductase are added to the buffered solution in the absorption cell,
no reduction of the cytochrome c takes place until a solution of the
reductase is added to complete the chain of enzymatic reactions.

Spectroscopic demonstration: The .apparatus consists of a simple
focusing illuminating lantern (Central Scientific Company) slightly modi-
fied to hold an absorption cell 2.5 cm. in diameter and 2 cm. thick.
A lamp with a ribbon type filament is used, and the filament, turned edge-
wise, is used as the slit. By placing a transmission grating in the light
path a spectrum is projected on the wall.

THE FLAVOPROTEINS 145

Then the cell is filled with buffer, and when all components of the
reducing system except the reductase are placed before the filament, no
bands appear. However, when the reductase is added, the two visible
bands of cytochrome c gradually appear as intense broad black lines.
The cytochrome is then reduced.

To oxidize the cytochrome c, H2O2 is added. No reaction occurs, i.e.,
the black bands do not disappear, until the enzyme cytochrome c peroxi-
dase is added. Upon the addition of this latter enzyme, the two bands
disappear and reappear again as soon as the HgO, is completely reduced.

Not only can this system be used to determine the concentration
of cytochrome c reductase, but it can serve as an analytical method
for the determination of any one of the constituents. In fact, my two
collaborators, Haas and Harrer (9), have only recently worked out
the conditions necessary for a relatively simple determination for
triphosphopyridine nucleotide. By using glucose and adenosine
triphosphate instead of hexose monophosphate, this system should
serve as a method for determining the relative concentrations of
either mutase, the enzyme that converts glucose-1-phosphate to
glucose-6-phosphate, or hexokinase, the enzyme that directly phos-
phorylates glucose-6-phosphate.

Haas and Harrer (9) have also made very accurate measurements
on the rate of oxidation of the reductase by cytochrome c and the
rate of reduction by dihydrotriphosphopyridine nucleotide under
a great variety of conditions, and as a result we have demonstrated
by these kinetic measurements that the reductase forms com-
plexes with both the dihydrotriphosphopyridine nucleotide and with
the cytochrome c. The approximate first-order reaction velocities
have been determined, as have also the heat of dissociation of the
complexes and the energy of activation of the first-order reactions.
The tentative results of these studies are embodied in Table 4. The
values given in the table are approximations. Although the data

Table 4.— Properties of cytochrome c reductase

Reaction

Dissociation

constant of

complex with

reductase 25°

First-order

velocity
constant 25°

Heat of
dissociation

Energy of
activation

Reduction of reduc-
tase by TPNH2

Oxidation of reduc-
tase by CyFe+++

1X10-6
moles per liter

7X10-8
moles per liter

ca. 2000
min-i

ca. 2000
min"!

2 kg. cal.
8 kg. cal.

12 kg. cal.
17 kg. cal.

146 A SYMPOSIUM ON RESPIRATORY ENZYMES

are at hand, the final calculations (which involve a series of ap-
proximations) have not been made. The dihydrotriphosphopyridine
nucleotide forms a stable complex with the reductase, but the com-
plex between the reductase and the cytochrome c is a more stable
one. In this latter case the complex is one in which both components
are proteins; the cytochrome c reductase, with a molecular weight of
about 75,000, combines with cytochrome c, with a molecular weight
of 13,000. In all probability both the dihydrotriphosphopyridine
nucleotide and the cytochrome c, as well as the riboflavin phosphate,
are simultaneously attached to the same protein molecule.

This picture of the oxidation-reduction complex inspires specula-
tion with respect to the manner in which the hydrogen atoms are
transferred from the dihydrotriphosphopyridine nucleotide to the
riboflavin phosphate constituents of the complex. Because of the
complexity of the molecules involved, it is difficult to imagine the
"active" positions of each of them approaching close enough for a
direct hydrogen transfer. The most likely mechanism seems to be
that of ionization, with a hydrogen ion dissociating into the solution,
this process followed by an electron transfer from one molecule to
the other, and this in turn followed by an attachment of another
hydrogen ion from the solution to the new position of the electron.
In the oxidation or reduction of cytochrome c we already regard the
change taking place as electronic.

Cytochrome c reductase bridges one gap in the hydrogen trans-
port system— that between cytochrome and triphosphopyridine
nucleotide. The other gap, between the cytochrome system and
diphosphopyridine nucleotide, is still open, although a beginning
toward the solution of this problem has been made. My two col-
laborators, Altschul and Persky (32), have found a soluble protein
in yeast that is capable of acting as an intermediate in the reduction
of cytochrome c by dihydrodiphosphopyridine nucleotide. It is
soluble; like other proteins it is precipitated by ammonium sulfate,
acetone, and alcohol; it is heat-labile, and can be dialyzed without
great loss of activity. It is not reactive toward triphosphopyridine
nucleotide. Since its enzymatic function is so nearly like that of
cytochrome c reductase, it, too, is probably a flavoprotein, although
there is not yet any direct evidence to this effect.

Of the flavoproteins which react with either dihydrodiphosphopyr-
idine nucleotide or dihydrotriphosphopyridine nucleotide, only
Warburg's old yellow enzyme and cytochrome c reductase react
directly, and to any appreciable extent, with physiological oxidizing

THE FLAVOPROTEINS 147

agents; the reaction between Haas's new yellow enzyme or Straub's
yellow enzyme and oxygen is very slow, unless methylene blue is
used as a "carrier." Possibly carriers other than methylene blue exist
in respiring cells. Xanthine oxidase and aldehyde oxidase react with
reducing agents which, while important, do not contribute greatly
to the production of energy. Lipmann (33), using the Warburg
separation technique, split from a soluble fraction obtained from
Bacterium Delbriickii (Lactobacillus delbrilckii) a protein portion
which was active as pyruvic acid oxidase only when both thiamine
pyrophosphate and alloxazine adenine dinucleotide were added.
From this result he postulated that a yellow enzyme possibly
oxidizes thiamine pyrophosphate. Green, Knox, and Stumpf (34)
have recently reported the finding of another yellow enzyme, the
function of which has not yet been determined.

Formerly it was assumed that cytochrome b, because of its poten-
tial, acted as one of the intermediaries between the flavoproteins and
cytochrome c, but the discovery of cytochrome c reductase has
obviated the necessity for any such intermediary, although in the
intact cell it may act as such (two or more paths of oxidation may be
in operation).

At present nine flavoproteins are known, not all of which react
with both the oxidizing and the reducing agents which are present
in the living cell; the discovery of more or these important enzymes
will undoubtedly follow. The relatively large riboflavin content of
the liver and the kidney and the multiple and complicated bio-
chemical functions of these organs alone indicate that we might
expect to find many more members of this class of enzymes.

REFERENCES

1. Stern, K. G., and Holiday, E. R., Ber., 67, 1104, 1442 (1934).

2. Warburg, O., and Christian, W., Biochem. Z., 254, 438 (1932); 263,
228 (1933); 287, 291, 440 (1936).

3. KuHN, R., et al, Ber., 66, 1034 (1933); 68, 1765 (1935); 69, 1557 (1936).

4. Karrer, p., Helv. Chim. Acta, 18, 69, 72, 426 ( 1935); Ber., 68, 216 ( 1935).

5. Theorell, H., Biochem. Z., 272, 155 (1934).

6. KuHN, R., Rudy, H., and Weygand, F., Ber., 69, 2034 (1936).

7. KuHN, R., and Wagner- J auregg, Th., Ber., 67, 361 (1934).

8. Haas, E., Biochem. Z., 290, 291 (1937).

9. Haas, E., Harrer, C, and Hogness, T. R. (unpublished).

10. Krebs, H. a., Biochem. J., 29, 1620 (1935).

11. Warburg, O., and Christian, W., Biochem. Z., 296, 294; 298, 150 (1938).

12. Haas, E., Biochem. Z., 298, 378 (1938).

13. Straub, F. B., Biochem. J., SS, ISl (1939).

14. Warburg, O., and Christian, W., Biochem. Z., 298, 368 (1938).

15. Ball, E. G., J. Biol. Chem., 128, 51 (1939).

148 A SYMPOSIUM ON RESPIRATORY ENZYMES

16. Fischer, F. G., and Eysenbach, H., Ann., 530, 99 (1937).

17. Fischer, F. G., Roedig, A., and Rauch, K., Naturwissenschaften, 27, 197
(1939).

18. Gordon, A. H., Green, D. E., and Subrahmanyan, V., Biochem. J., 34,
764 (1940).

19. Haas, E., Horecker, B. L., and Hogness, T. R., J. Biol. Chem., 136, 747
(1940).

20. Kalckar, H. M., Chem. Rev., 28, 72 (1941).

21. Green, D. E., Mechanisms of Biological Oxidations (Cambridge Univer-
sity Press, 1940).

22. Oppenheimer, C., and Stern, K. G., Biological Oxidation (W. Junk, The
Hague, 1939).

23. Barron, E. S. G., and Harrop, G. A., J. Exp. Med., 48, 207 (1928), J.
Biol. Chem., 79, 65 (1928).

24. Ball, E. G., and Ramsdell, P. A., J. Biol. Chem., 131, 767 (1939).

25. Morgan, E. J., Stevi'art, C. P., and Hopkins, F. G., Proc. Roy. Soc.
(London), B 94, 109 (1922).

26. Theorell, H., Biochem. Z., 275, 37 (1934).

27. Warburg, O., and Christian, W., Biochem. Z., 296, 294 (1938).

28. Warburg, O., and Christian, W., Biochem. Z., 295, 261; 298, 150 (1938).

29. Theorell, H., Biochem. Z., 288, 317 (1936).

30. Szent-Gyorgyi, A., Z. physiol. Chem., 249, 211 (1937).

31. Altschul, a. M., Abrams, R., and Hogness, T, R., J. Biol. Chem., 136,
777 (1940).

32. Altschul, A. M., Persky, H., and Hogness, T. R., Science, 94, 349 ( 1941).

33. Lipmann, F., Nature, 143, 436 (1939).

34. Green, D. E., Knox, W. E., and Stumpf, P. K., J. Biol. Chem., 138, 775
(1941).

Wilson and Kalckar:
'The manuscript deadline is

HoGNESs AND Elvehjem: "The respiratory eiiz\nie, alloxazine-
adenine dinucleotide, . . ." "The vitamin, riboflavin, . . ."

OcHOA, Wood, and Carson: "With hea\ v carbon .
"But with radioactive carbon . . ."

COMMENTS OFF THE RECORD

Cytochromes

ELMER STOTZ

Harvard University"*

THE SUBJECT OF THE iron-containing cellular respiration catalysts
has demanded the attention of many biochemists. One obvious
reason for this is the fact that the cytochromes are so readily
detectable by spectroscopic means that their wide distribution and
hence their apparent importance were early recognized (1, 2). With
increasing knowledge of cellular respiration the unique and funda-
mental position of the cytochromes in the respiratory scheme has
been emphasized more and more. Although the various substrates of
respiration may require many enzymes, coenzymes, and mediators,
the individual pathways appear to converge at the cytochrome sys-
tem. It is through the ferrous to ferric change of this system that
the electrons of the ultimate substrate, hydrogen, come to terms
with the ultimate oxidant, oxygen.

In proportion to its importance in respiration, perhaps less is
known of the cytochromes than of other respiratory components.
Cytochrome c has been isolated, but the peculiar linkage of its
prosthetic group with the protein is not yet fully understood. Cyto-
chromes a and b still remain bands in the absorption spectrum of
tissues with little appreciation of their function. Finally, the all-
important cytochrome oxidase is still httle more than the insoluble
ground residue of tissues.

Properties of the Cytochrome Components

Cytochrome c— Cytochrome c appears to be quantitatively the
most important of the three cytochrome constituents. It was defined
spectroscopically by Keilin (2, 3) as that component which in the
reduced state has an alpha-band at 5500 A. and a beta-band at
5200 A. It was first isolated in apparently pure form by Theorell
(4) in 1936 by a dilute sulfuric acid extraction of defatted beef
heart muscle; isolation was followed by ammonium sulfate precipi-
tation, barium sulfate adsorption, acetone precipitation, and finally
adsorption on cellophane. Shortly thereafter Keilin and Hartree (5)

* Contribution from the McLean Hospital, Waverly, Massachusetts, and the
Harvard Medical School, Boston.

149 ^

150 A SYMPOSIUM ON RESPIRATORY ENZYMES

described a simpler method of preparation involving a trichloracetic
acid extraction of the ground muscle, ammonium sulfate fractiona-
tion, and trichloracetic precipitation. Both groups of workers ob-
tained a product which had the same absorption bands as the
cytochrome c of the intact or phosphate-extracted muscle, and
which contained 0.34 per cent iron, resisted further fractionation,
and was therefore considered pure. Later Theorell and Akesson (6)
obtained by electrophoretic means a product containing as high as
0.43 per cent iron. It would be of interest to know whether this
product displayed an equal increase in catalytic activity over the
0.34 per cent iron product.

Zeile and Renter (7) calculated a molecular weight of 18,000 for
cytochrome c on the basis of its hemin content. Theorell (4), from
a study of its diffusion and sedimentation, determined a molecular
weight of 16,500. The iron content of 0.34 per cent also yields an
equivalent weight of 16,500. The isoelectric point of cytochrome c
is approximately at pH 9.8 (4).

Ferric cytochrome c is readily reduced by a variety of agents,
such as hydrosulfite, ascorbic acid, cysteine, adrenalin, hydroqui-
none, p-phenylenediamine, and many leuco dyes, as well as by
certain physiological reducing systems to be discussed later.

Ferro -cytochrome c is essentially non-autoxidizable in neutral
solution, the slow rate being largely inhibited by small amounts of
cyanide (8), indicating heavy metal catalysis. It is readily oxidized
by ferricyanide and aerobically by oxidase preparations. Below pH
4.0 and above 11.0 the spectrum changes and the substance becomes
autoxidizable.

A neutral solution of cytochrome c can be boiled, and upon cooling
the original spectrum and catalytic properties return. Cytochrome c
is likewise stable to dilute acid. It is stable to 0.1 normal potassium
hydroxide, but 1.0 normal alkali produces an irreversible change in
the spectrum. The substance is then autoxidizable and forms a light-
sensitive carbon monoxide compound (9).

According to Keilin (10), cytochrome c does not appear to com-
bine with hydrogen sulfide, hydrogen cyanide, sodium azide, or
hydroxylamine, nor, according to Stern (11), with carbylamine.
Ferri-cytochrome c does, however, form a compound with nitric
oxide (12). Keilin (13) was unable to detect any change in the
spectrum of the c component in the presence of carbon monoxide
except in solutions whose pH was above 13.0. Altschul and Hogness
(14), using an accurate photoelectric spectrophotometer (15) with a

CYTOCHROMES 151

narrow slit, have, however, found evidence of a ferro-cytochrome-
carbon monoxide compound throughout the entire pH range. The
change in spectrum was reversible; that is, the carbon monoxide
could be removed by nitrogen. The carbon monoxide compound
was light-sensitive. Keilin and Hartree (10) attribute this finding to
the presence of denatured cytochrome or other hematin compounds.
At neutral pH they were able to liberate and measure manometri-
cally only 10 per cent of the theoretical amount of carbon monoxide
that should combine with reduced cytochrome.

Potter (16) concluded that cyanide also, contrary to popular
belief, forms a complex with ferri-cytochrome c. This conclusion
was based not only on the fact that a change in the spectrum was
detected but also upon studies on the enzymatic reduction of cyto-
chrome c. Since the spectral shift is small, as with carbon monoxide.
Potter questions whether one can safely conclude from simple spec-
troscopic observation that a given inhibitor has not reacted with
cytochrome.

Considerable gains have been made in determining the structure
of the prosthetic group of cytochrome c and how this might explain
the peculiar stability of the heme-protein linkage. Hill and Keilin
(17) obtained a porphyrin by hydrochloric acid and sulfur dioxide
treatment of cytochrome c which, unlike most porphyrins, was solu-
ble in water. Zeile and Piutti (18), in extensive synthetic work, were
able to introduce various nitrogen bases into the unsaturated side
chains of protoporphyrin and obtain porphyrins whose solubility
was similar to that obtained from cytochrome c. When iron was
introduced into some of these compounds, they showed the charac-
teristic cytochrome c absorption band at 5500 A. Later Zeile and
Renter (7) isolated hematoporphyrin from a hydrobromic-acetic
acid degradation of cytochrome c. Theorell (19) isolated a sulfur-
containing porphyrin and postulated that the vinyl, groups of the
hemin are linked to amino acids of the protein by thio-ether bonds.
Upon demonstrating later that such a porphyrin could arise by con-
densation of hemato -porphyrin with cysteine during the course of
the cytochrome hydrolysis, Theorell (20) explained that either nitro-
gen or oxygen as well as sulfur might form the connecting link.
Zeile and Meyer (21) offer support to the sulfur-bridge theory in
obtaining the sulfur-containing porphyrin under conditions of
hydrolysis in which a condensation of porphyrin with free cysteine
would be very unlikely. A tentative structure of cytochrome c is
illustrated in Figure 1.

152

A SYMPOSIUM ON RESPIRATORY ENZYMES

Cytochrome c has, as compared with other biological systems,
a very high oxidation-reduction potential. As early as 1932 CooHdge
(22), using an impure preparation from yeast, reported a potential.
The rather unsatisfactory potential with the electrode could be
stabilized with hydroquinone. By adding oxidants or reductants to
a point where the spectrum of the cytoclirome changed, an Eo' of
+ 0.260 V. at pH 7.0 was recorded. Lower values were obtained at
pH 5.0. In 1934 Green (23) determined the potential of an impure

PROTEIN

H.G

rNJH2 COOH
CH

r^Hp COOH

C7H,oO±C±2H ± O

— I

CH-CH

CH2
COOH

Figure 1. — Structure of cytochrome c

CYTOCHROMES 153

yeast cytochrome c, but found a much lower value of Eg' — +0.127
V, between pH 4.6 and 7.1. Finally, Wurmser and Filitti-Wurmser
(24) in France and Stotz, Sidwell and Hogness (25) in Chicago,
measured the potential of pure cytochrome c isolated from heart
muscle. The former measured the equilibrium potential in mixtures
of reduced and oxidized cytochrome c, the proportion being deter-
mined spectrophotometrically. They obtained the value Eo' =
+ 0.254 V. between pH 5.0 and 8.0. Stotz et al. used a purely
spectrophotometric method and obtained a value of +0.262 v. in
the same pH range. The spectrophotometric method consisted in
measuring accurately the amounts of oxidized and reduced indicator
and cytochrome in equilibrium with each other. The potential of the
indicator being known, the potential of the cytochrome could be
readily calculated. The results recorded by the two groups were
reached independently and represent good agreement. They are
both in essential agreement with the potential of +0.27 v. reported
by Ball (26), who was able to estimate the potentials of the three
cytochromes as they existed in a heart muscle extract. At a physio-
logical pH, therefore, cytochrome c has about the same potential as
the hydroquinone-quinone system.

Cytochrome h.—in the reduced state this cytochrome component
possesses an alpha-absorption band at 5640 A. and a beta-band
at 5300 A. It appears to be more closely bound to the insoluble
material in tissue extracts than is cytochrome c. Nevertheless Ya-
kushiji and Mori (27) claim to have isolated cytochrome Z? in a
soluble form. It seems doubtful from their method of preparation
whether the product obtained could be an undenatured cytochrome
h. In some of the original extracts the reduced band is not at 5640 A.,
but as purification proceeds this band is shifted to the normal posi-
tion of reduced cytochrome h. They believe that the hemin portion
of their product is ordinary protohemin. Since several other proteins
combined with protohemin to form spectroscopically and catalyti-
cally similar hemochromogens, it is difficult to believe that these
workers actually obtained cytochrome h.

Judged from its behavior in tissue extracts, cytochrome h appears
to be a thermolabile hemin-protein complex. Unlike cytochrome c,
the h component is autoxidizable. Since this component reaches
equilibrium with other reversible systems in a heart muscle extract.
Ball (26) was able to estimate its potential as —0.04 v., the lowest of
the cytochrome components. It does not combine with carbon
monoxide or other respiratory inhibitors (10).

154 A SYMPOSIUM ON RESPIRATORY ENZYMES

Cytochrome a.— Cytochrome a is another component whose prop-
erties can be judged only in a crude tissue extract. It was originally
designated as the component which in the reduced state possessed
an alpha-band at 6000-6050 A. Upon more careful analysis, Ball
(26) and Keilin and Hartree (10) discovered that this band was not
homogeneous to reduction or to various reagents; that is, the
absorption in this region must be attributed to more than one sub-
stance. Their results might be interpreted to mean that the portion
of the band nearer 6050 A. is to be attributed to cytochrome a.
Because of this complication, the properties of cytochrome a have
not been definitely established. Keilin and Hartree (10) conclude
that it does not combine with carbon monoxide or cyanide. It is
reduced by the same agents as the other cytochromes. Its potential
has been estimated by Ball (26) as +0.29 v., a value which is perhaps
less certain than those of the other cytochromes.

Very recently Yakusizi and Okunuki (28) claim to have isolated
cytochrome a from heart muscle. The muscle pulp was extracted
with sodium cholate and alkaline phosphate. Ammonium sulfate
fractionations followed by redissolving in the cholate mixture yielded
a product which was, in the oxidized state, of a red-brown color.
When it was reduced it was green, indicating that the prosthetic
group was of the "mixed" or Spirographis hemin type. The reduced
compound showed a strong absorption at 6050 A. and a weak band
at 5130 A., carbon monoxide having no effect on the spectrum.
Reduced cytochrome c was partially oxidized by the oxidized form
of this compound. The latter properties are in agreement with our
concept of cytochrome a, and this important finding should be con-
firmed and extended.

There is some evidence that the potentials of the yeast cyto-
chromes differ from those of the heart cytochromes. But the only
measurement on a pure component has been with heart cyto-
chrome c. It will be recalled that Green (23) obtained a value of
Eo' = +.127 V. for yeast cytochrome c, and the recent work of
Baumberger (29) is in agreement with this finding. The latter work,
however, requires certain comments. Baumberger was able to meas-
ure simultaneously the light absorption at various wave lengths
(photoelectrically) and the Ei, levels of a yeast suspension. The
suspension was vigorously stirred by oxygen-nitrogen mixtures,
which likewise eventually established a constant Ei, level. By vary-
ing the gas mixture and hence the Eh level, the presence or absence
of the cytochrome bands could be determined by changing the wave

CYTOCHROMES 155

length of the incident hght and observing the galvanometer deflec-
tions of the photoelectric device. In this way he arrived at the above
potential for cytochrome c.

Perhaps more startling was his finding that all three cytochromes
appeared to have the same potential; that is, at a given Eh level all
were equally reduced. This is distinctly a contradiction of Ball's
finding that in a heart muscle extract the cytochrome potentials
diflfered markedly. In Ball's work, however, the relative degree of
reduction of the cytochromes was measured when in equilibrium
with systems of known potential and systems known to react with
the cytochromes, whereas in Baumberger's work the normal re-
ductants within a more organized structure establish the equilibrium.
A legitimate question concerning these experiments would be
whether it can be assumed that the potential recorded by a platinum
electrode in a yeast suspension is the same as that existing within
the cell. And, furthermore, is it not likely that different points in
the organized cells actually have very different potentials? The data
might indeed be taken as evidence for the latter hypothesis. Baum-
berger suggests the possibility of a molecular aggregate of the three
cytochromes which is oxidized or reduced as a whole or in which
the three cytochromes do have the same potential.

In a more disorganized structure, such as a heart muscle extract,
these relations apparently do not exist, and it is not unlikely that
this explains the great difference between the frequency of oxida-
tion and reduction of cytochromes in the intact yeast as compared
with that in extracts. Certain it is that in the study of tissue respira-
tion the problem of adsorption and dependence of function on
organized structure is met most frequently in the consideration of
the cytochrome system (see "Oxidation and Cell Structure" in Korr,
30).

Cytochrome Oxidase (Cytochrome a^).—\t may be recalled that in
1924 Warburg (31), upon observing the cyanide sensitivity of cellu-
lar respiration in conjunction with the catalytic behavior of the
hemin-charcoal model, gave the name Atmungsjerment to the cata-
lytically active iron compounds involved in cellular respiration.
This study continued with measurements of the inhibition of yeast
respiration by carbon monoxide and its reversibility by light (32, 33).
By measuring this effect at various light frequencies, the relative
carbon monoxide spectrum and later the absolute carbon monoxide
spectrum of the Atmungsferment were determined (34, 35). It
was renamed the Sauerstoffiibertragendes Ferment or oxygen-

156

CYTOCHROMES 157

transferring enzyme. The spectrum was obviously that of a hemin-
containing compound which resembled in type that of Spirographis
hemin (36). The alpha-band of the reduced carbon monoxide com-
plex lies at 5920 A. and the gamma-band at 4320 A. Finally, in the
highly respiring Bacterium Pasteurianum (Acetobacter pasteuria-
num), under anaerobic conditions, a weak band was observed at
5890 A. which was attributed by Warburg and Negelein (37) to the
reduced form of the oxygen-transferring enzyme itself, since carbon
monoxide shifted the band to 5920 A. On the other hand, cyanide
produced a band at 6390 A. which, since it may be observed even
in the simultaneous presence of the 5890 A. band, need not be a
derivative of the oxygen-transferring enzyme. Keilin (38) believes,
however, that the 5890 A. band is only a degradation product of
cytochrome a and is seen only in certain bacteria. In fact, Fujita
and Kodama (39) observed the 5890 A. band in bacteria only when
the cytochrome a band was absent and have named this band cyto-
chrome flj. Certainly the best criteria by which to establish the
identity of a compound with Warburg's oxygen-transferring enzyme
would be the positions of the carbon monoxide absorption bands
(5920 and 5320 A.).

Keilin's work (40) with "indophenol oxidase" pointed to the
identity of this enzyme with the Warburg enzyme. The oxidase
brought about the aerobic oxidation of the cytochromes, was in-
hibited by cyanide, and showed a Hght-reversible inhibition with
carbon monoxide.

Because of the similarity it has been generally believed that War-
burg's "oxygen-transmitting enzyme" and Keilin's presently-called
cytochrome oxidase are identical. Until recently, however, Keilin
had not observed a band that he could attribute to the carbon
monoxide complex of the oxidase.

In 1939 Keihn and Hartree (10) believed that they had identified
spectroscopically in heart muscle extracts a new cytochrome, a^,
which might be identical with the oxidase. They concluded that
the cytochrome a band at 6000-6050 A. is actually due to com-
ponents a and a^, since upon addition of carbon monoxide this band
divides and a new one appears at 5900 A. With the aid of strong
cane sugar or glycerine solutions, or bile salts, to clarify the solutions
for spectroscopic examination, they were able to examine further
the Soret or gamma-bands of the cytochromes. Simultaneously with
the above shift, a portion of the 4480 A. band is shifted to 4320 A.
These two new bands represent the carbon monoxide complex of

158 A SYMPOSIUM ON RESPIRATORY ENZYMES

the new component, a^. Since these positions correspond to those of
the Warburg enzyme, a^ may be identical with this enzyme. On the
other hand, the alpha-band of the compound itself is claimed to be
at 6000 A., whereas Warburg and co-workers (41, 42, 43) believe
their compound to have a band at 5890 A. This compound (also
called cytochrome a^) likewise reacts with oxygen, and formation of
its cyanide compound prevents reoxidation of the other cytochromes.
Furthermore, its carbon monoxide compound has an alpha-band at
5920 A.

Cytochrome a^, in either the reduced or the oxidized form, com-
bines with potassium cyanide. The cyanide complex of the reduced
form is readily autoxidizable, whereas that of the ferric form is not
easily reduced. Ferric cytochrome a^ also reacts with hydrogen
sulfide, sodium azide, and hydroxylamine. It is thermolabile and
easily destroyed by organic solvents, acids, or alkalies. Cytochrome
flg is reduced along with the other cytochromes by e.g., succinate.
It is also autoxidizable. It is therefore believed that a^ is also identi-
cal with cytochrome oxidase.

The authors have themselves offered certain objections to the
above conclusion:

1. It was impossible to demonstrate the reduction of cytochrome
flg by added reduced cytochrome c, but technically these experi-
ments were not satisfactory. In this connection it is interesting that
Ball (26) noticed that the portion of the 6000-6050 A. band at-
tributed to ^3 by Keilin has a higher potential than any of the
other cytochromes, an expected but not an essential condition for
oxidase function.

2. The carbon monoxide compound of ferro -cytochrome flg did
not appear to be sensitive to light anaerobically. The effect of light,
however, may become apparent only in the presence of oxygen,
which oxidizes the a^ component and thereby prevents its reaction
with carbon monoxide. Such an explanation of the light effect could
also explain its property of relieving inhibition of carbon monoxide.

3. Finally, it has been found possible in the presence of carbon
monoxide to oxidize cytochromes a, b, and c by air while the spec-
trum of the carbon monoxide complex of reduced a^ remains visible.
It is therefore difficult to explain the oxidation of a, h, and c through
the flg component.

In general, it may be said that the Keilin and Hartree paper by
no means clarifies the whole problem of the identity of cytochrome
oxidase and oxygen-transferring enzyme or of either one with the

CYTOCHROMES 159

new flg component. The situation is understandable, however, when
one considers the number of hematin compounds which exist in
tissue preparations and the fact that apparently the same component
may vary slightly in the position of its absorption band in diflFerent
biological materials.

An experiment that has been much needed has finally been pub-
lished as a short note by Melnick (44), namely, the photochemical
determination of the carbon monoxide spectrum of cytochrome
oxidase. This was accomplished by employing a phosphate extract
of heart muscle with succinate as substrate. On the assumption that
the oxidase is the only functional substance present in the prepara-
tion which forms a light-dissociable carbon monoxide complex, the
spectrum measured should be, by Keilin's own definition, cyto-
chrome oxidase. The spectrum obtained was that of a pheohemin
compound. The alpha-band was located at 5890 A., which agrees
very well with that of the oxygen-transferring enzyme in yeast and
bacteria, as well as with the carbon monoxide complex of cyto-
chrome Og seen directly. But in the case of the gamma- or Soret-
band, Melnick finds a band at 4500 A., which does not agree with
the carbon monoxide band of the oxygen-transmitting enzyme (War-
burg) nor with that of a^ (Keilin). The whole situation therefore
remains clouded and awaits chemical separation and identification
for its clarification.

Keihn and Hartree (10) believe that because of the association of
cytochromes a and a^ the two components are intimately related.
They may have an identical heme nucleus, since on alkali denatura-
tion and addition of pyridine they yield the same hemochromogen.
They are both sensitive to heat, alcohol, acetone, and extreme
changes in pH.

It has been suggested that cytochrome oxidase may be a copper
protein. The evidence is quite indirect, such as the wide distribution
of copper, the ability of copper salts to oxidize cytochrome c, and
certain similarities between cytochrome oxidase and the copper-
containing polyphenol oxidase (45, 46). It may be pointed out that
copper does appear to be essential in the formation of cytochrome
oxidase. Cohen and Elvehjem (47) have found it to be essential for
the regeneration of cytochrome a and oxidase in anemic rats, and
Yoshikawa (48) finds it to be a stimulus to the oxidase activity of
yeast cultures. Most conclusive is the recent work of Schultze (49,
50), who showed that copper was necessary for the maintenance
and formation of cytochrome oxidase in rat liver and heart, and that

160 A SYMPOSIUM ON RESPIRATORY ENZYMES

the regeneration of cytochrome oxidase in the bone marrow of
anemic rats was extremely rapid following administration of copper.
Important as these findings are, they do not constitute proof that
cytochrome oxidase contains the copper any more than similar find-
ings with hemoglobin regeneration.

Graubard (51) claims actually to have isolated a water-soluble
cytochrome oxidase from uterus. It is very labile and its action is
inhibited by copper inhibitors. He therefore claims that at least this
oxidase contains copper as the active metal. Certainly any claims
for the identity of a water-soluble or copper-containing oxidase with
cytochrome oxidase must be supported by many experiments giving
evidence that the material conforms with the existing definition of
cytochrome oxidase.

The work of Altschul, Abrams, and Hogness (52, 53) in connec-
tion with the oxidase is of particular interest. They first reported
the isolation from yeast of a soluble cytochrome oxidase which
aerobically oxidized cytochrome c, and which was inhibited by
cyanide and carbon monoxide. Upon concentration, it was noted,
the activity of the enzyme was inhibited by catalase. It was then
found that during reduction of the substrate, cytochrome c, hydro-
gen peroxide was produced as a contaminant and oxidation was
actually a catalyzed oxidation by peroxide. This enzyme, being
specific, is now called cytochrome c peroxidase. Because so little is
known about the physiological mechanism of the aerobic oxidation
of the cytochromes and of the function of hydrogen peroxide, this
very active enzyme may. be of no small importance physiologically.

From the standpoint of isolation, therefore, we still have only
cytochrome c. The other cytochromes and the oxidase are still as-
sociated with insoluble particles and have resisted separation. The
usual opalescent alkaline phosphate extract of heart muscle, which
contains these substances, has been the subject of a physico-chemical
investigation by Stern (54). Observations of this material, in the
ultracentrifuge and electrophoresis reveal properties similar to those
of other macro-molecular materials, such as fractions from Rous
chicken sarcomata. Such a dispersed suspension of particles con-
tains lipids, nucleic acid, hemin, and other constituents. Stern feels
that it is largely a matter of definition whether such a "mono-
dispersed" suspension should be called a mechanical dispersion or a
true solution, and he predicts that when the individual components
are isolated they will no longer display their characteristic biological
orientation. This view does not and should not discourage attempts

CYTOCHROMES 161

to isolate the individual components. Although such heart muscle
preparations have thus far resisted fractionation, one factor, the
diaphorase or coenzyme factor (a flavoprotein) has been separated
from such a preparation (55).

Euler and Hellstrom (56) claim to have efiFected a separation of
the cytochromes by an ammonium sulfate fractionation of a sodium
cholate clarified preparation. The first fraction to precipitate con-
tained cytochromes a and h, the second only cytochrome h, and the
third, cytochrome c. The activity of some of these fractions toward
succinate does not, however, bear out the claim for any extensive
fractionation (see Keihn and Hartree, 9). Such precipitates simply
resuspend in buffers to yield the usual opalescent preparations.
Nevertheless the clarifying or so-called peptizing action of the bile
salts is interesting. I have found (8) in a few experiments that
sodium desoxycholate at a neutral pH yields a virtually clear solu-
tion of the original turbid oxidase preparation. Fractional salt pre-
cipitations have not, however, yielded any striking results. In view
of the action of bile salts with lipids, it is possible that the diflBculties
of separating the cytochrome components may lie in their associa-
tion with or their presence as lipo-proteins.

Reduction of the Cytochromes by Other Respiratory Systems

The fundamental position of the cytochromes in cellular respira-
tion is emphasized by the fact that tissue respiration is so completely
blocked by cyanide (57), and by the experiment of Haas (58) in
which it was demonstrated that the rate of alternate oxidation and
reduction of cytochrome c in intact yeast cells could account for all
the oxygen consumption of the yeast.

The succinate-succinic dehydrogenase system has long been recog-
nized as a reducing system for the cytochromes, and this connects
the important Szent-Gyorgyi— Krebs cycle with the cytochromes. On
the other hand, the details of this reduction are by no means clear.
Thus in 1939 Hopkins, Lutwak-Mann, and Morgan (59) prepared
a succinic dehydrogenase from heart muscle which with succinate
did not reduce cytochrome c, but did nevertheless reduce methylene
blue. The preparation was made in such a way (with alcohol treat-
ments) that no cytochrome oxidase activity remained. Their result
suggests another intermediate between succinic dehydrogenase and
cytochrome c.

Stern and Melnick (54, 60) in their ultracentrifuge studies found
that the sedimented material showed typical succinic dehydrogenase

162 A SYMPOSIUM ON RESPIRATORY ENZYMES

activity toward methylene blue and oxidase activity toward
p-phenylenediamine or hydroquinone. It lacked, however, the ca-
pacity to oxidize succinate, apparently being unable to reduce
cytochrome c. When supernatant fluid from the ultracentrifuge run
was added, aerobic activity of the preparation toward succinate was
restored. The unknown material is evidently a substance of lower
molecular weight (estimated at 140,000), is heat-labile, and is re-
moved by trichloracetic acid, hence is probably a protein. It can-
not be identified with aluminum (61), catalase, or the Straub flavo-
protein (55).

Recently Keilin and Hartree (9) have tested and analyzed the
eflFects of several factors on succinate and p-phenylenediamine oxi-
dation by typical heart muscle extracts. Among their findings were
these: 1. Narcotics inhibited the oxidation of succinate by the cyto-
chrome system more strongly than the oxidation of methylene blue.
Since not only reduction of the cytochrome components was in-
hibited, but also oxidation of cytochrome h, it is possible that these
findings are related to the function of cytochrome h in succinate
oxidation. 2. Preparations treated with alcohol modified irreversibly
the spectrum of cytochromes a^, a, and h and destroyed oxidase
activity. Such preparations did not reduce cytochrome c, although
they retained their ability to reduce methylene blue. 3. Treatment
with acetic acid (pH 5.0 for one hour) did not affect p-phenylene-
diamine oxidation, but destroyed the ability of the succinate system
to reduce cytochrome c. Again, methylene blue reduction was still
possible. Spectroscopically, the absorption bands of the cytochromes
were normal, except that cytochrome h appeared to be no longer
autoxidizable. Evidently cytochrome h had undergone some change.
4. Treatment with pancreatin gave a preparation similar to the
acid-treated preparation.

The results suggest that cytochrome h may be the labile link
between succinic dehydrogenase and cytochrome c, although the
possibility that a flavin or another hematin is a link is by no means
excluded. It may be recalled that the potential of cytochrome h
(—0.04 V.) places it in a favorable position as such a link. Keilin
and Hartree have suggested as an alternative the possibility that
the failure to react with cytochrome c "may be due to an irreversible
change in the colloidal structure of the preparation accompanied
by a loss of accessibility of the succinic system to c, which is a non-
diffusible protein while it remains still accesible to small and dif-
fusible molecules of methylene blue."

CYTOCHROMES 163

It was long desirable to find a link between the di- and tri-
phosphopyridine nucleotides and the cytochrome system. For some
time it has been known that the old yellow enzyme, i.e., the
Warburg-Christian flavoprotein (62), was readily reduced by tri-
phosphopyridine nucleotide (63), but only very slowly oxidized by
cytochrome c (64). This link has recently been established by the
excellent isolation work of Haas, Horecker, and Hogness (65), who
isolated a flavo (mononucleotide) -protein which rapidly reduces
cytochrome c. It has been given the functional name "cytochrome
reductase." The coenzyme II dependent systems have thus been
satisfactorily linked to the iron-containing system. Cytochrome c
reductase does not hnk reduced coenzyme I with cytochrome c
(66). Haas, Horecker, and Hogness (65) believe that since the
reductase loses its power to reduce cytochrome c when subjected to
chemical treatments common to the preparation of the old Warburg-
Christian flavoprotein, it is very probable that the latter represents
a denatured product of cytochrome reductase.

The story is less satisfactory than in the case of coenzyme I. In
1937 Adler, Euler, and Hellstrom (67) discovered an enzyme which
they called "diaphorase," and independently Green and Dewan
(68) investigated what was apparently the same enzyme, which they
called "coenzyme factor." This factor appeared to link coenzyme
I with the cytochrome system. Of significance for the present discus-
sion is the fact that Green and Dewan (68) stated that cytochromes
a and b but not c were involved in the reaction. The absence of
cytochrome c in their preparation has been challenged by Haw-
thorne and Harrison (69) and by Lockhart and Potter (66). The latter
authors have in fact demonstrated that cytochrome c is a link in the
aerobic oxidation of coenzyme I. They also have used two types of
"diaphorase" preparation, one of which catalyzed the coenzyme I
reduction of cytochrome c and the other did not. Both, however,
could catalyze the reduction of methylene blue. This recalls the
findings in connection with succinate reduction of cytochrome c;
in fact, Lockhart and Potter have noted that the preparation unable
to reduce cytochrome c contained no cytochrome b. Thus comes
the suggestion but not the proof that cytochrome b may be involved
as a link between coenzyme I dependent systems and cytochrome c.

Straub (55) has isolated from heart muscle a flavoprotein which
is considered to be identical with the coenzyme factor or diaphorase.
Corran, Green, and Straub (70), in studying the catalytic proporties
of this flavoprotein, find that its reduced form is only slowly autoxi-

164 A SYMPOSIUM ON RESPIRATORY ENZYMES

dizable and that it does not react with cytochrome c but only with
"carriers" such as methylene blue. It is not yet known whether this
protein requires another link (such as cytochrome h) for cytochrome
c reduction, or whether it is actually a denatured product which
has lost its ability to reduce cytochrome c, analogous to the old
Warburg-Christian yellow enzyme and the newer cytochrome re-
ductase.

Catalytic Relations of the Cytochromes and Oxidase

"Indophenol oxidase" was long recognized as the substance in
tissues which produced the aerobic oxidation of Nadi reagent or
p-phenylenediamine (3). This name was retained in spite of the
finding of Keilin (71) that the addition of cytochrome c accelerated
the oxidation of cysteine by "indophenol oxidase" and a similar
finding by Stotz, Harrer, Schultze, and King (72) with respect to
ascorbic acid. When pure cytochrome c became available, it was
not difficult to study the relation of oxidase and cytochrome c in
the oxidation of various substrates. The high oxidation-reduction
potential of cytochrome c that had been noted suggested that the
action of indophenol oxidase was due to an unspecific reduction of
the cytochrome c which it contained, followed by an aerobic cata-
lyzed oxidation of the reduced cytochrome. KeiHn and Hartree (73),
upon noting the accelerating efi^ect of cytochrome c on the oxidation
of several substrates, renamed the oxidase "cytochrome oxidase."
Stotz, Sidwell, and Hogness (74) had come to the same conclusion
and had prepared an oxidase which was largely free of cytochromes

Table 1.— The sensitivity of hydroquinone and p-phenylenediamine
oxidations to cyanide*

(Reference 74)

Percentage

of inhibition

Cyanide concentration

mM X 106 total

Hydroquinone

p-phenylenediamine

oxidation

100

120

100

240

100

T = 38° C; pH, 7.15; 23.5 X 10"* mM cytochrome c total.

CYTOCHROMES

165

c and h. This preparation was essentially unable to oxidize either
hydroquinone or p-phenylenediamine without the addition of cyto-
chrome c.

A study of the cyanide sensitivity of hydroquinone and
p-phenylenediamine oxidation (see Table 1) suggested, because of
the redox potential relations of the cytochromes and the substrates,
that hydroquinone oxidation involved only the oxidase and cyto-
chrome c, whereas p-phenylenediamine could be independently
oxidized by cytochrome h as well. The cyanide-resistant portion of
p-phenylenediamine oxidation is probably due to the autoxidizable,
cyanide-resistant cytochrome h.

The oxidation of hydroquinone is a function of both the oxidase
and cytochrome c; hence its oxidation by tissue extracts is not an
absolute method for determining either substance. The effect of
cytochrome c in accelerating the rate of hydroquinone oxidation by
a heart muscle oxidase preparation is shown in Figure 3.

500

400

300

200

100

Velocity
(cmm. 02/hr.)

h)iS^o

-^Mr

O addition of cytochrome clone

A oddition of cytochrome to heated oxidase

■ addition of cytochrome to crude oxidase preparation

I I I I I I I I 1

0 10 20 30 40 50 60 70 80

Added cytochrome c (mM. X|0^)

90 100

Figure 3. — The oxidation of hydroquinone by the oxidase-cytochrome c system.
T = 38°C., pH 7.15, hydroquinone 0.033 mM. total.

166 A SYMPOSIUM ON RESPIRATORY ENZYMES

The reduction of cytochrome c by hydroquinone is very rapid;
hence the rate-controHing reaction here is the oxidation of the re-
duced cytochrome. Several curves such as those in Figure 3 have
been found to comply with the laws of a typical enzyme-substrate
complex (75), indicating that the oxidase and cytochrome c form
such a complex. A study of such curves in relation to the effect of
cyanide and carbon monoxide led to the conclusion that the action
of these inhibitors on the reaction was concerned with the oxidase
component (75).

On the other hand, in the presence of an excess of cytochrome
c the velocity of oxidation of hydroquinone (correcting for autoxida-
tion) was directly proportional to the amount of oxidase added. This
offers a method of estimating, in arbitrary units, the cytochrome
oxidase activity of tissues.

From hydroquinone and p-phenylenediamine tests it appears that
successive acetic acid precipitations remove the larger part of the
cytochrome c and some of the cytochrome h. By two precipitations
with acetic acid and a long dialysis an active oxidase preparation
can be obtained which shows very little cytochrome c or h, al-
though the oxidase activity is likewise greatly diminished.

Determination and Distribution of Cytochrome c and
Cytochrome Oxidase

Junowicz-Kochalaty and Hogness (76) have developed a method
for estimating cytochrome c in tissues. Relatively large amounts
(100 grams of tissue) are worked up through the initial steps of
Keilin's isolation procedure (5) to the point where traces of hemo-
globin and myoglobin are the principal colored impurities. The
cytochrome c is then measured spectrophotometrically. The use of
measurements at three wave lengths permits of calculations to cor-
rect for the hemoglobin and myoglobin. These authors found pigeon
breast muscle and beef heart muscle high in cytochrome c, tumor
tissue very low.

Stotz (77) has developed a method for determining cytochrome c
in rat tissues. The ground tissue is extracted with trichloracetic acid,
the extract neutralized to eliminate further inactive protein, and the
cytochrome precipitated by phosphotungstic acid. After solution in
dilute ammonia, the phosphotungstate is eliminated with barium.
The final solutions are tested manometrically for their power to
accelerate oxidation of hydroquinone by a heart muscle oxidase prep-
aration. A calibration curve must be prepared, pure cytochrome c
being used.

CYTOCHROMES 167

In applying this test to various rat tissues it was found that agree-
ment between animals was reasonably good; the order of activity
of the tissues was the same in all the rats studied. The average results
of the cytochrome c estimations in the tissues of ten rats are re-
corded in Table 2.

Table 2.— Comparison of cytochrome oxidase and cytochrome c
activities of rat tissues

(Reference 77)

Oxidase Cytochrome c

Units per

mg. dry ' mg. per g.

tissue dry tissue

Heart 9.7 Heart 2.34

Kidney 4.7 Kidney 1.36

Brain 3.5 Skeletal muscle 0 . 68

Skeletal muscle 2.3 Brain 0.35

Liver 1.7 Liver 0 . 24

Spleen 1.6 Spleen 0.21

Lung 1.3 Lung 0.14

Testis 1.1 Embryo (early) 0.03

Diaphragm muscle 0.72 Embryo (late) 0.18

Large intestine 0.36 Tumor R-256 0.02

Embryo (early, late) 1.1 Tumor R-39 0.03

Tumor R-256 2.9 Tumor spontaneous 0.01

Tumor spontaneous 2.4

Most striking is the low cytochrome c content of embryos and
the tumors studied. It may be noted that the cytochrome c content
of the embryos increases just before birth. With the present knowl-
edge of the importance of cytochrome c in respiration, the low c
content of these tissues may be at least one factor responsible for
the aerobic glycolysis of these tissues.

Potter and Dubois (78) have likewise developed a micro method
for estimating cytochrome c, the actual deteiTnination being made
with a photoelectric spectrophotometer; the light absorption is
measured before and after specific enzymatic reduction with succi-
nate. The values they obtained with rat tissues are in remarkably
good agreement with those recorded by Stotz. They have found a
low c content in several tumors thus far studied.

The manometric estimation of oxidase activity in rat tissues may
be carried out on ground dialyzed tissue in the presence of excess
cytochrome with hydroquinone as a substrate (77). Schultze (49)
uses in addition semicarbazide, which maintains a constant rate of
oxidation over a longer period, a helpful modification.

168 A SYMPOSIUM ON RESPIRATORY ENZYMES

The distribution of oxidase in rat tissues is also illustrated in Table
2. Perhaps the most interesting thing about these data is the fact
that the oxidase activity of the tissues parallels quite closely their
cytochrome c content, although in the case of the tumors and em-
bryonic tissue there is no lack of cytochrome oxidase. This paral-
lelism may indicate a close chemical similarity between, or common
origin of, cytochrome c and cytochrome oxidase.

A decrease in oxidase activity has been noted in various tissues
of the rat in anemia (49, 50) and in guinea pig tissues during acute
scurvy (79).

Physiological Functioning of the Cytochrome System

Flexner and Stiehler (80, 81) have made some very interesting
observations on the funct. nal development of the cytochrome sys-
tem. By histochemical methods they studied the changes in the
chorioid plexus of the fetal p'g, especially during the phase when
the spinal fluid changes from an ultra-filtrate to a secretion. Oxidase
activity was measured by blue-staining with dimethyl p-phenylene-
diamine and alpha-naphthol, a test which of course measures the
combined oxidase and cytochrome activity. Oxidation-reduction
potentials were estimated by introduction of oxidation-reduction in-
dicators intravascularly or supravitally. It was found that previous
to the secretory phase the concentration of "indophenol oxidase"
was the same in the epithelium and stroma and that there was no
potential difference between the two. In the secretory phase, how-
ever, the oxidase was concentrated in the epithelium and a poten-
tial difference developed, more positive in the epithelium. It was
concluded that the functional changes occurring with the onset of
secretion are correlated with the potential difference set up as a
result of the selective development of the oxidase (cytochrome) sys-
tem in the epithelium. The selective transference of dyes across the
secretory plexus was abolished by cyanide, and this was associated
with a loss of the potential difference between epithelium and
stroma.

Flexner, Flexner, and Straus (82) have likewise studied the cyto-
chrome system in the cerebral cortex of the fetal pig. During the
first half of gestation p-phenylenediamine was not actively oxidized,
but during the second half an active oxidation system was present.
Employing the test for cytochrome oxidase (using excess cyto-
chrome c), they found that there was no lack of oxidase in any
phase of gestation and hence the change in p-phenylenediamine

CYTOCHROMES 169

oxidation was due primarily to development of cytochrome c.
Nevertheless the Qo^ of the tissue increased little during this period
and at all times was over 90 per cent sensitive to 0.001 M. cyanide.
Thus there is an apparent anomaly in that cyanide, the inhibitoiy
action of which is generally considered to be on the cytochrome
oxidase system, is inhibiting a respiratory system which does not
even contain a complete cytochrome system. This is not the first
instance in which the "single point of attack theory" of cyanide has
been questioned. Parallel experiments with cyanide and azide (see
Stannard, 83) on intact tissues have also suggested that cyanide
probably combines with one or more other enzymes involved in
respiration. The question arises, therefore, what is the nature of the
"oxygen-activating" system in the absence of cytochrome c? If
cyanide can also block an enzyme systeoi near the "dehydrogenase
end" of respiration, then flavin enzymes as well as cytochrome b
(which is relatively cyanide-stable aij^cX autoxidizable) become pos-
sibilities for this role, despite the fact that their oxidation is gener-
ally considered to be through the oxidase system.

A somewhat similar situation arises in Stannard's experiments
(83) on the "resting" and "activity" oxygen consumption of frog
muscle. It was found that the resting metabolism was insensitive to
azide, although sensitive to cyanide. On the other hand, the activity
metabolism (caffeine or electrical stimulation) was greatly inhibited
by azide. When the azide concentration in the stimulated prepara-
tion was increased, the inhibition *came to an abrupt stop, leaving a
respiration equal to that of the resting state. The latter finding
excludes the possibility that the azide stability of the resting respira-
tion can be explained by failure of the inhibitor to penetrate or its
absence in a form capable of exerting its typical inhibition on the
oxidase system. Stannard (84, 85), who has studied the anaerobic
glycolysis of frog muscle, does not believe that the azide insensi-
tivity of resting muscle can be explained by the lack of substrate for
"saturation" of the oxidizing enzymes. He has concluded that the
oxygen transfer for the resting metabolism and for the extra metab-
olism resulting from stimulation are due to different systems. The
extra metabolism due to stimulation was apparently by way of the
cytochrome system, but the nature of the "oxygen-activating" en-
zymes of the resting metabolism remain unknown.

Recently Korr (30) has carried out experiments on the metabolism
of slices of mammalian tissues during rest and during a stimulated
phase. Salivary glands stimulated by acetylcholine or adrenalin,

170 A SYMPOSIUM ON RESPIRATORY ENZYMES

pancreas by secretin, and myometrium by oxytocin all show that
large increases of respiration attend the change from a state of
rest to one of activity. The finding of azide-stable and relatively
cyanide-stable respiration, combined with spectroscopic observa-
tion of the cytochrome bands, indicated that the resting metabolism
was not proceeding through the cytochrome system, but that the
extra metabolism that followed stimulation was mediated through
this system. It is important to note that in the resting state the
cytochrome system was nevertheless "available" for the oxidation of
p-phenylenediamine. It was postulated that the cytochrome system
becomes "geared" or "linked" to the substrate-dehydrogenase system
by an agent capable of reducing the cytochrome that is made avail-
able upon stimulation of the cells. Again there is no clue to the
nature of the oxidizing enzymes that replace the cytochrome sys-
tem in the resting cells.

Perhaps the most serious obstacle to the immediate acceptance of
Stannard's and Korr's conclusions is the possibility that at the low
levels of oxygen consumption the oxidase system is only partially
saturated with substrate and hence considerable amounts could be
blocked by inhibitors without affecting the oxygen consumption of
the tissue. Such a view has been expressed by Warburg (86), and
Commoner (87) has actually demonstrated that cyanide inhibition
of yeast respiration is dependent on substrate respiration.

The early work of Keilin showed that whereas in the resting
muscle the cytochromes were oxidized, when it became active the
bands of the reduced cytochromes appeared. Thus during activity
the ratio of reduced to oxidized cytochrome c is higher. It is known
that reduced cytochrome c forms a complex with the oxidase (74,
75). Since under conditions of activity the oxidase is relatively more
saturated with its substrate (reduced cytochrome c), the respiration
of this system should, according to the "under-saturation" concept,
be more sensitive to the same concentration of oxidase inhibitor.
The "activity" respiration should then be totally sensitive to azide
if azide only affects the rate of oxidation of the cytochrome. But
Stannard's experiment demonstrated that during activity only the
extra oxygen consumption caused by the activity was azide-
sensitive.

The experiments of Stotz, Altschul, and Hogness (75) on the rela-
tions of the oxidase and of cytochrome c on hydroquinone oxidation
showed that the rate of oxidation was a function of both compo-
nents. Thus at a fixed concentration of reduced cytochrome c (oxi-

CYTOCHROMES 171

dase under-saturated) cyanide does produce an inhibition of hydro-
quinone oxidation. But if the cytochromes are chiefly in the oxidized
state, as they are in the resting tissue, such a decrease in oxygen
consumption could be compensated for by increased reduction of
the cytochromes. It is therefore important to know whether in the
experiments of Stannard and of Korr azide does or does not pro-
duce an increased state of reduction of the cytochromes in the
resting tissue.

The differences between the characteristics of the respiration of
the eggs of various species before and after fertihzation have been
extensively studied and have been related to the function of the
cytochrome system. The results are at present very difficult to inter-
pret, largely because of the tests employed for detecting the oxidase
or the cytochrome and because of the problem of permeability of
the cells to inhibitors. For example, it appears that in the un-
fertilized eggs of the sea urchin cytochrome has never been ob-
served spectroscopically, and its respiration is not inhibited by
cyanide or azide. After fertilization, however, the respiration is
typical of one proceeding through the cytochrome system (see Sha-
piro, 88). Korr believes the respiration of the unfertilized sea urchin
egg to be another case of functional inactivity of the cytochrome
system, since these eggs can oxidize p-phenylenediamine (see Dis-
cussion in Shapiro, 88). Allen (89), on the other hand, using grass-
hopper eggs, has shown that the activity metabolism is definitely
connected with the new development, rather than with "gearing,"
of the cytochrome system.

A recent paper by Krahl, Keltch, Neubeck, and Clowes (90) on
the cytochrome system of sea urchin eggs demonstrates that there is
a complete absence of cytochrome c in the unfertilized eggs, but no
lack of cytochrome oxidase. On the other hand, even in the fertilized
eggs, which are relatively more cyanide- and azide-sensitive, they
were still unable to detect any cytochrome c. The establishment of
the presence of cytochrome oxidase in the unfertilized eggs, al-
though it does not constitute proof, is strong evidence that this com-
pound functions in the respiration of these cells. The authors favor
the hypothesis suggested earlier (91) and described by Ball (page
29) that the action of cyanide and azide on cellular respiration may
be related to the formation of a complex with the oxidase possessing
a lower potential than the original oxidase (92, 93). The sensitivity or
stability of a respiration to azide or cyanide then depends on the
potential of the oxidase-inhibitor complex formed and the potentials

172 A SYMPOSIUM ON RESPIRATORY ENZYMES

of the available reducing systems. With a given lower potential of
the oxidase-inhibitor complex, the lower the potential of the reduc-
ing system the less sensitive would be the respiration to the inhibitor.
On this basis, a system in which cytochrome c (with a relatively high
potential) is the normal reducing agent of the oxidase would be
expected to show a high sensitivity to oxidase inhibitors. In the
absence of cytochrome c this theory provides an explanation of
cyanide or azide stability even when the cyanide may be combining
with the oxidase component. Such a theory deserves consideration
in the interpretation of all experiments purporting to demonstrate
the "by-passing" or non-functioning of the cytochrome-cytochrome
oxidase system.

The frontiers of research in the cytochrome problem therefore ap-
pear to consist on the biochemical side in the isolation and proper-
ties of the individual components, and on the physiological side in
the mode of function or non-function of this system in various states
of metabolism of tissues.

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Phosphorylation of Carbohydrates

CARL F. CORI

Washington University School of Medicine, St. Louis

THE METABOLISM of Carbohydrate in animal tissues is made up
of a series of enzymatic reactions in which phosphate plays an
essential role. What is usually referred to as the phosphate cycle
can be divided into four parts: the uptake of inorganic phosphate,
the intramolecular migration of phosphate groups, the transfer of
phosphate groups from one molecule to another (transphosphoryla-
tion), and the regeneration of inorganic phosphate.

Uptake of Inorganic Phosphate

The only reaction leading to the uptake of inorganic phosphate
that is definitely known to be enzymatic is the phosphorylation of
glycogen and starch. The uptake of inorganic phosphate which is
associated with the oxidation of phosphoglyceraldehyde is presum-
ably non-enzymatic, and the same may be true of the uptake of
inorganic phosphate associated with the oxidation of pyruvate.

In the phosphorylation of glycogen the C— O— C bond of the 1—4
glucosidic chain is replaced by the C— O— P bond of glucose-1-
phosphate. This reaction is reversible, and from the position of the
equilibrium it may be calculated that the change in free energy is
very small. This may be interpreted to mean that the ester linkage
in glucose-1-phosphate is nearly equivalent to the glucosidic linkage
in the large polysaccharide molecule.

When phosphate is replaced by water, as in the hydrolysis of
glycogen or starch by diastase, the reaction seems to be largely
irreversible; that is, the end products of diastatic activity, maltose
and glucose, even when added to diastase in high concentrations, are
not polymerized to glycogen or starch. Glucose, in order to undergo
enzymatic polymerization, must first be phosphorylated. Reversi-
bility is thus clearly connected with the introduction of a phosphate
group into the polysaccharide molecule. The position of the equi-
librium at physiological pH is about 77 per cent to the glycogen
side (see Table 2), and is determined by the concentration of the
divalent ions of orthophosphate and glucose-1-phosphate. Since the

175 xs^^-^iy

176 A SYMPOSIUM ON RESPIRATORY ENZYMES

second dissociation constant of these two acids is different, the posi-
tion of the equihbrium changes with pH (1, 2).

The enzymatic phosphorylation of glycogen and starch is of in-
terest from the standpoint of the configuration of these polysac-
charide molecules. There is considerable evidence, based on the
hydrolysis of methylated starch and glycogen, that these polysac-
charides contain other than the prevalent 1—4 glucosidic linkages.
At points at which a branching of chains occurs, a 1—6 glucosidic
linkage has been postulated. Starch appears to be made up of
relatively straight chains consisting of 24 to 30 glucose units; this
explains its ability to assume a crystalline structure and to exhibit
well-defined x-ray diffraction patterns. Glycogen seems to be made
up of relatively short chains (consisting of 12 to 18 glucose units),
with many branchings which give it the properties of greater solu-
bility and lack of crystallizability. Judging from the properties of
various plant starches, transitions exist between these two extremes.
These various forms of polysaccharide raise the problem of enzyme
specificity. The questions are whether one and the same enzyme
splits (or builds up) both the 1—4 and the 1—6 glucosidic linkages
and what determines the special configuration of the polysaccharide
synthesized. It is to be noted here that phosphorylases cannot poly-
merize glucose-1-phosphate unless a small amount of polysaccharide
is added to prime the reaction (1). However, the nature of the poly-
saccharide synthesized seems to be solely determined by the type of
phosphorylase used and not by the nature of the activating poly-
saccharide. For example, muscle phosphorylase, when primed with
hver glycogen, synthesizes a typical starch in vitro, and liver phos-
phorylase, when primed with plant starch, synthesizes glycogen (3).
Important also in this connection is the fact that the same enzyme,
muscle phosphorylase, can synthesize both starch and glycogen, the
former in vitro and the latter in the intact cell. The difference in
activity of the muscle phosphorylase in the two situations has not
been explained, but it suggests that unknown environmental factors
and perhaps the physical state of the enzyme have something to do
with the nature of the polysaccharide which is formed.

As has been stated, the phosphorylation of glycogen is an enzy-
matic reaction which provides for the entrance of inorganic phos-
phate into the phosphate cycle. All other reactions leading to the
uptake of inorganic phosphate are linked with oxidations. The reac-
tion between orthophosphate and phosphoglyceraldehyde during
oxidation of the latter to phosphoglyceric acid has been elucidated

PHOSPHORYLATION OF CARBOHYDRATES 177

by Warburg and his school (4). Lipmann (5) has described a bac-
terial enzyme system in which inorganic phosphate is taken up and
acetylphosphate is formed as an intermediate of pyruvate oxidation.
In both cases the phosphate group taken up during oxidation is
transferred by the adenylic acid system to suitable phosphate ac-
ceptors.

The oxidation of pyruvate in various animal tissues is also linked
with the uptake of inorganic phosphate, and the same has been
shown to be true for certain steps of the citric acid cycle, particularly
for the oxidation of succinic to fumaric acid (6). The primary phos-
phorylation products formed in these cases have not been identified.
In dialyzed and suitably supplemented tissue dispersions or extracts,
the inorganic phosphate taken up during oxidation of pyruvate is
transferred by the adenylic acid system to glucose, which is con-
verted to hexosediphosphate. It was noted, however, that when no
glucose was added to the system, a small amount of an easily
hydrolyzable phosphorus compound was formed. This compound
has recently been identified in our laboratory in collaboration with
Dr. Ochoa. In large-scale experiments with dialyzed rat liver dis-
persion and with glutamate, pyruvate, or succinate as oxidizable
substrate we have isolated inorganic pyrophosphate as the crystal-
hne sodium salt. The orthophosphate which disappeared corres-
ponded in amount to the pyrophosphate formed.

It is too early to evaluate the significance of this observation. There
is the possibility that the pyrophosphate group occurs in some or-
ganic combination which is split during the process of isolation. It
is fairly certain, however, that the pyrophosphate does not originate
from adenosinetriphosphate, since we have found no enzyme in liver
preparations which splits added adenylpyrophosphate to adenylic
acid and inorganic pyrophosphate. Another possibility is that the
pyrophosphate group has nothing to do with the primary phos-
phorylation product which is formed during the oxidation of the
substrate, but is the result of phosphorylation of orthophosphate.

The phosphorylation of glucose and other phosphate acceptors
which is connected with the oxidation of pyruvate has been termed
"aerobic phosphorylation" to signify that the energy for the forma-
tion of the phosphate bond comes from oxidations. Table 1 illustrates
the quantitative relationship between oxygen consumption and
phosphorylation.

This experiment shows that the dialyzed heart extract supple-
mented with magnesium ions, inorganic phosphate, and a trace of

178 A SYMPOSIUM ON RESPIRATORY ENZYMES

adenylic acid has practically no basal oxygen consumption and that
addition of 2 micromoles of succinic acid, a catalytic amount, has
very little effect on oxygen consumption. The addition of 50 micro-
moles of glucose had a very marked effect on oxygen consumption,

Table 1.— Glucose balance in dialyzed heart muscle extract*

(1 cc. of extract supplemented with Mg"*"*" ions, inorganic phosphate, and a trace of

adenylic acid. Incubated 60 minutes at 37° C. All values are

expressed in micromoles.)

Addition

Glucose Oxygen Phosphate Total glucose

disappearing consumed esterified accounted for

None 1.5

2 succinate 2.6

50 glucose +

2 succinate 24.4 22.3 36.1 3.5+18 = 21.5

*J. Biol. Chem., 137, 343 (1941).

and there can be no doubt that glucose was the substrate under-
going oxidation. Detennination of the respiratory quotient in other
experiments showed that it was unity for added glucose and 1.25
for added pyruvate. It is to be noted that an oxygen consumption of
22 micromoles corresponds to one-sixth as much glucose, that is to
3.5 micromoles, while the glucose which actually disappeared ac-
cording to sugar analysis was 24.4 micromoles. The glucose which
disappeared without being oxidized was largely recovered as hexose-
diphosphate, 36 micromoles of phosphate esterified corresponding
to 18 micromoles of glucose. Lactic acid formation was not deter-
mined. The balance indicates that for each mole of glucose oxidized
an additional 6 moles of glucose disappear, 5 of which are present
as phosphate ester. This means that about one atom of phosphate is
esterified for each atom of oxygen consumed. Ochoa (7) and Belitzer
and Tsibakova (8) observed even higher ratios, namely, from 2 to 3
atoms of phosphate esterified for each atom of oxygen consumed.
This would indicate that not only the primary removal of hydrogen
from the substrate but also one or even two subsequent hydrogen
transfers over intermediate catalysts may cause phosphorylation.

Aerobic phosphorylation is a mechanism by which oxidative
energy is utilized in the cell. The oxidative energy is converted into
phosphate bond energy, to use Lipmann's (5) terminology, and the
adenylic acid system serves as the mediator of this energy transfer.
When glucose is the phosphate acceptor, the system, once started, is

PHOSPHORYLATION OF CARBOHYDRATES 179

self -perpetuating. The phosphorylation of glucose enables it to un-
dergo oxidation by way of triosephosphate and pyruvate, and this
oxidation causes further phosphorylation of glucose, thus providing
new substrate for oxidation and so on.

During recovery of muscle from work, oxidative energy is also
converted into phosphate bond energy; that is, the phosphocreatine
which breaks down during muscular contraction is reformed largely
at the expense of oxidation, and the phosphorylation of glucose
which is supplied by the blood stream provides the necessary sub-
strate for the resynthesis of the glycogen lost during contraction.

Intramolecular Migration of Phosphate Groups

The first enzymatic reaction of this type was described by Meyer-
hof and Kiessling (9), namely, the conversion of glyceric acid-3- to
glyceric acid-2-phosphate. This reaction was shown to be reversible,
and the assumption that one is dealing with an intramolecular mi-
gration of the phosphate group was confirmed by the use of radio-
active phosphorus.

Another reaction of this type is the conversion of glucose-1- to
glucose-6-phosphate. This reaction was at first regarded as irre-
versible, but more recent work has shovsni that it can be reversed
under suitable experimental conditions; that is, glucose-6-phosphate
can be converted to glucose-1-phosphate and then to glycogen (10).
To study the equilibrium of this reaction it was necessary to separate
the enzyme which catalyzes this reaction from interfering enzymes
which upset the equilibrium by acting either on glucose-1- or glu-
cose-6-phosphate. With such a purified enzyme preparation 94 per
cent of added glucose-1-phosphate is converted to an ester which was
isolated and identified as glucose-6-phosphate. Fructose-6-phosphate
was absent, because Lohmann's enzyme (11), which catalyzes the
reversible reaction between glucose-6- and fructose-6-phosphate,
had been removed. Conversely, when pure glucose-6-phosphate was
added to the enzyme, 6 per cent of glucose-1-phosphate was formed;
that is, the same equilibrium was reached from either side. From
the equihbrium constant (K = 15.7 at pH 7 and 25° C.) it may be
calculated that the change in standard free energy amounts to about
—1600 calories.

When barium ions and phosphorylase were added to this system,
glucose-6-phosphate was converted to glycogen. As shown in Table
2, the position of the equilibrium of the second reaction is unfavor-
able for glycogen synthesis, because only a smaU amount of glucose-

180 A SYMPOSIUM ON RESPIRATORY ENZYMES

Table 2.— Position of equilibria at pH 7 and 25°

Glycogen + inorganic phosphate (77 per cent)<=^gIucose-l-phosphate (23 per cent) (1)
Glucose-1-phosphate (6 per cent) i=^glucose-6-phosphate (94 per cent) (2)

Glucose-6-phosphate (70 per cent)<=^fructose-6-phosphate (30 per cent) (3)

1-phosphate is formed. The overall reaction cannot progress very
far to the glycogen side, but when barium ions are added, which
cause precipitation of the inorganic phosphate set free when re-
action 1 goes to the left, up to 40 per cent of added glucose-6-
phosphate can be converted to glycogen. Such an experiment is
shown in Table 3.

Table 3.— Glycogen formation from barium salt of glucose-6-
phosphate with dialyzed muscle extract

(Extract + Mg++ ions + catalytic amounts of glycogen and adenylic acid.
Incubated at 30° C.)

Type of extract

Time of incubation

6-ester converted
to glycogen

minutes

per cent

Original

5.0

180

15.1

Concentrated sevenfold

3.1

9.9

180

33.4

Concentrated, but without addition of

adenylic acid and glycogen

180

1.2

Muscle extract, prepared in the usual manner and dialyzed free
of inorganic phosphate, shows only a slight activity when the barium
salt of glucose-6-phosphate is added. When the same extract was
concentrated sevenfold by freezing and by drying in vacuo, the
activity was markedly increased, since up to 33 per cent of the
added glucose-6-phosphate was converted to glycogen. The glycogen
formation was measured by (1) the amount of inorganic phosphate
set free, which precipitates as the barium salt because of the addition
of barium ions; (2) the increase in glycogen, which is somewhat less
than the increase calculated from the inorganic phosphate because
of the presence of diastase in the extract; and (3) the color reaction
with iodine, which is blue for the polysaccharide formed by muscle
phosphorylase. As a control procedure, the addition of adenylic
acid and of the glycogen necessary for the priming of the reaction

PHOSPHORYLATION OF CARBOHYDRATES 181

was omitted, and it may be seen that no conversion of glucose-6-
phosphate to glycogen took place.

Colowick and Sutherland have also been able to convert glucose
to glycogen in vitro by the addition of hexokinase and adenosinetri-
phosphate to the concentrated muscle extract. It can be shown that
in this case glucose is first converted to glucose-6-phosphate at the
expense of the labile phosphate groups of adenosinetriphosphate.

Glucose-6-phosphate is thus an important intermediate of carbo-
hydrate metabolism; it is formed from glycogen via glucose-1-
phosphate and it can also be formed by direct phosphorylation of
glucose in position 6. The latter reaction is the so-called hexokinase
reaction which was described by Meyerhof (12) and v. Euler and
Adler (13).

Transphosphorylation

All known transphosphorylation reactions involve adenosinemono-
or adenosinediphosphate; these nucleotides act in catalytic amounts
as acceptors of phosphate from such substances as phosphopyruvate,
acetylphosphate, and 1,3-diphosphoglycerate and are thus converted
to adenosinedi- and triphosphate respectively. These polyphosphates
then serve in a second enzyme reaction as phosphate donors to a
number of organic molecules, such as glucose, fructose, mannose,
fructose-6-phosphate, glycerol, creatine, adenosine, and probably
others. The enzymes which transfer the phosphate group from the
polyphosphates are specific with respect to the acceptors. For ex-
ample, yeast contains enzymes that phosphorylate glucose, fructose,
mannose, and adenosine, but no enzyme that phosphorylates crea-
tine.

Extract of skeletal muscle has little or no hexokinase activity.
Since this can hardly be true of intact muscle, it may be due to
destruction, inhibition, or poor extractability of the enzyme. Other
tissues, such as brain, heart, liver, kidney, and retina yield active
extracts. The hexokinase in these tissue extracts has not been
separated from other enzymes, but in the case of yeast such a
separation has been effected.

Colowick and Kalckar (14) have recently studied the reaction
between adenosinetriphosphate and glucose with purified yeast
hexokinase. Only one of the labile phosphate groups of adenosine-
triphosphate is transferred to glucose; that is, the reaction products
that were identified are adenosinediphosphate and glucose-6-phos-
phate. When adenosinediphosphate is substituted for adenosine-
triphosphate, no reaction with glucose takes place, but when a heat-

182

A SYMPOSIUM ON RESPIRATORY ENZYMES

stable protein of muscle is added to the system, hexokinase is able
to transfer the labile phosphate group of adenosinediphosphate, the
reaction products in this case being adenylic acid and glucose-6-
phosphate. This is illustrated in Tables 4 and 5.

Table 4.— Reaction of adenosinetriphosphate with glucose

(Hexokinase and Mg++ in all samples. T = 30°C.)

Time Po Pio Pio-Po

Addition (min.) (7) (7) (7)

Adenosinetriphosphate 0 4.0 68.0 64.0

Adenosinetriphosphate 5 8.9 70.0 61.1

Adenosinetriphosphate+glucose, 2 mg 5 8.8 43.8 35.0

Adenosinetriphosphate 15 9.0 68.0 59.0

Adenosinetriphosphate+glucose, 2 mg 15 9.5 43.4 33.9

The diflFerence between the Pq and Pjo value (initial and ten-minute
hydrolysis values in normal sulfuric acid) corresponds to the amount
of labile phosphate added as adenosinetriphosphate. With a purified
hexokinase preparation of yeast, no appreciable reaction takes place

Table 5.— Necessity of heat-stable muscle protein for reaction of
adenosinediphosphate with glucose

(Hexokinase and Mg++ in all samples. Time, 5 minutes. T = 30°C.)

Addition

Pio— Po

(7)

AdenosineM'phosphate 51.8

Adenosine/rzphosphate-l- glucose 28.4

Adenosine<nphosphate-|-glucose-}-myokinase 7.6

Adenosine<ftphosphate 62.4

Adenosine^iphosphate+glucose 62.9

Adenosinec/?phosphate-|-glucose-|-myokinase 16.0

when adenosinetriphosphate alone is added; when glucose is also
added, approximately half of the labile phosphate of adenosinetri-
phosphate disappears. The reaction is a rapid one, since it is nearly
completed during five minutes of incubation at 30° C. Table 5 shows
the effect of a heat-stable protein of muscle which has been named

PHOSPHORYLATION OF CARBOHYDRATES 183

"myokinase" by Colowick and Kalckar and which, when added in
catalytic amounts, enables hexokinase to transfer the labile phos-
phate group of adenosinediphosphate to glucose. It may be seen
that with adenosinetriphosphate as phosphate donor approximately
half of the labile phosphate disappears when hexokinase alone is
added, and that with the further addition of a few micrograms of
muscle factor almost all the labile phosphate disappears. With
adenosinediphosphate as phosphate donor no reaction with glucose
takes place until the muscle protein is added.

In extracts of mammalian tissues, such as kidney, heart, and brain,
the hexokinase reaction is generally followed by a reaction between
adenosinetriphosphate and fructose-6-phosphate, yielding fructose-1,
6-diphosphate or Harden-Young ester. In the intact cell, however,
particularly in muscle, where this has been studied in detail,
fructose-6-phosphate does not react rapidly with adenosinetriphos-
phate. This is borne out by the fact that hexosemonophosphate, the
equilibrium mixture of glucose- and fructose-6-phosphate, is a nor-
mal constituent of muscle and that it can increase considerably
under certain experimental conditions without any increase in the
formation of lactic acid (15). This indicates that the reaction between
fructose-6-phosphate and adenosinetriphosphate in intact muscle is
a limiting factor as regards the rate at which lactic acid is formed
and carbohydrate is oxidized. It is not yet known whether the system
for the direct oxidation of glucose-6-phosphate which has been
found by Warburg in yeast is significant for mammalian tissues; it
would in any case lead to the formation of triosephosphate and
hence of pyruvic acid and thus join the main path of carbohydrate
breakdown.

The reaction between fructose-6-phosphate and adenosinetri-
phosphate has not been studied in detail, and the enzyme that
catalyzes this reaction has not been purified. The reaction has been
regarded as irreversible. Lohmann (11), however, has reported that
muscle extract splits oflF phosphate from position 1 when fructose-I,
6-diphosphate and magnesium ions are added. Recent experiments
carried out in our laboratory with Dr. Ochoa have shown that
Harden-Young ester added to liver extract is converted in quantita-
tive yield to glucose. This involves dephosphorylation in position 1,
conversion of fructose-6- to glucose-6-phosphate, and splitting of the
latter by liver phosphatase to glucose and inorganic phosphate. We
have repeatedly convinced ourselves that liver phosphatase forms

184

A SYMPOSIUM ON RESPIRATORY ENZYMES

only glucose from the equilibrium mixture of fructose- and glucose-
6-phosphate.

When fructose is added to liver or kidney extract, it is also con-
verted to glucose. This involves phosphorylation of fructose and
splitting of the glucose-6-phosphate in equilibrium with fructose-6-
phosphate. Table 6 illustrates an experiment in which an aerobic
phosphorylation of fructose took place with partial conversion to
glucose. In this experiment glutamate was used as oxidizable sub-
strate, which caused a considerable consumption of oxygen. The
respiration, as in other cases of aerobic phosphorylation, serves here
for the regeneration of adenosinetriphosphate, the phosphate donor

Table 6.— Aerobic conversion of fructose to glucose in dialyzed rat

hver dispersion

(All samples contained 0.2 mg. Mg++, a catalytic amount of adenosinetriphosphate,
and 0.025 M phosphate buffer of pH 7.3, in a volume of 1.4 cc. Incubated 60 minutes
at 37°C.)

Substrate

Phos-

Oxygen

Fructose

Glu-

Fructose

phate

NaF

con-

Phosphate

disap-

cose

converted

oxidation

acceptor

sumed

esterified

pearing

formed

to glucose

MX 10-3

cmm.

mg.

per cent

None

none

282

0.22

Glutamate

none

962

0.05

Glutamate

none

950

0.49

Glutamate

fructose

932

0.14

3.42

2.67

Glutamate

fructose

938

1.10

4.17

1.37

to fructose. When no fluoride is added only a small amount of
phosphate ester accumulates, and most of the fructose that disap-
pears is converted to glucose. Fructose and glucose were determined
by separate methods. In the presence of fluoride, dephosphorylation
is inhibited and consequently more hexosephosphate esters accumu-
late and less glucose is formed. When no phosphate acceptor is
added, a small amount of inorganic phosphate is esterified, espe-
cially when fluoride is added. This compound is almost exclusively
pyrophosphate, as has been mentioned previously.

The mechanism of the conversion of fructose to glucose is shown
in Table 7, the enzyme system consisting of adenosinetriphos-
phate, kidney phosphatase prepared by Albers' method, and yeast
hexokinase— which also contains Lohmann's enzyme (11). If hexo-
kinase is not added, neither adenosinetriphosphate nor fructose
disappears. When phosphatase is omitted, all the added adenosine-

PHOSPHORYLATION OF CARBOHYDRATES 185

triphosphate and a corresponding amount of fructose disappear, but
practically no glucose is formed. The formation of glucose is clearly
dependent on the addition of phosphatase. The conversion of fruc-
tose to glucose is another example of the utilization of oxidative
energy in the cell by way of the phosphate cycle.

Table 7.— Conversion of fructose to glucose by a purified enzyme

system

(The complete system consisted of 5 mg. of hexokinase, 0.05 mg. of "muscle factor,"
10 mg. of phosphatase, 0.2 rag. of Mg++, 3 mg. of fructose, 0.33 mg. of labile phosphate
(as adenosinetriphosphate), and 0.025 M veronal buffer of pH 7.5, in a total volume of
1.3 cc. Incubated 60 minutes at 37° C.)

Adenosinetri-

Fructose

Sample phosphate dis-

Fructose

Glucose

converted to

appearmg

disappearing

formed

glucose

mg. labile

mg.

per cent

phosphate

No adenosinetriphosphate 0

No hexokinase 0.07

No phosphatase 0.29

1.2

0.07

Complete 0.33

1.1

0.48

It may be emphasized at this point that all the reactions of the
phosphate cycle except the phosphorylation of pyruvic acid by
adenosinetriphosphate have now been shown to be reversible. When
lactic or pyruvic acid is converted to carbohydrate, phosphopyruvic
acid is apparently formed in an indirect way, probably from a four-
carbon dicarboxylic acid such as malate or fumarate, both of which
are assumed to be intermediates in the oxidation of pyruvate. Kalckar
(16) has shown that when malate or fumarate is added to kidney
extract under aerobic conditions, phosphopyruvic acid is formed.

Regeneration of Inorganic Phosphate

The formation of phosphopyruvate has just been mentioned. The
reverse reaction, the dephosphorylation of phosphopyruvate, was
originally shown to consist in a transfer of phosphate from phospho-
pyruvate to adenylic acid, with formation of pyruvate and adeno-
sinetriphosphate. The reaction proceeds rapidly with catalytic
amounts of adenylic acid, provided the adenylic acid is regenerated,
either by phosphate transfer from adenosinetriphosphate to some
suitable phosphate acceptor such as creatine, or by dephosphoryla-
tion of adenosinetriphosphate by adenylpyrophosphatase. It was

186 A SYMPOSIUM ON RESPIRATORY ENZYMES

later shown by Pillai (17), and confirmed by us and recently also by
Parnas (18), that there is apparently a second mechanism for the
dephosphorylation of phosphopyruvate. This conclusion is based on
the observation that dialyzed and aged muscle extract or an acetone
powder of muscle extract which is unable to split adenosinetriphos-
phate, and thus to regenerate adenylic acid, can still split phospho-
pyruvate when a catalytic amount of adenosinetriphosphate is added.
That one is dealing with a different type of reaction is shown by the
fact that in such extracts adenylic acid cannot replace adenosinetri-
phosphate.

Another reaction that leads to the regeneration of inorganic phos-
phate is the splitting of adenosinetriphosphate by adenylpyrophos-
phatase. There is reason to believe that the activity of this enzyme
is increased during muscular contraction (19). Adenylpyrophospha-
tase, which is found in most tissues, plays an important regulatory
function; by converting adenosinetriphosphate to adenylic acid it
can overcome the "bottleneck" which is created in the phosphate
cycle by a lack of phosphate acceptors. In addition, it is possible
that the reaction described by Pillai, the direct dephosphorylation
of phosphopyruvate, plays a physiological role.

In some experiments with tissue slices the phosphate cycle is so
perfectly adjusted that the concentration of inorganic phosphate re-
mains virtually unchanged, and this has given rise to the erroneous
assumption that one is dealing with a non-phosphorylating glycol-
ysis. Dr. Ochoa in our laboratory has recently investigated the
glycolysis in brain, which has been regarded by some workers as a
tissue with non-phosphorylating glycolysis. In the past most of the
work was done with brain slices or brei because with brain extracts
the formation of lactic acid was veiy feeble. Geiger (20) made the
significant observation that when a brain extract which forms little
lactic acid is diluted, a rapid lactic acid formation sets in. This is
due to the fact that an inhibitor is present in brain extract, the effect
of which is nullified by dilution. Ochoa (21), who confirmed Geiger's
observation, was able to show that all the reactions which are
characteristic for phosphorylating glycolysis occur in this dilute
brain extract. One illustrative experiment is shown in Table 8. The
amount of lactic acid formed for an extract corresponding to only
40 mg. of tissue is quite large, as good as or better than is obtained
with other tissue extracts. Glucose, hexosemono-, and hexosediphos-
phate form about equal amounts of lactic acid and the changes in
lactic acid and inorganic phosphate correspond to the equations

PHOSPHORYLATION OF CARBOHYDRATES 187

given at the bottom of the table. There can be no doubt that lactic
acid is formed here by a phosphorylating mechanism. This is not to
imply that such a mechanism is the only one that has been invented
by nature for the degradation of carbohydrate, but so far as animal
tissues are concerned it would seem that the burden of proof is on
those who claim that a non-phosphorylating glycolysis exists.

Table 8.— Glycolysis in rat brain extract

(0.2 cc. of extract (equivalent to 40 mg. of brain) were made up to 2 cc. with addition
of Mg"*""*", phosphate-bicarbonate buffer, and catalytic amounts of adenosinetriphos-
phate and cozymase. Incubated 90 minutes at 38° C. From J. Biol. Chem., IJtl,
245, 1941.)

Lactic acid formed

- Change in inor-

Substrate

Determined Determined
manometrically chemically

ganic phosphate

mg. mg.

mg.

None

0.15 0.19

Glucose (0.028 M)*

1.22 1.58

-0.36

Hexose monophosphate (0.010 M)t

1.17 1.38

+0.03

Hexose diphosphate

(0.017 M)t . .

1.15 1.20

+0.35

* 2 hexose + 2H3P04= 2 lactic acid + 1 hexose diphosphate.

t 2 hexose monophosphate = 2 lactic acid+1 hexose diphosphate.

% 1 hexose diphosphate = 2 lactic acid + 2H3P04.

Table 9 summarizes the essential reactions of the phosphate cycle.
One important feature of this scheme is that the concentration of
inorganic phosphate has a marked influence on the rate of enzymatic
reactions. This is obvious in the case of the first reaction, since
whether glycogen will be broken down or synthesized depends en-
tirely on the relative concentrations of inorganic phosphate and
glucose-1-phosphate. It may also be pointed out that the oxidation of
triosephosphate and of pyruvate cannot occur in the absence of
inorganic phosphate and that its concentration therefore has a
marked effect on the rate of oxidation. Another feature of the scheme
which needs to be emphasized is that the rate of oxidation is also
dependent on the availability of a phosphate acceptor, because of the
fact that oxidation and phosphate transfer are coupled reactions. It
is for this reason that the addition of adenylic acid is often found
to have a marked stimulating effect on carbohydrate oxidation in an
enzyme system. Finally, other phosphate acceptors, particularly
creatine, and the dephosphorylating enzymes (phosphatase, adenyl-

188

A SYMPOSIUM ON RESPIRATORY ENZYMES

pyrophosphatase), play an important role as regulators of the con-
centration of inorganic phosphate.

Table 9.— Reactions of the phosphate cycle

Glycogen+Phosphate

it,

Glucose + Phosphate^

Glucose-1 -phosphate

it,

Other "P"

acceptors

-Glucose-6-phosphate< Glucose <

Fructose-6-phosphate< Fructose < — ATP—

t .! Uf

Fructose-l-6-diphosphate ADP<^

it -4H it

2 Triosephosphate j AA <■

If +2 phosphate j

>2 Phosphate

2 Phosphoglycerate

2 Phosphopyruvate

Lactate;=^2 Pyruvate

Phosphate
transfer -

-8H

+4 phosphate

Summary

This brief presentation of the phosphate cycle is of course far
from a complete picture. Its elements are the individual enzymatic
reactions. If an enzyme has been isolated from the tissues, if the
reaction product or products have been identified, if the kinetics of
the reaction and the role of coenzymes, activators, and inhibitors
are known, we are, I believe, on solid ground. Several of the indi-
vidual enzymatic reactions comprising the phosphate cycle have
been studied in this manner; others remain to be studied in greater
detail.

It is also possible to combine a number of individual enzymatic
reactions in the test tube and to reproduce overall effects, such as the
polymerization of glucose to glycogen or the conversion of fructose
to glucose. The coupling between respiration and phosphorylation
has given us an insight into the mechanism of energy transfer in the
cell. There is still an essential element lacking in this picture, which
in the absence of a better definition might be called the regulatory
function of the cell. There can be no doubt that mechanisms exist
in the intact cell which regulate the rate and direction of individual
enzymatic reactions and which lead to a high degree of integration

PHOSPHORYLATION OF CARBOHYDRATES 189

of overall eflFects. The next approach is, perhaps, a study of the
mechanisms underlying this regulatory function. The mechanism of
action of a number of hormones is still obscure; all that can be said
at present is that they are part of the regulatory mechanism of the
cell rather than essential constituents of enzyme systems.

REFERENCES

1. Com, G. T., and Com, C. F., J. Biol. Chem., 135, 733 (1940).

2. Hanes, C. S., Proc. Roy. Soc. (London), B 129, 174 (1940).

3. Baer, R. S., and Com, C. F., J. Biol. Chem., 140, 111 (1941).

4. Warburg, O., and Christian, W., Biochem. Z., 303, 40 (1939).

5. LiPMANN, F., Advances in Enzymology, 1, 99 (New York, 1941).

6. CoLowicK, S. P., Kalckar, H. M., and Com, C. F., J. Biol. Chem., 137,
343 (1941).

7. OcHOA, S., J. Biol. Chem., 138, 751 (1941).

8. Belitzer, V. A., and Tsibakova, E. F., Biokimia, 4, 516 (1939).

9. Meyerhof, O., and Kxessling, W., Biochem. Z., 276, 239 (1935).

10. Sutherland, E. W., Colowick, S. P., and Com, C. F., J. Biol. Chem., 140,
309 (1941).

11. LoHMANN, K., Biochem. Z., 262, 137 (1933).

12. Meyerhof, O., Biochem. Z., 183, 176 (1927); Natunvissenschaften, 23,
850 (1935).

13. V. EuLER, H., and Adler, E., Z. physiol. Chem., 235, 122 (1935).

14. Colowick, S. P., and Kalckar, H. M., J. Biol. Chem., 137, 789 (1941);
140, xxix (1941).

15. Com, G. T., and Cori, C. F., J. Biol. Chem., 116, 119, 129 (1936).

16. Kalckar, H. M., Biochem. J., 33, 631 (1939).

17. PiLLAi, R. K., Biochem. J., 32, 1087 (1938).

18. Parnas, J. K., Handbuch der Enzymologie, II, 902 (Leipzig, 1940).

19. Needham, J., Shen, S. C, Needham, D. M., and Lawrence, H. S. C,
Nature, 147, 766 (1941).

20. Geiger, a., Biochem. J., 34, 465 (1940).

21. OcHOA, S., J. Biol. Chem., 141, 245 (1941).

Discussion on Phosphorylation

H. M. KALCKAR

Washington University, St. Louis, Chairman

Dr. Kalckar:

It seems advisable to begin this discussion on phosphorylation
with a brief survey of the major present-day problems in this field.
The mechanism of the compulsory coupling between oxidation of
triose and phosphorylation of adenosine nucleotides was solved
when Warburg and Christian isolated and crystallized the catalyst
of the phosphotriose oxidation and Negelein and Bromel isolated
the 1,3-diphosphoglyceric acid. Warburg and his collaborators also
demonstrated the transfer of the phosphate from the carboxyl group
to adenosinediphosphate. Both the oxidation process (1) and the
phosphate transfer are easily reversed reactions:

O 0 0

I II II II

(1) c=0-t-HO— P— 0'+ pyridinium ^ — C— O— P— 0'+ reduced pyridine

I I I

O' O'

0 0 o

(2) — C — P — 0'+ adenosinediphosphate "'<=± — C + adenosine?r?phosphate""

1 I I
O O' O'

These two reversible reactions explain completely the findings of
Needham, Meyerhof, and others that the utilization of the labile
phosphate in adenosinetriphosphate determines the extent of oxida-
tion of phosphotriose.

Lipmann's observations of the formation of acetylphosphate in
the bacterial pyruvate oxidation furnished another important ex-
ample of carbonyl oxidation coupled with phosphate uptake.

Carbonyl oxidation is not the only type of oxidation that is coupled
with phosphorylation. Oxidations of fumaric acid or even of succinic
acid are coupled with phosphorylations, but the mechanism of these
reactions has not been clarified.

It has been mentioned that the extent of triose oxidation was auto-
matically regulated by the removal of labile phosphate in adenosine-

190

DISCUSSION ON PHOSPHORYLATION 191

triphosphate and it is therefore of interest to discuss briefly the
problems relating to adenylpyrophosphate utilization. The utilization
of adenylpyrophosphate takes place by easily reversed reactions or
by irreversible degradation. The phosphorylation of carboxylate or
amidine ions represents easily reversed dephosphorylations of adenyl-
pyrophosphate. The phosphorylations of the hydroxy groups of
monohexoses or glycerol and the mineralization of phosphate repre-
sent the irreversible degradations of adenylpyrophosphate. These
irreversible strongly exergonic reactions are probably essential in the
energy transformations.

The phosphorylation of glucose by adenylpyrophosphate has been
observed by Euler and Adler in brewer's yeast, and by Meyerhof
in baker's yeast and in extracts of animal tissue. Strangely enough,
the enzyme called hexokinase does not occur in muscle extracts.
This may be related to the fact that the concentration of free glucose
inside the muscle cell is very low. Perhaps phosphorylation of glu-
cose in the skeletal muscles takes place in the so-called cell mem-
brane and only there. I think that there is every reason to look for
the hexokinase enzyme in the water-insoluble residue. Recently
Colowick and I found that phosphorylation of glucose by adenylpy-
rophosphate when adenosinerf/phosphate is the phosphate donor
needed, besides hexokinase, another protein which occurred only in
skeletal muscle and was therefore called myokinase. Myokinase is
active in amounts smaller than one microgram per milliliter and is
extremely resistant to acid treatment. I have found recently that my-
okinase is also necessary for the dephosphorylation of adenosine-
diphosphate in muscle tissue. The experiments were carried out with
suspensions of myosin as described by Engelhardt and Ljubimova.

The exact function of myokinase is not known. Dr. Johnson has
suggested that myokinase catalyzes a transfer of phosphate from
one molecule of adenosinediphosphate to another molecule, thus
forming from two moles of adenosinediphosphate one mole of adeno-
sinetriphosphate and one mole of adenosinemonophosphate. One
might also consider the formation of a dinucleotide between adeno-
sinetriphosphate and adenylic acid of the type that has been isolated
by Kiessling and Meyerhof. Myokinase might function not only as a
catalyst but also as a phosphate transfer system. The inhibition of
myokinase by adenylic acid may also indicate a reversed reaction
of myokinase""with adenosinediphosphate.

Adenylpyrophosphate phosphorylates glucose or fructose to

192 A SYMPOSIUM ON RESPIRATORY ENZYMES

hexose-6-phosphate. Sutherland, Colowick, and Cori have shown
recently that this ester can be converted to hexose-1-phosphate and
thus to starch or glycogen.

The phosphorylation of hexosemonophosphate to hexosediphos-
phate has been observed in several tissues and also in muscle
extracts. This reaction is probably strongly exergonic, hke the hexo-
kinase reaction.

Meyerhof and Lohmann have shown that the dephosphorylation
of adenylpyrophosphate to adenosinedi- or monophosphate and
orthophosphate is one of the strongest exergonic reactions in bio-
logical systems. The enzyme is present in large amounts in muscle
extracts but the myosin fraction contains also large amounts of this
enzyme, and Engelhardt and Ljubimova have found that even
highly purified myosin contains adenosinetriphosphatase.

The utilization of adenylpyrophosphate by these irreversible reac-
tions probably determines the extent of numerous important oxida-
tions. The great question is how the irreversible utilization of
adenylpyrophosphate is regulated. The existence of adenylpyro-
phosphate in resting muscles with very low metabolism clearly shows
that the enzymes which catalyze the breakdown of pyrophosphates
must be in a more or less inactivated state and are fully activated
only under certain conditions. The state of structural proteins such
as myosin might determine the extent of the activation of the en-
zymes which catalyze the degradations of adenylpyrophosphate.
Discharged structural proteins might even be enabled to use the
large amount of energy liberated by pyrophosphate degradations for
recharging by participating in the transfer of phosphate.

We do not yet know how irreversible enzymatic reactions are
regulated or how they are directed in order to transfonn chemical
energy into mechanic^ energy. It has become clear, however, that
the irreversible dephosphorylations of adenylpyrophosphate repre-
sent some of the most important degradations in biological systems.

Otto Meyerhof, University of Pennsylvania:

May I discuss three of the points that have been proposed by Dr.
Kalckar:

1. Because of the specific structure of the living cell, the metab-
olism can be regulated differently from the metabolic processes in
extracts or other preparations of dead cells. At least one such dif-
ference can easily be explained by the insufiicient stability of the
extracted enzyme. I refer to the accumulation of hexosediphosphate
in yeast preparations in contrast to the balance of formation and

DISCUSSION ON PHOSPHORYLATION 193

decomposition of this ester in living yeast. Indeed the occurrence of
the same phosphorylated intermediaries in hving cells as in extracts
is clearly demonstrated by the experiments of Miss Macfarlane,
Werkman, Dische, and others. But the adenylpyrophosphatase,
which is responsible for the fermentation of hexosediphosphate in the
absence of a stoichiometric amount of phosphate acceptors, is weak-
ened even by drying the yeast, more by incubation and extraction
of the dried yeast, and it can be completely destroyed by precipita-
tion with acetone.

On the other hand, we may assume that in the living cell excess
phosphorylation of sugar occurs in connection with growth. The
starting point of these syntheses may be some other ester instead of
hexosediphosphate; nevertheless the autocatalytical increase of this
ester in a fermenting yeast extract can be taken as a good model
for material growth brought about by the energy of fermentation;
indeed, such an extract, containing sugar, will not start fermentation
unless it is "inoculated" by a trace of hexosediphosphate, which then
"grows" at the expense of sugar.

2. At least some of the experiments of different authors quoted by
Dr. Lipmann seem not too reliable in regard to abnormally high
oxidation quotients. I refer especially to cases with Qo, values of
0 to 1. Under such conditions the manometric method is not accurate
enough, and a completely stationary state during the time of the
experiment is not assured.

Furthermore, it seems to me, the distribution of oxidized to re-
duced cozymase in toto cannot be used as the basis for deciding
how far the hydrogen transfer by means of cozymase is affected by
respiration. This transfer occurs by means of the bound cozymase of
specific enzymes, and the oxidative state of such a compound may
well be altered by the oxygen transfer from oxidative catalysts with-
out an appreciable change in the overall distribution of oxidized
to reduced cozymase.

3. The interesting finding of Dr. Kalckar that the action of hexo-
kinase of yeast is supplemented by a heat-stable enzyme of muscle,
"myokinase," by which adenosinediphosphate transfers its labile
phosphate group to glucose, points to a difference, already well
known, between the enzymes in muscle and yeast preparations.
While the former (extract, acetone powder, etc.) retains the ability
to use adenylic acid as the phosphorylating coenzyme even after
long dialysis and "ageing," in yeast preparations the reaction step,

(2) P-acceptor + adenosinediphosphate ±5 P-acceptor — phos-
phate + adenylic acid.

194 A SYMPOSIUM ON RESPIRATORY ENZYMES

is easily destroyed, and only the first reaction step of transphos-
phorylation,

(1) P-acceptor + adenosinetriphosphate ±^ P-acceptor — phos-
phate + adenosinediphosphate

is still occurring. A more detailed study of the different enzyme
proteins in these phosphorylating reactions along the Hnes suggested
by Colowick and Kalckar seems highly desirable.

M. J. Johnson, University of Wisconsin:

The present status of our knowledge on biological phosphoryla-
tions raises the question whether energy of phosphorylation is the
sole form in which energy from food oxidation (or fermentation) is
made available for metabolic reactions. In other words, is phos-
phorylation the only device employed by the cell to utilize oxidative
energy?

The only answer that can be made at present is that phosphory-
lation is the only mechanism we know of by which a part of the
energy derivable from the burning of food material can be made
available for endergonic life processes. There appears to be no a
priori reason to suspect the existence of another mechanism. In fact,
the simplicity of a single-mechanism hypothesis seems very attrac-
tive. On the other hand, we have no reason to doubt the existence
of other "energy-fixing" reactions. The question is entirely open.

There is, however, at least one fragment of evidence that definitely
favors the single-mechanism hypothesis. Yeast growing anaerobically
in a simple medium must derive all the energy for its growth reac-
tions from the conversion of glucose into ethyl alcohol and carbon
dioxide. Unless our present views of the mechanism of this conver-
sion are erroneous, no energy-fixing mechanism other than phos-
phorylation exists in alcoholic fermentation. Therefore the only form
in which energy from the fermentation process is available to the
organism is energy of phosphorylation. It follows, of course, that the
yeast cell is capable of utilizing, directly or indirectly, energy of
phosphorylation for all its metabolic energy requirements. Hence
any other energy-fixing reactions which might be postulated to occur
during aerobic sugar breakdown are at least not essential for yeast
growth.

DISCUSSION ON PHOSPHORYLATION 195

Fritz Lipmann, Massachusetts General Hospital:

On several occasions during this symposium acetylphosphate has
been mentioned as an intermediate in carbohydrate breakdown.
Since I have not yet published a complete account of my experi-
ments, I should like to take this opportunity to summarize the evi-
dence so far accumulated for the formation of acetylphosphate as an
intermediate in pyruvic acid oxidation. Early in my study of the
oxidation of pyruvic acid in lactic acid bacteria it was observed that
inorganic phosphate was an integral part of the pyruvic acid oxida-
tion system (1). In partial explanation of its necessity it was shown
that with oxidation of pyruvic acid, phosphate could be transferred
to adenylic acid to form adenosinepolyphosphate (2). In other words,
pyruvic acid oxidation generated energy-rich phosphate bonds
(terminology of ref. 3). In analogy to Negelein and Bromel's phos-
phoglycerylphosphate (4), acetylphosphate was then suspected to be
the phosphorylated intermediate between pyruvate and adenosine
polyphosphate. In confirmation it was found that synthetical acetyl-
phosphate enzymatically transferred phosphate to adenylic acid
with the formation of adenosinetriphosphate (5). This observation
led to a reinvestigation of the phosphate turnover in pyruvic acid
oxidation. In the bacterial metabolism pyruvate is fortunately not
oxidized farther than the acetate stage. Under favorable conditions,
therefore, one could hope to demonstrate the accumulation of a
phosphorylated precursor of acetic acid.

Earlier attempts had always failed to disclose any disappearance
of inorganic phosphate during the oxidation of pyruvate. Now that
acetylphosphate was suspected as intermediate, its stability was
studied, and it was found that its formation would have been over-
looked because in the course of all the known procedures of phos-
phate assay it would have been decomposed to inorganic phosphate.
Therefore the problem arose of finding a method for the detennina-
tion of inorganic phosphate in the presence of a compound of the
stability of acetylphosphate, since this compound does not with-
stand the alkalinity of the magnesia mixture tolerated by creatine-
phosphate, nor the acidity of the molybdate reagent. It appeared
possible, however, to completely precipitate inorganic phosphate as
calcium phosphate in dilute alcohol at a pH of 8, where acetylphos-
phate is stable. The use of this more delicate precipitation procedure
showed indeed that large amounts of inorganic phosphate disap-
peared during pyruvate oxidation with or without fluoride (6).
Phosphorylation, oxygen consumption, and pyruvate disappearance

196 A SYMPOSIUM ON RESPIRATORY ENZYMES

occurred in definite proportions and in good agreement with the
equation:

CH3 • CO • COOH + HO • PO3H2 + O - CH3 • CO •
OPO3H3 + CO2 + H,0

Recently the formation of a labile phosphate compound could be
demonstrated directly by the use of the method with which creatine-
phosphate was originally discovered both by Fiske and Subbarow
and by the Eggletons. This method consists in reading the develop-
ment of the blue color against a standard of inorganic phosphate,
the increase in color with time being proportional to the breakdown
of the labile phosphate compound. The half-decomposition time of
our phosphorylation product (and of acetylphosphate) is about one
minute at room temperature. This rapid decomposition of the nat-
ural as well as the synthetic product is due not merely to the acidity
of the solution but largely to a catalytic effect of molybdate. Re-
cently silver fractions containing the labile phosphate have been
obtained from trichloroacetic acid extracts. Although, as should be
emphasized, pure preparations of the phosphorylation product have
not yet been obtained,* the reported results may be taken as fair
evidence that the intermediate formed during pyruvic acid oxidation
is in fact acetylphosphate.

REFERENCES

1. LiPMANN, F., Enzymologia, 4, 65 (1937).

2. LiPMANN, F., Nature, 143, 281 (1939).
OcHOA, S., J. Biol. Chem., 138, 751 (1941).

3. LiPMANN, F., Advances in Enzymology, 1, 99 (1941).

4. Negelein, E., and Bromel, H., Biochem. Z., 300, 225 (1939).

5. LiPMANN, F., Nature, 144, 381 (1939).

6. LiPMANN, F., J. Biol. Chem., 134, 463 (1940).

* Since submitting this paper we have succeeded in isolating silver acetyl-
phosphate from the impure silver fractions.

SCHLENK

Stern

Werkmax Haas

CLOSE-UPS OF FOUR PARTICIPANTS

Metabolic Cycles and Decarboxylation

E. A. EVANS, JR.
University of Chicago^

IF I WERE to adhere rigidly to a discussion of the topic assigned
me I should be compelled to mention almost every aspect of
our present knowledge of intermediary metabolism. I am claim-
ing, therefore, the traditional privilege of discussing those matters
that seem of particular interest and importance.

From what we know of the chemical constitution of most cells,
it is to be expected that those interactions of cell constituents by
which the organism obtains the energy necessary for continued
existence should exhibit certain characteristics of continuity and
recurrence, as does the cell itself. The utilization of foodstuffs by
the cell frequently involves a cyclic chain of chemical transforma-
tions in which certain cell constituents, usually present in small and
apparently constant amounts, facilitate the transformation of larger
quantities of other metabolites in reactions releasing energy or
leading to the formation of the actual protoplasmic fabric of the
cell itself. The synthesis of urea, the transforaiation of glycogen into
lactic acid, and, in a broader sense, the transport of the respiratory
gases by the blood are familiar and typical examples of the cyclic
mechanisms by which the organism maintains the balance necessary
for its existence in the midst of the dynamic processes by which it
functions.

In the past few years such a cyclic series of reactions has been
proposed for the mechanism of oxidation of carbohydrate in various
tissues— more specifically for the oxidation of pyruvic acid in volun-
tary musculature. This scheme, the so-called citric acid cycle, was
proposed by Krebs (1, 2, 3, 4). In its original form the theory was
concerned with the oxidation of pyruvic acid by minced pigeon
breast muscle, and although the generalizations of the theory have
been extended in part to other tissues and species (5, 6), the most
convincing and complete data are those derived from suspensions of
muscle tissue.

* The original work reported in this paper was aided in part by grants
from tlie John and Mary R. Markle Foundation and from tlie Dr. Wallace C.
and Clara A. Abbott Memorial Fund of the University of Chicago.

197

198 A SYMPOSIUM ON RESPIRATORY ENZYMES

A great variety of chemical transformations involving pyruvic
acid has been demonstrated in different species of Hving cells, iso-
lated enzyme systems, etc. (representative reactions are listed in
Table 1 which is not, however, complete). In some cases several of
these diverse ways of treating pyruvic acid can be demonstrated to
occur in the same cell. In such circumstances there is considerable
advantage in a working hypothesis in which reactions involving the
same compound are regarded as components of an integrated sys-
tem. In a tissue such as muscle, where specialization of function
might be expected to reflect a similar specialization of metabohsm,
an hypothesis of this type should be especially fruitful. The citric
acid cycle represents, then, an attempt to summarize the available
information with respect to the metabolism of pyruvic acid in pigeon
breast muscle. Such a summary is of value only so long as it does
not conflict with experimental observations and to the extent that it
adequately represents the experimental foundation on which it rests.
At the moment there are no observations that can be regarded as
invalidating the theory, although objections to one or another feature
of the scheme have been made. At the risk of adding little that is
new to what you already know, I should like to discuss several
aspects of the citric acid theory, particularly in regard to its experi-
mental basis. The cycle itself is shown diagrammatically in Figure 1.

The essential experimental support for the theory can be sum-
marized in the following equations:

In the presence of malonate:

( 1 ) Fumarate + pyruvate + 2 O2 — > succinate + 3 CO2 + H2O

(2) Malate + pyruvate + 2 O2 -^ succinate -\- 3 CO2 + 2 H2O

(3) Oxalacetate + pyruvate -f- IJ2 O2 -^ succinate + 3 CO2 + H2O

(4) Citrate + O2 -^ succinate + 2 CO2 +H2O

(5) a-Ketoglutarate + )k O^-^ succinate + CO2 + H2O

In the absence of malonate:

(6) Succinate + M O2 -^ fumarate -|- H2O

( 7 ) Pyruvic acid + 2Yi O2 -> 3 CO2 -|- H2O ( Equations 1 -|- 6 )

In nitrogen:

( 8 ) 2 Oxalacetate -j- pyruvate — > citrate + CO2 + malate

(9) Oxalacetate + citrate — > a-ketoglutarate -|- CO2 + malate.

The demonstration, in suspensions of muscle tissue, of the stoi-
chiometric relationships expressed in these equations constitutes the
experimental proof on which the citric acid cycle is based. This
demonstration has two aspects— one concerned with the qualitative
presence of these reactions, i.e., with the existence of the necessary

METABOLIC CYCLES AND DECARBOXYLATION
Table 1.— Reactions of pyruvic acid (P.A.)

199

Reaction studied

Tissue used

P.A. -^ acetaldehyde + CO2

P.A. + ^02 -^ acetic acid + CO2

2P.A. + H2O —* lactic acid + acetic acid + CO2

P.A. + 2H ^ lactic acid

P.A. + oxalacetic acid — > citric acid

2P.A. -^ acetic acid + formic acid

P.A. + glutamic acid — > alanine + a-ketoglutaric acid

2P.A. — > acetoacetic acid

2 P.A. —* ^-hydroxybutyric acid

P.A. — > alanine

P.A. + acetate — > acetopyruvate — > acetoacetate

P.A. -tC02 —* a-ketoglutarate

2P.A. — » acetylmethylcarbinol

yeast

brain, gonococcus

gonococcus, brain, liver

muscle, tumor

muscle, liver, kidney

streptococcus

muscle

liver

muscle

liver

muscle

enzymic channels through which these reactions may flow, and the
other with their quantitative capacity to perform the task imposed
upon them by their postulated role in the cycle. To the extent that
a suspension of minced pigeon breast muscle retains the chemical
reactions involved in its respiration in the intact state, the reactions
summarized in equation 7 may be regarded as most closely approxi-

"TRIOSE"

i -2H-

PYRUVATE

OXALACETATE j | +Y^p-2H-

CITRATE

cii-ACONlTATE

ISO-CITRATE

+ H20-2H-

o(-KETOGLUTARATE

J. Pl_l ^

frUMARATE

SUCCINATE

FUMARATE
I H-MALATE

+ H^0-2H OXALACETATE '

U H-MALATE

-2H-1

-2H

-iQ2-

— ^
■^H,0

-2H-

Figure 1. — Scheme of the oxidative breakdown of carbohydrate
in pigeon breast muscle

200 A SYMPOSIUM ON RESPIRATORY ENZYMES

mating the physiological state, since they occur when pyruvate is
added to the tissue in the presence of an adequate supply of oxygen
and in a medium approximately physiological in ionic concentration
and pH. In these circumstances the stoichiometric relationships of
equation 7 are very closely realized, and it seems certain that the
total oxygen uptake of the tissue is utilized for the oxidation of
pyruvic acid completely to carbon dioxide and water. Pyruvic acid
will also disappear in the absence of oxygen. The quantity con-
cerned is about one-tenth that removed aerobically, and although
lactic acid, acetic acid, carbon dioxide, succinic acid, and beta-
hydroxybutyric acid have been recognized as products of this anaer-
obic reaction, the details are still obscure.

Equations 1 to 5 deal with reactions carried out in the presence
of malonic acid. Equation 1 is the most important reaction of the
theory, since it demonstrates unequivocally an oxidative formation
of succinic acid from fumaric acid. In the presence of proper con-
centrations of malonic acid the oxidation of pyruvate by pigeon
breast muscle can be almost entirely inhibited. When fumarate is
added, however, pyruvate is oxidized and in accordance with equa-
tion 1 we find, per mole of fumarate added, one mole of pyruvate
and 2 moles of oxygen consumed, and 1 mole of succinate and 3
moles of carbon dioxide formed. A direct reduction of fumarate to
succinate under these circumstances is inhibited by the malonic acid
present, as can be shown by anaerobic experiments; that is, we find
much more succinate formed from fumarate in the malonate-
poisoned muscle in oxygen than in nitrogen. Since it is impossible
to explain the conversion of fumarate to succinate in the presence of
malonate by anaerobic reduction, a second mechanism must exist
which is oxidative and unaffected by malonate and which results in
the transformation of fumarate into succinate. It should be em-
phasized that we find in this transformation the stoichiometric re-
lationships of equation 1. The citric acid cycle postulates that this
oxidative transformation involves the intermediate formation of
citric acid. Regardless of the nature of the intermediates, however,
any explanation of the oxidation of pyruvic acid by muscle must
account for reaction 1, i.e., what might be teiTned the Krebs reaction.

The demonstration of the stoichiometric relationships of equation
1 depends upon the recognition of essential experimental conditions.
In view of the fundamental nature of this reaction it may be per-
missible to discuss a few matters of experimental detail.

The working plan of the experiments summarized in equations 1

METABOLIC CYCLES AND DECARBOXYLATION 201

to 5 involves interruption of the cycle at the succinate-fumarate stage
by poisoning with malonic acid. Implicit here is the assumption that
malonate acts specifically on this particular reaction, i.e., the oxida-
tion of succinate to fumarate. The inhibition of succinic dehydro-
genase by malonic acid in isolated enzyme preparations has been
recognized and demonstrated by various investigators (7, 8). The
inhibition is competitive in nature; that is, it depends not upon the
absolute concentration of malonate but on the relative quantities of
succinate and malonate. Since the basis of this competitive inhibition
is the resemblance in chemical structure between succinic and
malonic acids, it follows that malonate will probably inhibit the
enzymic transformation of any substrate bearing some chemical
resemblance to its own structure. However, the available data
suggest that the effect of the poison is most pronounced with the
succinic dehydrogenase. Expressed quantitatively, with 1 : 10 suspen-
sions of pigeon breast muscle in calcium-free phosphate sahne, 0.001
M malonate inhibits pyruvate utilization about 20 per cent. Higher
concentrations of malonate, around 0.025 M, inhibit to more than
90 per cent.

As equation 1 indicates, fumaric acid will abolish the inhibitory
eflFect of malonate in muscle tissue with the simultaneous utilization
of one mole of pyruvate and 2 moles of oxygen to give 1 mole of
succinate and 3 moles of carbon dioxide. The extent of this fumarate
eflFect, however, depends upon the relative concentrations of malo-
nate and fumarate. Krebs, in a long series of experiments (2), has
shown very clearly that the demonstration of the stoichiometric
relationships of equation 1 depends upon the presence of an ade-
quate concentration of malonic acid. When the malonate concentra-
tion is too low, as, for example, with 0.001 M malonate in the
presence of 0.0025 M fumarate, the gradual conversion of fumaric
acid to succinic acid will so increase the succinic acid concentration
as compared with that of the malonate that succinic acid will be
oxidized to fumaric acid at a rate sufficient to provide for the
continuation of the cycle at the full rate. On the other hand, with
0.025 M malonate the stoichiometric relationships of equation 1 are
realized. It is obvious, then, that the relationships expressed in
equations 1 to 5 can be demonstrated only in the presence of ade-
quate concentrations of malonic acid. When this is done, the experi-
mental data are in fairly close approximation to the expected values.

In similar experiments with pig heart muscle, Smyth (5) has ob-
served an occasional failure with citric acid to obtain the quantities

202 A SYMPOSIUM ON RESPIRATORY ENZYMES

involved in reaction 4. He oflFers several possible explanations for
these occasional failures, although the matter must still be con-
sidered unsettled.

In terms of the postulated cycle, the possible participation of
intermediate steps such as those listed in equations 1 to 5 can also
be critically examined from the standpoint of the minimum rates at
which they occur. This has aheady been discussed in considerable
detail elsewhere (9). If we assume that the citric acid cycle repre-
sents the entire respiratory process involved in the oxidation of
pyruvic acid in pigeon breast muscle, then the total oxygen uptake
of the tissue represents a total of five consecutive reactions of the
cycle. Under circumstances in which individual reactions of the
cycle are isolated— for example, when alpha-ketoglutarate is added
to the malonate-poisoned tissue— the rate of oxygen uptake for the
conversion of alpha-ketoglutarate to succinate cannot be less than
one-fifth that for pyruvate oxidation. If the reaction velocity is below
this minimum requirement, the step can be excluded as an inter-
mediate in the overall reaction. On the other hand, if the isolated
reaction proceeds with a velocity greater than the minimum rate,
it may still be considered as an intermediate, since in the intact
system the concentration of the particular enzyme concerned may
exceed the quantity of the substrate normally available. When the
reactions summarized in equations 1 to 5 are examined from this
viewpoint, we find in each case that the rate of the isolated step
proceeds with the necessary velocity consonant with its composing
part of the cycle.

The existence of what I have termed the Krebs reaction is gener-
ally accepted: most critical comment on the citric acid hypothesis
centers around the postulated intermediate foi*mation of citric acid.
It has been argued that failure of this substance to accumulate in
large amounts during pyruvate oxidation in various tissues is con-
trary to its postulated intermediate role. However, the accumulation
of an intermediate must represent a balance between its synthesis
and its removal, and the accumulation of large amounts of citric
acid would be much more difiicult to reconcile with the premise
that it is an active intermediate substance than would the fact that it
accumulates to only a limited extent. In addition, Krebs has demon-
strated an anaerobic reaction between citrate and oxalacetate to yield
alpha-ketoglutarate and malate (equation 9). Although there is
reason to question the validity of such anaerobic experiments as the
basis for assigning the role of hydrogen carriers to the oxalacetate-

METABOLIC CYCLES AND DECARBOXYLATION 203

malate system in the aerobic cycle, this reaction may also explain
why citrate does not accumulate to any extent. On the other hand,
the only reason we have for including citric acid in the cycle is the
rapidity of its formation from oxalacetate and pyruvate as indicated
in equation 8 and the fact that its oxidative breakdown will yield
alpha-ketoglutarate and succinate at the required rate. It has been
suggested, first by Breusch (10) and later by others, that citrate
formation represents a side reaction in pyruvate metabolism— a
means for disposing of excessive amounts of the dicarboxylic acids.
There is, however, no experimental support for this concept at the
present time; the simpler hypothesis, in which citric acid is regarded
as an intermediate in the cycle, is probably to be preferred as a basis
for further experiments. It should be emphasized that succinate is
formed in malonate-poisoned muscle at the proper rate only by
those compounds listed as intermediates, although a great number
of other possible substances have been examined for their ability
to do this. It seems improbable, therefore, that the quantitative
formation of succinate from citrate in the malonate-poisoned muscle
is without significance for the role of citrate in muscle metabolism;
Smyth's failure to obtain consistent results with citrate in pig heart
muscle may be due to our ignorance of the essential conditions for
citrate oxidation, as he suggests.

With other tissues the evidence for similar mechanisms of pyru-
vate oxidation is much less complete. It has been shown in pigeon
hver, for example, that the enzyme systems necessary for the reac-
tions of the citric acid cycle are present (6). Whetlier or not they
play any considerable role in its metabolic function remains a
question for further study. We do have evidence that alpha-
ketoglutaric acid formation can occur in this tissue independently
of the reactions of the citric acid cycle (9, 11, 12).

Demonstration of the synthesis of alpha-ketoglutaric acid from
pyruvic acid in pigeon liver has been followed by efforts to ascer-
tain the mechanism involved. The reaction proceeds in the presence
of malonic acid and without the addition of any of the four-carbon
dicarboxylic acids, presumably by a direct utilization of the three-
carbon compound. The scheme of Toeniessen and Brinkmann (13)
for succinate synthesis from pyruvate in muscle, involving the inter-
mediate formation of diketo-adipic acid, could be disregarded, since
pigeon liver is incapable of metabolizing formic acid at a rate neces-
sary for this mechanism to operate (6).

Since pigeon Hver can form citric acid from oxalacetic acid and

204 A SYMPOSIUM ON RESPIRATORY ENZYMES

can also oxidize citric acid to alpha-ketoglutaric acid, the reaction
of Wood and Werkman involving the carboxylation of pyruvic
acid to yield oxalacetate (14) offers an attractive hypothesis for
alpha-ketoglutarate synthesis, i.e., oxalacetate + pyruvate -^ citrate
-> alpha-ketoglutarate. Slotin and I carried out experiments to test
this hypothesis, using carbon dioxide containing radioactive C"
and found that the succinic acid formed on the addition of pyruvate
to malonate-poisoned pigeon liver, presumably through the inter-
mediate formation of alpha-ketoglutaric acid, was devoid of radio-
activity. However, when we extended these experiments to the iso-
lation of alpha-ketoglutaric acid as the dinitrophenylhydrazone, we
could clearly observe the utilization of carbon dioxide in the forma-
tion of radioactive alpha-ketoglutarate (15). As the earlier succinate
experiments suggested, and as was confirmed later by direct exami-
nation of the isolated keto acid, the radioactivity is confined entirely
to the carboxyl group of this compound.

The fact that carbon dioxide is used in the synthesis of alpha-
ketoglutarate from pyruvate in pigeon liver and that the assimilated
carbon dioxide is present entirely in the carboxyl group alpha to
the carbonyl oxygen, is now firmly established in view of the iden-
tical results of Wood, Werkman, Hemingway, and Nier (12), using
the stable C^^, and of Slotin and me, with the short-lived radioactive
C" (11, 15). These data cannot be reconciled with the intermedi-
ate formation of citric acid, since any symmetrical intermediate mole-
cule of this type would yield alpha-ketoglutarate with radio-
activity at both carboxyls. Therefore the original conception of
alpha-ketoglutarate synthesis occurring by way of pyruvate and
carbon dioxide condensation to oxalacetate, and the subsequent
reaction of this dicarboxylic acid with another mole of pyruvate
to yield citric acid, must be abandoned. Support for such a view
is also derived from experiments in which non-radioactive citrate
was added to liver suspension in radioactive bicarbonate medium
during the synthesis of alpha-ketoglutaric acid. Table 2 lists ex-
periments showing that the addition of 25 mg. of sodium citrate
affects neither the yield of alpha-ketoglutaric acid nor the ratio of
activity per mg. carbon of the alpha-ketoglutarate to that of the
medium. If citrate is an inteiTnediate in the formation of alpha-
ketoglutaric acid from pyruvate and carbon dioxide, the addition
of non-radioactive citrate to the synthesizing tissue should yield
alpha-ketoglutaric acid in which the radioactivity had been con-
siderably diluted. Failure to demonstrate such an effect may be

METABOLIC CYCLES AND DECARBOXYLATION 205

considered additional evidence for the nonparticipation of citric
acid in the synthesis of alpha-ketoglutaric acid from pyruvate and
carbon dioxide.

Table 2.— The e£Fect of citrate on the radioactivity of synthesized
alpha-ketoglutaric acid*

Experiment

Sodium
citrate added

a-Ketoglutarate
synthesized

Activity of
a-ketoglutarate

mg.
0

mg.
31.4

per mg. C
0.148

28.8

0.113

36.9

0.116

39.8

0.116

* Experimental conditions were similar to those in the experiments reported in
Table 3. The sodium citrate was added simultaneously with the pyruvic acid.

Wood and Werkman and their collaborators (12) have suggested
that isocitric acid rather than citrate serves as an intermediate, such
a scheme retaining in skeleton form the original suggestion that
alpha-ketoglutarate was produced by way of the citric acid cycle.
In view of the demonstrated equilibrium, in most tissues, between
citrate and isocitrate (16), additional evidence is required to estab-
lish this hypothesis. The exclusion of citric acid as an intermediate
in alpha-ketoglutarate synthesis might well lead to an examination
of whether oxalacetate itself is formed as the primary product of
carbon dioxide assimilation in pigeon liver. The great chemical
reactivity and instability of this compound make it improbable that
it should accumulate to any appreciable extent under our experi-
mental conditions. Though we have studied a wide variety of
conditions, we have failed to obtain any evidence that the compound
was present during the course of alpha-ketoglutarate synthesis; or,
to express the matter more precisely in experimental terms, we have
failed to observe the accumulation of any substance that would
release radioactive carbon dioxide on treatment with aniline citrate.
Certainly it would be desirable to have more convincing evidence
that oxalacetic acid is the primary product of carbon dioxide as-
similation in pigeon liver.

There seems little doubt that the utilization of carbon dioxide by
pigeon liver is a process of considerable magnitude. Table 3 gives
data from a series of typical experiments which indicate that as
much as 5 per cent of the original activity of the added inorganic

206

A SYMPOSIUM ON RESPIRATORY ENZYMES

carbonate appears in the synthesized alpha-ketoglutaric acid. Un-
fortunately these data are difficult to interpret in terms of any
precise mechanism of alpha-ketoglutarate formation. Since all the

Table 3.— Synthesis of alpha-ketoglutarate in radioactive
bicarbonate medium*

Terminal

Ratio

a-Keto-
Exp. glutarate
synthe-
sized

Activity t of
a-ketoglutarate

Total
activity

of
medium

activity
of medium

per mg.
inorganic

carbon

a-ketoglutarate
activity

medium activ-
ity per mg. C

Total

per

mg.C

original activ-
ity of medium

a-ketoglutarate
per mg. C

mg.
1 31.4

0.471

0.036

8.74

0.078

0.054

2.0

2 27.5

4.09

0.362

67.0

1.13

0.061

3.1

3 55.0

3.85

0.171

0.161J

0.178i

79.8

1.17

0.048

6.9

* 7.6 gm. minced pigeon liver in 50 ml. calcium-free bicarbonate saline, pH. 7.4,
containing radioactive C"; 1.7 ml. 0.1 M malonate; 10 ml. 0.02 M pyruvate; malonate
added directly to tissue; pyruvate added after 10 minutes; temperature, 40° C; gas
phase, 5 per cent carbon dioxide; experimental period, 40 minutes.

t Activities are expressed in divisions per second (Lauritsen electroscope) ; and are
corrected for decay so that the values are comparable.

X Activities after successive recrystallizations of alpha-ketoglutarate dinitrophenyl-
hydrazone.

radioactivity is lost on oxidation of alpha-ketoglutaric acid to suc-
cinic acid, we can definitely conclude that not more than one carbon
atom of the five of alpha-ketoglutarate is derived from the medium.
From the quantity of radioactivity present in the alpha-ketoglutarate
we can calculate (the details of these very approximate calculations
are given elsewhere [11]) that approximately one carbon atom in
ten of the alpha-ketoglutarate is derived from the inorganic car-
bonate of the medium. Since this figure involves very approximate
calculations, and is of value only in suggesting the order of magni-
tude of the reaction, we are inclined to believe, as a working
hypothesis, that one mole of carbon dioxide from the medium is
assimilated per mole of alpha-ketoglutarate synthesized.

The synthesis of alpha-ketoglutarate is by no means the limit of
carbon dioxide assimilation. Under our experimental conditions we
find that alpha-ketoglutaric acid represents only about 25 per cent
of the radioactivity which has been assimilated, that is, activity
which can no longer be released as carbon dioxide on the addition

METABOLIC CYCLES AND DECARBOXYLATION 207

of strong acid. Part of this non-a-ketoglutarate radioactivity can be
released as carbon dioxide on treatment with ninhydrin at 100° C,
and with chloramine-T (Table 4). This suggests that part of the
assimilated carbon dioxide has been converted into an amino acid.
Beyond this, however, we know nothing of the chemical nature of
the compounds concerned.

Table 4.— Effect of ninhydrin and chloramine T on non-a-
ketoglutarate radioactivity*

Total
a-Keto- Total activity Activity of Activity of

Experiment glutarate activity of medium CO2 released CO2 released

synthesized of a-keto- after CO2 by ninhydrin by chloramine-T
glutarate removal

mg.

1 35.0 5.18 31.8 7.8 —

2 39.0 6.2 31.2 — 10.39

3 53.0 1.2 4.2 — 0.78

4 31.6 0.22 0.929 0.209 —

* Experimental conditions are similar to those described in Table 3. The experiments
with chloramine-T and with ninhydrin were carried out on aliquots of the metaphos-
phoric deproteinized reaction mixture under the conditions described, respectively,
by P. P. Cohen in Biochem. J., 33, 551 (1939) and by D. D. Van Slyke and R. T. Dillon
in Comp. rend, du Lab. Carlsberg, Ser. Chim., 22, 480 (1937).

In all experiments involving the use of radioactive isotopes or
tracer elements it is a matter of primary concern whether the ap-
pearance of the tagged atom in a product represents a true meta-
bolic reaction or has been introduced by a nonspecific exchange
reaction. Oxalacetic acid, for example, breaks down to pyruvic acid
and carbon dioxide in tissues, and it might be argued that any
reaction involving this compound would yield a radioactive end
product if the reaction were carried out in the presence of radio-
active carbon dioxide. Such a possibility is difficult to control. How-
ever, the very large amounts of carbon dioxide assimilated argue
against this explanation of the presence of radioactivity in the
alpha-ketoglutaric acid. Also suggestive are experiments in which
the possibility of an exchange between the carboxyl group of oxal-
acetic acid and carbon dioxide of the medium has been examined.
In muscle, alpha-ketoglutaric acid is synthesized by the condensation
of oxalacetate with pyruvate to form citrate and the subsequent
oxidation of this compound to alpha-ketoglutarate; i.e., in terms of
the citric acid cycle this is what occurs. Now, a formation of oxal-

208 A SYMPOSIUM ON RESPIRATORY ENZYMES

acetic acid from pyruvate and carbon dioxide in pigeon breast
muscle does not occur, inasmuch as malonate will completely inhibit
pyruvate utilization in the absence of added oxalacetic or other
dicarboxylic acids. Under these circumstances the synthesis of
muscle alpha-ketoglutarate in a radioactive bicarbonate medium
would give rise to radioactive alpha-ketoglutarate only if a process
of exchange between intermediates in the reaction (such as oxal-
acetate) and the carbonate of the medium took place. If the quantity
of radioactivity found in muscle alpha-ketoglutarate is similar in
magnitude to that found in the alpha-ketoglutarate synthesized by
liver, it would eliminate the necessity for proposing a stoichiometric
utilization of carbon dioxide in the process. When this reaction is
studied in radioactive bicarbonate medium, the alpha-ketoglutarate
formed is devoid of radioactivity. There is apparently no appreciable
interchange between the carbon dioxide of the medium and the
carboxyl group of oxalacetic acid or other intermediates. A similar
negative result was obtained in studies of the equilibrium between
pyruvic acid and carbon dioxide in the presence of carboxylase in
which radioactive carbon dioxide and pressures as high as 300
atmospheres were used. Under these circumstances no interchange
of carbon dioxide and the carboxyl of the pyruvate could be demon-
strated. In view of these data, it seems most probable that the
assimilation of carbon dioxide by minced liver is a true metabolic
reaction, although the mechanism is still obscure.

The most promising line of attack of these problems lies probably
in the attempt to isolate or simplify the enzyme systems involved.
Recently attention has been directed again (17, 18) to the enzymic
formation of acetylmethylcarbinol from pyruvate as a possible step
in pyruvate oxidation. This reaction has two features of interest:
first, it represents an anaerobic, i.e., non-oxidative, decarboxylation of
pyruvate; secondly, it poses again the question of acetaldehyde as an
intermediate. It is still too early to determine the relative importance
of this reaction in pyruvate metabolism of muscle and other tissues.
If it should prove to be of significance, the resulting clarity will
undoubtedly be suflBcient compensation for the revision that will
have to be made in our current beliefs.

REFERENCES

1. Krebs, H. a., and Johnson, W. A., Enzymologia, 4, 148 (1937).

2. Krebs, H. A., and Eggleston, L. V., Biochem. J., 34, 442 (1940).

3. Krebs, H. A., Biochem. J., 34, 460 (1940).

4. Krebs, H. A., Biochem. J., 34, 775 (1940).

METABOLIC CYCLES AND DECARBOXYLATION 209

5. Smyth, D. H., Biochem. J., 34, 1046 (1940).

6. Evans, E. A., Jr., Biochem. J., 34, 829 (1940).

7. Thunberg, T., Skand. Arch. Physiol., 24, 23 (1910).

8. QuASTEL, J. H., and Wooldridge, W. R., Biochem. J., 22, 689 (1928).

9. Evans, E. A., Jr., BuU. Johns Hopkins Hosp., 69, 225 (1941).

10. Breusch, F. L., Biochem. J., 33, 1757 (1939).

11. Evans, E. A., Jr., and Slotin, L., J. Biol. Chem., 141, 439 (1941).

12. Wood, H. G., Werkman, C. H., Hemingway, A., and Nier, A. O., J. Biol.
Chem. 139, 483 (1941).

13. ToENiEssEN, E., and Brinkmann, E., Z. physiol. Chem., 187, 137 (1930).

14. Wood, H. C, and Werkman, C. H., J. Bact, 30, 332 (1935); Biochem. J.,
30, 48 (1936); 32, 1262 (1938); 34, 129 (1940).

Wood, H. C, Werkman, C. H., Hemingway, A., and Nier, A. O., J. Biol.
Chem., 135, 789 (1940); 139, 365 (1941); 139, 377 (1941).

15. Evans, E. A., Jr., and Slotin, L., J. Biol. Chem., 136, 301 (1940).

16. Johnson, W. A., Biochem. J., 33, 1046 (1939).

17. Tanko, B., Munk, L., and Abonyi, I., Z. physiol. Chem., 264, 91 (1940).

18. Green, D. E., Westerfeld, W. W., Vennesland, B., and Knox, W. E.,
J. Biol. Chem., 140, 683 (1941).

Transamination

PHILIP p. COHEN

University of Wisconsin

IN VIEW OF THE FACT that Braunstciii (1) has recently reviewed in
some detail the results of experiments on transamination in his
laboratory, no attempt will be made here to present tabular
details of these studies. Unfortunately most of the publications
issued since the review by Braunstein have not been available to
the writer except in Chemical Abstracts, to which reference is made
in the bibliography.

Many of the papers published in this field, being of a preliminary
sort, are of uncertain value. Most investigators seem to be interested
merely in demonstrating that transamination does or does not occur
in a given tissue. Unfortunately the analytical methods usually em-
ployed permit little more than a qualitative demonstration of the
reaction. Thus, in spite of a widespread interest in transamination,
few reliable quantitative data are available that can be used for
evaluating the role of this reaction in intermediary metabolism.

Types of Transamination

Transamination is a reaction between an alpha-amino and an
alpha-keto acid resulting in the transfer of the amino group from
the former to the latter. When the reaction is enzymatically cata-
lyzed, the end products formed are an alpha-amino and an alpha-
keto acid, the former corresponding in structure to the original
alpha-keto acid, and the latter to the original alpha-amino acid
(Reaction 1).

(1) Ri-CH(NH2)-COOH-f-R.COCOOH^RrCOCOOH

+ R2-CH (NH2)-COOH

When carried out at boiling temperatures, the end products of
transamination are usually an aldehyde corresponding to the
original alpha-amino acid, an alpha-amino acid corresponding to
the original alpha-keto acid, and carbon dioxide (Reaction 2).

(2) Ri-CH(NH2)COOH + RrCOCOOH^R,-CHO

-f- RjCHNHj-COOH -f- CO2

Reaction 2 has been studied in some detail by Herbst, who has
recently reviewed this subject (2).

210

TRANSAMINATION 211

The present review will deal chiefly with the system represented
by Reaction 1, and the use of the term "transamination" will be
Hmited to this reversible, enzyme-catalyzed system.

Discovery of Transamination Reaction

Transamination as a biological reaction was first recognized by
Needham "(3), who found that glutamic and aspartic acids disap-
peared anaerobically when added to pigeon breast muscle without
a decrease in amino nitrogen. She could find no increase in urea or
ammonia nitrogen, but did observe an increase of succinic acid.
She suggested that "possibly a combination of the amino group
with some reactive carbohydrate residue takes place; then when
splitting and oxidation occur the amino group is retained in the
form of a new amino acid."

Unrecognized evidence for transamination was published from
Szent-Gyorgyi's laboratory in 1936 and 1937. It was observed that
the rate at which oxalacetic acid disappeared was greatly increased
when glutamic acid was added to pigeon breast muscle (4) and to
certain enzyme preparations from the same tissue (5).

Credit for the discovery of transamination goes to Braunstein and
Kritzmann (6), who carried out the first detailed investigation of
this reaction. They succeeded in showing that the reaction, termed
"Umaminierung" by them, took place in pigeon breast muscle. The
system represented in Reaction 3 was studied, and they were able
to show its reversibility by isolation and chemical identification of
the alanine and glutamic acid formed.
(3) Z( + ) -glutamic acid -j- pyruvic acid <^ a-ketoglutaric acid -f Z(-j-) -alanine.

Substrates Aciive in Transamination

Investigations by Braunstein and Kritzmann (7) led them to con-
clude that the enzymatic transfer of amino groups in pigeon breast
muscle takes place between any alpha-amino acid (with the possible
exception of glycine) and the dicarboxylic acids, alpha-ketoglutaric
and oxalacetic, as well as between the dicarboxylic amino acids,
glutamic and aspartic, and various alpha-keto acids. No amino group
transfer was observed in the following systems: (1) between mono-
carboxylic alpha-amino acids and monocarboxylic alpha-keto acids;
(2) from amines and peptides; or (3) from alpha-amino dicarboxylic
acids to ketones, hydroxy-ketones, or aldehydes. The analytical
method employed in this study for measuring the formation or dis-

212 A SYMPOSIUM ON RESPIRATORY ENZYMES

appearance of glutamic acid was an adaptation of the Foreman (8)
method, which has recently been shown by Zorn (9) to be unreliable
for studying transamination.

In contrast to certain of the above findings Cohen (10) found that
of twenty-one different alpha-amino acids studied in pigeon breast
muscle Z(— )-aspartic acid and Z( + )-alanine were the most active in
forming glutamic acid in the presence of alpha-ketoglutaric acid.
Alpha-aminobutyric acid and ?( + ) -valine were slightly active, but
none of the remaining amino acids was appreciably active. Of a
series of alpha-keto acids studied, oxalacetic and pyruvic acids were
the most active in causing the anaerobic disappearance of glutamic
acid. Alpha-ketobutyric and mesoxalic acids were slightly active,
but no activity was observed with alpha-ketovaleric, alpha-keto-
caproic, acetoacetic, and laevulic acids. Moreover, no transamination
was observed between alpha-ketoglutaric acid and a variety of
amino compounds other than alpha-amino acids. The formation and
disappearance of glutamic acid were determined by the method of
Cohen (11). The specificity and accuracy of this method has recently
been confirmed (12, 13).

d-Amino Acids.— Amino acids of the d series are not active in
transamination (10, 14, 15, 16). Activity with (i-amino acids has been
reported by Braunstein (1), Braunstein and Azarkh (17), and Euler
et at. (18).

Peptides.— The role of peptides as transamination substrates is of
considerable interest. As noted above, Braunstein and Kritzmann (7)
observed no activity with various peptides. In the case of trans-
aminase preparations Cohen (14) was unable to demonstrate any
appreciable transamination between glutathione and oxalacetic acid.
On the other hand Agren (19) reported that in minced cattle
diaphragm muscle transamination takes place between alpha-
ketoglutaric acid and the peptides glycylaminobenzoic acid and
valylglycine. Valylglycine was found to be as active as alanine
(about 30 per cent transamination), while glycylaminobenzoic acid
was less active. Agren employed the same method as Braunstein and
Kritzmann for measuring transamination. In view of the latter work-
ers' observation that glycine interfered with this detemiination (1)
by being carried down in the dicarboxylic acid amino nitrogen
fraction, it is possible that the results obtained by Agren with
glycine dipeptides are due to this fact. No control experiments of
glycine dipeptides plus tissue are reported.

"Primary" and "Secondary" Substrates.— According to Braunstein

TRANSAMINATION 213

the substrates available for transamination can be considered as
consisting of two types:

1. "Primary" or "active" substrates. These have a high affinity
for the enzyme and react with mono- or dibasic acids after adsorp-
tion. Primary substrates are the dibasic alpha-amino or alpha-keto
acids, and include compounds such as cysteic acid.

2. "Secondary" or "passive" substrates. This group includes all the
alpha-amino and alpha-keto monobasic acids which have no direct
affinity for the enzyme and serve only as "reaction partners" for the
primary substrates.

This idea of Braunstein can be extended to mean that only the
dibasic alpha-amino or -keto acids are activated by the enzyme.
This would suggest that the dibasic alpha-amino and -keto acids act
as prosthetic groups, which in the presence of the enzyine react
with the secondary substrates. That is, the secondary substrates have
an affinity for the enzyme only when the "primary" substrates have
become activated. This concept is not unreasonable, but it appears
unnecessary to classify the substrates as active or passive. From
Braunstein's point of view this is essential, since he has to account
for the activity of the large number of monobasic alpha-amino and
-keto acids which he reports to be active in transamination. From the
writer's point of view this concept is unnecessary, since his experi-
mental data lead to the conclusion that transamination is essentially
a limited reaction and concerned chiefly with the dibasic alpha-
amino and -keto acids. The activity of other compounds can be
explained on the basis of different affinities for the enzyme. Thus
with large amounts of tissue and long incubation periods other
amino acids show a small amount of activity (20).

"Catalytic" Transamination.— Braunstein (1) and Braunstein and
Kritzmann (21) have reported that the addition of small amounts of
a dibasic alpha-amino or alpha-keto acid (as little as M/16000)
causes transamination to take place in pigeon breast muscle brei
between lysine and pyruvic acid. The latter system alone is inactive.
With glutamic acid, the reaction is pictured as proceeding in the
following manner:

( 3 ) glutamic acid + pyruvic acid — > a-ketoglutaric acid + alanine

( 4 ) a-ketoglutaric acid -f lysine —> glutamic acid -f 2-keto-6-aminocaproic acid
The net effect of this would be:

( 5 ) lysine + pyruvic acid —> 2-keto-6-aminocaproic acid -|- alanine

The effectiveness of so low a concentration as M/16000 is remark-

214 A SYMPOSIUM ON RESPIRATORY ENZYMES

able in view of the fact that, as Braunstein himself points out, pigeon
breast muscle brei has a much higher content of dibasic alpha-
amino and -keto acids normally present (11). Yet Braunstein reports
that reaction 5 will not take place without the addition of small
amounts of catalyst. The writer (10, 14) was unable to demonstrate
a similar catalytic eflFect with either pigeon breast muscle or purified
transaminase by using the system:

( 6 ) l{ — )-aspartic acid + pyinivic acid ^ oxalacetic acid + H + ) -alanine.

Braunstein (1) and Bychkov (22) have reported that cysteic acid
and phosphoserine are active in transamination in pigeon breast
muscle. Using purified transaminase, the writer confirmed the ac-
tivity of cysteic acid, but phosphoserine was found to be inactive.
Of interest, however, is the report by Braunstein (1) that neither of
these two compounds is active with purified enzymes (glutamic and
aspartic aminopherases).

Preparation and Properties of Transaminating Enzymes

Kritzmann (23, 24, 25) has described in some detail the prepara-
tion and properties of purified transaminating enzymes from pigeon
breast and pig heart muscles. According to her, two distinct systems
exist, one of which is concerned with glutamic acid (and alpha-
ketoglutaric acid) and the other with aspartic acid (and oxalacetic
acid). The former enzyme is called glutamic aminopherase and the
latter aspartic aminopherase. Both enzymes are reported to require
co-factors, present in muscle kochsaft, whose chemical constitutions
are still unknown but which are similar if not identical for the two
systems. Aspartic aminopherase is thought to be a more labile
system, since it is claimed that muscle suspensions lose their trans-
aminating activity on dilution more rapidly with aspartic acid than
with glutamic acid.

The following are some of the properties of the aminopherases
found by Kritzmann (25): 1. Purification by adsorption, salting out,
or dialysis results in inactivation. 2. Reactivation follows on addi-
tion of boiled muscle extracts or ultrafiltrates. 3. To be effective,
the glutamic-aspartic aminopherases must contain a thermostable,
low molecular weight activator or coenzyme. 4. Denaturation by
acetone, ethyl alcohol, or methyl alcohol leads to an irreversible in-
activation. 5. Heating at 80° C. for five minutes causes a 50 per cent
decrease in activity. 6. pH activity range is 5.5-8.5, with an optimum

TRANSAMINATION 215

at 7.4. 7. Glutamic aminopherase is best obtained from pig heart
muscle, aspartic aminopherase from coarsely ground pea seedlings.
If the latter are finely ground, both aspartic and glutamic amino-
pherases are obtained.

Transaminating enzyme preparations from pig heart and pigeon
breast muscle have recently been studied by Cohen (14, 26). It was
found that the activity of these preparations was greatest with the
systems, Z( + ) -glutamic acid plus oxalacetic acid, and alpha-keto-
glutaric acid plus /(—) -aspartic acid. That is, the enzyme was most
active in catalyzing a reaction in which both glutamic and aspartic
acids (and the corresponding alpha-keto acids) were substrates. The
addition of pigeon breast muscle kochsaft was without influence on
the rates of reactions 3, 6 or 7.

(7) /( + ) -glutamic acid -f- oxalacetic acid ^ a-ketoglutaric acid

-f Z( — ) -aspartic acid

Reaction 7 was catalyzed at a rapid rate, the Qt values being
of the order of 1600.* The position of equilibrium for this system
was far to the right, with an equilibrium constant of about 3. Re-
action 3 was catalyzed at a much slower rate by transaminase, the
Qt values being of the order of 300. The equilibrium constant was
about 1. Reaction 6 was not catalyzed by transaminase.

The following are some properties of transaminase: 1. The enzyme
is best prepared from pig heart muscle and pigeon breast muscle.
2. Transaminase can be dried by rapid lyophilization at low tem-
peratures. Such preparations remain active for as long as six weeks
at room temperature (63). 3. Purification by salting out or dialysis
results in inactivation. However, solutions of the enzyme can be
further purified by adsorption on calcium phosphate (63). 4. Trans-
aminase has an optimum activity at 40° C. and at pH 7.5. The
Michaelis constant with the substrates glutamic and oxalacetic acids
is 0.0138 M. 5. Muscle kochsaft, diphosphopyridine nucleotide, thia-
min, and cocarboxylase are without influence.

V. Euler et al. (27) have stated that the transaminating enzyme
does not require coenzymes or apparently any other cofactor dis-
sociable at a neutral reaction.

Substrate Specificity.— Ex-periments with purified enzyme prepara-
tions (transaminase) showed essentially the same substrate specificity
as with pigeon breast muscle (14). Thus with alpha-ketoglutaric

* Qt = Qtransamiiiation = microlitcrs substratc transaminated per mg. dry
weight per hour.

216 A SYMPOSIUM ON RESPIRATORY ENZYMES

acid only Z(— )-aspartic acid, Z(+) -alanine, and Z( — )-cysteic acid
were active, while with oxalacetic acid only Z( + ) -glutamic and
Z(— )-cysteic acids reacted. A few experiments have been reported
by Braunstein (1) in which glutamic acid formation from alpha -
ketoglutaric acid and diflferent amino acids was measured with
both pigeon breast muscle and purified enzyme preparations. Of
interest are the findings that while Z( + ) -alanine shows a slightly
smaller activity with the pigeon breast muscle, /( + ) -valine, Z( — ) -leu-
cine, and Z( + )-isoleucine react to only about one-third the extent
with purified enzyme. These differences, no doubt, reflect the in-
adequacy of the analytical method employed in these investiga-
tions.

From the studies with purified transaminase (12, 14) it appeared
that the chief substrates for this enzyme are those represented in
reaction 7. Aside from pyruvic acid and /(-{-) -alanine, no monobasic
alpha-amino or alpha-keto acids were found to be active. However,
dibasic alpha-keto and alpha-amino acids other than those shown
in reaction 7 are active. Thus ?(— )-cysteic acid will react with both
oxalacetic and alpha-ketoglutaric acids. Glutathione does not react
with oxalacetic acid.

Mechanism of Transamination

The mechanism by which the intermolecular transfer of the
amino group takes place is not known. Following Herbst's (2) idea
of the mechanism of non-enzymatic transamination, Braunstein and
Kritzmann (6) have postulated the formation of an intermediate
Schiff's base, and picture the reaction as follows:

SCHEME I
R R. R R

HpO.

CO + HpN-C-H -^^^^ C=N-C-H

I I I I

COOH COOH COOH COOH

R R, R R,

H-C-NH2 + CO ^^^^ H-C-N=C

I I II

COOH COOH COOH COOH

TRANSAMINATION

217

The inability of purified transaminase preparations to dehydrogen-
ate glutamic or aspartic acids in the presence of suitable hydrogen
acceptors, and the absence of free ammonia during the course of
the reaction suggest that the amino group is transferred through
an intermediate complex of the SchiflF's base type.

Karrer et al. (28) have investigated the metabolism of octopin,
following the suggestion of Knoop and Martins (29) that octopin
represents a naturally occurring intermediate compound of trans-
amination. Karrer et al. found that fresh Hver brei (which they
state contains an Z-amino acid dehydrogenase) was capable of de-
hydrogenating octopin. d-Amino acid dehydrogenase preparations
were not active, indicating that the alanine was of the Z-form. Since
the biological synthesis of octopin involves an intermediate reduc-
tion of a SchifiF's base to an a, a'-imino dicarboxylic acid, the authors
conclude that transamination may involve intermediate reduction of
the Schiffs base with subsequent dehydrogenation. The reaction is
pictured as follows:

SCHEME II

R R,

I I

H-C-NHp + 0=0

I I

COOH COOH

-^^ H-C— N=C

COOH COOH

+ H;

R R,

I I

C=0 + HpN-C-H

I I

COOH COOH

H-C— N— C-H

I I

COOH COOH

-Hz

R R,

+H2O I I

^-^- C=N— CH

I I

COOH COOH

It has the advantage of providing a mechanism for the explana-
tion of the double bond shift between the nitrogen and the two
alpha carbon atoms.

As previously pointed out, the evidence for the existence of a

218 A SYMPOSIUM ON RESPIRATORY ENZYMES

cofactor for transaminase is still inconclusive. Should an oxidizable
and reducible cofactor prove to be involved, its function might be
that of acting as the hydrogen acceptor and donator in the scheme
of Karrer et al. (Scheme II). However, as has been said, none of the
known cofactors has any influence on transaminase activity, not-
withstanding the fact that the method of its preparation is such
as to remove practically all the known cofactors with the possible
exception of flavinadenine dinucleotide. Of interest in this connec-
tion is the writer's unpublished observation that the activity of
transaminase preparations, fractionated by various methods for pur-
poses of purification, is associated with those fractions showing a
green fluorescence, similar to that obtained with flavoproteins.

Transamination in Different Tissues

As has been said, much of the available data on transamination
is of a qualitative nature. Not only must it be demonstrated that
transamination occurs in a given tissue, but the rate of the reaction
in terms of unit weight of that tissue must be known. Thus a sig-
nificant amount of transamination may be shown to take place in
certain cases with large amounts of tissue and long incubation
periods, but calculation of the rates in terms of Qt would reveal
a value so low as to cast doubt on the significance of this reaction
in the metabolism of that tissue. It is thus essential to have accurate
data on the rate of transamination in different tissues before as-
signing to it a role in intermediary metabolism. Unfortunately very
few such data are available.

Animal Ti55we5.— Transamination in different animal tissues was
first studied by Kritzmann (30). Using the system glutamic acid plus
pyruvic acid, she reported transaminase activity in liver, kidney,
skeletal muscle, heart muscle, and brain, but none in smooth muscle
(chicken gizzard), lung, erythrocytes, and yeast. There was ques-
tionable activity in the case of malignant tissue. Values of Qt
calculated from these data are of the order of 1.5-2.0 for the more
active tissues. Similar studies were carried out on a variety of
tissues by Cohen (31).

A quantitative study of the rate of transamination in different
rat tissues was recently carried out by Cohen and Hekhuis (15). As
can be seen from Table 1, the rates, expressed in terms of Qt, are
very high in most tissues with the substrates glutamic acid plus
oxalacetic acid. The Qt values are higher than the succinoxidase

TRANSAMINATION 219

Q values for the same tissues (32) and in most instances exceed the
cytochrome oxidase Q values (32, 33).

The Qt values with the systems glutamic acid plus pyruvic acid,
and aspartic acid plus pyruvic acid, are comparatively low in all
tissues studied except liver. The higher rates of transamination in
liver are probably due to the fact that pyruvic acid is converted
into oxalacetic and alpha-ketoglutaric acids in this tissue (15, 34,
35, 36).

Table 1.— Values of Qt in difiFerent rat tissues

Glutamic acid

Aspartic acid

Tissue

Oxalacetic acid

PjTuvic

acid

Pyruvic acid

Heart muscle . . ."'

. . 425

Skeletal muscle . .

. . 316

Brain

. , 260

Liver

. . 245

Kidney

. . 245

It is apparent from the data in Table 1 that transamination is
chiefly concerned with the substrates glutamic acid plus oxalacetic
acid. The metabolic importance of this will be discussed later.

Breusch (37) reported some experiments on transamination in
different tissues with the system glutamic acid plus oxalacetic acid.
The following Qt values for various minced cat tissues were cal-
culated from his data: muscle, 19.7; liver, 14.1; kidney, 22.5; lung,
22.5; embryonic muscle, 14.1; brain, 19.7; and washed erythrocytes,
2.8. These values are all much lower than those reported for cat tis-
sues by Cohen and Hekhuis (16). Breusch claims that lung tissue
is an excellent source of transaminase, since it is free of many
dehydrogenase systems. Cohen and Hekhuis, on the contrary (15),
found lung to have a low transaminase activity. The method of
oxalacetic acid determination used by Breusch (4) for measuring
transamination is far from satisfactory and probably accounts for
the results obtained.

Karayagina (38) found that ?(— )-aspartic acid reacts with pyruvic
acid to form alanine in skeletal and cardiac muscles, in liver, kidney,
and brain, but not in testes.

Malignant and Embryonic Tissues.— Euler, Giinther, and Fors-
man (39) and Braunstein and Azarkh (17) reported low transamina-
tion values for tumors. The former workers used a quaUtative ana-

220 A SYMPOSIUM ON RESPIRATORY ENZYMES

lytical method for measuring the disappearance of oxalacetic acid.
The latter workers studied chiefly the reactions, glutamic acid plus
pyruvic acid, and alpha-ketoglutaric acid plus diflFerent amino acids.

Quantitative studies of the rate of transamination in different
tumors and embryonic tissue have been carried out by Cohen and
Hekhuis (16). In their study a series of mouse tumors and cat em-
bryonic tissues were shown to have low rates of transamination as
compared with normal adult tissue. Further, the writer has shown
(63) that the transaminase activity of liver from rats fed dimethyl-
aminoazobenzene decreases progressively to a low value in the
hver tumors arising in these animals. It thus appears that rapid
growth, or increased protein synthesis, is associated with a low
transaminase activity.

Plant Tissues.— Euler et al. (27) reported transaminase activity in
higher plants, but no data were pubhshed in support of this state-
ment. Virtanen and Laine (41) state that transamination between
oxalacetic acid and alanine takes place in crushed pea plants.
Transaminase activity in extracts prepared from pea, lupine, and
pumpkin seedlings has been reported by Kritzmann (24). Cedran-
golo and Carandante (42) studied transamination in leguminous and
graminaceous plants. Dialyzed extracts of seeds and sprouts were
used and were prepared by the same method as that employed by
Adler and Sreenivasaya (43) for the preparation of formico-
dehydrogenase. The systems Z(— )-aspartic acid plus pyruvic acid,
Z(— )-aspartic acid plus alpha-ketoglutaric acid, and alpha-keto-
glutaric acid plus Z( + ) -alanine were investigated. Experimental
data are not presented in this paper, but rather the relative veloci-
ties of the above reactions in graminacae seeds and sprouts as com-
pared with leguminous seeds and sprouts, which are assigned an
arbitrary value of 100. The graminacae extracts are reported to be
1.5 to 2.5 times as active as those from legumes. According to these
authors, the lack of aspartic acid utilization by graminacae, re-
ported by Virtanen, cannot be due to the lack of a transaminating
enzyme. Of interest is the finding of these workers that dialyzed
plant extracts are active. As previously mentioned, Kritzmann (25)
reported that dialysis inactivates transaminating enzymes from
plant sources.

Experiments with Chlorella (63) by the author failed to show
any evidence of transaminase activity with the systems alpha-
ketoglutaric acid plus Z(+) -alanine, and alpha-ketoglutaric acid plus
Z( — ) -aspartic acid.

TRANSAMINATION 221

Wyss (44) observed formation of considerable amounts of aspartic
acid when oxalacetic acid was added to crushed pea nodules. The
amount of aspartic acid found was somewhat greater when alanine
was also added. Whether the aspartic acid formation is due to
transamination or to some other reaction is not certain.

Yeast and Bacteria.— Adler, Giinther, and Everett (45) reported
transaminase activity in yeast extracts. Adler et al. (46) state that
Bacterium coli {Escherichia coli) suspensions form oxalacetic acid
from alpha-ketoglutaric acid and aspartic acid, and that lactic acid
bacilli are capable of transamination, though at a slower rate than
Esch. coli. No experimental data are given in either of the above
papers.

Experiments by the writer (63) showed that Lebedev juice pre-
pared from brewer's yeast was active in catalyzing the reaction
alpha-ketoglutaric acid plus Z(—) -aspartic acid, but not the reaction,
alpha-ketoglutaric acid plus Z(-f )-alanine. Baker's yeast showed no
activity with either system. Experiments with Esch. coli suspensions
(63) demonstrated that no transamination occurred between Z( + )-
glutamic acid and pyruvic acid, but did take place between
Z( + ) -glutamic acid and oxalacetic acid. Qt for the latter reaction
was 17.2.

Transamination in uiuo. —Kritzmann (40) found that intravenous,
intramuscular,- or intraperitoneal injection of glutamic acid into
rabbits, pigeons, and white mice causes the rapid appearance of
alanine in the blood and the tissues. The pyruvic acid necessary
for this reaction is endogenous. The injection of alpha-ketoglutaric
acid plus alanine resulted in the formation of glutamic acid. No
transamination occurs in the blood itself. The failure of blood cells
to catalyze transamination has also been observed by the writer (63).

Influence of Various Substances on Transamination

Inhibitors.— None of the well-known inhibitors has any marked
effect on transamination with the exception of cyanide, which in
high concentrations (0.05 M) causes up to 80 per cent inhibition.
This is in all probability due to the formation of cyanohydrin. How-
ever, at 0.001 M concentration cyanide still causes an inhibition of
about 30 per cent, indicating that an effect on the enzyme system
may occur. Malonate, pyrophosphate, sodium fluoride, iodoacetate,
bromoacetate, arsenious oxide, and octyl alcohol have little or no
influence on transamination in pigeon breast muscle (10).

With transaminase no inhibition of transamination between glu-

222 A SYMPOSIUM ON RESPIRATORY ENZYMES

tamic acid and oxalacetic acid was noted in the presence of malo-
nate, succinate, pyrophosphate, and citrate (14).

In contrast to the above findings with di- and tribasic acids
Braunstein (1) reported that small concentrations of dibasic acids
competitively inhibit "catalytic" transamination. The latter refers
to the catalytic effect of small concentrations of a dibasic alpha-
amino or alpha-keto acid on transamination between a monobasic
alpha-amino acid and a monobasic alpha-keto acid. The author's
attempts to corroborate this finding of Braunstein's have not been
successful (10, 14).

Vysshepan (47) found that the activity of glutamic aminopherase
was inhibited by the following reagents (molar concentrations):
quinone (0.01); potassium cyanide (0.01); glutathione (0.002-0.004);
cations of calcium, barium, and strontium (0.02); mercury and silver
(0.0001). Reagents which are relatively harmless are narcotics, so-
dium fluoride, monoiodo- and monobromo-acetic acids, arsenite,
arsenate and selenite; the anions, chloride, bromide, iodide, acetate,
nitrate, carbonate, sulfate (0.01); ascorbic acid (0.01), hydrogen
sulfide, cysteine, ferrous ion, semicarbazide, phenylhydrazine and
hydroxylamine. Sober and Cohen (64) observed no inhibition of
transaminase by glutathione (0.005 M.).

It is apparent that thus far no specific inhibitor is available for
transaminase. The discovery of such a compound would aid greatly
in elucidating the physiological role of transamination.

JF/ormones.— Transamination with purified transaminase and the
system glutamic acid plus oxalacetic acid is uninfluenced by high
concentration of the following: insulin (crystalline and zinc com-
pounds), desoxycorticosterone, cortical extract, anterior pituitary
extract, estradiol, androsterone, testosterone, and stilbestrol (63).

Carcinogens.— No effect on transaminase activity was observed
with methylcholanthrene and dimethylaminoazobenzene (63).

Vitamins.— Thiamin and cocarboxylase are without influence on
transaminase activity (63). However, a decrease in activity was ob-
served in minced breast muscle from B^ deficient pigeons with the
system glutamic acid plus pyruvic acid (63). Similar findings have
recently been published by Kritzmann (48). Barron (49), on the
other hand, found no decrease in transamination in liver from Bj
deficient rats. These results are not necessarily contradictory, since
Ban'on and his coworkers employed somewhat different experi-
mental conditions (personal communication). Investigations are at

TRANSAMINATION 223

present under way by the writer to determine the influence of other
vitamin deficiencies on this reaction.

Role of Transamination in Intermediary Metabolism

The exact role which transamination plays in intermediary metabo-
lism is still not clear. The substrates of this reaction are highly
reactive and participate in many diflFerent rapid metabolic proc-
esses. Obviously if transamination is to play a significant role in
cellular metabolism it must be shown to proceed at a rate rapid
enough to be quantitatively significant. From Table 1 it is apparent
that truly rapid rates are seen only with the system glutamic acid
plus oxalacetic acid. The rapid rates at which oxalacetic, alpha-
ketoglutaric, and pyruvic acids participate in non-transaminating
reactions make it highly doubtful whether reactions 3 and 6 ever
proceed fast enough to participate in the metabolism of these com-
pounds. This may not apply to liver and pigeon breast muscle.

Among the possible metabolic reactions which transamination may
influence are those of protein and amino acid synthesis and degrada-
tion, glycolysis, and hydrogen transport.

protein and amino ACm SYNTHESIS AND DEGRADATION

Animal Tissues.— An attractive theory of amino acid synthesis and
degradation in plant and animal tissues has been proposed by
Braunstein (1). According to this theory amino acids are synthesized
or degraded by the transamination reaction in conjunction with the
glutamic dehydrogenase system of Euler et al. (27) and Dewan (50).
The latter system serves the two functions of synthesizing glutamic
acid for transamination with alpha-keto acids to yield new amino
acids, and of oxidizing the glutamic acid formed from alpha-
ketoglutaric acid and different amino acids. Both enzyme systems
are present in most tissues, although in varying amounts. Thus
transaminase is higher in muscle than in liver, whereas glutamic
dehydrogenase is higher in liver than in muscle.

Braunstein and Bychkov (51) have reported production of am-
monia from Z( + ) -alanine when the latter is incubated with alpha-
ketoglutaric acid, glutamic aminopherase, glutamic dehydrogenase,
pyocyanine, and cozymase. About 12.5 per cent of the theoretical
yield was realized after three hours' incubation. These workers
pointed out that the above system is a cell-free model of Z-amino
acid dehydrogenase, which to date has not been demonstrated to

224

A SYMPOSIUM ON RESPIRATORY ENZYMES

represent a single enzyme system, as is the case with ci-amino acid
dehydrogenase.

The mechanism of deamination of Z-amino acids in Hver and
kidney can be explained on the basis of the above theory. However,
the failure of the other tissues to oxidize amino acids other than
glutamic acid to any appreciable extent is difficult to explain, since,
as was said above, the two necessary enzyme systems are present in
most tissues. Inasmuch as the theory rests in large measure on
Braunstein's claim that all amino acids are active in transamination,
unequivocal proof for this would seem desirable. As previously
pointed out, experiments by the writer have shown transamination
to be a limited reaction rather than a general one. Further, the
author has found that homogenized liver and kidney, fortified with
cozymase and methylene blue, failed to show any appreciable
yields of ammonia from various combinations of alpha-ketoglutaric
acid and Z- amino acids (63).

A possible metabolic relationship between transamination and
protein synthesis in animal tissues has been pointed out by Linder-
str0m-Lang (52). On the basis of the plausible assumption that the
synthesis of protein occurs by a metabolic mechanism other than
the reversal of proteolysis, he postulates the following scheme:

0 OH

SCHEME III

-H2O

R-C-C-OH + HpNR,

I
H

O OH

li I
^ R-C-C-N-R,

I I
H H

-H2
O OH

NHp O H
\^ II I
R-C-C-N-R,

I
H

R-C-C=N-R,

.Glutamic
, Acid
NH2 OH

I /

R-C-C=N-R,

ex Ketoglutaric
Acid

TRANSAMINATION 225

As can be seen from this scheme, a keto-aldehyde reacts with an
amino acid to form a ketonic SchifiTs base. The latter then reacts
with glutamic acid via the transamination reaction to yield a pep-
tide. Agren (19) has recently published unconvincing evidence in
support of this.

On the basis of the above reaction it would be expected that in
tissues where rapid protein synthesis was taking place, e.g., embry-
onic and tumor tissue, the transaminase activity would be higher
than in normal adult tissues. Actually the reverse has been found
to be the case (16). Thus it was observed that in tumors and em-
bryonic tissue the transaminase activity was low as compared with
normal adult tissues. This apparent inverse relationship between
protein synthesis and transamination suggests that the latter re-
action may serve as a controlling mechanism in protein synthesis.

Plant Tissues.— The possible role of transamination in plant pro-
tein synthesis is suggested from the following scheme of Virtanen
and Laine (41) for leguminous plants:

SCHEME IV

Ng ^ Hydroxylamlne^^

^(Oxime of Oxalaceiic Acid

Carbohydrate — ^Oxalacefic Acidj^ +H2

\ \p

l(-)Aspar+ic Acid

+ a Keto Acid

'Oxalaceiic Acid

+ a Amino Acid

Experimental evidence for the above scheme has been reported
by Virtanen and Laine (41, 53) and has been critically examined
by Wilson (44). However, careful quantitative studies on trans-
amination in plant tissues have not as yet been carried out. Until
this is done the role of transamination in plant tissues will continue
to remain obscure.

OTHER REACTIONS

Transamination and Glycolysis— Krehs (54) and Weil-Malherbe
(55) observed that glycolysis in retina and brain tissue was in-

226 A SYMPOSIUM ON RESPIRATORY ENZYMES

hibited by glutamic acid. This eflFect has been studied in more
detail recently by Grodzensky (56), who was able to show that
anaerobic glycolysis in pigeon breast muscle was also inhibited
(20-50 per cent) by glutamic acid. Further, he was able to demon-
strate that the inhibition was due to the conversion of pyruvic
acid to alanine by transamination of the former with glutamic acid.

The influence of glutamic acid on glycolysis in tumors has been
investigated by the author (63). Inhibitions of the order of 10-15
per cent were observed. These results are in keeping with the finding
that tumors have a low transaminase content (16).

Transamination and Hydrogen Transporf.— Transamination is not
only a very rapid reaction but it is also concerned chiefly with those
substances that are known to play key roles in intermediary metabo-
lism. Thus oxalacetic, alpha-ketoglutaric, glutamic, and aspartic
acids all catalytically influence respiration (3, 4, 57, 58, 59). Further,
glutamic acid has been found to function as a hydrogen carrier not
only because its dehydrogenase can act with both di- and triphos-
phopyridine nucleotides (27, 60) and so can couple with other di-
and triphosphopyridine nucleotide-catalyzed systems (60, 61), but
also because of its role in a dismutation reaction involving alpha-
ketoglutaric acid and ammonia (62).

It would thus appear that the chief role of transamination may be
that of rapidly interconverting certain of the respiratory mediators.
That the transamination reaction is fast enough to compete suc-
cessfully with other metabolic reactions involving the same sub-
strates has been previously indicated.

REFERENCES

1. Braunstein, a. E., Enzymologia, 7, 25 (1939).

2. Herbst, R. M., Symposia on Quantitative Biology, 6, 32 (1938).

3. Needham, D. M., Biochem. J., 24, 208 (1930).

4. Annau, E., Banga, I., Blazso, A., Bruckner, V., Laki, K., Straltb, F. B.,
and Szent-Gyorgyi, A., Z. physiol. Chem., 244, 105 (1936).

5. Banga, I., and Szent-Gyorgyi, A., Z. physiol. Chem., 245, 118 (1937).

6. Braunstein, A. E., and Kritzmann, M. C, Enzymologia, 2, 129 (1937).

7. Braunstein, A. E., and Kritzmann, M. G., Biochimia, U.S.S.R., 3, 603
(1938).

8. Foreman, F. W., Biochem. J., 8, 463 (1914).

9. ZoRN, K., Z. physiol. Chem., 266, 239 (1940).

10. Cohen, P. P., Biochem. J., 33, 1478 ( 1939).

11. Cohen, P. P., Biochem. J., 33, 551 (1940).

12. Macara, T. J. R., and Plimmer, R. H. A., Biochem. J., 34, 1431 ( 1940).

13. Woodward, G. E., Reinhart, F. E., and Dohan, J. S., J. Biol. Chem., 138,
677 (1941).

14. Cohen, P. P., J. Biol. Chem., 136, 565 (1940).

TRANSAMINATION 227

15. Cohen, P. P., and Hekhxhs, G. L., J. Biol. Chem., 140, 711 (1941).

16. Cohen, P. P., and Hekhuis, G. L., Cancer Research, J, 620 (1941).

17. Braunstein, a. E., and Azarkh, R. M., Nature, 144, 669 (1939).

18. V. EuLER, H., Hellstrom, H., Gunther, G., Elliott, L., and Elliott, S.,
Z. physiol. Chem., 259, 201 (1939).

19. Agren, G., Acta Physiol. Scand., 1, 233 (1940).

20. Cohen, P. P., Proc. Am. Soc. Biol. Chem., J. Biol. Chem., 133, xx ( 1940).

21. Braunstein, A. E., and Kritzmann, M. G., Biochimia, U.S.S.R., 4, 168
( 1939). Chem. Abstr. 34, 1694 ( 1940).

22. Bychkov, S. M., Biochimia, U.S.S.R., 4, 189 (1939). Chem. Abstr., 34,
1694 (1940).

23. Kritzmann, M. G., Biochimia, U.S.S.R., 3, 603 (1938).

24. Kritzmann, M. G., Nature, 143, 603 (1939).

25. Kritzmann, M. G., Biochimia, U.S.S.R., 4, 667 (1939). Chem. Abstr., 34,
5865 (1940).

26. Cohen, P. P., J. Biol. Chem., 136, 585 (1940).

27. V. EuLER, H., Adler, E., Gijnther, G., and Das, N. B., Z. physiol. Chem.,
254, 61 (1938).

28. Karrer, p., Koenig, H., and Legler, R., Helv. Chim. Acta., 24, 127
(1940).

29. Knoop, F., and Martius, C., Z. physiol. Chem., 254, I ( 1938).

30. Kritzmann, M. G., Enzvmologia, 5, 44 (1938).

31. Cohen, P. P., Am. J. Physiol., 126, 467 (1939).

32. Elliott, K. A. C, and Grieg, M. E., Biochem. J., 32, 1407 (1938).

33. ScHULZE, M. O., J. Biol. Chem., 129, 727 (1939).

34. Evans, E. A., Jr., and Slotin, L., J. Biol. Chem., 136, 301 (1940).

35. Krebs, H. a., and Eggleston, L. V., Biochem. J., 34, 1383 (1940).

36. Wood, H. G., Werkman, C. H., Hemingway, A., and Nier, A. O., J. Biol.
Chem., 139, 483 (1941).

37. Breusch, F. L., Biochem. J.. 33, 1757 (1939).

38. Karayagina, M. K., Biochimia, U.S.S.R., 4, 168 (1939). Chem. Abstr.,

34, 1694 (1940).

39. V. EuLER, H., GiJNTHER, G., and Forsman, N., Z. f. Krebsforsch., 49, 46
(1939).

40. Kritzmann, M. G., Biochimia, U.S.S.R., 4, 184 (1939). Chem. Abstr., 34,
1694 (1940).

41. ViRTANEN, A. I., and Laine, T., Nature, 141, 748 (1938).

42. Cedrangolo, F., and Carandante, G., Boll. soc. ital. biol. sper., 15, 482
(1940).

43. Adler, E., and Sreenivasaya, M., Z. physiol. Chem., 249, 24 (1937).

44. Wyss, O., quoted by Perry W. Wilson in The Biochemistry of Symbiotic
Nitrogen Fixation ( University of Wisconsin Press, Madison, Wisconsin,
1940), p. 175.

45. Adler, E., GiJNTHER, G., and Everett, J. E., Z. physiol. Chem., 254, 27
(1938).

46. Adler, E., Hellstrom, V., GIjnther, G., and v. Euler, H., Z. physiol.
Chem., 255, 14 (1938).

47. Vysshepan, E. D., Biochimia, U.S.S.R., 5, 271 (1940). Chem. Abstr., 35,
4788 (1941).

48. Kritzmann, M. G., Biochimia, U.S.S.R., 5, 281 (1940). Chem. Abstr.,

35, 4788 (1941).

49. Barron, E. S. G., Ann. Rev. Biochem., JO, 15 (1941).

50. Dewan, J. G., Biochem. J., 32, 1378 (1938).

51. Braunstein, A. E., and Bychkov, S. M., Nature, 144, 751 (1939).

228 A SYMPOSIUM ON RESPIRATORY ENZYMES

52. Linderstr0m-Lang, K., Ann. Rev. Biochem., 7, 37 (1939).

53. ViRTANEN, A. I., and Laine, T., Biochem. J., 33, 412 (1939).

54. Krebs, H. a., Biochem. J., 29, 1951 (1935).

55. Weil-Malherbe, H., Biochem. J., 32, 2257 (1938).

56. Grodzensky, D. E., Bull. biol. med. expd. U.S.S.R., 9, 116 (1940). Chem.
Abstr., 35, 2535 (1941).

57. Krebs, H. A., Biochem. J., 34, 775 (1940).

58. Krebs, H. A., and Eggleston, L. V., Biochem. J., 34, 442 (1940).

59. Baumann, C. a., and Stare, F. J., J. Biol. Chem., 133, 183 (1940).

60. Adler, E., v. Euler, H., GiJNTHER, G., and Plass, M., Biochem. J., 33,
1028 (1939).

61. Dewan, J. G., Biochem. J., 33, 549 (1939).

62. Krebs, H. A., and Cohen, P. P., Biochem. J., 33, 1895 (1939).

63. Cohen, P. P., unpublished studies.

64. Sober, E. K., and Cohen, P. P., unpublished studies.

BuRK AND Elliott
continue the tumor discussion.

LlPMANN AND MeYEHHOF

Whv is the Pasteur effect?

Neuberg and Cori
Who is con\incing whom?

DISCUSSIONS NOT ON THE AGENDA

Discussion on Tumor Respiration

C. A. BAUMANN, University of Wisconsin, Chairman

CHARACTERISTICS OF TUMOR RESPIRATION

K. A. C. ELLIOTT
Institute of the Pennsylvania Hospital, Philadelphia

Having been out of the cancer research field for some time, I am
not in a position to discuss many recent developments. But it may
be useful to start this discussion with a resume of the generahzations
and theories concerning the metabolism of cancer tissue that have
been proposed from time to time. My remarks may appear somewhat
pessimistic, for it does not seem to me that any definite and peculiar
characteristic of tumor metabolism has been proved. However, later
speakers who are familiar with more modern work will perhaps
strike a more optimistic note.

Warburg's First Theory.— As is well known, Warburg and his
school discovered that, whereas slices of a number of normal tissues
produce lactic acid from glucose or glycogen rapidly in the absence
of oxygen (anaerobic glycolysis), cancer tissue slices show an un-
usual ability to continue production of lactic acid from glucose in
the presence of oxygen (aerobic glycolysis). In his early studies
Warburg happened to study tumors which showed very low oxygen
uptake rates. He therefore concluded that in cancer tissue the
respiratory mechanism was impaired and that glycolysis took its
place as a means for producing energy. Later, however, Warburg
observed, and emphasized, the fact that the oxygen uptake rate of
most tumor tissues under good conditions is not usually lower than
that of many normal tissues.

Warburg's Second Theory.— Warburg concluded that since the
respiration rate of cancer tissue might be normal while rapid aerobic
glycolysis continued, there must be something wrong with the
type of respiration in the tumors. Some sort of damage to the respira-
tory mechanism must have occurred in the production of tumor cells
which caused a loss of eflBciency of respiration in suppressing gly-
colysis. High anaerobic glycolysis appeared to be a general property
of growing or multiplying tissues, since it was found in embryo tissue
and testis, but in these normal tissues glycolysis was largely abolished
when respiration occurred, that is, in the presence of oxygen. In

229

^30 A SYMPOSIUM ON RESPIRATORY ENZYMES

the preface to the Enghsh edition of The Metabolism of Tumors ( 1),
which should be consulted for details of Warburg's work on tumors,
Warburg stated that "interference with the respiration in growing
cells is, from the standpoint of the physiology of metabolism, the
cause of tumors. If the respiration of a growing cell is disturbed, as a
rule the cell dies. If it does not die, a tumor cell results. This is no
theory, but a comprehensive summary of all the measurements at
present available."

At present, however, the idea that respiration itself rather than
the presence of oxygen inhibits glycolysis is frequently questioned.
High anaerobic glycolysis is not a property of growing tissue alone,
since it is found, for instance, in adult brain and in glycogen-rich
livers. More important, the high aerobic glycolysis is not found in
tumors alone. Warburg himself showed that mammalian retina
glycolyzes very rapidly, aerobically as well as anaerobically, though
he considered that some sort of damage occurred to this delicate
tissue in preparing it for in vitro work. Gyorgy and co-workers (2)
and Dickens and Weil-Malherbe (3) found high aerobic glycolysis
in kidney medulla, and the latter authors (4) have recently found
the same for jejunal mucous membrane. Testis and several other
non-cancerous tissues are now known to show a moderately high
aerobic glycolysis, and my co-workers and I have found that a
fairly high rate of aerobic glycolysis occurs during the first few
minutes of an experiment with various other normal tissues. Murphy
and Hawkins (5), on the other hand, reported little or no aerobic
glycolysis with some spontaneous tumors occurring in mice. Con-
cerning the normal tissues which show continuous aerobic glycolysis,
that is, an "anaerobic type of metabolism" by which energy is pro-
duced by the anaerobic method even in the presence of oxygen,
Dickens (3) considered that "the cause of an anaerobic type of
metabolism is in all such cases merely a disparity between blood
supply, i.e., oxygen supply, and energy requirements in vivo." Con-
sideration of aerobic glycolysis as opposed to anaerobic glycolysis
does not enter into the following theory of Dickens.

Dickens' Theory.— Dickens and Simer (6) arranged the normal
tissues into three groups. One group, which included kidney cortex
and liver, showed little anaerobic glycolysis and respired with a
rather low respiratory quotient and so apparently metabolized little
carbohydrate. Another group, including brain, retina, chorion, and
embryo tissue, showed high anaerobic glycolysis and a respiratory
quotient of unity in glucose-containing medium. These tissues appar-

DISCUSSION ON TUMOR RESPIRATION 231

ently respired at the expense of carbohydrate, and in the absence
of oxygen the carbohydrate metaboHsm led to lactate accumulation.
A third group, including spleen, testis, and submaxillary gland,
formed an intennediate group. But cancer tissue seemed to consti-
tute a separate class, having a high glycolysis and a low respiratory
quotient. It was concluded that in cancer tissue the mechanism for
starting carbohydrate metabolism is present, even over-developed,
but the mechanism for oxidizing the split products of carbohydrate
is lacking.

However, Dr. Baker and I (7) found specimens of tumors which
respired with respiratory quotient values ranging up to unity and
pointed out that other workers, including Dickens himself, had
found similar high values. On the other hand, we found values
of somewhat less than unity for retina and brain. We concluded
therefore, that Dickens' generalization was not valid.* Dickens
himself, with Weil-Malherbe (4), has now found that normal
jejunal mucosa, a rapidly respiring tissue, has a low respiratory
quotient with high aerobic and anaerobic glycolysis. A similar type
of metabolism was found with synovial membrane by Bywaters
(8). Dickens has therefore abandoned his generahzation, since these
are normal tissues showing the behavior that was believed to be
specific for tumors.

It thus appears that no characteristic of metabolism is truly spe-
cific for cancer tissue. Nevertheless, most cancer tissues show only
a moderately high respiration rate, a moderately low respiratory
quotient, and a definitely high sustained aerobic and anaerobic
glycolysis, whereas few normal tissues show all these characteristics
together. Orr and Stickland (9) very recently found that tumors oc-
curring in the livers of rats fed butter yellow possess the power,
as do most cancer tissues, to form lactate from glucose, anaerobi-
cally and aerobically, and differ distinctly in this respect from non-
cancerous liver tissue. Dr. Dean Burk [see below, page 242] has

* Dickens at first defended his tlieory by attacking our value for the respiratory
quotient of brain sUces, while ignoring the rest and ridicuhng "a complicated
theory of salt eflFects" which did not appear in our paper. Our values for the
respiratory quotient of brain slices were supposed to disagree with otliers in the
literature, but careful study showed that objections could be raised to all the
results cited, except perhaps Dickens' own. Varying results could be due to the
fact that early aerobic glycolysis by brain liberates carbon dioxide from the
medium, and this must be carefully controlled if incorrectly high or low respira-
tory quotient values are to be avoided. Observing this precaution, we ourselves
later obtained slightly higher values ( 18 ) . Our figure for retina has been con-
firmed by Dixon (19).

232 A SYMPOSIUM ON RESPIRATORY ENZYMES

expressed the view that, even as the histology of cancer is suflBciently
characteristic to enable a pathologist to recognize the tissue as cancer
usually, but not always, so also the experienced student of tissue
metabolism could almost always state correctly what is or is not
cancer tissue from results of metabolic measurements on tissue sHces.

Impaired Respiration Mechanisms.— Warburg's idea that the res-
piration of cancer tissue is in some manner deranged may still be
true, and the idea has inspired a number of studies of individual
respiration mechanisms in cancer tissue. Dr. Benoy, Dr. Baker, and I
(10) found the succinic oxidase system inactive in sHces of certain
tumors, and Dr. Greig and 1(11) showed that most tumor suspensions
tested were low in succinic dehydrogenase and the cytochrome-
cytochrome oxidase systems. I (12) found that tumor breis added to
liver or other tissue breis (except heart) rapidly destroyed the suc-
cinic oxidase system of the liver or other tissues. Stotz (13) and
Potter and DuBois (14) found low cytochrome c content in a number
of cancer tissues. Banga (15) found that certain tumors are scarcely
able to reduce added oxalacetate; this and the lack of succinic
dehydrogenase would indicate impaired catalysis by the mechanism
postulated by Szent-Gyorgyi.

However, Dr. Greig and I found some tumors with a fair amount
of succinic dehydrogenase and some normal tissues with little. Some
normal tissues, especially pancreas, and also commercial trypsin,
would inhibit liver succinoxidase; the inhibition might be a purely
in vitro effect and auto-digestion might partly account for low suc-
cinoxidase values found for tumor tissues. Potter and DuBois found
at least one normal tissue (lung) with as low a cytochrome c content
as tumor tissue, and Breusch (16) found that the rate of oxalacetate
reduction was negligible also with some normal tissues, namely
spleen, lung, placenta, and peripheral nerves.

Dr. Baker and I (17) found that the effects of a number of dyes on
the metabolism of tumor tissue were different from their effects on
any of the normal tissues tested. Nothing further has come of these
observations.

Altogether the various results suggest that cancer tissue tends to
differ from normal tissues in its respiratory mechanisms, but no
very well-defined difference has yet been disclosed.

REFERENCES

1. Warburg, O., The Metabolism of Tumors, translated by F. Dickens ( Richard
Smith, Inc., New York, 1930).

2. Gyorgy, p., Keller, W., and Brehme, T., Biochem. Z., 200, 356 (1928).

DISCUSSION ON TUMOR RESPIRATION 233

3. Dickens, F., and Weil-Malherbe, H., Biochem. J., 30, 659 (1936).

4. Dickens, F., and Weil-Malherbe, H., Biochem. J., 35, 7 ( 1941).

5. Murphy, J. B., and Hawkins, J. A., J. Gen. Physiol., 8, 115 (1925).

6. Dickens, F., and Simer, F., Biochem. J., 24, 1301 (1930); 25, 985 (1931).

7. Elliott, K. A. C, and Baker, Z., Biochem. J., 29, 2433 (1935).

8. Bywaters, E. G. L., J. Path. Bact., 44, 247 (1937).

9. Orr, J. W., and Stickland, L. H., Biochem. J., 35, 479 (1941).

10. Elliott, K. A. C., Benoy, M. P., and Baker, Z., Biochem. J., 29, 1937
(1935).

11. Elliott, K. A. C., and Greig, M. E., Biochem. J., 32, 1407 (1938).

12. Elliott, K. A. C., Biochem. J., 34, 1134 (1940).

13. Stotz, E., J. Biol. Chem., 131, 555 ( 1939).

14. Potter, V. R., and DuBois, K. P., J. Biol. Chem., 140, cii (1941).

15. Banga, I., Z. physiol. Chem., 244, 130 (1936).

16. Breusch, F. L., Biochem. J., 33, 1757 (1939).

17. Elliott, K. A. C, and Baker, Z., Biochem. J., 29, 2396 ( 1935).

18. Elliott, K. A. C, Greig, M. E., and Benoy, M. P., Biochem. J., 31, 1003
(1937).

19. Dixon, M., Biochem. J., 31, 924 ( 1937).

PHOSPHORYLATION THEORIES AND TUMOR

METABOLISM

VAN R. POTTER
McArdle Memorial Laboratory, University of Wisconsin

I should like to submit briefly for your consideration a working
hypothesis concerning the metabolism of tumor tissue. This hypothe-
sis is grounded in the Embden-Meyerhof scheme of carbohydrate
breakdown, the Warburg descriptions of tumor metabolism and the
concept of phosphate energy transfer recently put forth by Johnson.
It is difficult if not impossible actually to prove such theories, and
hence one must be satisfied with data that do not prove but are
merely compatible with the given concept. Only after a great mass
of circumstantial evidence has accumulated can we begin to have
confidence in the theory. During the accumulation of these data the
hypothesis is necessarily modified in the light of incompatible data.
It is such incompatible data which probably will soon be brought to
bear upon the hypothesis I am about to present.

According to this hypothesis, tumor tissue uses its adenosine tri-
phosphate (ATP) reservoir for but two main purposes, growth and
glucose phosphorylation, in contrast with most other tissues, which
in addition have function and thus do work and presumably split
ATP in doing it. Since the growth stimulus is ever present in tumor
tissue, inorganic phosphate is released in large enough quantities
to permit rapid glycolysis (in the sense of carbohydrate cleavage),

234 A SYMPOSIUM ON RESPIRATORY ENZYMES

yet the growth does not deplete the ATP to such an extent that
glucose cannot be phosphorylated and hence glycolyzed. In other
tissues glycolysis is slowed down either by a depletion of inorganic
phosphate, as in resting tissue, or by a depletion of ATP, as in dying
tissue. (Liver and kidney under anaerobic conditions deplete ATP
by tending to maintain function and are hence unable to phos-
phorylate glucose; therefore glycolysis stops and death occurs.) In
the tumor tissue the glycolytic rate is so rapid in relation to the
oxidative mechanisms that lactate accumulates. The oxygen uptake
is limited by the amount of the oxidative enzymes present, but
since there is an excess of substrates, the Q02 is higher than might
be expected on the basis of the Qoo of normal tissue, in which the
oxidative enzymes are present in excess and the Q02 is limited by
the amount of substrate furnished by glycolysis. In the tumor tissue
the growth process outpaces the synthesis of the oxidative enzymes,
and the latter become diluted as compared with their concentration
in other active tissues. From this it would follow that growth may
not require as high a level of oxidative enzymes as does function.
Our experimental results are being reported elsewhere. At this
point it may be said that one component of the oxidative mech-
anism, namely, cytochrome c, appears to have been established as
definitely lower in the various types of tumor tissue than in normal
tissues. Assays on the succinoxidase system are at present being
carried out. Preliminary experiments with rapidly growing hver
support the idea that growth outpaces the synthesis of the oxidative
enzymes temporarily in this tissue.

DISCUSSION ON TUMOR RESPIRATION 235

ON THE SPECIFICITY OF GLYCOLYSIS IN MALIGNANT

LIVER TUMORS AS COMPARED WITH HOMOLOGOUS

ADULT OR GROWING LIVER TISSUES*

DEAN BURK

National Cancer Institute, National Institute of Health, U. S. Public Health

Service, and Cornell University Medical College

In this discussion I wish to focus attention upon one particular
aspect of tumor and growth metabohsm that is simple but far-
reaching in implication. It is a problem more of comparative bio-
chemistry than of intermediate metabolism proper, and concerns
the origin of tumor metabolism. The question I wish to pose, and
hope to succeed in answering here, is whether the large glycolysis
of tumors is necessarily an expression and requirement of their
extensive and usually rapid growth. It has been widely held, since
the middle period of Warburg's tumor work (1925), that growing
tissues in general have a high anaerobic (and sometimes aerobic)
glycolytic activity. This very active metabolism has in turn been
attributed to extra and special metabolic requirements of the growth
process. If it could be found that certain growing tissues do not
exhibit marked glycolysis, then it might well be said that the glycol-
ysis of tumors is not necessarily a consequence merely of extensive
growth, but that it has a more specific and characteristic significance
for tumor metabolism than has been recognized or acknowledged.

Before presenting data bearing directly on the foregoing question,
a related aspect of the problem of the origin of tumor glycolysis, and
of suitable criteria for ascertaining significant differences between
normal and tumor metabolism, should be discussed by way of back-
ground (cf. also ref. 17). In recent years Berenblum, Chain, and
Heatley (1) have made the claim that "valid comparisons can only
be made between any particular tumor and the normal tissue from
which it is derived" (la, p. 370). This emphatic assertion, which I
believe it is very desirable to contravert at this early stage in its
possible development, is surely dogmatic and arbitrary to say the
least, for there are many valuable comparisons to be drawn between
tumor materials and adult tissues widely separated from them
embryologically, as well as between tumors and tissues as closely
homologous as possible. I for one would not undertake to say which
type of comparison would, in fact, be the more profitable in the
long run, let alone advocate the exclusion of either one. Certainly

* For much valuable help in the preparation of this manuscript I am greatly
indebted to Miss Juliet M. Spangler, Senior Cancer Aide,

236 A SYMPOSIUM ON RESPIRATORY ENZYMES

both types of comparison, involving non-homologous as well as
homologous contrasts and similarities, must be made.

In furtherance of their position, Berenblum, Chain, and Heatley
advance the view that "the tumors which have hitherto been found
to have a glycolyzing type of metabolism associated with a low
R.Q, possess these properties in virtue of their origin from noiTnal
tissues which also possessed these metabolic characters" (Id, p. 138).
In experimental support of this view they reported, following Crab-
tree's earlier measurements showing that whole skin undergoes little
alteration of metabolism when it becomes papillomatous, that noniial
skin epithelium and Shope papilloma of the domestic rabbit also
possess essentially the same quantitative metabolism in regard to
aerobic and anaerobic glycolysis, respiration and respiratory quo-
tient. Unfortunately these data, although interesting enough in
themselves, have no great bearing on the really pertinent problem
as to the difference (or similarity) between a definitely malignant
tumor and a closely homologous normal tissue; indeed the support-
ing experiments are themselves somewhat unsatisfactory, being diffi-
cult to analyze because of the unorthodox technical method em-
ployed and the fact that Q values were based on nucleic acid-
phosphorus content instead of on dry weight. Certainly there are
advantages in the use of the nucleic acid-phosphorus criterion, but
it is unfortunate that the dry weight values were not at least reported
so that the reader could make Q value comparisons by the standard
methods and check, in particular, the bare and doubtful state-
ment (lb, le) that the normal skin epithelium and Shope papilloma
metabolic values were "very similar to those for many skin carci-
nomas quoted in the literature." (By certain inferences, the anaerobic
glycolysis of the skin epithelium and papilloma studied would appear
to have been at most Q^^a = 1 to 3, or quite low for the usual malig-
nant tumor.) The two criticisms of the unorthodox (however correct)
procedures employed are admittedly minor as compared with the
fact that the Shope papilloma, as such, is not malignant, nor was it
so described.

Table 1, now presented for discussion, provides, in regard to
primary rat liver tumors, not merely one but several types of homol-
ogous tissue, including adult normal liver and two types of growing
liver, regenerating and embryonic. These materials will provide, I
believe, as pertinent cases as are yet available for the comparison of
a malignant tumor with an homologous, in fact identical, tissue of
origin (liver).

DISCUSSION ON TUMOR RESPIRATION 237

The data in Table 1 are the result of the collaboration of many
investigators, as indicated in the footnotes, and have been or are
being detailed elsewhere under respective authorships. They are
brought together here for the purpose of a broad and unified inter-
pretation and discussion at this meeting.

Adult Liver

The azo dye tumors reported upon in the table ofiFer an excellent
opportunity to determine not only whether their metabolism is dif-
ferent from the tissue of origin but also, if it is, to ascertain at what
stage or stages of tumor development the altered metabolism ap-
pears. The hepatomas, adenocarcinomas, metastases therefrom, and
necrotic material thereof, obtained from rats fed butter yellow, and
also the mouse tumor transplant derived originally from o-amino-
azotoluene feeding, all show the high anaerobic glycolysis and
low or intermediate respiratory quotient characteristic of malignant
tumors, and a considerably increased aerobic glycolysis as compared
with either normal rat or mouse liver. The respiration is not changed
significantly. There is a definite but relatively small anaerobic lactic
acid formation in "prc-cancerous," cirrhotic hver as compared with
normal liver; there is likewise a slight but quite definite increase
in "normal" lobes adjacent to tumor-bearing lobes and in livers of
rats protected against tumor formation and extensive liver damage
by butter yellow feeding. The aerobic glycolysis increases rather
abruptly, essentially at the onset of gross tumor formation, and cer-
tainly more abruptly than in the case of the anaerobic glycolysis,
where there is a small, but perfectly definite, several-fold increase
in the pre-cancerous liver as compared with the normal. The results
on tumor in Table 1 in good part confirm and extend the well-known
results of Nakatani, Nakano, and Ohara ( 10), who obtained, in fact,
relatively more pre-cancerous change in anaerobic glycolysis, though
hkewise none in the aerobic. For comparison with absolute and
not relative Q values, it is necessary to reduce the Japanese values
by four- to five-tenths to put them on an initial dry weight basis
corresponding to those in Table 1.

Orr and Stickland ( 13, and previous preliminary communications)
reported, contrary to the results of Nakatani et al. and our own,
that they did not observe any change in the glycolytic metabolism
of liver tissue in the pre-cancerous stage of butter yellow treatment.
For the basis of their comparisons, however, they reported that
their normal hvers yielded anaerobic glycolysis Q values of 2-16

238

A SYMPOSIUM ON RESPIRATORY ENZYMES

(presumably 1-8-12, if corrected to initial dry weights). These
results for normal liver, with the possible exception of some very
early work of Rosenthal ( 14), are practically unique in the literature
of liver metabolism. In our own experiments, Nakatani et ah, and (so
far as I know) essentially all others, anaerobic glycolysis Q values
of more than 2 (initial dry weight basis) have never been consistently
reported. In our experience with many hundreds of normal rat livers
under a great variety of dietary conditions, Q^^a values of 1 or
considerably less were regularly obtained in rats weighing over 50
grams (using the ordinary manometric methods, and with varying
periods of oxygenation between the killing of the rat and the estab-
lishment of anaerobiosis). Without attempting to account at this

Table 1.— A comparison of the metabolism* of various rat liver

tumors with various homologous liver tissues (normal,

embryonic, aged, cirrhotic, regenerating)

Rat

Tissue

Weight
(grams)

Q02

R.Q.

QN^A

M.O.Q

Average Normal Liver (2-24 mos.)

50-350

1.5

6.0

0.70

1.0

-0.3

-11.0

"Butter Yellow" Liverf

75-250

"Normal," yeast protected

1.6

4.9

0.98

2.1

0.3

- 7.7

"Normal" lobe, adjacent to tu-

mor lobe

1.3

7.7

0.82

2.3

0.4

-13.1

Cirrhotic

1.5

6.5

0.82

3.1

0.7

- 9.9

Necrotic tumor

3.9

4.2

0.77

6.0

1.5

- 2.4

(Mouse transplantf)

2.1

4.7

0.66

7.7

1.2

- 1.7

Metastases (to omentum and

mesentery)

2.5

5.0

0.83

8.7

3.7

- 1.3

Adenocarcinoma-hepatoma

3.3

7.3

0.84

10.0

2.8

- 4.6

Hepatoma

6.0

6.4

0.87

12.1

2.9

- 0.7

* Q values based on initial dry weights (original data of Tamiya (16) based on final
dry weights and here factored by 50 per cent to reduce to approximate initial dry
weight; cf. refs 5 and 8c).Qoj,Q'^2AandQ°2^ = inm.' oxygen consumption, anaerobic and
aerobic acid production/mg. initial dry weight of tissue per hr.; R.Q. = respiratory
quotient = Qo2/Qc02; M.O.Q. = Meyerhof oxidation quotient = 3(QN2A.— Q°2a)/Qoj;
tJ = fermentation excess = Q'^'^a — 2Q02.

t Sample of average data taken with O. K. Behrens and K. Sugiura (cf. ref. 4 et seq.).
Up to 30 specimens of each tissue type examined metabolically. Rats fed 0.06 per cent
p-dimethylaminoazobenzene (butter yellow) for 150-200 days on brown rice-carrot
diet. "Yeast-protected" with 5-15 per cent added dried brewer's yeast.

J 33d generation, subcutaneously transplanted, o-aminoazotoluene-induced liver
carcinoma 1 in dba strain. Mouse obtained from Dr. H. B. Andervont through Dr
P. M. West; liver of same animal: QN'a = 0.8; QO2a = 1.0.

DISCUSSION ON TUMOR RESPIRATION
Table 1. — continued

239

Rat

Tissue

Weight

Q°U

Qo2 RQ-

QN^A

M.O.Q. U

(grams)

Regenerating Livers§

Days Per cent regeneration

of unexcised livers

2 180

361

0.8

4.5 0.38

0.6

-0.1

- 8.4

11 280

300

0.5

5.2 0.72

0.7

0.1

- 9.7

10 330

205

0.7

2.3 0.67

0.6

-0.1

- 4.0

1 170

3.6 0.88

0.7

- 6.5

3 280

1.5

6.4 0.64

1.8

0.1

-11.0

Embryonic and Post-embryonic

Liver**

Liver Per cent

Rat age weight body

(grams) weight

Foetal 0.060 7.4

0.80

8.9

Foetal 0.093 8.1

1.09

10.0

Foetal 0.110 8.7

1.26

0.6

6.0 1.00

8.1

1.3

- 3.9

Foetal 0.185 8.7

2.12

0.6

6.6 0.98

8.0

1.2

- 5.2

8 hours 0.240 5.3

4.6

6.4

Iday 0.186 3.7

4.7

2.7

1 day 0.215 3.9

5.5

1.2

6.9 0.66

2.3

0.4

-11.5

3 days 0.262 4.8

5.4

1.7

9 days 0.350 2.5

14.0

1.0

21 days 1.052 3.5

30.0

1.7

5.7 0.52

0.8

0.1

-10.6

Embryonic Chicken Liver (16)

Embryo age Liver weight

5 days 0.048 mg.

9.5

0.093

7.6

0.131

0.7

7.7

5.4

1.8

-10.0

6 days 0.398

5.0

0.580

0.0

6.8

4.0

1.8

- 9.6

7 days 0.720

4.3

0.960

0.0

6.1

4.0

2.0

- 8.2

8 days 1 . 40

3.4

2.25

2.8

(Adult) 8000.00

0.0

7.2

1.4

0.6

-13.0

Chick Bone Marrow Erythro-

blaststt

1.9

4.2 0.84

8.2

4.5

- 0.2

Rabbit Bone Marrow Erythroid

Cells (nucleated) (18)

0.0

9.0

7.0

2.3

-11.0

§ Sample of data taken with J. Blanchard, C. Povolny, J. Norris, and J. Saxton (12).
Rats 10-600 days old (15-400 grams body wt.); regeneration 1-11 days after original
65 per cent hepatectomy (left and median lobes extirpated).

** Data taken with J. Norris (11, 12).

tt Average of data taken with H. Sprince, E. A. Kabat, and J. Furth on chickens
treated with acetylphenylhydrazine to produce hyperplastic (not leukotic) bone mar-
row, with some leukogenic cells but mainly erythroblasts. To be published.

240 A SYMPOSIUM ON RESPIRATORY ENZYMES

time for the exceptional results of Orr and Stickland with normal
hvers, it can very definitely be stated on the basis of our results
and those of Nakatani et ah, that there is a one or more fold increase
in the anaerobic glycolytic metabolism (and likewise glucolytic, for
those interested in this distinction) of liver tissue in the pre-cancerous
stage of butter yellow treatment. That this increase, unless due to
a rather inconceivable difference in rat strains, "must have been due
to chance," as proposed by Orr and Stickland (13, p. 486), is out
of the question, and we prefer the alternate view that the very
unusual magnitude and spread of normal liver values obtained by
Orr and Stickland have served to confuse rather than to clarify
the results they observed with the pre-cancerous livers. A second
and regular point of difference in the pre-cancerous livers observed
by us was that the initial rate of glycolysis was better maintained
over a period of several hours, whereas in the normal livers the
Q^^A values dropped to zero or a few tenths in the course of an
hour or two, and this relative effect was even more striking when
the Q values were based on chemically measured lactic acid rather
than on manometric acid production. It is conceivable, in the absence
of information to the contrary, that the Orr and Stickland determina-
tions on normal liver do not refer to measurements over sustained
periods of time (hours), and that in some way the high normal
values reported by them involve incidental aspects of initial or
preparatory phases of technique, in some measure connected, to be
sure, with the glycogen content of livers, as they demonstrated; I
hesitate to suggest explicitly the trite explanation of extensive damage,
but evidently some factor is operating to give them profoundly
atypical (this is not to say incorrect) values for normal liver that
certainly make comparisons with other kinds of liver material difficult.
Regardless of the foregoing discrepancy of result in regard to
pre-cancerous livers, all investigators agree that lactic acid formation
from glucose by malignant tumors induced by butter yellow is
strikingly different from that of normal liver and definitely or con-
siderably increased over any form of pre-cancerous liver, and that
the same is true in less marked degree with respect to aerobic lactic
acid formation. In general, the anaerobic and aerobic lactic acid
productions by these hepatomas are, on an absolute basis, inter-
mediate between those of most rat, human, and chicken malignant
tumors, on the one hand, studied by Warburg and many others
afterward, and, on the other hand, those of certain mouse tumors
studied originally by Murphy and Hawkins, and by Crabtree and

DISCUSSION ON TUMOR RESPIRATION 241

Cramer and others later. The fermentation excess, U, is in fact not
positive but zero or shghtly negative, and the Meyerhof oxidation
quotient is nearer 3 than 6.

In regard to the low or intermediate respiratory quotient of the
azo dye tumors of Table 1, I might comment, in view of the frank
discussion and expression of personal opinion desired here, that in the
recent discussions in Nature on the metabolism of tumors by Dick-
ens (7), Boyland (2), Berenblum, Chain, and Heatley (lb), and
Dickens and Weil-Malherbe (8b), I agree in general with the com-
ments of Dickens and disagree with the other commentators where
they take exception, for in my judgment they fail to introduce the
proper quantitative perspective. However, I do not feel that Dickens
has been correct, during the past decade, in his view that "cancer
tissue has a respiratory quotient indicating that the oxidation of
carbohydrate is abnormal" (7, p. 512). I prefer to regard the low
or intermediate respiratory quotient exhibited by the majority
of malignant tumors as being unchanged from the similar low or
intermediate respiratory quotient values of the great majority of
normal adult tissues, those, in fact, cited by him over a decade ago.
So why refer to them as "abnormal"? Why not consider them as
simply unaltered? It is the glycolytic capacity, not the respiratory
quotient, of tumors which by and large has changed or is "abnormal"
or different from normal adult tissue; it is in most growing normal
tissues that the respiratory quotient has tended to rise to or attain
unity, and the oxidation of carbohydrate to become relatively more
pronounced, and also the capacity for glycolysis (mainly anaerobic).
General confusion on these matters has led some, including Dr.
Elliott, in his intentionally pessimistic comments this morning, to
suggest, with reference to the very recent paper of Dickens and
Weil-Malherbe (8c) that the high glycolysis and low respiratory
quotient found by them for jejunum mucosa put the metabolism of
this tissue into the class of malignant tumor metabolism; but the
very high absolute Q value for respiration (about equal to the high
anaerobic glycolysis Q value) and the absence of a Pasteur effect
make this designation, in my opinion, quite impossible. Likewise, if
not one or two but a sufficient number of metabolic criteria (absolute
and relative) are considered, it is impossible to agree with the recent
tendency (la, and possibly 7) to regard cartilage (data, 6, 8a) and
synovial membrane (data, 6b) as, like the alleged skin epithehum (1),
providing rather good examples of normal tissues with "mahgnant
tumor metabolism," even after appropriate correction for inert ma-

242 A SYMPOSIUM ON RESPIRATORY ENZYMES

terials in these tissues and, I must add, also in the tumors taken
for comparison! I know of no normal tissue whose metabolism, fully
regarded, need as yet be confused with that of malignant tumors.*
Further background for the foregoing interpretation of quite recent
data is detailed elsewhere (3, 4, 5).

Growing Liver

One type of homologous tissue has been presented, but it might
still be argued that the normal liver, although homologous, was not
a growing tissue and not as comparable with liver tumor as might

* I may reiterate a statement I have already made on many occasions, namely,
that I believe that tlie metabolic diagnosis of malignant tumor as compared with
normal tissue may be correlated with pathologic diagnosis in well over 95 per
cent of tested cases (and, I venture to say, as yet untested cases), upon due
consideration of the absolute as well as the relative magnitudes of, first and fore-
most, anaerobic glycolysis (8-20 ± ) and of respiratory quotient (0.75 - 0.9 ± ) ;
secondly, respiration (2 - 10 ± ), and aerobic glycolysis (0 - 15 ±); and
thirdly the derived quotients, absolute Pasteur effect (8 - 15 ± ), Meyerhof
oxidation quotient (M.O.Q. ) (3-6 ±), fermentation excess (U) ( — 5 to +
25 ± ) etc., (Q values based on initial dry weights); and fourthly quite possibly
the new criterion developed by Salter et al. (15) in regard to separation of
certain tumors from their homologues on the basis of differential oxidation of
glucose and succinate. Non-tumor tissues can be excluded from malignant tumor
tissue designation by one or more of these metabolic criteria; thus, to consider
previously debated cases: for the kidney medulla, too high an R.Q.; cartilage,
synovial membrane, and (presumably) skin epithelium, too low an anaerobic
glycolysis ( Q^^a ) or respiration ( Q02 ) even with reasonable correction for inter-
cellular substance and inert components; retina and jejunal mucosa, too high
a respiration and in tlie latter case also M.O.Q. =: 0 (no Pasteur effect).

The recent discussion, pro or con ( Ic, Id, 7, 2, lb, 8b), and emphasis laid,
on aerobic glycolysis would in my opinion be much better transferred to anaero-
bic glycolysis, which without exception, to my knowledge, is always considerable
in malignant tumors. From my point of view aerobic glycolysis is almost in-
variably merely an expression ( consequence ) of how much anaerobic glycolysis
goes on in relation to how much oxygen consumption is occurring in tlie par-
ticular tissue under examination (3b, 4). In malignant tumors for example, it
can be said that the anaerobic glycolysis values are so high relative to the respira-
tion that the latter is unable to inhibit completely the glycolysis under aerobic
conditions, even with extensive operation of the Pasteur effect ( M.O.Q. = 3 — 6)
( 5 ) ; tlie aerobic glycolysis thus resulting is a quantity dependent upon two rather
independent functions, oxygen consumption and anaerobic glycolysis. I might
add, parenthetically, that most of the aerobic glycolysis values reported as zero
in the literature ( including the often quoted mouse data of Murphy and Hawk-
ins ) are in fact definitely positive due to a methodological error of not correcting
the calculations for the fact that the R.Q. is ordinarily definitely less than unity,
and hence the aerobic glycolysis greater than otherwise calculated. Unfortunately
I cannot go here more deeply into details of elaboration needed to treat adequately
the subjects discussed in this footnote and the two sentences tliat gave rise to it,
but shall do so when the butter yellow tumor data summarized in Table 1 are
described at length.

DISCUSSION ON TUMOR RESPIRATION 243

be desired. Two more types of homologous tissue are presented in
Table 1 by the data on regenerating hver and embryonic hver, both
of which tissues may at certain stages attain growth rates even
greater than that of liver tumor. The data on regenerating liver are
very striking in that they show, as compared with normal adult liver,
no appreciable alteration in any of the metabolic values studied. In
the case of the very young rats, the regenerating Q^^a value is
slightly increased, but mainly as a matter of neonatal age rather
than of regeneration. Regenerating liver is indeed a remarkable case,
demonstrating that tissue growth may take place without appreciable
glycolysis, and at the expense, even, of unchanged oxygen consump-
tion; for the growth increase in liver tissue (on a water-free basis)
may attain 50 to 100 per cent per day during the most active phases
of regeneration at about two to three days after partial hepatectomy,
when mitotic figures are most numerous, several being visible on a
high power field, or even more than would be found with a butter
yellow liver tumor. Orr and Stickland (13) reported that the glycoly-
sis of regenerating liver was not appreciably different from that
of the normal livers they examined, but in these data the issue is
again confused and rendered indefinite by their exceedingly high and
variable normal liver values.

A third type of homologous tissue is the rapidly growing embry-
onic Hver. Tamiya (16) showed over a decade ago that there is a
marked rise in the anaerobic glycolysis of chicken embryo livers
the younger the embryo and the smaller the liver (at least back to a
very early stage or microscopic size). The recent experiments of
J. Langdon Norris (11, 12) give essentially the same results for
embryonic rat livers as for the embryonic chicken livers. Contrary,
however, to opinion held since the work of Tamiya (16) and Hawkins
(9) that embryonic liver in particular and growing tissues in general
show considerable anaerobic glycolysis, the histopathologic sections
taken by Norris (illustrated elsewhere, 11, 12) show that the smaller
the embryonic livers and the greater the anaerobic glycolysis, the
greater and in parallel manner is the extent of haematopoesis, which
in the extreme may amount to an estimated 70 to 80 per cent of the
liver, involving mainly erythropoietic cells (with some megakary-
ocytes and myelogenic cells). The metabolism of these nucleated
erythropoietic cells has not yet been measured directly, but two
comparable types of nucleated erythroid cells are available for com-
parison, namely, the chick bone marrow erythroblasts produced by
acetylphenylhydrazine injections and the normal rabbit bone mar-

244 A SYMPOSIUM ON RESPIRATORY ENZYMES

row erythroid cells. In both these cases the anaerobic glycolysis
Q values are of the same order of magnitude (7-10) as that of the
embryonic Hvers containing a high percentage of red cells, and the
other metabolic values are hkewise comparable. Direct measurement
of the metabolism of the red cells in the embryonic livers offers con-
siderable diflBculty experimentally; moreover, when they are ob-
tained, the measurements might still be somewhat uncertain because
of possible secondary effects of the technical methods employed to
separate them from the liver. But it is felt that in the light of the
two quite comparable cases offered and a considerable background
of knowledge regarding the metabolism of blood cells generally,
there is no reason to doubt that the rather high glycolytic metabohsm
of embryonic hvers is due to the erythropoietic element and not to
the true liver cells. In other words, neither the embryonic liver per
se, nor the regenerating Hver, nor adult normal liver, nor in fact any
healthy liver, growing or otherwise, possesses a noteworthy glycolysis,
in contrast with the various malignant hepatomas, where a large
glycolytic capacity obtains. The case of Berenblum, Chain, and
Heatley ( lb) that "when a tumour is compared with the tissue from
which it is derived, there are no metabolic characteristic differences
or pecuHarities between the carbohydrate metabolism of the two" is
clearly not valid; nor, for lack of evidence, is the more general
contention quoted earlier that tumors glycolyze by virtue of their
origin from normal tissues which also possess this metabohc charac-
ter* (Id, p. 138).

The question whether the increased formation of lactic acid in
the hepatic tumors— or in tumors generally— is necessarily an expres-
sion or requirement of the growth involved, as is commonly believed,
is thus answered in the negative by the experiments briefly described
in Table 1. It may be concluded, more generally, that growth does
not necessarily require glycolysis, and may on occasion be main-
tained at the highest levels on an essentially aerobic non-fermenta-
tive metabolism.

If it should be asked why malignant tumors possess glycolysis
if not because of growth, I would venture the opinion that the
glycolysis is better correlated with the more primitive organization
or lesser differentiation involved.

Most of the foregoing discussion has been concerned with com-

* I do agree with Berenblum et al. — and not with Dickens, as aheady indi-
cated— tliat the medium-low respiratory quotient of tumors may well be derived
from the tissues of origin, in the sense that it remains by and large unchanged
in tumors.

DISCUSSION ON TUMOR RESPIRATION 245

parative biochemistry, not intermediate metabolism. The studies
made in recent years by Elhott and by Potter, and especially this
year by Salter and collaborators (15), on the deficient (cytochrome-
succinate) oxidation systems in many tumors as compared with
tissues of varying degrees of homologousness, will undoubtedly lead
the way in indicating by what mechanisms, if any, the respiration
systems of tumors permit high glycolysis to occur. But that is a
story for the future. The paper of Mr. Kensler now to follow, based
on his study with Dr. C. P. Rhoads (9a) on the action of butter
yellow intermediates (free radicals) in correlating metaboHsm and
carcinogenic action, will present an exceedingly promising pioneer
work that opens up an entirely new approach to the connection be-
tween tumor metabolism and tumor genesis, an approach that will,
I believe, command the admiration of us all.

REFERENCES

1. Berenblum, I., Chain, E., and Heatley, N. G., (a) Amer. J. Cancer, S8,
367 (1940); (b) Nature, 145, 778 (1940); (c) Ann. Rep. Brit. Emp. Can-
cer Campaign, 16, 215 (1939); (d) ibid., 17, 135 (1940); (e) Abstr. 3d
Inter. Cancer Congress, Atlantic City, p. 127 ( 1939).

2. BoYLAND, E., Nature, 145, 512 (1940).

3. BuRK, Dean, (a) Occas. Publ. Amer. Assoc. Adv. Sci., No. 4, 121 (1937);
(b) Cold Spring Harbor Symposia on Quantitative Biology, 7, 420 (1939).

4. BuRK, Dean, Behrens, O. K., and Sugiura, K., Cancer Res., 1, 733 (1941)
et seq.

5. Burk, Dean, Springe, H., Spangler, J. M., Kabat, A. E., Furth, J.,
and Claude, A., J. National Cancer Institute, 2, 201 (1941).

6. Bywaters, E. G. L., (1) Nature, 138, 30 (1936); (b) J. Path. Bact, 44,
247 (1937).

7. Dickens, F., Nature, 145, 512 (1940).

8. Digkens, F., and Weil-Malherbe, H., (a) Nature, 138, 125 (1936); (b)
ibid., 145, 779 ( 1940); and (c) Biochem. J., 35, 7 ( 1941 ).

9. Hawkins, J. A., J. Gen. Physiol., 9, 111 ( 1926).

9a. Kensler, C. J., Dexter, S. O., and Rhoads, C. P., Cancer Res. 2, 1 (1942).

10. Nakatani, M., Nakano, K., and Ohara, Y., Gann, 32, 240 (1938).

11. Norris, J. Langdon, "Metabolism of rat livers, with particular reference to
embryonic liver and malignancy," Polk Prize Paper, Cornell University
Medical College, Sept., 1941.

12. Norris, J. L., Blanchard, J., and Povolny, C, "Regeneration of rat liver
at different ages and metabolism of embryonic neonatal and regenerating
rat liver," Amer. J. Path., Opie Dedication Number (in press).

13. Orr, J. W., and Stickland, L. H., Biochem. J., 35, 479 ( 1941).

14. Rosenthal, O., Biochem. Z., 207, 263 (1929).

15. Salter, W. T., Craig, F. N., and Bassett, A. M., Cancer Res., 1, 751
(1941); cf. 1, 869 (1941).

16. Tamiya, C, Biochem. Z., 189, 175 ( 1927).

17. Voegtlin, Carl, Physiol. Rev., 17, 92 (1937).

18. Warren, C, Amer. J. Physiol., 131, 176 (1940).

246 A SYMPOSIUM ON RESPIRATORY ENZYMES

THE EFFECTS OF CERTAIN DIAMINES ON ENZYME SYS-
TEMS, CORRELATED WITH THE CARCINOGENICITY
OF THE PARENT AZO DYES

C. J. KENSLER
Memorial Hospital, New York

In 1935 Hashimoto (1) reported the isolation of acetyl-2-methyl-p-
phenylenediamine from the urine of rats fed the carcinogen, o-
aminoazotoluene. Another azo carcinogen, dimethylaminoazoben-
zene (butter yellow), the metabolism of which has been studied by
Stevenson, Dobriner, and Rhoads (2), has also been found to be
split in vivo at the azo linkage. The urine of animals fed butter
yellow contained aminophenol and p-phenylenediamine. The free
and acetylated forms of both compounds were found. No dimethyl-p-
phenylenediamine was isolated, but it may be assumed until evi-
dence to the contrary is presented that it is a precursor of the
excreted p-phenylenediamine. Orthoamidoazotoluene and dimethyl-
aminoazobenzene are the only carcinogenic azo compounds whose
metabolic breakdown has been studied.

A previous report (3) from this laboratory presented evidence that
the concentration of diphosphopyridine nucleotide (Coenzyme I)
in the livers of rats fed dimethylaminoazobenzene is 60 per cent
less than in the livers of rats fed the same basal diet without the
carcinogen. This fact suggested that the administered dimethyl-
aminoazobenzene, or some metabolic breakdown product of it, might
depress in vitro the activity of a fermenting system from yeast in
which diphosphopyridine nucleotide is the limiting factor. Experi-
ment proved the validity of this suggestion. The methods and the
results obtained are presented in detail in a report which will appear
shortly (4).

In brief, it was found that whereas the original carcinogen, butter
yellow, did not inhibit fermentation at all, the isolated derivative
of butter yellow, p-phenylenediamine, was strongly inhibitory, and
dimethyl-p-phenylenediamine, the supposed precursor of the simpler
compound, had an even more powerful toxic eflFect.

In view of the results obtained with butter yellow and its break-
down products, it seemed desirable to test on the same feraienting
system the eflFect of chemically related substances. Dr. Leonor
Michaelis, who had previously prepared a large number of methyl
derivatives of p-phenylenediamine as a part of his general study
of two-step oxidations, generously provided samples of these com-
pounds for use in this investigation. Their inhibitory eflPects on the

DISCUSSION ON TUMOR RESPIRATION 247

rate of fermentation in the yeast system were measured. In Table 1
are presented the results of tests of only those substances of which
the carcinogenic potency of the primary azo compound for rat
livers has been tested. However, in further tests of the inhibitory
effect on the yeast system of seventeen of the methyl derivatives of
p-phenylenediamine, as well as of those hsted, the inhibition was
found to correlate closely with the stabihty of the semiquinone
intermediary oxidation product as reported by Michaelis, Schubert,
and Granick (5).

In the experiments with the yeast system it was noted, further-
more, that those compounds which were toxic and which formed
stable free radicals (semiquinones) were oxidized readily by the
yeast apozymase used. The substances which were shown by
Michaelis and his associates not to form stable free radicals were
not so oxidized. This suggested, therefore, that the stable free radi-
cal or some further oxidation product of it was responsible for the
inhibition observed.

Among the possible end products of the oxidation that might be
responsible for the inhibition are quinone, methylamine, dimethyl-
amine, and formaldehyde. Of these compounds only quinone was
found to have significant toxicity. This compound, however, was less
than half as toxic as the diamine in equivalent concentrations. Hydro-
gen peroxide, which can be formed under certain conditions of
oxidation, was also proved to be non-toxic. The acetylated p-phenyl-
enediamine, which is quite stable and is a metaboHte of dimethyl-
aminoazobenzene, was found to be non-toxic.

This inhibition of the diphosphopyridine nucleotide system by
the p-aromatic diamines made it desirable to test another yeast
enzyme system. Similar results were obtained when a carboxylase-
cocarboxylase system was employed, except for one difference. The
addition of reducing agents such as cysteine, glutathione, and as-
corbic acid reduced markedly the toxicity of the p-aromatic diamines
for the diphosphopyridine nucleotide system. In the carboxylase-
cocarboxylase system, on the other hand, to which the same sub-
stances are inhibitory and where there is no complicating catalytic
oxidation of the inhibiting agent, the addition of the reducing sub-
stances prevents any inhibition at all.

In experiments with both the diphosphopyridine nucleotide sys-
tem and the cocarboxylase system, where the inhibition by the toxic
compounds was large in the presence of low coenzyme concentra-
tions, it was found that the addition of large amounts of coenzyme

Table 1*

Parent molecule
X=CH3

Carcino-
genic

potency

(rat

liver)

Ref.

Split product
X=CH3

■N=N

•N<

4,5'-Dimethyl-iV-iV-diinetliylamino-
azobenzene

H2N

N<^

-N=N-
Aminoazobenzene

-NH,

HoN

NH2

X X

2,3'-Dimethylaminoazobenzene

NH2

H2N

NH2

N=N'

■N<

4,6'-Dimetliyl-iV-A^-dimethylamino-
azobenzene

H2N

■N<

•N-:N-

iV-A^-dimethylaminoazobenzene

H2N

•N<

+ +

4-Methyl-A^-iV-dimethylaminoazo-
benzene

H2N-

CH3OC

N-

* The table on these facing pages presents the results of tests showing the
apparent correlation between the carcinogenic properties of the parent azo com-
pounds and the toxic properties of the p-aromatic diamine split product of these
molecules.

248

Stability

free

radical

of split

product

Oxidation by
cytochrome-
oxidase
system*
cmm. O2 per
10 minutes

Percentage Inhibition of Enzyme Systems

diphospho-

pyridine
nucleotide t

cocarboxy-
lasef

respirationj

oxygen
consump-
tion**

5 minutes

4-8 hours

160

4-8 hours

164

2 days

176

7 days

165

7 days

165

very
unstable

* M/50 concentration used. No inhibition of the cytochrome-oxidase system with
M/50 p-phenylenediamine results with any of the split products at concentrations of
5X10-*M.

t System: washed yeast; compounds at a concentration of 5X10"'* M.

t System: rat liver slices, 5-hour experiment; compounds at a concentration of
1X10-3 M.

** System: rat liver brei, 2-hoiu- experiment; compounds at a concentration of
5X10-«M.

249

250 A SYMPOSIUM ON RESPIRATORY ENZYMES

(500-1000 micrograms) prevented the inhibition. But when large
amounts of coenzyme were added, the inhibition was prevented
only in the system in which it was the limiting factor; that is, the
addition of large amounts of diphosphopyridine nucleotide main-
tained activity only in the yeast-fermenting system and did not
reduce the toxicity of the diamino compounds in the cocarboxylase
system; and, conversely, the addition of large amounts of cocar-
boxylase maintained activity only in the carboxylase system. These
experiments indicate that the inhibition in both systems is a com-
petitive one and further suggest that the action of the diamino
compounds is on the protein enzyme component rather than on
the coenzyme.

Rat liver cell (slice) and rat hver suspension (brei) oxidations are
also inhibited by the p-aromatic diamines. The toxicity gradient
of these compounds to surviving liver tissue is similar to that ob-
served in the yeast systems. To detect the protection of these rat
liver systems by the addition of reducing agents is not practicable,
for it was observed that the presence of these diamino compounds
in the crude liver enzyme systems catalyzed the oxidation of the re-
ducing agents, cysteine and ascorbic acid. Those diamino compounds
that are most toxic act as stronger catalysts of the oxidation of the re-
ducing agents than do the less toxic compounds in the presence of a
liver suspension. It was important to establish the fact that the
p-aromatic diamines are not simply non-specific enzyme poisons.
The (Z-amino acid oxidase, tyrosinase, cytochrome oxidase, and acid
and alkaline phosphatase enzymes were not inhibited by dimethyl-
p-phenylenediamine in equivalent concentrations (5 X 10"^ molar).

In these experiments (see Table 1) the gradations of the toxicity
of the diamino split products of methyl derivatives of aminoazo-
benzene parallel the carcinogenic potency of the parent molecules.
But inasmuch as the list of compounds of this series which have been
tested for carcinogenic power is small, further animal experiments
with other related compounds are needed to determine whether this
apparent correlation is a true one. Furthermore, the evidence that
the production of p-aromatic diamino split products from the parent
azo molecule in the liver of the rat is concerned in the resulting
production of hepatic cancer is entirely indirect.

Although the mechanism of the inhibition of the protein enz)Tnes
in these several systems by the p-aromatic diamine is obscure, it
appears that the formation of an oxidation product or products of
the reduced compounds is essential to secure this effect. The indirect

DISCUSSION ON TUMOR RESPIRATION 251

evidence available suggests, in addition, that the inhibition may be
produced by oxidation of, or combination with, sulfhydryl groups on
the protein enzyme. For example, in the diphosphopyridine nucleo-
tide system iodoacetate and alloxan, which are known to react with
sulfhydryl groups, also inhibit fermentation. The alloxan inhibition
is, like that caused by the p-aromatic diamines, a competitive one.
Vassel (6) has found that dimethyl-p-phenylenediamine will con-
dense with cysteine in strongly acid solutions in the presence of
Fe+++ and ZnCL to yield a colored (blue) product. In several
experiments done in our laboratory we found that in the presence
of a liver suspension (brei) an orange product is formed when both
dimethyl-p-phenylenediamine and cysteine are added. This product
is not formed on the addition of either alone.

The work of White (7) has shown that organic sulfur (cystine,
methionine) can counteract the growth-inhibiting properties of both
the azo and hydrocarbon carcinogens and supports the view that
the carcinogen may combine with the sulfhydryl groups of proteins.
Further evidence is provided by the studies of L. F. Fieser and
his collaborators (8). These concern experiments with the hydro-
carbon carcinogens in which an entirely different approach was fol-
lowed. The results suggest that the action of the hydrocarbon carcin-
ogens may be on an S— S link of a protein and serve to emphasize
the need for further information on the mode of action of the
p-aromatic diamines in catalytically active systems.

REFERENCES

1. Hashimoto, Y., Gann, 29, 306 (1935).

2. Stevenson, E. S., Dobriner, K., and Rhoads, C. P., in press.

3. Kensler, C. J., SuGiuRA, K., and Rhoads, C. P., Science, 91, 623 (1940).

4. Kensler, C. J., and Rhoads, C. P., Journal of Cancer Research, 2, 1
(1942).

5. Michaelis, L., Schubert, M. P., and Granick, S., J. Amer. Chem. Soc, 61,
1981 (1939).

6. Vassel, R., Jour. Biol. Chem., 140, 323 (1941).

7. White, J., J. Natl. Cancer Inst., 1, 337 (1940).

8. Fieser, L. F., Production of Cancer by Folynuclear Hydrocarbons ( Univer-
sity of Pennsylvania Press).

9. Nagao, N., Gann, S5, 20 ( 1941 ) .

10. Sasaki, R., and Yosida, T., Virchow's Archiv., 295, 175 ( 1935).

11. Yosida, T., Virchow's Archiv., 283, 29 (1932).

12. KiNosiTA, R., Trans. Soc. Path. Jap., 27, 665 (1937).

Discussion on Bacterial Respiration

W. H. PETERSON, University of Wisconsin, Chairman

CRITERIA FOR EXPERIMENTS WITH ISOTOPES
H. G. Wood, Iowa State College:

The criteria that are to be used in considering the rehabihty of
isotopic work will vary considerably with the type of investigation.
For example, in studies with carbon of atomic weight 13 in which
one wishes to determine qualitatively whether or not carbon dioxide
is fixed in a biological reaction, the criteria are relatively simple.
The reaction is simply conducted in an atmosphere containing car-
bon dioxide in which the content of C^^ has been increased artifi-
cially above that of carbon found in nature. Carbon in nature
contains about 1.1 per cent C^^. The carbon dioxide used for a tracer
usually contains from 5 to 15 per cent C^^, the concentration
depending on the method used to obtain the heavy isotope. To
determine whether or not carbon dioxide is incorporated into an
organic compound, it is necessary only to free the reaction mixture
of carbon dioxide, convert the organic compounds to carbon dioxide,
and determine its content of C^^ in a mass spectrometer. If the C^^
content of the reaction mixture is significantly above 1.1 per cent
(the natural complement of C^^), carbon dioxide has been incor-
porated into organic compounds. This is obviously the case, since
there is no other source of carbon than carbon dioxide with a content
of C^^ above the normal.

The question is, what reliable conclusion can be drawn from such
an experiment? Clearly, the only conclusion that can be made is
that carbon dioxide is fixed, but no idea is given as to the reaction
involved or the compounds concerned. In the early isotopic work
such experiments were given undue significance. For example, this
type of experiment was conducted by Ruben and Kamen with pigeon
liver, Escherichia coli, and a number of other heterotrophic systems
and it was found that a small amount of radioactive carbon dioxide
was fixed. These experiments have been cited as examples of assimila-
tion of carbon dioxide by heterotrophic organisms. There is no general
agreement on a definition of assimilation, but it is certain that to

252

DISCUSSION ON BACTERIAL RESPIRATION 253

many the term implies construction of cell material, enzymes, and
perhaps organic compounds involving a carbon chain. Clearly this
simple demonstration of carbon dioxide fixation is not a reliable cri-
terion of assimilation so defined. Obviously the carbon dioxide could
have been fixed in the simple one-carbon compound urea or formic
acid, a fixation far different from that which the term "assimilation
of carbon dioxide" suggests to most people.

Assuming that one has completed a simple demonstration of fixa-
tion of carbon dioxide in a biological process, what additional criteria
must be met then to determine the mechanism of fixation? It is here
that the real problems of isotopic work are met, and the problems
are by no means simple. Isotopes are a very valuable tool to the
investigator, but even isotopes involve many uncertainties. Frankly,
the field of isotopic investigation is not fully enough developed to
permit a clear understanding of all the criteria that must be met.
Therefore, in this short presentation only a few of the possible
sources of error will be presented as a starting point for discussion.

In any investigation, whether it involves isotopes or not, the gen-
eral experimental procedure must be reliable if results are to be
valid. In fact, assuming that one is cooperating with a competent
physicist, the actual isotopic analysis and separation of isotopes will
be the minor problem; the general experimental procedure will offer
the real problems. A specific experiment may be cited as an example
of a case in which the error resulted from the general procedure and
not from any isotopic considerations. In the propionic acid fermen-
tation, propionic acid is formed in which carbon dioxide-carbon is
incorporated. To obtain information on the possible mechanism of
the reactions concerned in the fixation of carbon dioxide in propionic
acid, it was necessaiy to determine the location of the fixed carbon
in the propionic acid molecule. Thus the problem was to select a
reliable chemical reaction for the degradation of the molecule. In
doing this Carson and his co-workers degraded radioactive propionic
acid obtained from the propionic acid fermentation by alkaline
permanganate oxidation and obtained oxalic acid and carbon diox-
ide:

CH3CH2COOH -> COOHCOOH + CO2

From 70 to 75 per cent of the radioactive carbon was found in the
oxalate fraction and 25 per cent in the carbonate. Since the workers
believed that the carbonate arose from the carboxyl carbon and the
oxalate from the alpha and beta carbons of propionic acid, it ap-

254 A SYMPOSIUM ON RESPIRATORY ENZYMES

peared that the carbon dioxide was equally distributed among the
three carbons in the chain. In other words, it seeemed that the pro-
pionic acid might be synthesized entirely from carbon dioxide, which
would be a most remarkable accomplishment for a typically hetero-
trophic propionic acid organism. It was soon proved, however, that
the reaction was not a reliable method of decarboxylating propionic
acid. This was done by checking the reaction with synthetic pro-
pionic acid, which contained C^^ in the carboxyl group. Further, by
use of a rehable degradation reaction, all the fixed carbon in the
biologically formed acid was shown to be in the carboxyl group.
Carson and his co-workers obtained the same results when they re-
checked their previous procedures.

With this example we may pass on to consideration of the case, in
which it will be assumed the criteria described above have been
met, i.e., ( 1) a reliable determination of C^^ has shown there is fixa-
tion of carbon dioxide; (2) the experimental procedure has been a
good one for demonstrating the desired results; and (3) the com-
pound or compounds containing the fixed carbon have been isolated
and degraded by reliable chemical reactions; thus the location of the
fixed carbon in the respective compounds is known.

It is true that only by the use of isotopic carbon could such in-
formation be obtained, but even then there may be much uncer-
tainty respecting the mechanism of fixation of carbon dioxide. For
example, in bacterial glucose fermentations by Staphylococcus,
Streptococcus, and Proteus, it has been shown by Slade et al. that the
carbon dioxide is fixed in the carboxyl groups of the lactate and suc-
cinate; with Aerohacter and Clostridium welchii, there is fixation in
the carboxyl group of the acetic acid as well. With pigeon liver on
pyruvate, there is fixation in the carboxyl groups of malate, fumarate,
succinate, lactate, and alpha-ketoglutarate. Many of these fixations
are believed to occur initially by three- and one-carbon addition
through the following reaction:

CO2 -1- CH3 • CO • COOH = COOH • CH2 • CO • COOH

Particularly it is believed that this reaction is instrumental in the
formation of four-carbon dicarboxylic acids. The mechanism of the
fixation in lactate and acetate is still largely unknown. But what
criteria can be used in determining the reliability of the suggested
fixation by three- and one-carbon addition? At present there is no
completely reliable criteria; but this represents our interpretation of
the present known experimental facts.

Thus far the synthesis of oxalacetate from pyruvic acid and carbon

DISCUSSION ON BACTERIAL RESPIRATION 255

dioxide has not been accomplished, but Krampitz in our laboratories
has done the next best thing. With a preparation of Micrococcus,
which decarboxylates oxalacetate to pyruvate, he has shown the
catalysis of the exchange of C^'^-carbon dioxide with the carboxyl
group of oxalacetate, i.e., incubation of oxalacetate with C^^-carbon
dioxide and the enzyme gave oxalacetate containing C^^ in the
carboxyl group. During this exchange the oxalacetate is apparently
broken down to a three-carbon compound and again resynthesized,
permitting the entrance of C^ ^-carbon dioxide. It is suggested that
the three-carbon compound involved in the fixation reaction is not
pyruvic acid, as such, but a derivative of this compound that is
foraied during the decarboxylation of oxalacetate. This is the first
direct evidence, i.e., evidence with oxalacetate as such, that has been
obtained as proof of the reaction.

Finally, I should like to comment on one further consideration.
Assuming that we know the mechanism of the reactions concerned
in fixation of carbon dioxide, we are still faced with one very impor-
tant question, namely, is the fixation reaction an essential step in the
dissimilation or is it the result of an exchange reaction, for example?
Time does not permit full development of this subject. Furthermore,
since the problem has not yet been given much consideration in most
investigations, satisfactory criteria have not been devised for de-
termining whether a reaction is essential or not. An example may
serve to illustrate the point. In the Krebs cycle, as Dr. Evans pointed
out in his lecture, the pyruvate is believed to be oxidized after
union with oxalacetate through a cyclic conversion. The oxalacetate,
it is believed, arises from the fixation reaction through union of pyru-
vate and carbon dioxide. The fixation reaction would then be an
essential reaction in the oxidation of pyruvate, since it would supply
the necessary oxalacetate. It is possible, however, that the oxalacetate
cannot be formed by this reaction but is formed by some other
reaction of pyruvate. The oxalacetate thus formed might react with
carbon dioxide by an exchange reaction as studied by Krampitz. In
this case one would arrive at oxalacetate containing heavy carbon
just as he would if the oxalacetate was formed by union of pyruvate
and carbon dioxide. Isotopic analysis would not serve to differentiate
the mechanisms. It is evident that a false importance may be ascribed
to carbon dioxide fixation reactions, for fixed carbon dioxide may
result from a non-essential exchange reaction. Frankly, I do not
believe this to be the case, but I can cite only indirect criteria to
support my opinion, such as the fact that carbon dioxide is necessary
for growth of bacteria and in the reduction of methylene blue by

256 A SYMPOSIUM ON RESPIRATORY ENZYMES

dehydrogenase. If carbon dioxide was concerned only in a non-essen-
tial exchange, it would not be required.

R. H. BuRRis, Columbia University:

The criteria that must be met in the use of isotopes as tracers have
been well covered by Dr. Wood in the preceding discussion, so we
may consider certain other problems connected with isotopic tracers.
The question is frequently heard, "Which tracers are more suitable
for biological problems, radioactive or stable isotopes?" There is no
blanket answer, for the suitability of a given tracer depends not only
upon the properties and availability of the given element, but also
upon the type of experiment in which the tracer is to be employed.

In quahtative studies the radioactive isotopes of reasonably long
half -life possess many advantages; they can usually be detected in
greater dilution and with greater facility than can the stable isotopes.
In addition, they may often be traced directly in vivo without la-
borious fractionation of the organism. For example, shortly after
the ingestion of a radioactive sodium salt radioactivity may be de-
tected in any part of the body if it is brought into proximity to a
Geiger counter chamber. Radioactive substances with sufficiently in-
tense radiations will reveal their distribution by forming radiographs
on photographic plates. In this manner the pattern of radioactive
substances in bones has been demonstrated following the feeding of
isotopes.

Radioactive isotopes have been prepared in much greater variety
than have the stable isotopes. More than two hundred radioactive
isotopes have been produced. Many, of course, are of no biological
significance; others have not been prepared in suitably high con-
centrations for tracer studies; and unfortunately some of the ele-
ments of greatest biological interest form isotopes of very short half-
life. The following radioactive elements have been used in biological
investigations:

Isotope Half-life Isotope Half-life

,W 85 days i,CP« 37 minutes

eC^i 20.5 minutes j^K" 12.4 hours

eC^^ 100-1000 years 2oCa*^ 2.5 hours

tN^^ 10.5 minutes scFe^^ 47 days

9F18 112 minutes 3,Br8o 44 j^q^j-s

nNa^* 14.8 hours g.Br^^ 34 j^q^^s

15P'' 14.3 days ggP^s 25 minutes

leS^^ 88 days

DISCUSSION ON BACTERIAL RESPIRATION 257

The chief feature in favor of the stable isotopes is, of course, their
stabihty. The extreme speed necessary in handhng radioactive iso-
topes of short half-hfe, such as C" and W^, has led to some un-
fortunate errors, as has been pointed out by Dr. Wood. With the
stable isotopes time is not a factor, and compounds can be separated
and analyzed at leisure. When isotopes become available for general
distribution, the stable forms may be stocked vi^ithout decomposition.

The objection that radiations from radioactive tracers may injure
tissues is scarcely a serious one, since in most cases there is a wide
margin between the level of radioactivity necessary for measurement
and the level that will be injurious. With the stable tracers the ques-
tion of radiation does not arise. Only in the case of deuterium, with
its marked differences in properties from its analogue, have injury
eflFects been noted when high concentrations of a stable isotope were
present.

Although the stable isotopes have not been prepared in as great
variety as the radioactive isotopes, the elements of chief interest to
the biologist have been concentrated. H- (deuterium), C^^, N^^, O^^,
and S^* have been concentrated by Urey. Deuterium is an item of
commerce, and N^^ and C" may be on the market soon.

For quantitative experimentation the stable isotopes are far su-
perior to the radioactive isotopes. Rittenberg has found that in the
analysis of N^^ with the mass spectrometer he can expect a precision
of ± .003 atom per cent N^^. To obtain comparable precision in meas-
uring a radioactive substance with a Geiger counter it would be
necessary to count in the order of a million impulses. If a Geiger
counter capable of handling a thousand impulses a minute were
available, and the sample under examination were suflBciently con-
centrated to give this output of charged particles, sixteen and two-
thirds hours would be required to register a million counts. It is
obvious that radioactive nitrogen with a half -life of 10.5 minutes or
radioactive carbon with a half-life of 20.5 minutes could not be
measured with precision. Nor is short half -life the only factor that
interferes with the quantitative measurement of radioactive isotopes;
many of the radioactive isotopes with a long half -life (C", S^^, Fe^^,
etc.) emit such soft radiations that measurement is extremely diffi-
cult, and background counts constitute a considerable percentage of
the total counts. Radioactive phosphorus is an ideal tracer with a
long half -life and an intense radiation, and in this case precise quan-
titative measurements can be obtained. But with the elements of
chief interest to the biologist— carbon, hydrogen, and nitrogen— the

258 A SYMPOSIUM ON RESPIRATORY ENZYMES

stable isotopes are much more favored for quantitative studies than
the radioactive elements.

The application of isotopes to problems involving respiratory en-
zymes has not been extensive. The metabolism of lactic acid and the
assimilation of carbon dioxide have been traced with stable and
radioactive carbon isotopes. The use of radioactive P^^ in the study
of the role of phosphorus in respiration has attracted a number of
investigators. In the near future, undoubtedly, isotopes will be em-
ployed in many other studies of this nature.

MECHANISMS FOR THE COMPLETE OXIDATION OF
CARBOHYDRATES BY AEROBIC BACTERIA
C. H. Werkman, Iowa State College:

This topic is a broad one that has not been exhaustively inves-
tigated because it offers serious technical diflBculties. Bacteria
seem to possess a distinct and quite troublesome cell wall whose
behavior as regards changes in permeability is far from clear. Realiz-
ing the danger of drawing conclusions from the use of unnatural
systems, we have attempted, by correlating the results obtained
with juices and whole cells, to circumvent the pitfall. But to do so
would apparently require an uncanny ability, since neither the work
with juices nor the cell suspension could be accepted as portraying
processes that occur in the living, reproducing organism. In the face
of this difficulty of preparing juices or cell suspensions that behave
naturally, the task of elucidating the natural processes in bacterial
respiration is laden with danger. Our present purpose is to encour-
age a free discussion of the possibilities in bacterial respiration and
to outline a working hypothesis.

There is abundant evidence that phosphorus plays an important,
if not essential, role in bacterial respiration. Early workers, including
Virtanen, have shown the ability of bacteria to form phosphorylated
esters. Wiggert in our laboratory showed an uptake of phosphorus
by living bacteria. That the principles of the Embden-Meyerhof
scheme of glucolysis operate in bacterial metabolism was first given
substantial support in 1936, when the characteristic intermediate of
that scheme, phosphoglyceric acid, was isolated from representa-
tive types of bacteria, both aerobic as well as anaerobic (Werkman
et al., Stone and Werkman). Utter in our laboratory has shown the
occurrence of the aldolase reaction by means of bacterial juices. In
this reaction hexosediphosphate is converted into phosphoglyceralde-
hyde and dihydroxyacetone phosphate. Furthermore, he has estab-

DISCUSSION ON BACTERIAL RESPIRATION 259

lished the dissimilation of phosphoglyceric to pyruvic acid through
phosphopyruvic.

Pyruvic acid has been shown to be a general intermediate in the
dissimilation of glucose by bacteria. In the case of anoxybiontic
metabolism the pyruvic acid is converted into a variety of products;
in the case of oxybiontic (aerobic) metabolism, however, it appears
that pyruvic acid initiates the changes of terminal respiration. These
are the changes of the whole aerobic dissimilation, and provide for
the oxidation of pyruvic acid to carbon dioxide and water. With
respect to bacteria there is evidence that the final processes of
respiration involve the cytochrome-cytochrome oxidase system. Both
cytochrome and its oxidase have been shown to be present in many
aerobic bacteria, and in no case known has cytochrome failed in an
aerobic species. There is no doubt that bacteria possess a cytochrome
mechanism, although many questions respecting the details of its
operation remain to be answered. We have found, for instance, an
acetone-resistant cytochrome oxidase not reported in animal tissues,
and bacterial cytochrome may have a lower potential than that in
animal tissue.

Between the terminal stage of glycolysis and the initial stage of
the cytochrome mechanism there is a portion in the spectrum of
respiration that has received only passing investigation so far as
bacteria are concerned. This may be referred to as the four-carbon
dicarboxylic acid portion. The Szent-Gyorgyi and Krebs schools
have pioneered in this work as it relates to animal tissue, and our
lead is taken from their work.

It is this four-carbon acid portion which we wish to discuss first.
Mr. Krampitz in our laboratory has been wrestling with these secrets
of nature. A few experiments and results will be cited here for pur-
poses of discussion. It is hardly necessary to mention that our remarks
are preliminary.

Shortly after Szent-Gyorgyi formulated his theory of the role
of the four-carbon acid in cellular physiology, an attempt was made
to apply it to bacteria. Before any catalytic eflFect of the four-
carbon acids can be shown, the preparation of enzymes must be
made deficient in four-carbon acids by washing bacteria free from
these acids. Using Micrococcus lysodeikticus to make our prepara-
tions, we did obtain stimulation with fumaric acid varying from 35
to 120 per cent as measured by oxygen uptake. Such stimulation was
not, however, obtained consistently, and therefore we attempted to
increase the cell permeabihty by acetone treatment, hoping to re-

260 A SYMPOSIUM ON RESIRPATORY ENZYMES

move the four-carbon acids more effectively. But this preparation
did not function in all respects like normal cells, failing to oxidize
glucose and succinate among others, although it did oxidize the four-
carbon acids (fumaric and malic) and lactate and pyruvate to acetic
acid. Since succinate was not oxidized, we believed that the cyto-
chrome system might have been injured, inasmuch as the hydrogen,
according to Szent-Gyorgyi, is transmitted through succinate to the
cytochrome system and thence to oxygen. When methylene blue or
cresyl blue was added to the system as carriers of hydrogen to re-
place the cytochrome system, no oxidation of succinate occurred.
Injury to Havoprotein action appeared ruled out because of the rapid
turnover of the substrates that were oxidized, i.e., fumarate, malate,
etc. Since acetone is known to destroy cytochrome oxidase, it was
difficult to understand the rapid attack on fumarate, malate, lactate,
and pyruvate. It was shown spectroscopically that the preparation
contained a cytochrome oxidase resistant to the acetone treatment.
These results were enough to indicate that investigations of bacterial
respiration were to prove interesting as well as a bit troublesome.

One of our principal objectives has been to integrate the hetero-
trophic assimilation of carbon dioxide and respiration. We have
therefore run a number of experiments to gain a better insight into
the mechanism of bacterial respiration. The evidence so far ac-
cumulated seems to indicate that the Szent-Gyorgyi cycle does not
function in bacterial respiration. Glucose, for instance, is not at-
tacked by Micrococcus lysodeikticus anaerobically by cell suspen-
sions or the acetone preparation. If the Szent-Gyorgyi system were
operating, malic acid present would be oxidized anaerobically, since
fumarase would provide fumaric acid as a hydrogen acceptor for the
system. This does not occur.

Thus far the evidence favors the occurrence in principle of the
Krebs citric acid cycle in bacteria. As the Krebs cycle is presented,
every alpha-keto acid with the single exception of pyruvic acid is
oxidatively decarboxylated. This point should be further investigated
to determine whether pyruvic acid is not also oxidatively decar-
boxylated, not necessarily to acetic acid, but to some two-carbon
compound which is able to condense with oxalacetic acid to initiate
the citric acid cycle. Acetic acid is commonly found as an end-prod-
uct when juices or perhaps injured cells are employed. Maintenance
of an adequate supply of oxalacetic acid is a requirement of the
Krebs scheme, and this is assured by regeneration in the cycle and
by the utilization of carbon dioxide through the Wood and Werk-

DISCUSSION ON BACTERIAL RESPIRATION 261

man reaction. We have found some evidence for the formation of
citric acid in the presence of acetic acid by Micrococcus lysodeik-
ticus.

On the other hand, many bacteria do not appear to metaboHze
citric acid; this is difficult to reconcile with the Krebs cycle as pro-
posed, although it is probable that it is not citric acid as such which
is the intermediate.

At present we are at work on C" acetic acid as a tracer. Certain
experimental evidence with the four-carbon acids has at times sug-
gested that we are dealing with phosphorylated compounds, prob-
ably of a very labile nature. Thus an acetone preparation is made
magnesium- and cocarboxylase-deficient by alkaline phosphate
washing of the cells as determined by testing on pyruvic acid. The
deficient preparation does not decarboxylate oxalacetic acid; how-
ever, the addition of magnesium ions completely restores the ac-
tivity. The reaction yields carbon dioxide and pyruvic acid. When
malic acid replaces oxalacetic acid, the deficient preparation under
the same conditions does not oxidize malic acid to carbon dioxide
and pyruvic acid, but only to oxalacetate, which accumulates and
does not inhibit the bacterial malic dehydrogenase as it does tissue
dehydrogenase. The complete preparation (deficient plus magnesium
ions or the unwashed acetone preparation) oxidizes malate to carbon
dioxide and pyruvic acid with traces of oxalacetic acid. Thus with
laboratory oxalacetic acid the reaction goes to carbon dioxide and
pyruvate, whereas with "physiological" oxalacetic acid (from malate)
the reaction appears to maintain an equilibrium.

Is the "physiological" oxalacetic acid different from that prepared
in the laboratory, possibly a phosphorylated compound? We investi-
gated the problem and at one time thought that it was. Definite
stimulation by phosphate has been demonstrated for fumaric or
malic acid. As yet we have not shown a phosphate uptake, or isolated
an organic phosphate; however, we may be dealing with a labile
carbonyl phosphate in the sense of Lipmann. It is suggested that the
carbonyl group is bound in the physiological oxalacetate formed
from malic acid, since traces of oxalacetate are known to inhibit
malic oxidation in tissue. If malate is oxidized in an atmosphere of
C^^Oa and O2, the oxalacetic acid contains C^^ in the carboxyl group
adjacent to the methylene group. No chemical exchange takes place.
The enzymatic decarboxylation of oxalacetic acid in the presence of
heavy carbon dioxide also yields heavy carbon oxalacetic acid. This
is a form of carbon dioxide utilization.

262 A SYMPOSIUM ON RESPIRATORY ENZYMES

E. S. GuzMAPf Barron, University of Chicago:

Until a few years ago seme investigators in the field of oxidation-
reductions tended to devote their efforts exclusively to animal, plant,
or bacterial oxidations. Many papers published recently showing the
variety of oxidation mechanisms, even in the oxidations involving
simply an electron transfer, have demonstrated the necessity of in-
tegrating the facts obtained with these different kinds of living be-
ings. For such comparative studies, work with bacteria has been
fruitful not only because it is possible to obtain suspensions or ex-
tracts with which quantitative studies can be perfoiTned but also
because in a single species, say hemolytic streptococci, a variety of
oxidation mechanisms may be found in different strains.

If we take the component of oxidation enzyme systems closest to
molecular oxygen, the iron porphyrins, we may divide bacteria into
two groups: cytochrome-containing bacteria (including most of the
so-called aerobic bacteria) and cytochrome-lacking bacteria (the so-
called anaerobic bacteria). Species of the two groups may produce
identical oxidations. They may oxidize, for example, lactate or
glycerol. The rxidation of lactate and glycerol by cytochrome-con-
taining bacteria (Staphylococcus) is completely inhibited by cyanide,
whereas the same oxidations by cytochrome-lacking bacteria are
cyanide-insensitive. Obviously iron porphyrins take part in the
oxidation of lactate and glycerol by cytochrome-containing bacteria,
whereas in cytochrome-lacking bacteria the oxidation proceeds
through different channels (flavin nucleotides).

In the field of phosphorylative oxidations the laboratory of Werk-
man has demonstrated that there exist in bacteria the different
phosphorylations observed in the breakdown of carbohydrate by
muscle or yeast extracts. In our laboratory it has been found that
the oxidation of glycerol by hemolytic streptococci does not take
place in the absence of phosphates. This does not mean that phos-
phorylation is essential for glucose oxidation; it is known that the
breakdown of carbohydrate by molds proceeds without phosphoryla-
tion.

It is assumed that in animal tissues carbohydrate metabolism starts
with the fermentation process ending in lactate, whereas in yeast it
ends in the fonnation of alcohol. In bacteria the fermentation process
may end in the formation of either lactate or of alcohol or in the
formation of both end products, as Friedemann has shown. Fermen-
tation may be absent altogether, as in glucose-non-fermenting bac-

DISCUSSION ON BACTERIAL RESPIRATION 263

teria. These bacteria either oxidize glucose directly without previous
phosphorylation {Pseudomonas aeruginosa) or oxidize directly phos-
phorylated hexose (hexose monophosphate and diphosphate).

The same variety of mechanisms is found in the oxidation of pyru-
vate (CH3COCOOH + 'AO^ = CH3COOH + CO2): it requires an
iron porphyrin catalyst in gonococci; it proceeds without iron por-
phyrin in Bacterium Delbriickii, as Lipmann has shown. d-Amino
acid oxidase, isolated by Warburg and Christian, is an alloxazin
dinucleotide protein, the oxidation of alanine to pyruvate being
cyanide-insensitive; this oxidation when performed by cytochrome-
containing bacteria requires iron porphyrin as a component of the
enzyme system because the oxidation is completely inhibited by
cyanide.

These examples are presented as proof of the existence of multiple
mechanisms of oxidation. A comprehensive study of biologic oxida-
tion-reduction demands, therefore, a continuous and simultaneous
attention to the oxidation mechanisms throughout living cells.

P. W. Wilson, University of Wisconsin:

A discussion of oxidations by aerobic bacteria should certainly
include reference to the fact that one of the most actively respiring
tissues known belongs to this group of organisms. I refer, of course,
to the extremely high rate of respiration possessed by certain cultures
of Azotobacter, the free-living nitrogen-fixing bacteria. One of our
distinguished guests and participants. Dr. Otto Meyerhof, first called
attention to this several years ago when he reported Q02 values of
500 to 8600 for Azotobacter chroococcum on glucose at 28° C. (1, 2).
The extremely high values were obtained with very young cultures
so diluted that the total dry weight involved was less than 10 micro-
grams. Its estimation may have been subject to some error, but it is
probable that young cultures of this organism have a Q02 value of
at least 5000.

Since Meyerhof and his collaborators made these experiments,
important advances have been made toward developing cultural
conditions that are optimum for growth and nitrogen fixation by
Azotobacter. Bates of fixation are consistently obtained in experi-
ments today which are several times greater than those reported
several years ago. For example, Azotobacter vinelandii can fix as
much as 20-30 mg. of nitrogen in 24r-36 hours instead of 4-5 mg. in
one or two weeks which was the characteristic result of most of the

264 A SYMPOSIUM ON RESPIRATORY ENZYMES

earlier studies (3). It is of interest to examine such cultures for re-
spiratory activity. My associate. Dr. R. H. Burris, has determined the
Q02 values of a 12-hour culture of Azotobacter agilis on a number of
substrates; his findings were as follows: endogenous, 28; glucose,
29; lactate, 129; arabinose, 29; acetate, 1109; ethyl alcohol, 1240.
When a 48-hour culture was used, these values were greatly reduced.

More recently Mr. Joe Wilson in our laboratory has made similar
observations with Azotobacter vinelandii. In most of these studies a
24-hour culture was diluted, and the oxygen uptake measured im-
mediately for a period of 60 minutes. The substrate was sucrose, the
temperature 30° C, the pH 7.0. Air was used as the gas phase so
that opportunity for growth existed, but no detectable fixation of
nitrogen occurred during this short experimental period. To avoid
the error attached to estimation of dry weight, he calculated the
rate of respiration on the basis of cell nitrogen (4). Since these cells
of Azotobacter contain about 10 per cent nitrogen, such Q02 (N)
values are on the average about 10 times as great as the Qoo based on
dry weight. Under these conditions the values of the Q02 (N) ranged
from 25,000 to 30,000.

These data emphasize the extremely high rate of respiration of
difiFerent species of Azotobacter and suggest that this organism may
well provide an excellent source for the preparation and isolation of
different enzyme systems concerned with tlie transfer of hydrogen
from substrate to molecular oxygen. With this in mind we are in-
vestigating methods for growing Azotobacter on a scale considerably
greater than any previously attempted. In a pilot plant designed for
yeast production we have succeeded in producing several pounds of
moist azotobacter cells during a growth period of 24 to 30 hours.

REFERENCES

1. Meyerhof, O., and Burk, D., Z. physik. Chem., 139A, 117 (1928).

2. Meyerhof, O., and Schulz, W., Biochem. Z., 250, 35 (1930).

3. Wilson, J. B., and Wilson, P. W., Jour. Bact., 42, 141 (1941).

4. Burris, R. H., and Wilson, P. W., Proc. Soc. Exp. Biol. Med., 45, 721 ( 1940).

REACTIONS IN CELL-FREE ENZYME SYSTEMS
COMPARED WITH THOSE IN THE INTACT CELL
F. F. NoRD, Fordham University:

In interpreting the results of investigations of bacterial metabolism
a few principles should be mentioned which, in addition to the very
recent use of tracers, appear to have been applied in approaching

DISCUSSION ON BACTERIAL RESPIRATION 265

the problems with which we are confronted in extracts and in Hving
cells:

1. In extracts a disturbance of the ratio of the various components
and an effect upon the total enzyme system occurs automatically,
which may cause an accumulation and even a stabilization of tran-
sient products.

2. By selective poisoning of parts of the enzyme system numerous
facts have been established.

3. In hving cell processes, because of the introduction of reagents
not akin to the whole system, a supposed or possible intermediary
product is removed and thereby excluded from the reaction se-
quence. For example:

1. Amino acid oxidase is capable of deaminating I- or d-amino
acids in tissue slices. In the case of injured or denatured tissues,
however, natural amino acids are no longer deaminated.

2. The extent to which the carrier enzymes are dispersed may
change under the influence of various factors. Moreover, in the cells
disperse particles of the various protoplasmic substances actually
possess widely differing pH values.

3. Even Dr. and Mrs. Cori declare, in accordance with the afore-
mentioned fact, that the conditions for glycogen synthesis are much
more favorable in the intact cell than in tissue extracts, where, they
state, they have obtained starch.*

4. Experiments show that in the case of Corynebacterium diph-
theriae, strains gravis and mitis, conditions are comparable.

5. There is no stoichiometrical relationship between the carbon
dioxide evolved and the actual decrease in inorganic phosphorus
(in the living cell).

6. When carbon dioxide evolution was compared with energy
liberated as heat by living yeast cells and by Lebedew extract, it
was noted that the heat of reaction in the course of fermentation
changed continually, indicating that fermentation with living cells
does not proceed according to a fixed scheme.

The thermochemical course of fennentation with juices shows, in
contrast, that at least two different conversions occur: (1) fermenta-
tion of free sugar in the presence of free phosphate (inhibited by
phloridzin) and (2) the subsequent fermentation of the residual
substrate in the absence of free phosphate (not inhibited by phlorid-
zin).

* Compare G. J. Goepfert, Brewers Digest, 16, No. 6 (1941).

266 A SYMPOSIUM ON RESPIRATORY ENZYMES

P. W. Wilson, University of Wisconsin:

It is only recently that most investigators of bacterial metabolism
have had the opportunity to choose between cell extracts and intact
cells. Because of their small size, bacteria as a group have resisted
attempts to destroy their cellular integrity. In the past, if studies on
bacterial enzyme systems were to be made, the investigator was in
the position of Kipling's thief who took the hot stove because there
was nothing else that season— he had to use intact cells. Although this
limitation has undoubtedly complicated the investigations and the
interpretation of the results, it has not been insurmountable. By use
of the so-called "resting cell" technique, great strides have been
made toward an understanding of the biochemistry of bacteria. With
respect to individual enzyme systems the greatest success has at-
tended studies in which isolation of the reaction is made possible
through choice of substrate rather than enzyme system, for example,
hydrogenase and hydrogenlyase. When less specific substrates are
employed, great care must be exercised that the interpretation is
not oversimplified by applying information gained in what appears
to be an analogous study made with the more purified enzyme prep-
arations.

With certain bacterial enzyme systems the investigator is denied
even the use of non-proliferating cells. For example, studies on
nitrogen fixation by Azotobacter must usually be made with growing
cultures : on the symbiotic system with a very complex association of
bacteria and host plant. Despite these technical handicaps, the de-
velopment of certain methods (1, 2) during the last decade has
enabled investigators to secure what Burk has recently described as
"the most intimate information that we possess on the mechanism of
fixation, and in particular on the nature of the first crucial step in-
volved" (3).

In recent years special techniques have been developed which
allow cell-free extracts containing a variety of enzymes to be pre-
pared from bacterial species. While it is gratifying that this first step
toward isolation of individual enzymes has been made, the imme-
diate practical value of the achievement has not been great. In most
cases the net result of the separation has been the verification of a
portion of the knowledge previously obtained with the resting cells.
Recently we have prepared a cell-free Azotobacter "juice" which
contains among other enzymes very powerful preparations of hydro-
genase and oxalacetic decarboxylase. Our satisfaction over this ac-

DISCUSSION ON BACTERIAL RESPIRATION 267

complishment is somewhat lessened by our recognition that we can
do Httle with the extract that we have not already done with intact
resting and acetone-treated cells. Nevertheless, we continue studies
with it in the hope that a clue to the mechanism of nitrogen fixation
may be furnished by the extract that has been successfully hidden in
the intact organism. Meanwhile, however, the physical-chemical
studies on the intact growing bacteria will not be neglected, since
they have already proved their value.

REFERENCES

1. BuRK, D., Ergebnisse d. Enzymforschung, 3, 23 (1934).

2. Wilson, P. W., The Biochemistry of Symbiotic Nitrogen Fixation (University
of Wisconsin Press, 1940 ) .

3. BuRK, D., and Burris, R. H., Ann. Rev. Biochem., 10, 587 (1941).

Discussion on Animal Tissue Respiration

C. A. ELVEHJEM, University of Wisconsin, Chairman

FACTORS AFFECTING THE PREPARATION OF TISSUE FOR
METABOLIC STUDIES

EPHRAIM SHORR
Cornell Medical College, New York

My comments will be restricted to certain difficulties encountered
in the preparation of tissue for in vitro studies of metabolism. They
touch on the various methods of preparing tissue, such as the slice
and mince method and on the influence of certain chemical changes
that are inevitable during the handling of the tissue prior to the
experimental run.

The Slice Method— Most workers use the limiting formula of War-
burg without testing the permissible thickness of the specific tissue
with which they are working. The general tendency is to get a slice
as thin as possible. Histological studies, as well as comparative
studies of the rate of respiration, show that this is not altogether wise.
The superficial layers can be shown to undergo degeneration to
variable depth. The thinner the slice the larger is the proportion of
damaged tissue. This is particularly important for tissues such as
cardiac muscle, where a whole large cell unit at the surface must
inevitably undergo degeneration. Thicker slices can be shown to
have a higher rate of respiration than very thin ones. The maximum
thickness which is permissible is therefore better. Not infrequently
thicknesses which exceed the formula behave very well. This points
to the possibility that there may be mechanisms for maintaining
oxygen pressure other than the gradient set up by the tension in the
solution— perhaps the iron-carrying compounds of the tissue, which
serve as a storehouse. The slice method is of course best adapted to
parenchymatous organs, least well to muscle. Unfortunately cardiac
muscle does not lend itself to dissection as does skeletal. However,
the individual muscle cells are much shorter than skeletal muscle
cells, hence the degeneration occurring at the surface is not extensive
enough to do much harm. As regards the brain, there seems to be
little evidence that the more convenient method of chopping the
tissue with a razor is less good than slicing.

268 X

DISCUSSION ON ANIMAL TISSUE RESPIRATION 269

The Muscle Strip Technique.— In most experiments reported in
the literature where skeletal muscle has been employed, it has either
been minced or chopped with scissors if larger animals have been
used, or the diaphragm of a small animal, such as the rat, has been
employed intact. The chopping or mincing is extremely destructive,
sets up abnormal chemical processes in the presence of oxygen, such
as aerobic glycolysis, and permits brief survival. The rat diaphragm,
while excellent for many purposes, is small, permits of few con-
comitant chemical measurements, and has only a limited usefulness
because of the fact that many metabolic conditions, such as diabetes,
cannot be brought about in this animal. The dog is a much better
experimental animal in these respects. The neck muscles of the dog
are ideal for obtaining, by careful dissection, long muscle strips for
in vitro studies. Individual fibers can be teased out intact. These
maintain their histological integrity for long periods. They respond
to electrical stimulation for hours unchanged, and give reproducible
results as far as work and heat production. Furthermore, enough
material can be obtained to allow for extensive chemical balances.
It is, I am sure, the method to be used when this type of tissue is
employed with in vitro studies.

Minced Tissue.— Here the choice lies between using a Latapie
mincer or a homogenizing apparatus described by Potter. Histo-
logical examination of the tissue obtained by these two methods
shows that with the homogenizer the tissue is completely and uni-
formly disintegrated, whereas with the Latapie the destruction is
not complete and the tissue is a mixture of disintegrated and intact
cells. The amount of destruction differs with the organ, and these
differences are paralleled by differences in the rate of respiration.
For example, skeletal muscle because of its long fiber is completely
destroyed by both methods and the rate of respiration is the same.
With the parenchymatous organs, such as the liver and kidney, a
definite difference in respiration results from the two methods, the
respiration of the homogenized tissue being lower than that of the
Latapie. The same is true of cardiac muscle. The higher respiration
may be due to the presence of the intact cells in the tissue put
through the Latapie. Obviously tissues minced by these two methods
are not comparable with respect to respiration, and other differences
may exist. This discrepancy should be borne in mind whenever the
two methods yield different results. It would certainly seem desir-
able to decide on one or the other. My preference would be homog-

270 A SYMPOSIUM ON RESPIRATORY ENZYMES

enization because of its uniformity. With this latter method, over-
heating of the solution during the stirring should be rigorously
avoided. With minced tissue from the Latapie, two methods of
obtaining aliquots are commonly employed. The minced tissue may
be weighed out as such or stirred up in a cool solution and pipetted
out. My preference is for the latter method, which is a simple one if
a wide-mouth pipette is used. It is also much more rapid and yields
somewhat more uniform respiration in duplicates and triplicates.
With the other method it seems difficult to keep all the tissue under
the same conditions of cooling, a desideratum when things are hap-
pening as rapidly as they do in such a tissue. Furthermore, I have no
confidence in single experiments and so would urge that triplicate de-
terminations be carried out.

Complicating Factors Arising between Removal of the Tissue and
the Experimental RMn.— During this inevitable unphysiological pe-
riod in which the tissue is anaerobic, many breakdown processes
occur. These lead to the accumulation of a number of metabolites
which can influence the results, especially during the early part of
the experiment. Whatever the speed of preparation, these changes
cannot be avoided. Those I have had to deal with have been the
accumulation of lactic acid, and striking changes in the hexosemono-
phosphates, adenosinetriphosphate, and phosphocreatine. The ac-
cumulation of lactic acid influences markedly the initial rate of res-
piration, and may suppress the effect of added lactate. In short
experiments it may lead to erroneous conclusions respecting the rates
of respiration of individual tissues. The literature contains not a few
instances of such misconceptions. It can be dealt with either by reduc-
ing the lactate content prior to the experiment, by aerating the tissue
in a Ringer solution long enough to bring the content to the normal
value, or by allowing the excess lactic acid to be dealt with in the
micro respiration vessels for an hour or so before the experimental
run. The former appears to be much less time-consuming and just as
effective. Oxygen is bubbled vigorously through the solution con-
taining the tissue. This is kept at room temperature. As regards
changes in the organic phosphate compounds during this interval of
preparation, it is very important to restore the normal relationships
existing prior to the experiment; otherwise the recovery process may
be taking place during part of the experimental period, confusing
the results of concomitant chemical balance experiments. This is
particularly important for muscle tissue, and it is necessary to de-
termine, for each tissue, how long an equilibration is required to

DISCUSSION ON ANIMAL TISSUE RESPIRATION 271

permit complete recovery. Undoubtedly other accumulations and
disturbances occur which we have not detected. This is worthy of
investigation for any specific system under study.

The Escape Phenomenon.— Where in vitro experiments are pro-
longed, another type of phenomenon must be borne in mind. We
have shown that diabetic tissue on prolonged survival in vitro grad-
ually regains its ability to oxidize carbohydrate. A complete restora-
tion takes place in four hours at 41° C, in ten hours at 37.5° C. With
cardiac muscle a definite elevation in the respiratory quotient of
normal tissue is found as early as the second hour. This change has
been attributed to the release of the tissue from certain influences
carried over from the intact animal. The change can be checked by
the use of other than inorganic phosphate buffers. Among these is
beta-glycerophosphate. In prolonged experiments with any tissue
this phenomenon should be borne in mind, since, it may be asso-
ciated, as in this instance, with an entirely different type of me-
tabolism toward the end of the experiment than at the start.

Miscellaneous.— In obtaining respiratory quotients it has been our
experience that for vessels of any given size more reliable results
are obtained when the respiratory exchange is large. When the total
oxygen consumption and carbon dioxide production is small, the
results are likely to be unreliable, and generally the respiratoiy
quotient is erroneously high. In vessels of 20 to 24 cc. capacity, if
the oxygen consumption in the period of observation is less than
150 cmm., we are likely to obtain incorrect respiratory quotients. It
is recommended that for each size and type of vessel used, studies
be made to determine the amount of oxygen consumption that yields
a reliable respiratory quotient.

COMPARISON OF SLICES AND HOMOGENIZED SUSPEN-
SIONS OF BRAIN TISSUE

K. A. C. ELLIOTT
Institute of the Pennsylvania Hospital, Philadelphia

Warburg introduced the technique of using slices of tissue for
metabolic studies, and the method has been used by many other
workers. Slices of many tissues can be prepared without disrupting
the majority of the cells; gases and substrates can diffuse in and out
of thin slices rapidly enough not to limit the rates of metabolic
processes. With various mashed and ground preparations, respiration
has been found to occur less rapidly, and it is commonly believed
that a closer approach to physiological conditions is obtained with

272 A SYMPOSIUM ON RESPIRATORY ENZYMES

slices than with tissue breis. While slices have been used in attempts
to determine what a tissue actually does, breis have been useful in
discovering essential substances and the interrelation of reactions.
The disintegration of tissue permits various essential materials to be
more readily diluted by the suspending medium or destroyed by
enzymes, so that their concentration falls below the optimal and
their addition produces striking efiFects often not found with slices.

The preparation of slices from some tissues, especially brain, is
slow and delicate work, sampling is inaccurate, and one cannot be
sure that the individual slices do not vary in activity. Dr. Libet and
I (1) have recently studied the respiration of brain suspensions and
we find that two types of suspension can be obtained. One type
behaves as disintegrated tissue; the other type, when prepared under
proper conditions, behaves very similarly to slices, shows a com-
parable respiration rate, and is a suitable and very convenient
preparation for the study of brain metabolism.

The first type of suspension is obtained when brain tissue is
homogenized by the apparatus of Potter and Elvehjem (2) in hypo-
tonic medium, dilute phosphate buffer solution. Such suspensions
respire at a low rate which may be increased up to 65 per cent by
adding salt or sugar after homogenization. With such preparations
it is therefore necessary that the osmotic pressure be equal in the
control and experimental flasks when the effect of added substances
is tested. These hypotonic suspensions have largely lost the power to
utilize glucose. They show considerable effects when tissue extract
or substances like fumarate are added. The rate of respiration per
unit weight of tissue increases with increasing tissue concentration.

The second type of suspension is obtained when the medium in
which the brain is homogenized contains sufficient salt, sucrose, or
glucose to make the osmotic pressure equal to that of serum. The
respiration rate of such suspensions is up to 400 per cent greater than
the rate of tissue homogenized in hypotonic medium. (Isotonic urea
behaves like hypotonic solution.) Suspensions of whole brain (con-
taining a large amount of white matter, which respires only slowly),
prepared in 0.13 M sodium chloride-0.017 M phosphate buffer solu-
tion, respire on the average at 71 per cent of the rate of an equal
tissue weight of slices of pure gray matter in the same medium.
Such suspensions are much less affected by the addition of tissue
extract, fumarate, etc., than are hypotonic suspensions, and their
respiration rate per unit weight is practically independent of the
tissue concentration. Isotonic suspensions respire with the same res-

DISCUSSION ON ANIMAL TISSUE RESPIRATION 273

piratory quotient as slices, and are similarly affected by additions of
various substrates and by variations in the ionic content of the med-
ium. An exception to this rule is that the respiration of slices, unlike
that of suspensions, is less well maintained in isotonic sucrose than in
isotonic saline solution. When isotonic suspensions are used, air may
be used in reaction flasks instead of oxygen. This is an advantage,
since it has been found that, while the respiration rates in the presence
of air and in the presence of oxygen are identical for 90 minutes, the
rate in the presence of oxygen falls off much more rapidly there-
after. A similar slowly appearing toxic effect of oxygen can also be
demonstrated with slices, but with slices the presence of oxygen is
necessary, since otherwise the initial respiration rate is limited by
inadequate diffusion of oxygen into the tissue.

After homogenizing fresh brain in isotonic medium, the rate of
respiration falls off very rapidly at first, thereafter less rapidly. Slices
and isotonic suspensions prepared from slices do not show the initial
very rapid decrease in rate. This suggests that a specially labile part of
the respiratory activity can be observed with suspensions of fresh
tissue, but is lost during the slow process of preparing for experi-
ments on slices.

Salts induce specific as well as osmotic effects. Isotonic solutions of
sodium chloride, nitrate, sulfate, and especially phosphate increase
the initial respiration rate appreciably when added to suspensions in
isotonic sucrose. Bicarbonate in physiological concentration has no
special effect. Calcium and magnesium ions have inhibitory effects
on the respiration but cause better maintenance of rate. Potassium
in high concentrations is also inhibitory. The initial inhibitory ef-
fect of magnesium in the concentration found in serum is fairly
small, and this ion is known to take part in reactions of carbohydrate
metabolism. For studies of brain respiration, tissue homogenized in
isotonic sodium chloride medium containing magnesium is therefore
recommended.

However, respiratory activities measured on suspensions or slices
in any given medium cannot be considered to represent the true
physiological activity of brain tissue in vivo until more is known of
the effective concentrations of ions in the cells' immediate environ-
ment in vivo and until more is known of the differences that may
exist between brain in its normal physiologically active condition
and the tissue which has been subjected to the abnormal injuries
and stimuli of in vitro work.

The marked difference between tissue homogenized in isotonic

274 A SYMPOSIUM ON RESPIRATORY ENZYMES

and in hypotonic media occurs to some extent with certain tissues
other than brain, but not with all tissues. PreHminary studies would
have to be made on each tissue to which the method of isotonic
suspensions is to be applied before it could be assumed that the
behavior of suspensions would be comparable to that of slices of the
particular tissue.

REFERENCES

1. Elliott, K. A. C, and Libet, B., in press.

2. Potter, V. R., and Elvehjem, C. A., J. Biol. Chem. 114, 495 (1936).

THE HOMOGENIZED TISSUE TECHNIQUE, THE DILUTION
EFFECT AND ION EFFECTS

VAN R. POTTER
McArdle Me7norial Laboratory, University of Wisconsin

We have developed the homogenized tissue technique for the
study of isolated phases of metabolic activity. Since the enzymes
that catalyze biological oxidations are in many cases extremely la-
bile, we have used the device of homogenization to effect a physical
isolation of a particular enzyme system by dilution. We believe that
by so doing we can retain the original activity of the tissue and thus
develop assay methods for specific enzymes, where separation by
chemical treatment, such as fractional precipitation, could not result
in 100 per cent yields and hence would be useless for assay purposes.
Since certain components of various enzyme systems are readily
soluble and are capable of diffusing away from each other or from
solid phases, such as cytochrome oxidase, it is necessary to fortify
the homogenate with these diffusible components. Whether it is
necessary to add these accessoiy factors is determined by measur-
ing the enz)Tne activity at various dilutions. If the measured effect is
proportional to the amount of enzyme used, fortification is unneces-
sary. With regard to choice of buffer, it should be pointed out that
since we are now dealing in tenns of intracellular components, buf-
fers based on extracellular fluids (such as serum) may not necessarily
be optimum.

A system which may illustrate these points is the succinoxidase
system. It is inhibited by chloride ions, hence these are omitted from
the buffer medium. It requires at least three soluble components for
maximal activity, namely, cytochrome c, calcium ions, and aluminum
ions. The dehydrogenase and the cytochrome oxidase appear to be
associated with solid particles of protoplasm. When the dissociable

DISCUSSION ON ANIMAL TISSUE RESPIRATION 275

factors are supplied, the activity of the system is just as great as that
of intact tissue.

More complex are the coenzyme systems in which two additional
factors are soluble, namely, the dehydrogenase and the coenzyme.
However, by means of fortification with the appropriate coenzyme
and inhibition of the coenzyme nucleotidase it should be quite
feasible to study these complicated systems in tissue homogenates.

THE STIMULATORY EFFECT OF CALCIUM UPON THE
SUCCINOXIDASE ACTIVITY OF RAT TISSUES

A. E. AXELROD

University of Wisconsin

Variations in the ionic composition of the medium in which the
surviving tissue respires are known to exert profound eflFects upon
the extent of the respiration. Our attention was drawn to these
ionic effects by the observation that calcium (as calcium chloride)
stimulates markedly the succinoxidase activity of minced rat liver.
This phenomenon was investigated further in the succinoxidase sys-
tem of tissue homogenates prepared according to Potter and Elve-
hjem. The following results were obtained. In the absence of added
cytochrome c the succinoxidase activity of minced liver was in-
creased 43 to 80 per cent by the addition of 20 micrograms of cal-
cium. With homogenized liver (40 mg. per flask) the addition of
20 micrograms of calcium resulted in increases of 93 and 48 per
cent in the absence and presence, respectively, of added cytochrome
c (3 X 10'* mole per flask). The succinoxidase activity of homoge-
nized kidney cortex (20 mg. per flask) was stimulated 40 per cent in the
presence of added calcium. Added cytochrome c did not affect the
magnitude of the stimulatory effect of calcium in this tissue. The
most pronounced effect of calcium was observed in the case of
homogenized heart tissue (20 mg. per flask), in which the addition
of 20 micrograms of calcium in the presence of 3 X 10"* mole of
cytochrome c caused an increase of 200 per cent in the succinoxidase
activity. Under our experimental conditions the addition of 20
micrograms of calcium always yielded the maximum stimulatory
effect. In many cases the addition of smaller amounts of calcium (as
little as 1 or 2 micrograms) resulted in a marked acceleration of
succinoxidase activity. In only a few isolated cases was a calcium
effect observed in brain and skeletal muscle.

The apphcation of the homogenized tissue technique to the study

276 A SYMPOSIUM ON RESPIRATORY ENZYMES

of isolated enzyme systems has been discussed by Dr. Potter, who
pointed out that all precautions must be taken to assure complete
restoration of the diffusable factors necessary for the maximal ac-
tivity of the enzyme system under consideration. The stimulatory
eflFect of calcium upon the succinoxidase system in tissue homo-
genates necessitates the addition of calcium to such a system if the
maximal activity is to be attained. A dissociable complex involving
calcium is indicated. The addition of aluminum ions has been shown
by Dr. Potter to overcome an effect resulting from further dilution
of the tissue. It thus becomes apparent that when either minced liver
preparations or tissue homogenates are employed as the source of
the succinoxidase system, it is necessary to add calcium and, under
certain conditions, aluminum in order to eliminate these ions as
possible limiting factors which may affect the validity of the succin-
oxidase assay.

TISSUE METABOLISM IN VITRO AND IN VIVO

FREDERICK BERNHEIM

School of Medicine, Duke University

The work of Battelli and Stern in the early part of the century may
be said to have begun the work on tissue metabolism in vitro which
has culminated in the isolation of a number of dehydrogenases. Some
of the enzymes which they showed to be present in tissue suspensions
have since been shown to be of importance in the economy of the
animal. In particular, all subsequent work on the physiology and
pharmacology of alcohol has shown that its fate in the body can be
accounted for by the activity of the alcohol oxidase of liver which
they discovered. Some time later Warburg studied the effect of
cyanide on isolated tissues, and from this work came the discovery
of cytochrome oxidase and the cytochromes. Again, work on the
pharmacology of cyanide has shown that its action on the animal
can be explained on the basis of its inhibition of the cytochrome
oxidase. In these two early groups of experiments the correlation
between in vitro and in vivo results is good. With more recent work
sirnilar correlations have either not been made or have not been
satisfactor)^

The justification for working with broken cell suspensions or cell
extracts is that it is only by this means that intracellular enzymes can
be studied. The results thus obtained can indicate only that the cell
or cell catalyst has certain potentialities under the given set of condi-
tions. Under no circumstances are such results in themselves evi-

DISCUSSION ON ANIMAL TISSUE RESPIRATION 277

dence for the normal activity of the cell. Attempts to make the condi-
tions more "normal" by adding physiological salt solutions to tissue
suspensions is essentially paradoxical, for it is known that the ionic
environment inside the cell diflFers markedly from that outside, and
thus, for the study of intracellular enzymes, physiological salt solu-
tions are unphysiological. If better results are obtained by the addi-
tion of such solutions to broken cell suspensions, it may simply mean
that the salts are acting on the residual penneabilities of partially
damaged cells rather than on the enzymes directly.

The physiological significance of results obtained in vitro must be
obtained by physiological means, i.e., by experiments on the whole
animal. The classical method for studying intermediary metabolism
in which substance x is fed and substance y is isolated in the urine
does not ordinarily give enough detailed information about the fate
of the substance. Correlation with the data obtained with isolated
tissue suspensions is therefore often impossible. With the increasing
use of labeled atoms animal experiments are yielding more precise
results and thus offer promise of better correlation between in vitro
and in vivo results. The story of sarcosine illustrates this point. When
it is fed to a rabbit with benzoic acid, an increased amount of hip-
puric acid is excreted. This shows that sarcosine in some way gives
rise to glycine. When sarcosine with labeled nitrogen is fed, the
hippuric acid excreted contains the labeled nitrogen. This shows
that sarcosine must be demethylated in the body but not deaminated.
When sarcosine is added to liver suspensions an extra oxygen uptake
occurs and glycine is formed. The sarcosine undergoes an oxidative
demethylation. In this series of experiments the in vitro results serve
to elucidate the mechanism of a reaction known to occur in the intact
animal. Only in this way is the physiological significance of results
with tissue experiments established.

Workers in tissue respiration tend to look for reactions of general
significance. Results obtained with yeast or hashed pigeon breast
muscle are applied with great facility to mammalian tissue. The
omnipresence of the cytochrome oxidase and succinoxidase encour-
ages this attitude, but does not justify it. Animals that differ so
profoundly, physiologically, pharmacologically, and in all other
ways, undoubtedly show differences in their respiratory mechanisms.
Perhaps in the future more information will be obtained if these
differences between animals are emphasized rather than minimized
in an attempt to provide universal mechanisms that may have only
superficial similarity.

278 A SYMPOSIUM ON RESPIRATORY ENZYMES

PATHWAYS OF CARBOHYDRATE METABOLISM

E. S. GUZMAN BARRON
University of Chicago

Whether the metabohsm of carbohydrates by animal tissues is
always accomplished according to Embden-Meyerhof's and Cori's
schemes of phosphorylations and oxidation-reductions has not yet
been established. In fact, if the normal pathway is obstructed, the
breakdown of carbohydrates might proceed through other pathways.
Direct oxidation of glucose, of hexosemonophosphate, and of phos-
phoglycerate might be the accessory pathways, although none of
these has yet been shown to occur in animal tissues. The oxidation of
a single substance may proceed via different enzyme systems; thus
in sea urchin eggs (with no succinodehydrogenase and no cyto-
chromes) the metabolism of carbohydrates undoubtedly proceeds
through different pathways than in sperm (with succinodehydro-
genase and cytochromes). The interesting findings of Korr (oxida-
tions not inhibited by azide in resting cells and inliibited by azide
when the cells are in active work) can be presented as examples in
favor of this opinion.

The existence of these multiple pathways makes possible the
orientation of reactions which occur continually in living cells, the
metabolism of pyruvate being the clearest example. This is illus-
trated in the scheme shown on page 279 in which only the pertinent
steps are reproduced.

Pyruvate is an extremely reactive substance, and seventeen differ-
ent pathways of its metabolism are known to exist in living cells.
In animal tissues, in the absence of oxygen, part of the pyruvate
formed during the breakdown of carbohydrate is reduced by dihy-
drodiphosphopyridine nucleotide (Py(P04)2Ho) to lactate, a reac-
tion the extent of which represents the degree of anoxia; part of it
may be reduced to alanine (Warburg and Christian's d-amino acid
oxidase) or may be used for transaminations or dismutations. In the
presence of oxygen, pyruvate activated by diphosphothiamine-
protein may be oxidized to acetylphosphate; may, through conden-
sation reactions, be responsible for the synthesis of alpha-ketoglu-
tarate, cisaconitate, acetoacetate, acetylmethylcarbinol, or carbo-
hydrate. It might, as postulated by Wood and Werkman, combine
with carbon dioxide to give oxalacetate, an important reaction still
eluding direct demonstration, which is the base of many hypotheses
for the breakdown and synthesis of carbohydrate. Oxalacetate
formed in this way may be reduced by Py(P04)8Hg to malate, thus

DISCUSSION ON ANIMAL TISSUE RESPIRATION

279

starting the oxidative pathway through Szent-Gyorgyi's cycle, or it
may produce phosphopyruvic acid and thus start the synthesis of
carbohydrate. These manifold reactions of pyruvate, all of them
present in different degrees in animal tissues, show that the orienta-
tion of reactions during the metabolism of carbohydrates is ex-
tremely complex, and certainly diflFerent from tissue to tissue.

^, oxidation
Glucose >

,^ II 1 , . oxidation
Hexose monophosphate

it
Hexose diphosphate

it
Phosphoglyceraldehyde + Py (PO4) 2

it
Phosphoglycerate + Py (PO4) 2H2

it t I

Phosphopyruvate <

Py(P04)2H2 i
Lactate ^^ Pyruvate

oxidation

Diphosphothiamine-proteins

4- '■ *^ -

>> c3

-a

is ^

9, 03

S 4> H

^ ."S o a

< ^■

Malate

Fumarate

Succinate

Fe^"*"*" cytochrome c

Cytochrome oxidase

IT
o.

280 A SYMPOSIUM ON RESPIRATORY ENZYMES

THE CITRIC ACID CYCLE IN TISSUE METABOLISM

FREDRICK J. STARE

Washington University School of Medicine, St. Louis

This is an appropriate time to mention the reasons why some of us
do not believe in the importance, or even the presence, of citric acid
as a component of the metaboHc cycle generally termed the "citric
acid cycle." The citric acid cycle is based on carefully established
experimental facts. Some of these were mentioned this morning by
Dr. Evans. We question, however, the application of these facts,
particularly the presence of citrate, to a cycle of importance in re-
spiring muscle.

It was only with high concentrations of pyruvate that Krebs was
able to demonstrate increases in citrate, and these increases were of
a small order, varying from 1 to 15 per cent. In tissue and body
fluids, pyruvate occurs in a far lower concentration.

It was only with high concentrations of pyruvate, citrate, and
malonate that an increase in succinate was detected. I know of no
evidence, but I believe that a high concentration of glutamate, and
other related compounds not included in the citric acid cycle, would
also yield increases in succinate in the presence of a high malonate
concentration.

The citric acid cycle assumes that malonate completely prevents
the anaerobic reduction of oxalacetate to succinate, but there is no
adequate proof for this assumption.

Citrate is definitely a weaker substance, as compared with the
other members of the cycle, in increasing or prolonging the oxygen
consumption of respiring muscle or in eflFecting pyruvate removal
by respiring muscle. In fact, citrate frequently inhibits such effects.

Malonate in a concentration of 0.005 M which is 5 to 6 times less
than the concentration used by Krebs, inhibits oxygen uptake and
pyruvate utilization on an average of 70 to 75 per cent (pigeon breast
muscle). It completely and always inhibits any catalytic effect that
citric acid may show. Yet any other member of the cycle when
added in an equal concentration of 0.005 M will completely over-
come the malonate inhibition of oxygen uptake and of pyruvate
removal. If the latter two depend in any way on a mechanism in-
volving citric acid, the malonate should stop It because citric acid
activity is always inhibited by malonate.

Recently, in Dr. Barron's laboratory, Lipton, Goldinger, and I
have studied pyruvate and citrate utilization in respiring pigeon
muscle tissue. According to the citric acid cycle, each molecule of

DISCUSSION ON ANIMAL TISSUE RESPIRATION 281

pyruvate is converted to citrate in the course of its oxidation. The
cycle imphes that either citrate should be oxidized as rapidly as
pyruvate, or that if citrate is not oxidized as rapidly as pyruvate, but
still is a stage in the removal of pyruvate, it should accumulate in
quantities suflBcient to account for the difference in the rates of
utihzation of the two compounds. We found that pyruvate is oxidized
at a far greater rate than citrate, and that citrate does not accumu-
late.

Our experimental observations favor a cycle involving a conver-
sion of pyruvic acid to alpha-ketoglutaric acid, without citrate as an
intermediary, followed by the Szent-Gyorgyi series of conversions
of the dicarboxylic acids to oxalacetate. The occasional catalysis of
respiration observed when citrate is added to muscle is probably
not due to citrate itself but rather to alpha-ketoglutarate and the
four-carbon acids which may be formed from it. Citrate may serve
as a "stockroom" for the essential catalysts, exerting an effect on
respiration only when they are low. Its synthesis from pyruvate may
represent an unusual side reaction which under normal conditions
is of little significance. However, under specific experimental condi-
tions in vitro, such as large amounts of pyruvate, the rate of their
reaction may be accelerated. A similar condition, with a high level
of pyruvate, appears to exist in vivo. Thus Sober, Lipton, and Elve-
hjem found that in the recovery from acute thiamine deficiency large
amounts of citrate are excreted.

In concluding these remarks, may I emphasize that these criti-
cisms of the citric acid cycle are directed against a citric acid cycle
which contains citric acid; they do not apply to a citric acid
cycle which contains no citric acid. Proponents of the citric acid cycle
quite properly spend most of their energy proving that some sort
of a cycle exists rather than attempting to answer the question
whether citric acid is, or is not, a member of it.

Ifo '

'^i

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