THE DYNAMICS OF LIVING MATTER
Columbia JEmfoersitjj Biological %>eiitQ
EDITED BY HENRY FAIRFIELD OSBORN
AND
EDMUND B. WILSON
I. FROM THE GREEKS TO DARWIN
By Henry Fairfield Osborn
II. AMPHIOXUS AND THE ANCESTRY OF THE VERTEBRATES
By Arthur Willey
III. FISHES, LIVING AND FOSSIL. An Introductory Study
By Bashford Dean
IV. THE CELL IN DEVELOPMENT AND INHERITANCE
By Edmund B. Wilson
V. THE FOUNDATIONS OF ZOOLOGY By W. K. Brooks
VI. THE PROTOZOA
By Gary N. Calkins
VII. REGENERATION
By T. H. Morgan
VIII. THE DYNAMICS OF LIVING MATTER By Jacques Loeb
IX. STRUCTURE AND HABITS OF ANTS. (In preparation)
By W. M. Wheeler
X. BEHAVIOR OF THE LOWER ORGANISMS. (In preparation}
By H. S. Jennings
6
COLUMBIA UNIVERSITY BIOLOGICAL SERIES. VIII.
THE DYNAMICS OF LIVING
MATTER
BY
JACQUES LOEB
PROFESSOR OF PHYSIOLOGY IN THE UNIVERSITY OF CALIFORNIA
, 1754- 1
COLUMBIA!
1893 1
THE COLUMBIA UNIVERSITY PRESS
THE MACMILLAN COMPANY, AGENTS
LONDON: MACMILLAN & CO., LTD.
I9O6
All rights reserved
COPYRIGHT, 1906, BY THE MACMILLAN COMPANY.
Set up and electrotyped. Published March, 1906.
J. S. Gushing tt Co. — Berwick & Smith Co. Norwood, Mass., U.S.A.
ANNE LEONARD LOEB
PREFACE
THIS book owes its origin to a series of eight lectures delivered upon the invitation of Professor E. B. Wilson and Professor H. F. Osborn at Columbia University in the spring of 1902. The aim of the lectures was to give a presentation of my researches on the dynamics of living matter and the views to which they had led me. In preparing the book I have tried to give a somewhat more com- plete survey of the field of experimental biology than was possible in the lectures, without, however, trying to alter their character. In the introductory lecture use was made of my address at the Inter- national Congress at St. Louis.
To Dr. S. S. Maxwell, who has undertaken the main burden of reading the proof and preparing the index, and to Professor J. B. MacCallum, who also assisted me in the reading of the proof, my sincere thanks are due.
BERKELEY, CALIFORNIA, January I, 1906.
vn
CONTENTS
LECTURE I
INTRODUCTORY REMARKS
PAGE
I
LECTURE II CONCERNING THE GENERAL CHEMISTRY OF LIFE PHENOMENA
1. Historical Remarks ............ 7
2. Reversible Enzyme Action — Lipase Action — Reversible Enzyme Action in the
Carbohydrate Group — The General Occurrence of Protein-splitting Enzymes 9
3. Respiration as a Catalytic Process 13
O) The Oxidases 13
(£) Further Remarks on the Significance of Oxygen in Life Phenomena . . 1 6
(<:) Death in Lack of Oxygen and the Protective Action of Oxygen . . 18
(</) Changes of Structure in Lack of Oxygen 19
4. The Production of CO2 through En/.ymes 21
5. Concerning the Theory of Enzyme Action 24
(a) Stereochemical Attempts .24
(<$) The Theory of Intermediary Reactions 26
LECTURE III THE GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER
1. The Limits of Divisibility of Living Matter ... 29
2. Foam Structures and Emulsions ..... . . . 31
3. The Colloidal Character of Living Matter 33
4. The Formation of Surface Films and Traube's Membranes of Precipitation -
Overton's and Meyer's Work on Narcotics and the Nature of Surface Films . 38
5. Osmotic Pressure and the Exchange of Liquids between the Cells and the Sur-
rounding Liquid .......... • 41
6. Further Limitations of Traube's Theory of Semipermeability 45
7. The Antagonistic Effects of Salts 46
LECTURE IV ON SOME PHYSICAL MANIFESTATIONS OF LIFE
1. Hypotheses of Muscular Contraction
2. Quincke's Theory of Protoplasmic Motion
3. Concerning the Theory of Cell Division ......
4. The Origin of Radiant Energy in Living Organisms
5. Electrical Phenomena in Living Organisms .
113
53 55 58 66 68
CONTENTS
LECTURE V
THE ROLE OF ELECTROLYTES IN THE FORMATION AND PRESERVATION OF
LIVING MATTER
PAGE
1. On the Specific Difference between the Nutritive Solutions for Plants and Animals
— Protective Solutions and Nutrient Solutions . . . . . .71
2. Concerning a Theory of Irritability and the Role of Na, K, and Ca for Animal Life
— Rhythmical Contractions in Skeletal Muscle, in Medusa?, and the Ven- tricle of the Heart — Contact Irritability in Muscle — Analogies to the Role of Salts in Coagulation of Milk — Significance for the Understanding of Functional Nervous Diseases — The Action of Purgatives .... 78
3. The Reaction of Living Matter and the Role of Bicarbonates in the Preservation
of Life -95
4. Electrical Stimulation .• 9&
LECTURE VI THE EFFECTS OF HEAT AND RADIANT ENERGY UPON LIVING MATTER
I. Effects of Heat — Upper Temperature Limit of Life — Influence of Reaction- velocity upon Biological Processes — Lower Temperature Limit — Other Biological Effects of Heat 106
?.. General Effects of Radiant Energy upon Living Matter — Chemical Action of Light
upon Organisms . . . . . . . . . . • .112
LECTURE VII HELIOTROPISM
1. The Heliotropism of Sessile Organisms 117
2. Heliotropism of Free-moving Animals . . . . . . . . .124
3. The Control of the Precision and Sense of Heliotropic Reactions in Animals . . 130
4. The Reaction of Animals to Sudden Changes in the Intensity of Light . . 135
LECTURE VIII
FURTHER FACTS CONCERNING TROPISMS AND RELATED PHENOMENA
1. General Theory of Tropisms ... 138
2. Galvanotropism .........•••• I4l
3. Geotropism r47
4. Chemotropism and Related Phenomena *52
5. Stereotropism • • J55
6. Concluding Remarks concerning Tropismlike Reactions 158
LECTURE IX
FERTILIZATION
1. The Specific Character of the Fertilizing Power of the Spermatozoon — Hybrid
Fertilization . . . . 161
2. Artificial Parthenogenesis and the Theory of Fertilization . . . 164
CONTENTS xi
LECTURE X HEREDITY
PAGE
1. The Hereditary Effects of the Spermatozoon and Egg — The Prevailing Influence
of the Egg in the Early Stages of Development — Merogony — Toxicity of
the Blood of Forms not closely Related — Mendel's Experiments . . .179
2. The Determination of Sex and the Secondary Sexual Characters .... 186
3. Egg Structure and Heredity . . . . . . . . . . . 191
LECTURE XI
ON THE DYNAMICS OF REGENERATIVE PROCESSES
1. Sachs's Hypothesis of the Formation of Organs 199
2. Heteromorphosis and Regeneration in Tubularia ....... 2OI
3. Regeneration in an Actinian {Cerianikus membranaceus) ..... 207
4. Regeneration and Heteromorphosis in Planarians . . . . . . 210
5. On the Influence of the Central Nervous System upon Regeneration and on Phe-
nomena of Correlation in Regeneration ........ 213
6. The Effect of Some External Conditions upon Regeneration and the Transforma-
tion of Organs . . . . . . . . . . . .217
7. The Role of Reversible Processes in Phenomena of Regeneration — The Distribu-
tion of the Power of Regeneration in the Animal Kingdom . . . .218
LECTURE- XII CONCLUDING REMARKS 223
INDEX 227
LECTURE I INTRODUCTORY REMARKS
IN these lectures we shall consider living organisms as chemical machines, consisting essentially of colloidal material, which possess the peculiarities of automatically developing, preserving, and reproducing themselves. The fact that the machines which can be created by man do not possess the power of automatic development, self-preservation, and reproduction constitutes for the present a fundamental difference between living machines and artificial machines. We must, however, admit that nothing contradicts the possibility that the artificial produc- tion of living matter may one day be accomplished. It is the purpose of these lectures to state to what extent we are able to control the phe- nomena of development, self-preservation, and reproduction.
Living organisms may be called chemical machines, inasmuch as the energy for their work and functions is derived from chemical pro- cesses, and inasmuch as the material from which the living machines are built must be formed through chemical processes. It is therefore only natural that the dynamics of living matter should begin with an analysis of the specific character of the chemical processes in organisms. It is neither our intention nor is it possible for us to give an exhaustive analysis, and we shall only go far enough to satisfy ourselves that no variables are found in the chemical dynamics of living matter which cannot be found also in the chemistry of inanimate nature.
The material of which living organisms consist is essentially col- loidal in its character. Graham introduced the discrimination between colloidal and crystalloidal substances : the latter diffuse easily, the former only with difficulty, or not at all, through animal membranes. The colloidal substances may be in solution or fine suspension, or they may appear in a jellylike or coagulated or precipitated form. In the former case wheje they are liquid we speak of sols, in the latter of gels. The structures which we find in living matter originate mostly through a gelation or coagulation of liquid colloids. We shall see in these lectures that liquefactions and gelations or coagulations may possibly play a great role in various physical manifestations of life ; but as the physics of colloids is still in its beginning, we must not be surprised that it is as yet impossible to carry its application to life phenomena very far.
2 DYNAMICS OF LIVING MATTER
As the chemical character of life phenomena and the physical struc- ture of living matter form the basis for the understanding of the dynamics of living matter, it is natural that they should be the starting-point in our lectures.
As far as the specifically biological phenomena are concerned, namely, the phenomena of development, self-preservation, and repro- duction, it will be our aim to analyze them as much as at present pos- sible from a physicochemical point of view. It may perhaps be desirable before undertaking this task for us to state, as simply as possible, some of the individual problems that will present themselves for discussion, and the general method of their solution.
We know that the eggs of the majority of animals cannot develop unless they are fertilized, I.e. unless they are entered by a spermato- zoon. We do not know how the spermatozoon causes the egg to develop, but it is not to be expected that we shall gain an insight into these causes except by trying to imitate by purely chemical and physical means the effects which a spermatozoon has upon the egg. We shall see that this has been accomplished in some forms. Under ordinary condi- tions, the egg of Strongylocenlrotus purpuratus, a sea urchin of the Pacific coast, does not develop unless a spermatozoon enters it; but the fer- tilizing effect of a spermatozoon can be imitated in all essential details by putting the egg for a minute into sea water to which a certain amount of a fatty acid has been added and by subsequent exposure of the egg for about half an hour to sea water whose concentration has been raised by a certain amount. Similar results can be obtained in other forms.
From a given egg can arise a specific organism only, the morpho- logical and physiological qualities of which can be predicted with cer- tainty, if we know the organism from which the egg is derived. We call this fact heredity. Modern embryology has shown that the com- plicated adult forms develop gradually from simpler forms, and by following the development from the egg we readily understand how it happens that the adult form is so much more complicated than the egg from which it arises. The question which has recently puzzled biolo- gists is whether the egg has any structure which can be related to the adult form. This seems to be true in the eggs of some forms, to the extent at least that from the various regions of the egg somewhat differ- ent parts of the embryo arise. We do not know the causes which deter- mine this relatively slight differentiation inside of the egg, but we shall see that everything indicates that these causes may be of a simple physicochemical order. In studying the mechanism of heredity, it is perhaps of importance to realize that as far as the heredity of the earliest embryonic stage is concerned, it is almost, or exclusively, determined
INTRODUCTORY REMARKS 3
by the egg. This is beautifully illustrated in the case of the hybridiza- tion of the egg of the sea urchin with the sperm of the starfish. The development of the pure breed of the sea urchin is after a certain stage (the gastrula stage) typically different from the development of the star- fish, inasmuch as the sea urchin's egg forms at that point a skeleton and goes into the characteristic pluteus form, while the starfish egg forms no skeleton, but undergoes a different development. When the sea urchin's egg is fertilized with starfish sperm, the egg always develops into a pluteus, never into the corresponding starfish form. If we exam- ine the adult forms of hybrids, however, we find that it makes no differ- ence which of the two forms furnishes the spermatozoon or the egg. This difference in the influence of the spermatozoon and egg upon the early embryonic and the adult form of the offspring is possibly due to a difference in the mass of the egg and the spermatozoon, the latter being as a rule much smaller than the former. As soon, however, as the embryo grows and its mass becomes large in comparison with that of the egg, the original difference in the hereditary effects of the two sex cells must diminish or disappear.
The foundations of a theory of heredity in the adult were laid by Gregor Mendel in his treatise on the hybrids of plants, and this theory is atomistic in its character. He showed that certain simple character- istics of plants, e.g. the round or angular form of the seeds of peas, or the color of their endosperm, must already be represented in the germ by definite determinants. His experiments on the hybridization of various forms of peas indicate that each hybrid contains two kinds of sexual cells, one possessing the determinants for the discriminating fatherly characteristic only, the other for the discriminating motherly characteristic. Both kinds of sexual cells seem to exist in equal numbers in such a hybrid. A similar fact has been discovered in other cases of hybridization, although it does not hold good for all cases. Hugo de Vries and others have begun to build up the physiology of heredity on the basis of Mendel's discovery.
It is obvious that no theory of evolution can be true which disagrees with the fundamental facts of heredity. It is the merit of De Vries to have shown that a mutation of species can be directly observed in cer- tain groups of plants, and he has further shown that the changes occur by jumps, not gradually. This fact harmonizes with the consequence to be drawn from Mendel's experiments that each individual char- acteristic of a species is represented by an individual determinant in the germ. This determinant may be a definite chemical compound. The transition or mutation from one form into another is therefore only possible through the addition or disappearance of one or more
4 DYNAMICS OF LIVING MATTER
of the characteristics or determinants. If this view can be applied generally, it is just as inconceivable that there should be gradual varia- tions of an individual characteristic and intermediary stages between two elementary mutations, as that there should be gradual transitions between one alcohol and its next neighbor in a chemical series.
The fact that, as a rule, at a definite stage of development, larger masses of sexual cells are formed, is one of the automatic phenomena of development. The mechanism of this formation is unknown. Miescher tried to solve this problem in the salmon. In this animal the sexual cells seem to be formed at the expense of the substances of the muscles, and it was the disappearance of the muscles at the time the sexual organs began to grow which aroused Miescher's interest. But this seems to be after all of only secondary importance, inasmuch as with our present knowledge of the chemistry of living organisms it is immaterial whether the animal's own muscles furnish the material for the sexual cells or the muscles of the animal it devours. The real problem is, how it happens that at a certain stage in the development of the animal the sexual glands take away so much material from the blood. From our present knowledge we must suspect that the mechan- ism of such a process is a transformation of liquid constituents of the blood into solid constituents inside of those cells which show a rapid growth or a transformation into different compounds.
We possess a little more knowledge concerning sexual dimorphism. It has been known for a long time that it is possible to produce in plant lice (Aphis) either females exclusively, or both sexes, at desire. In bees and related forms, as a rule, only males originate from unfertil- ized eggs, and females only from fertilized eggs. It is known, more- over, that in higher vertebrates such twins as originate from the same eggs are also uniform in sex, while twins originating from different eggs may be different in sex. All that we know thus far concerning the origin of sex seems to indicate that the sex of the embryo is already determined in the unfertilized egg, or is determined very soon after the impregnation of the egg.
Sexual maturity is sooner or later followed by death. Is death determined just as automatically by the processes of development pre- ceding it, e.g. the maturation of the sexual products, as are these pro- cesses by the previous processes of development, or as is the develop- ment of the egg by the entrance of a spermatozoon? The fact that most higher animals, at least, die by bacterial infection, and that cer- tain plants, e.g. the Sequoia, which are more free from bacteria, can reach an almost fabulous age, renders the answer to this question some- what uncertain or prevents the generalization of an answer. . It is not
INTRODUCTORY REMARKS 5
impossible that further experiments on the egg may aid us in solving this problem. In certain forms, e.g. the starfish, the mature egg, if not fertilized, dies rapidly, while the fertilized egg continues to live. For this egg the act of impregnation is a life-saving act.
We shall now attempt to give a short sketch of the phenomena and problems of self-preservation similar to that just given of the phe- nomena of development. Among the phenomena of self-preservation are such facts as the existence of automatic mechanisms for the union of the sexual cells wherever there exist separate sexes, or the existence of automatic mechanisms for the deposition of eggs in places which furnish food for the young larvae, and so on. What do we know con- cerning the nature of these automatic mechanisms? Metaphysics has supplied us in these cases with the terms "instinct" and "will." We speak of instinct when an animal, apparently unconsciously, executes motions or actions which are necessary for the preservation of the in- dividual or the species; while we speak of will when motions are exe- cuted consciously. We call it instinct when the female fly deposits her eggs on meat on which the young larvae can feed. An analysis of the instinctive actions has yielded the result that the purposeful motions of animals frequently depend upon mechanisms which are a function of the symmetrical structure and the symmetrical distribution of irrita- bility on the surface of the body of the organisms. Symmetrical points on the surface of an animal have usually an equal irritability, i.e. if such spots are stimulated equally the same amount of motion, but in opposite directions, is produced. Points on the surface which are nearer the oral pole of the animal usually have a higher irritability than, or a different irritability from, points which are nearer the aboral pole. If lines of force, e.g. rays of light, current curves, lines of gravi- tation, lines of diffusion, strike one side of an animal in greater density than the other side, the tension of the muscles, or contractile elements, on both sides of the organism does not remain the same, and if the animal moves, a tendency to turn to one side must result. This will continue until symmetrical points on both sides of the animal are again struck at the same angle by the lines of force. As soon as this occurs the ten- sion of the muscles, or contractile elements, on both sides of the animal becomes equal, and there is no more reason why the animal should deviate toward the right or the left ; it will therefore continue to move in the original direction. This automatic orientation in a field of force toward or away from the center of force is called a tropism. It has been possible to dissolve a number of animal instincts into tropisms. The analysis of various tropisms has shown that there exists a great variety, and often a great complexity due to the combination of several
6 DYNAMICS OF LIVING MATTER
forms of tropisms in the same individual, and to the changes of these tropisms under the influence of internal and external factors. In these lectures we shall discuss some of the elementary tropisms and their role in the animal instincts and in the preservation of the indi- vidual and the species.
The will actions of animals, i.e. those motions which are executed consciously, will not be discussed here as I have already analyzed them in another book.* I will simply state here that I consider conscious- ness the function of a definite machine or mechanism, which we may call the mechanism of associative memory. Whatever the nature of this machine may be it has one essential feature in common with the phonograph, namely, that it reproduces impressions in the same chrono- logical order as that in which they were received. The mechanism of associative memory seems to be located - - in vertebrates - - in the cere- bral hemispheres. It follows from the experiments of Goltz that one of the two hemispheres is sufficient for all the phenomena of memory and consciousness. As far as the chemical or physical mechanism of memory is concerned, we have at present only a few vague data. H. Meyer and Overton have pointed out that substances which are easily soluble in fat are also, for the most part, strong anaesthetics, e.g. ether, chloro- form, etc., and that the ganglionic cells are especially rich in lipoids. It is possible that the mechanism of associative memory depends in part upon the properties and activities of the fatty constituents of the cerebral hemispheres. Another fact which may be of importance is the observation of Speck that if the partial pressure of oxygen in the air is lowered to below one third of its normal value, the fundament of mental activity, namely, memory, is almost instantly interfered with, and total loss of consciousness rapidly follows.
In those animals which possess the mechanism of associative memory, a number of the automatically regulated processes may become con- scious. Respiration is purely an automatic process, yet we may at any time become conscious of it. This has misled a number of authors to believe that such automatic processes as in ourselves may become conscious must be accompanied by consciousness in any animal in which they occur. These authors overlook the fact that the automatic mech- anisms of self-preservation must occur in every organism, while an apparatus for associative memory may be found only in a limited num- ber of organisms. Without such an apparatus, consciousness is impos- sible. The fact that a physiological process may become conscious in ourselves does not therefore prove that it is accompanied by conscious- ness in every organism.
* Loeb, Comparative Physiology of the Brain and Comparative Psychology. G. P. Put- nam's Sons, New York.
LECTURE II
CONCERNING THE GENERAL CHEMISTRY OF LIFE PHENOMENA
i. HISTORICAL REMARKS
TO-DAY every one who is familiar with the field of chemical biology acknowledges the fact that the chemistry of living matter is not spe- cifically different from the chemistry of the laboratory. We owe the certainty of this fact essentially to three publications, which may be mentioned briefly. The first contained the proof furnished by Lavoi- sier and La Place in 1780, that animal heat is produced by a process of slow combustion, and that for a certain amount of heat produced a certain amount of oxygen is consumed in the production of CO2. A measurement of the quantity of COs formed and the amount of heat produced gave approximately identical results in the case of a burning candle and a living guinea pig.*
A second step in this direction was taken when Woehler showed that an organic substance like urea, which is a product of metabolism, can be made artificially in the laboratory. "j" To-day so many of the compounds produced in the living body can be produced artificially that we can hardly understand that in 1828 Woehler's discovery was considered sensational.
The discovery of Lavoisier and La Place left a doubt in the minds of scientists as to whether after all the dynamics of oxidations and of chemical reactions in general is the same in living matter and in inani- mate matter. The oxidation of food stuffs could indeed be imitated outside the body, but only at such temperatures as were incompatible with life; phenomena of digestion could be imitated, but only with the aid of acids too strong for life to continue. The way out of the difficulty was shown in a remarkable article by Berzelius.^ He pointed out that in addition to the forces of affinity, another force is active in
* Lavoisier et De la Place, Memoire sur la Chaleur, 1780. (Euvres de Lavoisier, Vol. 2. (Also in Ostwald's Klassiker der Naturwissenschaften, Nr. 40.)
f Woehler, Ueber kunstliche Bildung des Harnstoffs. Poggendorfs Annalen, Vol. 12, p. 253, 1828.
I Berzelius, Einige Ideen iiber eine bei der Bildung organischer Verbindungen in der lebenden Natur wirksame aber bishtr nicht bemerkte Kraft. Berzelius u. Woehler, Jahres- bericht, 1836.
7
8 DYNAMICS OF LIVING MATTER
chemical reactions: this he called catalytic force. As an example he used Kirchhoff's discovery of the action of dilute acids in the hy- drolysis of starch to dextrose. In this process the acid is not consumed, hence Berzelius concluded that it did not act through its affinity, but merely by its presence or its contact. Another instance quoted by Berzelius was the decomposition of EL-Ch which had been investigated by Thenard. In acid solution this body is stable ; in alkaline solution, or in the presence of platinum, silver, or gold, or in the presence of fibrin of the blood, it is rapidly decomposed. In this decomposition apparently neither the fibrin, the gold, nor the platinum acted through the force of affinity, but catalytically. He then suggests that the spe- cific and somewhat mysterious reactions in living organisms might be due to such catalytic bodies as act only by their presence, without being consumed in the process. He quotes as an example the action of diastase in the potato. "In animals and plants there occur thousands of catalytic processes between the tissues and the liquids." The idea of Berzelius has proved fruitful, and the catalytic agencies which in his opinion are responsible for the characteristic reactions in living matter are the enzymes of modern biological chemistry. In some details, however, Berzelius's idea was erroneous. We now know that we have no right to assume that the catalytic bodies do not participate in the chemical reaction because their quantity is found unaltered at the end of the reaction. On the contrary, we shall see that it is probable that they can exercise their influence only by participating in the reaction, and by forming intermediary compounds, which are not stable. The catalyzers may be unaltered at the end of the reaction, and yet participate in it.
In addition we owe to Wilhelm Ostwald* the conception that the catalyzer does not as a rule initiate a reaction which otherwise would not occur, but only accelerates a reaction which otherwise would indeed occur, but too slowly to give noticeable results in a short time.
Thus the existence of catalytic agencies, the so-called enzymes in living matter, explains the fact that chemical changes may occur very rapidly in the body at a comparatively low temperature and at a prac- tically neutral reaction. Catalyzers are used extensively in chemical factories, e.g. in the manufacture of sulphuric acid, so that it is impos- sible to see in their presence in living matter a specific difference between the chemistry of living and inanimate nature. The only difference is, perhaps, that living matter manufactures its own catalyzers. This, however, is part of that peculiarity mentioned in the introductory lec- ture, that living machines possess the peculiarity of automatically pre- serving themselves.
* W. Ostwald, Lehrbuch Jer allgemeinen Chemie, Vol. II, 2d part, p. 248, 1902.
GENERAL CHEMISTRY OF LIFE PHENOMENA
2. REVERSIBLE ENZYME ACTIONS
Reversible chemical processes are characterized by the fact that the reaction comes to a standstill before all the substances on one side of the equation are transformed into those on the other side. The reason is, that a point is reached when, in the unit of time, the change in one direction is just as great as the change in the opposite direction. When this occurs we say that chemical equilibrium has been established. Inasmuch as, according to Ostwald, enzymes do not inaugurate chem- ical reactions, but only accelerate them, it follows that the action of enzymes must also be reversible, if the process itself is reversible. It is the merit of Arthur Croft Hill to have first shown a few years ago that an enzyme, maltase, which accelerates the hydrolysis of maltose into dextrose, also accelerates the synthesis of dextrose into maltose when added to pure dextrose. It is no exaggeration to say that Hill's paper entirely changed the conceptions of the physiology of metabolism. We shall return to Hill's experiments later, and first discuss the revers- ible action of a fat-splitting enzyme, lipase.
It had been known for some time that the pancreas secretes an enzyme which digests fat in the intestinal canal. Kastle and Loeven- hart* showed that in all tissues and liquids of the body which contain fat, lipase can be found. A watery extract of pancreas contains a sub- stance in solution which is capable of hydrolizing fats, i.e. of splitting fats into fatty acid and alcohol. Kastle and Loevenhart showed, more- over, that the watery extract of any tissue which contains fat acts in a similar way. The chemical character of this catalytic substance is unknown, except that its efficiency is rapidly destroyed if it is heated in water. According to Taylor, f it does itself, at a high temperature, undergo a hydrolytic cleavage.
Kastle and Loevenhart showed that lipase not only accelerates the hydrolysis of fat, but also the synthesis of fat, when added to a mixture of fatty acid and alcohol. Their experiments were made on ethylbuty- rate. If an extract from the pancreas or liver was added to a mixture of ethylalcohol and butyric acid, ethylbutyrate was formed. This reversible action of lipase has the effect that the process of digestion of fat can only be completed if the products of digestion are removed. In the intestine this occurs through absorption.
The velocity of the hydrolysis of ethylbutyrate was found to be in proportion to the concentration of the lipase. This explains the fact
* Kastle and Loevenhart, Am. Chem, Journal, Vol. 24, p. 491, 1900.
t A. E. Taylor, University of California Publications, Pathology, Vol. I, p. 33, 1904.
10 DYNAMICS OF LIVING MATTER
that those tissues which possess most fat, as a rule, also possess most lipase.* The more lipase a cell possesses the quicker it will be able to convert the fatty acid and alcohol, which diffuse or are absorbed into it from the blood, into fat; and hence more fatty acid and alcohol must diffuse into such a cell in the same length of time from the blood than into a cell with less lipase.
We understand how it happens that in times of abundant fat supply our tissues are able to store up fat, while in times of want fat disappears from them. If the blood receives no fat from the intestine, and if the other sources of fat formation, which we shall mention later, cease, the digestive effect of the lipase in the cells must outweigh its synthet- ical action.
The experiments of Kastle and Loevenhart were not tried with the fats occurring in the body. A. E. Taylor f has filled this gap by showing that the lipase extracted from the castor bean, which digests fats, is able to produce synthetically the triglyceride of oleic acid. The pro- cess is a very slow one, inasmuch as in six months only 3.5 g. of the fat were formed. Taylor concludes that in the body other agencies than mere enzymes must contribute toward the acceleration of the hydrolytic, as well as the synthetical processes. These conditions are, however, not of a vitalistic character, but may be due to the pres- ence of certain other substances. Thus Hewlett has recently found in Taylor's laboratory that the addition of lecithin to lipase accelerates the hydrolysis of fat considerably. Taylor found also that the lipase from the castor bean cannot synthetize every fat, but only the triglycer- ide of oleic acid. Experiments with palmitic and stearic acid and glycerine as an alcohol gave negative results, as gave also experiments with oleic acid and mannit or dulcit as alcohol. This is in harmony with the theory of intermediary reactions, which will be discussed later.
It is, however, worth mentioning that fat may be produced in the body from carbohydrates, and that lipase. has, as far as we can tell, nothing to do with this mode of fat formation. The most striking case of such an origin of fat is found in the leaves of the olive tree, which synthetize it from the carbohydrates formed from the CO2 of the air. The fact that a reduction must form part of the process of the forma- tion of fat from carbohydrates may explain why so often a hypertrophic heart has a tendency to fatty degeneration, inasmuch as the hyper- trophic heart is as a rule an overworking heart, and is thus liable to suffer from lack of oxygen.
* Loevenhart, Am. Jour. Physiology, Vol. 6, p. 331, 1902.
t A. E. Taylor, University of California Publications, Pathology, Vol. I, p. 33, 1904.
GENERAL CHEMISTRY OF LIFE PHENOMENA \ r
Neilson* has shown that the catalytic action of lipase on ethylbuty- rate can be imitated by platinum-black. The latter not only acceler- ates the hydrolysis, but also the synthesis of ethylbutyrate. Kastle and Loevenhart found that certain poisons like hydrocyanic or sali- cylic acid weaken the action of lipase. Neilson found that these poisons act similarly on the digestion of ethylbutyrate by platinum-black.
The study of the reversible action of enzymes in the carbohydrate group is complicated by the fact that the digestion of starch to sugar occurs in a series of successive stages, and that apparently each stage requires a different catalyzer. According to Duclaux,f we possess specific enzymes for the transformation of solid into liquid starch ; the liquid starch is then split by amylase or diastase into a disaccharide, i.e. maltose. Maltose is split by the enzyme maltase into d-glucose. In case another disaccharide is formed in the place of maltose, other enzymes are required, e.g. in the case of cane sugar, invertase, whereby dextrose and laevulose are formed. In the animal body, glycogen is formed in the place of starch. It is obvious that the synthesis of dex- trose into glycogen or starch requires the presence of several catalyzers. The action of these catalyzers must be studied individually.
Hillij: found that the hydrolysis of maltose in the presence of the enzyme maltase is retarded if dextrose is added to the maltose; that, moreover, the hydrolysis of maltose under the influence of maltase is complete only in very dilute solutions, while the reaction otherwise comes to a standstill before all the maltose is transformed into glucose. The following table shows the point at which the hydrolysis comes to a standstill at various concentrations: —
„ ,. PERCENTAGE OF MALTOSE WHICH
CONCENTRATION OF MALTOSE ,s spUT ,NTO GLUCOSE
40% 84%
10% 94-5%
4% 98%
2 % 99%
Hill convinced himself that if the enzyme maltase is added to a solu- tion of glucose, maltose is formed, and that, moreover, equilibrium is reached at the same point as in the case of hydrolysis. When he added fresh maltase to a 40 per cent solution of dextrose in one experiment, 14.5 per cent, and in another 15.5 per cent, maltose was formed. It has since been shown that the synthetical product formed in this case was isomaltose instead of maltose, but this slight deviation does not alter the principal result.
* Neilson, Am. Jour. Physiology, Vol. IO, p. 191, 1903. t Duclaux, Traite de microbiologie, Vol. 2, Paris, 1899. } A. C. H\\\,Joitr. Chem. Society, Vol. 73, p. 634, 1898.
12 DYNAMICS OF LIVING MATTER
Hill raises the question, as to whether or not a synthetical forma- tion of maltose under the influence of maltase may occur in the living cell. He points out that for such a result a high concentration of dextrose in the blood is by no means necessary; that it is sufficient if the product of the synthesis is removed immediately, possibly through a further synthesis into a higher carbohydrate by another enzyme in the cell. In this way the concentration of the maltose is kept at zero, and the tendency toward the establishment of the chemical equilibrium must favor the further synthesis.
The synthesis of sugar into glycogen is of general importance, inas- much as glycogen is the form in which the carbohydrates are stored in our liver and muscles. Max Cremer* immediately after the appear- ance of Hill's paper published the important observation, that the juice pressed out from yeast, which had previously been rendered free from glycogen, is capable of forming glycogen from sugar. When 10 per cent of a fermentable sugar was added to the juice, the latter gave the glycogen reaction after from twelve to twenty-four hours, but this result was not obtained in all cases.
It may perhaps not be unnecessary to call attention to the fact that in all the cases we have discussed the enzymes are soluble substances, which can be extracted from the cells, and therefore can exist inde- pendently of the life or structure of the cell from which they are obtained.
In the group of proteins we not only meet with the same difficulties which are found in the group of carbohydrates, but also with the addi- tional difficulty, that we know considerably less about the constitution and configuration of the various protein molecules than of the carbo- hydrates. These two conditions probably account for the fact that a direct reversion of the action of a hydrolytic enzyme has not yet been satisfactorily proven for proteins.
It is hardly necessary to mention especially the fact that hydro- lytic enzymes, e.g. of the type of trypsin, acting on proteins, are found not only in the intestine, but also in tissues, probably generally. Thus Salkowski has shown that if yeast cells or muscles are kept aseptically in an incubator, an autodigestion occurs in which leucin and tyrosin, i.e. typical end products of proteolysis, are formed.
Kutscher f has completed the proof by showing that, in addition to the acids, the other end products of proteolysis are formed in the autodigestion of yeast; namely, the hexonbases, e.g. arginin and lysin. He found, moreover, that in starving yeast the above-mentioned end
* Cremer, Ber. der deut. chem. Gesell., Vol. 32, p. 2062, 1899.
f Kutscher, Hoppe-Seyler's Zeitsch. fur physiolog. Chemie, Vol. 32, p. 59, 1901.
GENERAL CHEMISTRY OF LIFE PHENOMENA 13
products of tryptic hydrolysis are found in considerable quantities, while these products could be obtained in fresh and well-nourished yeast only in minimal quantities. In this he sees, and probably cor- rectly, an indication of the reversible action of the proteolytic enzymes. There is, however, one essential link missing, i.e. the proof that the hydrolytic action of the proteolytic enzymes is retarded, and finally inhibited, when the products of digestion are not removed.
R. O. Herzog has recently made an attempt to prove the reversible action of enzymes in a very interesting way upon the discussion of which, however, we cannot enter here.
3. RESPIRATION AS A CATALYTIC PROCESS. OXIDATION AND OXIDASES
By respiration we mean the taking up of oxygen and the giving off of CO2. We shall see later that the latter process can exist indepen- dently of the taking up of oxygen.
Since the days of Lavoisier and La Place the real problem of oxida- tion has consisted in the explanation of the fact that at the body tem- perature our food stuffs are not oxidized at all, or only infinitely slowly, outside the body, while in the body they are oxidized rapidly. The solution of the problem was found in the discovery of "oxidizing fer- ments" in living organisms. This conception is chiefly due to Moritz Traube,* who was also the first to recognize that the oxidations occur in the cells, and not, as had been assumed before, in the lungs or the blood.
Traube's idea was that there exist in the cells autoxidizable sub- stances, i.e. substances which bind loosely the free oxygen at a com- paratively low temperature, and which are capable of giving off their oxygen to disoxidizable substances, such as our food stuffs. It is obvious that Traube's idea of the action of an oxidizing ferment was that of intermediary reactions. He realized also that these oxidizing ferments exhibited no effects which could not be produced in inanimate nature, as the following quotation shows: "The ability to transfer oxygen . . . is found in many, even inorganic bodies. There are substances like nitrogenoxide, platinum, various coloring matters, copper salts, which are capable of transferring free oxygen upon neighboring substances." f
* M. Traube, Ueber die Beziehiing der Respiration zur Muskelthatigkeit und die Bedeu- tung der Respiration uberhaupt, 1861. Gesammelte Abhandlungen von M. Traube, p. 157, Berlin, 1899. (In this paper Traube showed also that the work of the muscle is normally done at the expense of carbohydrates. His arguments induced Pick and \Vislicenus to try the classical experiment by which this theory was proved.)
t M. Traube, Die Chemische Theorie der Ferment wirkttngen und der Chemismus der Res- piration, 1878. Gesammelte Abhandl., p. 384, Berlin, 1898.
I4 DYNAMICS OF LIVING MATTER
I think Jacquet* was the first to separate a "ferment of oxidation " from the living organism, and to obtain it in a watery extract from tissues. The oxygen of the air oxidizes benzylalcohol (C6HSCH OH) only slowly to benzoic acid (C6H5COOH) at body temperature; the
OTT
same is true for the oxidation of salicylaldehyde C6H4rn-H-to salicylic
OH acid C6H4TT' Schmiedeberg had already shown that the animal
tissues accomplish this oxidation comparatively rapidly. Jacquet proved that this energetic oxidation of benzylalcohol is not dependent upon living protoplasm, as he found that it occurred also in dead tissues. Tissues poisoned with carbolic acid continue to accelerate these oxida tions, and even tissues which have been preserved in alcohol are capable of so doing. Nor are these oxidations dependent upon the structure of the cells, as watery extract from the cells also had oxidative effects upon benzylalcohol. The action of the oxidizing enzymes is annihi- lated when they are heated to a temperature of about 100° — pos- sibly through a hydrolysis of the enzyme itself.
Engler and Wild f have found that there exists a group of substances which behave like Traube's autoxidizable substances. These sub- stances have the peculiarity of easily forming peroxides of the following
./<?
<!>
These peroxides are capable of giving off one atom of oxygen to dis- oxidizable substances. Through the loss of this atom of oxygen the peroxides are transformed into oxides. This view is supported by an important observation which was first made by van't Hoff and Jorissen. If the quantity of oxygen which disappears in the oxidation of a known quantity of a disoxidizable substance is measured, it is found to be in most cases exactly twice as large as the quantity required for the oxida- tion of the disoxidizable substance. % This finds its explanation in the fact that for every molecule of oxygen which is taken up by the autoxi- dizable substance, only one atom is transferred to the disoxidizable substance.
The view of Engler and Wild is also supported by the investigations of Kastle and Loevenhart § on the oxidizing effects of plant tissues, e.g. the potato, and their watery extracts. They found that organic
* Jacquet, Arch, fur experimentelle Pathologie und Pharmakologie, Vol. 29, p. 386, 1892. f Engler und Wild, Ber. der deutsch. chem. Gesellsch., Vol. 30, p. 1669, 1897. Engler und Weissberg, Kritische Studien ilber die Vorgange der Autoxydation, Braunschweig, 1904. J See also Manchot, Zeitsch. fur anorganische Chemie, Vol. 27, p. 420, 1901. § Kastle and Loevenhart, Am. Chem. Journal, Vol. 26, 1901.
GENERAL CHEMISTRY OF LIFE PHENOMENA 15
peroxides, e.g. benzoylperoxide, phthalylperoxide, and succinylperox- ide, or inorganic ones, like lead peroxide and manganese peroxide, pro- duced the same blue color in the tincture of guaiacum as the tissues of plants, or watery extracts from the same. The production of this blue color is due to oxidation. The same authors showed, moreover, that the same poisons which in plants prevent the action of oxi- dases - - this is the name given to the enzymes of oxidation — prevent also the oxidizing action of the above-mentioned organic and inorganic peroxides upon tincture of guaiacum. Kastle and Loevenhart there- fore conclude that the oxidases, or oxidizing enzymes, in the tissues of animals and plants, are organic peroxides.
Here we meet with a difficulty, however. Oxidations occur inces- santly on a large scale in the living body, especially at the temperature of the warm-blooded animals. The peroxides, however, are not capable of transferring oxygen in unlimited quantities to disoxidizable sub- stances, but as soon as a peroxide molecule has given off one atom of oxygen, its oxidizing power is at an end. This difficulty can be over- come in the following two ways: it is possible that new autoxidizable substances are formed incessantly in the body; the second possibility is the existence of a second class of oxidizing enzymes which act more in the sense of true enzymes than the peroxides, inasmuch as they are able to take up and give off oxygen indefinitely. Haemoglobin is capable of binding and setting free oxygen indefinitely, but it is not capable of transferring its oxygen to disoxidizable substances, and hence does not act as an oxidase.
The question of localization of the oxidizing enzyme in the cell was raised by Spitzer.* He found that the proteins which can be extracted from the cells do not possess the qualities of Jacquet's oxidase, but that these qualities are found in such extracts as contain nucleoproteids. The nucleoproteids are typical constituents of the cell nucleus, and they differ from the proteins proper in that they contain PO4 and Fe. Spitzer was able to show, moreover, that of the products of cleavage of nucleoproteids only those constituents were able to act as oxidases which contained the Fe group. Two years ago I pointed out that if Spitzer's researches are correct, the cell nucleus must be regarded as the essential respiratory or oxidizing organ of the cell.f
It is possible that we have two groups of oxidizing catalyzers in the tissues : first, those of the type of peroxides which are possibly present in the protoplasm; and second, substances which can act indefinitely as oxidases, and are found in the nucleus. There are, indeed, a number
* Spitzer, Pflitgtr's Archiv, Vol. 67, p. 615, 1897.
t Loeb, Zeitsch. fur Entwickelungsmechanik, Vol. 8, p. 689, 1899.
1 6 DYNAMICS OF LIVING MATTER
of facts which seem to harmonize with the idea that the nucleus is the main oxidizing organ of the cell. Processes of regeneration demand oxygen. Nussbaum* has shown that if an Infusorian be cut in such a way as to divide it into two pieces, one containing the nucleus, and one without any nuclear matter, only the former is capable of regenerat- ing the lost parts. The other piece lives but a comparatively short time and is not capable of regeneration ; it dies under symptoms which are rather similar to death from lack of oxygen.
Ralph Lillie t tried to test the idea that the nucleus is the main oxi- dizing organ with the aid of staining substances which diffuse into the cell, and change color when they are oxidized. He worked with the cells of the blood, the liver, and the kidneys of frogs, and found that the oxidation seems to occur most rapidly in the nucleus and on its surface. He found, moreover, that the oxidations were most rapid in those organs and those regions of organs where the nuclei were densest.
It has been noticed that if cells containing chlorophyll are deprived of their nucleus, they keep alive longer if exposed to the light than if kept in the dark. This may be connected with the fact that in the light the chlorophyll is capable of liberating the oxygen from the CO2.
It is stated, as a rule, that the role of the oxygen is to supply the energy for the production of heat and of mechanical work, but it is evident that this statement does not take into consideration the fact that sessile plants, in which the loss of heat does not need to be com- pensated by a production of heat, and in which no energy is spent in mechanical motion, are in need of oxygen and possess oxidases. With the exception of a limited number of anaerobic bacteria, the statement can be made that oxidations and the presence of oxidases is a general characteristic of living matter. There are, however, other vital processes which are more general than those of locomotion and heat production, which also require oxygen, i.e. cell division and growth. Pasteur made the fundamental discovery J that with lack of oxygen the yeast cells continue to produce lively fermentations of sugar, - - in fact, Pasteur stated that their fermentative action is more energetic than in the presence of oxygen, - - but that they grow and multiply very little or not at all. If, however, oxygen is added freely, the yeast cells multiply and grow considerably, provided the necessary nutritive salts are present. According to Hoppe-Seyler and Duclaux, the absence of oxygen favors the formation of the catalyzer for the alcoholic fermenta-
* M. Nussbaum, Arch, fur mikroscop. Anatomic, Vol. 26, 1 886. t Ralph Lillie, Am. Jour. Physiology, Vol. 7, p. 412, 1902. \ Pasteur, Etudes sur la Iricre, Paris, 1876.
GENERAL CHEMISTRY OF LIFE PHENOMENA 17
tion in the yeast cell, the zymase ; * but we are here chiefly concerned with the fact which nobody has failed to confirm, that the presence of oxygen favors cell division and growth in yeast cells; that without oxygen these processes soon come to a standstill. The same is true for animals. I made a large number of experiments on the effects of lack of oxygen on the newly fertilized eggs of sea urchins and fishes (Fundulus and Ctenolabrus).t The eggs were kept in small Engel- mann gas chambers through which a current of hydrogen was sent to drive out the oxygen and the CO2 formed by the eggs. In the eggs of sea urchins and Ctenolabrus, the segmentation stopped in less than an hour after the beginning of the current of hydrogen. When the air was again admitted, the eggs began to divide, provided they had not remained too long without oxygen. The eggs of Fundulus, also a marine fish, do not respond as quickly, inasmuch as it required about twelve hours before they stopped segmenting in the current of hydrogen. Similar results were obtained by Godlewski | on the eggs of frogs. As far as growth and regeneration are concerned, I have found that without oxygen both are impossible in Hydroids (Tubularia).§ In plants, conditions are the same; seeds require a comparatively abundant supply of oxygen for germination. || The question arises, as to what connection exists between the oxidations in living tissues, and cell divi- sion and growth. We cannot answer this question as we do not know into which form of energy chemical energy must be transformed in order to produce cell division and growth. But another point may be settled. For the process of growth an increase in the quantity of living matter is required, and this requires synthetical processes. Schmiedeberg has called attention to the fact that oxygen is especially fitted to serve as a connecting link between organic radicals, and that through the intervention of oxygen a great many syntheses in the body may occur. He mentions, as an example, the combination in which sulphuric acid may appear in the urine. When a dog is fed with benzol, the benzol appears in the urine as benzolsulphate, provided that enough free oxygen is present. According to Schmiedeberg, this synthesis occurs in the following way : —
+ 2 C6H6 + 02 = 2 SO- + 2 H O.
* H. Buchner denies that lack of oxygen increases the rate of alcoholic fermentation by yeast, although the facts seem to speak in favor of Pasteur's statement. E. Buchner, H. Buchner, und M. Hahn, Die Zymasegarung, Miinchen und Berlin, 1903.
t Loeb, Pfiuger's Archiv, Vol. 62, p. 249, 1895.
\ Godlewski, Zeits. flir Entwickelungsmechanik, Vol. II, p. 585, 1901.
§ Loeb, Untersuchungenzur physiologischen Morphologic der Tiere, II, Wiirzburg, 1891.
|| M. Traube, Gesammelte Abhandlungen, p. 148. C
1 8 DYNAMICS OF LIVING MATTER
It is quite possible that the idea of Schmiedeberg will prove ex- tremely fertile in further work in this direction.*
But even this addition does not exhaust the role of oxygen in life phenomena; there are indications which make it appear as though the oxygen acted as a protective substance. When respiration is interrupted for but a short time in mammals or birds, loss of conscious- ness, and very soon death, follow. Lack of oxygen therefore affects first the cerebral hemispheres, and especially the ganglion cells. The blood supply to the nerves is either lacking, or is so meager that we must conclude that the functions of the nerves require very little oxygen. The experience in drowning shows that lack of oxygen leads in a limited number of minutes to death. In the case of death through lack of oxygen, the respiratory ganglia evidently undergo irreversible changes, which make attempts at revival futile. The heart retains its irritability much longer than the respiratqry ganglia. Kuliabko has recently shown that the heart of a child can be caused to beat, or to show fibrillary contractions, eighteen hours after death. f This shows that death was not due to the inability of the heart to resume its beat, but to the inability of the respiratory ganglia to work properly. It has often been observed in my laboratory that in dying larvae of fish or frogs the respiration stopped sooner than the heartbeat. The irreversible changes which mark death occur with unequal rapidity in the various tissues of an animal.
When the egg of a Fundulus is kept in an atmosphere of pure hydro- gen, segmentation comes to a standstill in about twelve hours, but permanent death occurs much later. The time required for permanent death to occur in the absence* of oxygen is the longer the younger the egg. When eggs were deprived of oxygen immediately after fertiliza- tion, they remained alive in the absence of oxygen for three or four days (at a temperature of about 22°). When embryos of three days were exposed to the same condition, death occurred in about thirty- four hours.
The higher the temperature the sooner lack of oxygen seems to cause death. I make this statement on the basis of casual observations, and I do not know whether or not any definite experiments exist on this point; should further investigation confirm this idea, it would seem to indicate that the changes in the tissues which cause death are produced by noxious substances formed in the absence of oxygen. Araki \ found that in the active muscle dextrose and lactic acid are found when the
* O. Schmiedeberg, Archiv fur experiment. Pathologic und P/iarmakologie, Vol. 14, pp. 288 and 379, 1881.
t Kuliabko, P/tiger's Archiv, Vol. 97, p. 539, 1903.
\ Araki, Zeitsch. fur physiol. Chemie, Vol. 15, p. 335, 1891.
GENERAL CHEMISTRY OF LIFE PHENOMENA 19
muscle works in lack of oxygen, while these substances are not found in the presence of abundant oxygen. They are probably formed in the latter case also ; but if atmospheric oxygen is present, are immediately oxidized, while in the case of lack of oxygen they remain in the muscle. This may serve as an example of the fact that metabolism in the presence of abundant oxygen is different from that in lack of oxygen. Richet and Broca have shown that if an excised muscle is stimulated in the presence of oxygen until fatigue sets in, it will recover, but not if stimu- lated in the absence of oxygen. It stands to reason that in the latter case the recovery is prevented by noxious substances which would have been oxidized and rendered harmless in the presence of oxygen.
Bacteriology furnishes examples of the fact that in the case of lack of oxygen more virulent substances may be formed, or exist, than in the presence of atmospheric oxygen. Kastle quotes the statement that the toxin of the diphtheria bacillus is weakened under the influence of light in the presence of free oxygen, while the light has no such effect in the absence of oxygen. Pasteur observed that cultures of the anthrax bacillus and of chicken cholera become less poisonous when exposed to the air. Recent experiments by Kastle and Elvove* have shown that substances which have a high reducing power are especially toxic, and these authors are inclined to assume that many toxins belong to the group of reducing poisons.
The fact that lack of oxygen is capable of producing irreversible changes, and thus death, is rendered more easily comprehensible through the direct observation of physical changes of living matter under such conditions. I have made such observations in the segmenting egg of a teleost fish, Ctenolabrus.f When these eggs are deprived of oxygen at the time they reach the 8 or 16 cell stage, it can be noticed that the membranes of the blastomeres are transformed into small droplets within half an hour or more, according to the temperature. These droplets begin to flow together, forming larger drops. Figures i to 5 show the successive stages of this process. When the eggs are exposed to the air in time, segmentation can begin again ; but if a slightly longer time is allowed to elapse, the process becomes irreversible and life becomes extinct. Such clear structural changes cannot be observed in the eggs of other animals under the same conditions. Are these changes of structure (apparently liquefactions of solid elements) responsible for death under such conditions? In order to obtain an answer to this question, I investigated the effect of the lack of oxygen upon the heart- beat of the embryo of Ctenolabrus. The egg of this fish is perfectly trans-
* Kastle and Elvove, Am. Chetn. Journal, Vol. 31, p. 195, 1904. t Loeb, Pfluger's Archiv, Vol. 62, p. 249, 1895.
2O
DYNAMICS OF LIVING MATTER
parent and the heartbeat can easily be watched. When such eggs are put into an Engelmann gas chamber and a current of pure hydrogen is sent through, the heart may cease to beat in fifteen or twenty minutes; the heart stops beating suddenly before the number of heartbeats has diminished noticeably: it ceases beating before all the free oxygen may have had time to diffuse from the egg. In one case the heart beat ninety times per minute before the hydrogen was sent through; four minutes after the current of hydrogen had passed through the gas chamber, the rate of the heartbeat was eighty-seven per minute, three
FIG. i.
FIG. 2.
FIG. 3.
FIG. 4.
FIG. 5.
FIGS. 1-5. Liquefaction of the cell walls of the egg of Ctenolabrus due to lack of oxygen. (From Nature.) The eggs were exposed to a current of hydrogen. The liquefaction of the cell walls and the formation of droplets began when the egg was in the 8 cell stage (Fig. 2). These droplets fuse into larger drops and finally nothing but these drops indicates the existence of the germinal disk. Figures 2, 3, and 4, are drawn in intervals of 15 minutes.
minutes later it was seventy-seven, and then the heart suddenly stopped beating. It is hard to believe that this standstill could have been caused by lack of energy. Hydrolytic processes alone could furnish sufficient energy to maintain the heartbeat for some time, even if all the oxygen had been used up. The suddenness of the standstill at the time when the rate had hardly diminished seems to correspond much more to a sudden collapse of the machine ; it might be that liquefactions or some other change of structure occurs in the heart or its ganglion cells, comparable to that which we mentioned before. In another fish, Fundulus, where the cleavage cells undergo no visible changes in the case of lack of oxygen, the heart of the embryo can continue to beat for about twelve hours in a current of hydrogen. In this case the rate of the heartbeat sinks during the first hour in the hydrogen current
GENERAL CHEMISTRY OF LIFE PHENOMENA 21
from about one hundred to twenty or ten per minute : then it continues to beat at this rate for ten hours or more. In this case one might believe that during the period of steady diminution of the tension of oxygen in the heart (during the first hour), the heartbeat sinks steadily, while it keeps up at a low but steady rate as long as the energy for the beat is supplied solely by hydrolytic processes ; but there is certainly no change in the physical structure of the cells noticeable in Fundulus, and consequently there is no sudden standstill of the heart.
Budgett has observed that in many Infusorians visible changes of structure occur in the case of lack of oxygen ; * as a rule the membrane of the Infusorian bursts or breaks at one point, whereby the liquid contents flow out. Hardesty and I found that Paramcecium becomes more strongly vacuolized when deprived of oxygen, and at last bursts. Amcebas likewise become vacuolized and burst under these conditions. Budgett found that a number of poisons, such as potassium cyanide, morphine, quinine, antipyrine, nicotine, and atropine, produce struc- tural changes of the same character as those described for lack of oxygen. As far as KCN is concerned, Schoenbein had already observed that it retards the oxidation in the tissues, and Claude Bernard and Geppert confirmed this observation. For the alkaloids, W. S. Young has shown that they are capable of retarding certain processes of autoxidation. This accounts for the fact that the above-mentioned poisons produce changes similar to those observed in the case of lack of oxygen.
I.
4. THE PRODUCTION OF CO2 THROUGH ENZYMES
It seems that organisms are pretty generally capable of producing CO2 from certain organic compounds, without the presence of free oxygen. The classical case of the production of CO2 through a process of cleavage is the alcoholic fermentation of sugar under the influence of yeast cells. In this case one molecule of dextrose is split into two molecules of CO2 and two of ethylalcohol. The process occurs in the absence of oxygen as well as in its presence, or, according to Pasteur, even better in the absence of oxygen than in its presence. The catalyzer in this case is an enzyme, the zymase, which Buchner succeeded in liberating from the yeast cell. This discovery is of special interest, as for years it was impossible to separate this enzyme from the cell. Pasteur even went so far as to maintain that the process of alcoholic fermentation was of an altogether different kind from that of the inver- sion of cane sugar, as the latter was due to an enzyme, soluble in water, which could easily be extracted from the cell ; while this was not so in
* Budgett, Am. Jour. Physiology, Vol. I, p. 210, 1898.
22 DYNAMICS OF LIVING MATTER
the case of the alcoholic fermentation. Buchner * showed that Pasteur f was mistaken, and that the only difference was a technical one, inasmuch as it requires a greater pressure to force the zymase out of the yeast cell than other enzymes, e.g. invertase. Through the discovery of Buchner, Biology was relieved of another fragment of mysticism. The splitting up of sugar into CO2 and alcohol is no more the effect of a "vital principle" than the splitting up of cane sugar by invertase. The history of this problem is instructive, as it warns us against considering problems as beyond our reach because they have not yet found their solution. The enzyme for the alcoholic fermentation of sugar is not confined to yeast, but seems to occur more generally. Thus Pasteur had already mentioned that certain kinds of fruit in the absence of air produced alcohol besides CO2 ; it is possible, however, that in fruits alcohol forms only an intermediary product which is oxidized further, or undergoes further changes, in the presence of oxygen, while it remains unaltered in the absence of oxygen. Godlewski and Polzeniusz demon- strated an alcoholic fermentation in seeds of plants which germinated in the absence of oxygen. J The fact that in these cases the alcoholic fermentation occurs only in the absence of oxygen seems to favor Pas- teur's statement, that lack of oxygen increases the velocity of the fermen- tative action of yeast in the case of alcoholic fermentation.
Stoklasa § and his pupils showed that in a number of plants and germinating seeds CO2 and alcohol are formed in the absence of oxygen in the same proportion in which these substances appear in the alcoholic fermentation of sugar, and that just as much dry substance from the plants disappeared as corresponded to the sugar that was fermented. They succeeded in extracting from these plants (roots of sugar beets, potatoes, seeds of peas, seedlings of barley) an enzyme which acted like Buchner's zymase.
As far as animals are concerned, G. von Liebig had already shown that the muscles continue to produce CO2 in the absence of air, and that the production of CO2 is increased when the muscle becomes active. Hermann repeated these experiments, and made sure that the muscle continues to produce CO2, even if it does not contain any free oxygen which can be extracted in the vacuum. Inasmuch as glycogen disappears during activity, it looks as if the CO2 formed in the absence
* E. Buchner, H. Buchner, und M. Hahn, Die Zvmaseivirkung, Munchen und Berlin, 1903.
t Pasteur, Etudes sur la biere, Paris, 1876. Annales de chimie et de physique, Vol. 58, p. 323, i860. See also Liebig, Ueber Gahrung, uber Quelle der Muskelkraft und Ern'dh- rung, Leipzig und Heidelberg, 1870.
t Godlewski et Polzeniusz, Btdletin de rAcad. de Cracovie, 1901.
§ Stoklasa, Hofmeister 's Beitrage zur chemischen Physiologie, Vol. 3, p. 460, 1902. Pfluger's Archiv, Vol. 101, p. 311, 1904.
GENERAL CHEMISTRY OF LIFE PHENOMENA 23
of oxygen were produced from glycogen or sugar. If this be correct, a process must, under such conditions, occur which bears a certain resemblance to the alcoholic fermentation of sugar, although in the place of alcohol another product may be formed.
It was remarkable that in spite of these observations and the well- known fact that the muscles are the main seat for the production of CO2, nobody was able to show that a muscle extract is able to decompose dextrose. This gap seems to have been filled recently by Cohnheim.* Von Mering and Minkowski had found that extirpation of the pancreas causes the most serious type of diabetes. This fact and subsequent discoveries suggested that the pancreas must secrete a substance into the blood, by which the oxidation or cleavage of sugar is accelerated. The place of the decomposition of the sugar must evidently be the muscles. Starting from these arguments, Cohnheim tested whether the muscle and the pancreas together do not contain a glycolytic power which neither contains alone. Cohnheim succeeded in showing that, by a process similar to that used by Buchner, liquids free from cells can be extracted from muscles and pancreas which, if mixed, cause dextrose, when added to the mixture, to disappear from it. The liquid extract from the muscle or the pancreas alone has no such action. "This observation may be analogous to the discovery of Pawlow, that the mucous membrane of the intestine secretes a substance, enterokinase, which activates the trypsinogen of the pancreatic juice, or to the observa- tion made in the case of the hemolysins by Bordet and Ehrlich, that this process requires two different substances, - - the so-called comple- ment and Zwischenkoerper."
All these facts show that the production of CO2 in the body may occur without the presence of free oxygen, that these processes are evidently accelerated by special enzymes.
In regard to the energetics of these processes, it may be said that the energy which can be obtained by the complete oxidation of dextrose is about ten times as large as that which can be obtained from it by alcoholic fermentation. Bunge has calculated that it would not be possible for a man to do the average amount of muscular work at the expense of energy derived solely from the alcoholic fermentation of sugar. For this process the oxidation of dextrose is necessary, and therefore the presence of oxygen is required.
* O. Cohnheim, Hoppe-Seyler's Zeitsch. fur physiol. Chemie, Vol. 39, p. 336, 1903.
24 DYNAMICS OF LIVING MATTER
5. CONCERNING THE THEORY OF ENZYME ACTION
a. Stereochemical Attempts.
Liebig expressed the idea that ferments or catalytic substances, in general, were bodies in a process of decomposition, and that their condition of motion was communicated to the fermentable body. Schoenbein, Pasteur, and Traube showed the untenability of this view by pointing out that platinum or the yeast cell cannot be well considered as bodies in a condition of rapid decomposition. To-day we do not value it so highly if an author tries to explain phenomena by vague statements concerning the vibration of atoms. Now and then an author still makes the statement that "life is motion," but as Driesch has pointed out, this statement is about as valuable as the information that the philosopher Kant was a vertebrate.
Certain observations by Pasteur and Emil Fischer seemed, for a time at least, to arouse the hope that the theory of enzymatic action might be found in the field of stereochemistry. Pasteur had observed in the beginning of his scientific career that while the right-handed tartaric acid is easily decomposed by fermentation, the same was not true for left-handed tartaric acid. Pasteur assumed that the geometri- cal shape of the tartaric acid molecules exercises an influence upon the fermentability of their solution. From the point of view of stereo- chemistry the forms of the right- and left-handed tartaric acid molecules show the same relation of symmetry as our right and left hand, or some asymmetrical object and its mirror image. It appears from Pasteur's biography that he expected important discoveries to be made concern- ing the nature of life from this relation between the form of the asym- metrical molecules and their biological effect. Pasteur's discovery found little consideration until it was taken up by E. Fischer.* He found that the alcoholic fermentation through yeast, e.g. Saccharomyces cerevism, depends upon the molecular constitution and configuration of the various sugars. Saccharomyces cerevisia brings about an alcoholic fermentation only with triose and hexose, possibly also with nonose. Tetrose, pentose, heptose, and octose undergo no alcoholic fermentation with this form of yeast. It is evident that only those monosaccharides are fermentable by yeast which have three or a multiple of three atoms of carbon in the molecule. As far as the influ- ence of the Stereochemical configuration of the sugars and glucosides upon their fermentability is concerned, a similar relation as that found by Pasteur exists. Of the hexoses or hexaldoses there exist sixteen
* E. Fischer und Thierfekler, Berichte der deutsch. chem. Gesellsch., Vol. 27, pp. 2036 and 2985, 1894. Fischer, Zeitsch. fur physiolog. C/iemie, Vol. 26, p. 60, 1898.
GENERAL CHEMISTRY OF LIFE PHENOMENA 2$
stereoisomeres, of which, however, only three are fermentable by Saccharomyces cerevisia, namely, (/-glucose, (/-mannose, and (/-galactose. The relation between fermentability and configuration will be rendered a little clearer by the following diagrams : —
H H OH H
1. CH2OH C C C C COH ^/-glucose (fermentable)
OH OH H OH
OH OH H OH
2. CH2OH C C C C COH /-glucose (nonfermentable)
H H OH H
H H OH OH
3. CH2OH C C C C COH ^-mannose (fermentable)
OH OH H H
HO HO H H
4. CH2OH C C C C COH /-mannose (nonfermentable)
H H OH OH
H OH OH H
5. CH2OH C C C C COH ^-galactose (fermentable)
OH H H OH etc.
Fischer gave a metaphorical illustration of these facts which was taken rather literally by some biologists, and which has had a decided influence upon the formation of biological hypotheses. For this reason it may be mentioned here. "Inasmuch as the enzymes are in all proba- bility proteins, and inasmuch as the latter are formed synthetically from carbohydrates, it is probable that their molecules also have a dissym- metrical structure, and one whose dissymmetry is, on the whole, com- parable to that of hexoses. Only if enzyme and fermentable substance have a similar geometrical shape can the two molecules approach each other close enough for the production of a chemical reaction. Meta- phorically we may say that enzyme and glucoside must fit into each other like key and lock."
Max Cremer* has expressed the idea that in all these cases in reality one and the same sugar undergoes alcoholic fermentation; namely, (/-glucose. It is, indeed, not impossible that the alcoholic fermentation of c?-mannose and d-galactose occurs in two stages, the first stage con- sisting in the transformation of these two substances into dextrose. This would be in harmony with the observations concerning the alcoholic fermentation of disaccharides, e.g. cane sugar, which must first be
* Max Cremer, Zeitsch.fur Biologic, Vol. 32, p. 49, 1895.
26 DYNAMICS OF LIVING MATTER
hydrolized into dextrose and laevulose by a special enzyme, namely, invertase. The dextrose and laevulose undergo alcoholic fermentation by zymase. It has indeed been shown by Lobry de Bruyn that d-man- nose, d-galactose, and d-fructose can easily be transformed into dextrose. If this view is correct, the relation between stereochemical configuration and fermentability is only an apparent one.
b. The Theory of Intermediary Reactions.
The facts that the catalyzer is unaltered at the end of the reaction, and that apparently a small quantity of the enzyme can catalyze infi- nitely or comparatively large quantities of the fermentable substance, harmonize with the assumption of intermediary reactions. As an example of the theory of intermediary reactions of enzymes, the oxida- tion of sulphurous acid to sulphuric acid in the presence of nitric acid may be mentioned. Without the presence of nitric acid, sulphuric acid is oxidized but slowly ; but in the presence of nitric acid the latter gives off oxygen to the sulphurous acid, and afterward takes up oxygen again. The intermediary processes seem to be rather complicated and are perhaps not fully known, but it seems that the successive transfer of oxygen from the nitric acid to the sulphurous acid and the reoxida- tion of nitrous acid to nitric acid occur with much greater velocity than the direct oxidation of sulphurous acid by free oxygen.
Inasmuch as the chemical nature of the enzymes is unknown, it is impossible to ascertain positively whether or not their efficiency is due to intermediary reactions. But there are inorganic catalyzers whose action resembles that of the enzymes, e.g. platinum and other metals, like iridium, osmium, silver, etc. We have already mentioned the fact that platinum acts like lipase in the hydrolysis of ethylbutyrate. The oxidation of alcohol to acetic acid is accelerated by Bacterium aceti as well as by platinum.* A striking analogy between the catalytic action of platinum and enzymes exists in regard to hydrogenperoxide. O. Loew has shown that there is a specific enzyme catalase, which is very general, and which accelerates the decomposition of H2O2. If this decomposi- tion occurs in the presence of oxidizable substances, the latter, too, are often oxidized. As a rule the process is represented by the equation —
H2O2 = H2O + O
The free atom of oxygen is said to be responsible for the oxidizing action of H2O2, although Kastle and Loevenhart have expressed a different view.f They quote a number of observations made by previous authors,
* Bredig enumerates these analogies in his interesting pamphlet on " Anorganischi Fermente" Leipzig, 1901.
t Kastle and Loevenhart, Am. Chem. Journal, Vol. 29, p. 563, 1903.
GENERAL CHEMISTRY OF LIFE PHENOMENA 27
which indicate that H2O2 is capable of adding itself to a large number of compounds and forming bodies like the following: BaO2H2O2 or K2O22 H2O2. Jones and Carroll found that certain acids and salts show in water with solutions of H2O2 an abnormal depression of their freezing point. They conclude that in this case a combination between the molecules of the salts and the H2O2 is formed. From this and similar observations Kastle and Loevenhart draw the conclusion that those substances which like platinum accelerate the decomposition of H2O2, first combine with H2O2, and that this combination is unstable and rapidly falls apart into molecular oxygen, water, and the catalyzer, i.e. platinum. If this occurs in the presence of reducing bodies which do not act directly on H2O2, they may be oxidized in this process. The platinum may afterwards combine again with another molecule of H2O2, and the process be repeated.
These views find a nice confirmation through the investigation of the action of certain poisons like HCN on the decomposition of H2O2 by platinum or catalase. It is well known that HCN kills warm-blooded animals rather rapidly under the symptoms of lack of oxygen. Schoen- bein had already shown that prussic acid inhibits the decomposition of H2O2 through animal tissues (or the catalase contained in them). Gep- pert showed that HCN prevents them from consuming the free oxygen, hence the animals die under symptoms of asphyxiation. HCN prevents also the decomposition of H2O2 through platinum. Bredig has con- tinued the experiments of Schoenbein. As the velocity of the catalytic action of platinum must be in proportion to the surface of the metal, Bredig, in order to get a maximal surface, made colloidal solutions of platinum and other metals. He showed that extremely small doses of HCN are sufficient to prevent the catalytic action of colloidal platinum upon H2O2. Kastle and Loevenhart made it probable that this "toxic" effect of HCN upon the catalytic action of platinum upon H2O2 is deter- mined by the fact that platinum forms an insoluble combination with HCN. The formation of a film of this insoluble compound on the surface of the platinum prevents the latter from forming the unstable combination with H2O2 which must precede the decomposition of the latter. The reason why so little of the poison is required for this effect is due to the fact that the film which is formed on the surface of the metal may be infinitely thin. Kastle and Loevenhart were able to put their hypothesis to a test. Silver and thallium act much like plati- num upon the hydrogenperoxide, inasmuch as both accelerate its decom- position. While silver forms a combination with HCN which is insoluble in water, thallium forms a soluble combination. Kastle and Loeven- hart showed that while hydrocyanic acid inhibits the decomposition
28 DYNAMICS OF LIVING MATTER
of H2O2 by silver, the catalytic action of thallium upon H2O2 is not diminished by HCN.*
The same authors could show, in general, that the anions of those salts which form insoluble compounds with the metal diminish also the catalytic action of this metal, while the same salts have no inhibiting effect if the catalyzer is a metal which forms soluble compounds with the anion of the salt.
Kastle and Loevenhart found that certain salts accelerate the action of platinum on the decomposition of hydrogenperoxide. They are inclined to assume that this class of salts acts directly upon the H2O2, and not upon the catalyzer.
Everything seems to indicate that the enzymes accelerate the reac- tions in the body by forming intermediary, unstable combinations with the bodies whose reactions they accelerate. These unstable compounds are rapidly decomposed, and this makes the catalyzer free to repeat the action. This makes it clear that a small quantity of the catalyzer can decompose indefinite quantities of the substance. The fact that many enzymes act specifically also harmonizes well with this view. The enzymes, being themselves organic compounds of a complex character, will not form unstable compounds equally well with any organic com- pound.
Inasmuch as the enzymes are necessary for the chemical processes in living matter, the formation of enzymes is one of the essential functions living matter has to perform. Spitzer and Friedenthal were inclined to assume that the nucleo-proteids act as enzymes. This view, while possible, is not yet proven.
* Kastle and Loevenhart, Am. Chem, Journal, Vol. 29, p. 397, 1903.
LECTURE III THE GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER
i. THE LIMITS OF DIVISIBILITY OF LIVING MATTER
THE preceding lecture has shown that living matter is a mixture of various compounds, namely proteins, fats, carbohydrates, and salts. The fact that the reaction velocity for a number of oxidative and hydrolytic processes is so great, in spite of the low temperature and the practically neutral reaction of the tissues, has found its explanation through the presence of specific enzymes and the intermediary reactions determined by them. If we ask whether it would suffice for the purpose of making living matter to try to find a mixture of the above-mentioned substances, including the enzymes, the answer would have to be no, for the reason that living matter is characterized by another peculiarity not yet men- tioned, namely, a definite structure.
Whatever may be the physical structure of living matter, it is certain that in most cases its complete destruction means the cessation of life phenomena. A brain or kidney which has been ground to a pulp is no longer able to perform its functions; yet it is evident from the facts mentioned in the previous lecture that certain chemical functions can still be performed by such pulps, e.g. the catalytic processes. The question now arises as to how far the divisibility of living matter can be carried without interfering with its functions. Are the smallest particles of living matter which still exhibit all its functions of the order of magni- tude of molecules and atoms, or are they of a different order? The first step toward an answer to this question was accomplished by Moritz Nussbaum,* who found that if an Infusorian be divided into two pieces, one with and one without a nucleus, only the latter will continue to live and perform all the functions of self-preservation and development which are characteristic of living organisms. This shows that not only more than two definite substances, but two different structural elements, are needed for life. We can understand partly from this why an organ after being reduced to a pulp, in which the differentia-
* Nussbaum, loc. cit. 29
3O DYNAMICS OF LIVING MATTER
tion into nucleus and protoplasm is definitely and permanently lost, is unable to accomplish its functions.*
The observations of Nussbaum and those who repeated his experi- ments showed that although two different structures are required, not the whole mass of an Infusorian is needed to maintain its life. I tried to solve the question as to how small a fraction of the original cell must be preserved in order to maintain life in the sense of the definition given at the beginning of these lectures. Will the smallest possible element be of the order of an aggregate of a few molecules, or will it be of the order of a small fraction of the original mass ? I tried to decide this question in the egg of the sea urchin, immediately after its fertiliza- tion. The egg divides and reaches successive larval stages, first a bias- tula, then a gastrula, and finally a pluteus stage. Without especial efforts the eggs cannot be raised beyond this stage in the laboratory. I had found a simple method by which the unsegmented eggs of the sea urchin (Arbacia) can easily be divided into smaller fragments. When the egg is brought from five to ten minutes after fertilization (long before the first segmentation occurs) into sea water which has been diluted by the addition of an equal part of distilled water, the egg takes up water and the membrane bursts. Part of the protoplasm then flows out, in one egg more, in another less. If these eggs are after- ward brought back into normal sea water those fragments which con- tain a nucleus begin to divide and develop. f In this case the degree of development such a fragment reaches, is clearly a function of its mass; the smaller the piece, the sooner on the whole its development ceases. The smallest fragment which is capable of reaching the pluteus stage possesses the mass of about one eighth of the whole egg. Boveri has since stated that it was about one twenty-seventh of the whole mass. Inasmuch as only the linear dimensions are directly measurable, a slight difference in measurement will cause a great discrepancy in the calculation of the mass.
These results are in harmony with experiments made by Driesch J for a different purpose. Driesch isolated the first blastomeres of the segmented egg of a sea urchin by shaking the egg and thus bursting its membrane. He found that an isolated cell of the two- or four-cell stage of the egg of a sea urchin is still capable of developing into a normal pluteus, but that an isolated blastomere of the eight- or sixteen- cell stage no longer possesses this power. This experiment, however,
* It must not be overlooked that in bacteria and the blue algae no distinct differentia- tion into nucleus and protoplasm can be shown. To these organisms, therefore, the experi- ments of Nussbaum cannot be applied.
t Loeb, P/l'tiger's Archiv, Vol. 55, p. 525, 1893.
J Driesch, Zeitsch. f'iir wisse nschaftli sche Zoologie,\o\. 53, 1891.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 31
FIG. 6. — AFTER BOVERI.
Structure of the unfertilized egg of a sea urchin. The contents of the egg are divided into three dis- tinct layers.
cannot be used as an unequivocal answer to our question, inasmuch as the possibility exists that in later stages of segmentation the different cells undergo different chemical changes, whereby they no longer remain equal in quality.
If we raise the question why such a limit exists in regard to the divisibility of living matter, the answer is possibly given by Boveri's observation that the unsegmented egg of the sea urchin (Strongylocentrotus limdus] possesses three different layers.* It is possible that these three layers contain chemically different material, and that only those fragments of an egg are capable of development which contain material of each of the three layers. If this be correct, it will certainly not suffice to mix the chemical con- stituents of the egg in order to produce the phenomena of development; but we must pro- vide for a definite arrangement or structure of this material. We shall see later on that this structure may be very simple and capable of a physicochemical definition. The limits of
divisibility seem therefore to depend upon the physical structure of the cells or organs. These limits vary for different organisms and cells. The smallest piece of a sea-urchin egg that can reach the plu- teus stage is still visible with the naked eye, and is therefore consider- ably larger than bacteria or many algae, which also may be capable of division.
2. FOAM STRUCTURES AND EMULSIONS
Living matter seen through the microscope invariably offers the same characteristic appearance which has caused biologists to desig- nate it with one general term; namely, protoplasm. Yet the common physical features of living "protoplasm" are still a matter of contro- versy. Some authors maintain that the protoplasm is a network of fine fibers, while others say, and apparently justly, that the network does not occur in living protoplasm, but is caused by the coagulation of the colloids contained in the cells and liquids of the tissues. It is a fact that the proteins which are dissolved in the living body are pre- cipitated by the fixing reagents of the histologists, and when they are precipitated they form net structures which do not exist during life when the proteins are held in solution. f
* Boveri, Die Polaritat des Seeigeleies. Verhandl. der physik.-med. Gesellsch., Wiirzburg, Vol. 34, 1901. t Hardy, Jour, of Physiology, Vol. 24, p. 158, 1899.
32 DYNAMICS OF LIVING MATTER
Biitschli * has expressed a view concerning the structure of living protoplasm which is more probably correct than the assumption of the net structure. According to him, living protoplasm has the structure of a microscopic emulsion. His view is shared by E. B. Wilson and other authors, while it is accepted, but not unconditionally, by Hardy and Pauli. Biitschli's conception, however, seems to harmonize with a great many facts, and therefore we may discuss some features of the theory of emulsions. Emulsion and foam are, according to Quincke, only different names for the same (diphasic) physical system. An emulsion like milk consists of a large number of spherical droplets of fat, which are distributed in a watery liquid. f We speak of a foam in the case of an emulsion of a gas in a liquid. The foam in a soap solu- tion consists of spherical masses of air which are distributed in a watery liquid. Foams and emulsions have therefore the same physical struc- ture. The peculiarity of emulsions and foams which interests us in this connection is their durability, and the theory of foams is concerned with this side of the problem. According to Lord Rayleigh, an ab- solutely pure liquid cannot form a durable foam.J When a gas bubble rises in pure water it is surrounded by a liquid film which, however, does not possess any durability, and therefore bursts. If, however, the water is contaminated by another substance, these liquid films become more durable. This influence upon the durability varies with the nature of the contaminating substance. It is known that the addi- tion of small quantities of colloidal material, e.g. soap, saponin, a solu- tion of gelatine, is capable of making the liquid films very durable.
This influence of the contamination is due to its effect upo'n surface tension. Experiments have shown that those substances which make these liquid films more durable decrease the surface tension which ex- ists at the limit between water and the emulsified substance. In con- sequence of this latter fact, these substances have a tendency to collect at the common surface between water and air, or whatever the emulsi- fied substance may be. The surface acts like a trap on such particles, inasmuch as it requires an outside force to bring them back to the interior, if they have once collected at the surface. § The consequence is that a film of this contaminating material is formed at the surface, between the water and the oil droplets, or whatever the substance held in emulsion may be. In the case of oil emulsions in water, the contami- nating substance is a trace of soap formed through the hydrolysis of fat
* Biitschli, Untersuchungcn uber tnicroscopische Schaume und das Protoplasma, Leipzig, 1892.
1 Quincke, Pfiuger's Archiv, Vol. 19, p. 129, 1879.
| Lord Rayleigh, Scientific Papers, Vol. 3, p. 351, 1902.
§ F. G. Donnan, Ze itsch. fur physikal. Chemie, Vol. 31, p. 42, 1899.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 33
or oil. Soap solution diminishes the surface tension between water and oil. The question arises as to how this circumstance can make the emulsion more durable. The answer is as follows: If a film of water separates two oil drops, the soap particles gather at the surface on either side of the water film. If these soap particles be now brushed aside on one spot of this watery film, the water will come in direct con- tact with the oil. The surface tension between oil and water is greater than that between soap solution and water. Hence, in a spot where the soap is removed the surface tension or tendency to contract will be greater than in the rest of the film, and the consequence will be that this spot will contract and thus mend the hole in the layer of soap. In this way the diminution of the surface tension by the contaminating substance makes the emulsion more durable, and prevents the fusion of two neighboring droplets in an emulsion. It is not impossible that the existence of such a film may explain why it is that the cleavage cells of an egg do not easily fuse.
As far as the thickness of the contaminating layer is concerned, Lord Rayleigh has measured it, and found that the contaminating layer of oil on the surface of water which suffices to prevent the motion of particles of camphor on the surface of water, need be only Yg^VoTro mm. thick. It is hardly necessary to mention that such a film is far below the limits of microscopic visibility.
Biitschli assumes that living protoplasm is an emulsion of two liquids : a viscous one, which is insoluble in water ; and a watery liquid, which possesses little viscosity.*
3. THE COLLOIDAL CHARACTER OF LIVING MATTER
Hardy as well as Biitschli assumes that living matter is essentially liquid. The most common observations on living organisms, which are sufficiently transparent, show that this is undoubtedly true for a large part of the material contained in such organisms. Certain ele- ments, however, are apparently solid, e.g. the surface films of cells and nuclei, and possibly certain structures in the interior of the cell, such as centrosomes. The observations of Traube, as well as Hardy, show how solid constituents can be formed from liquids in living matter. We are dealing here with a chapter of the physics of colloids, which is just at present the object of many investigations.
The substances in living matter which occur in a liquid as well as in a solid condition are the colloids. The name was given by Graham, who discriminated between two kinds of soluble substances, — crystal-
* Butschli, Archiv fur Entwickelnngsmechanik, Vol. II, 499, 1901. D
34 DYNAMICS OF LIVING MATTER
loids and colloids. The former diffuse easily through animal mem- branes, the latter only with difficulty, or not at all. It is, however, well to remember that there exist transitions between both groups. According to Krafft,* the colloidal character of sodium soaps increases with the size of the acid molecule. Thus sodium acetate possesses the qualities of a crystalloid in a watery solution, while sodium stearate belongs to the colloids. The proteids, certain carbohydrates like starch or glycogen, and higher fats, belong to the colloids; while their products of cleavage, e.g. dextrose, may belong to the crystalloids. According to Krafft, there is therefore a steady transition from the crystalloids to the colloids, f
In physical chemistry, as a rule, a different idea of the colloidal solution is given, i.e. that they are no real solutions, but suspensions of small particles in a liquid, or a system of two phases. We have already mentioned the fact that not only organic substances may form colloidal solutions, but also many inorganic substances, and even pure metals, such as platinum, gold, silver, etc. All these colloidal sub- stances alter the freezing point or boiling point of the liquid not at all or but little. From this the conclusion is drawn that no work, or but little, is required to separate the solvent from the dissolved colloidal particles. It must, however, be stated that according to some authors the proteins dissolved in the blood serum have a definite osmotic pres- sure which is far from being a " quantite negligeable." Starling J found by a direct measurement — the freezing-point determinations fail in such cases --an osmotic pressure of the colloids of as much as 30 to 40 mm. of mercury. This pressure plays, according to Starling, a definite and important role in phenomena of lymph formation, oedema, etc. In view of Starling's observations it is doubtful whether we still have a right to maintain that colloidal solutions behave like suspensions, inasmuch as the latter differ from real solutions through the fact that they possess no measurable osmotic pressure. §
A second argument in favor of a principal difference between col- loidal and crystalloidal solutions lies in the fact that dissolved particles in a colloidal solution have, as a rule, a definite electrical charge. The same is often found in the particles which form suspensions in water. The existence of the electrical charge can be demonstrated if an
* Krafft, Zeitsch. fur physiologische Chemie, Vol. 35, pp. 364 and 376, 1902.
t This idea receives still further support from the fact that if a salt solution is exposed to the action of a centrifuge, the concentration at the periphery becomes larger than at the center.
J Starling, Jour, of Physiology, Vol. 19, p. 312, 1895.
§ More recently Reid has reached the conclusion that the colloids in Starling's experi- ments were not free from salts, and that he in reality measured the osmotic pressure of the latter.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 35
electrical current be sent through a colloidal solution ; in this case, the particles move in the direction of the negative or positive current, ac- cording to the chemical character of the colloid. This charge of the colloidal particles is generally held to be due to the formation of a double layer of electricity at the surface, between particle and water. A similar explanation was given to the charge of particles suspended in water; and this is considered another argument in favor of the idea, that colloidal solutions are diphasic systems. It is, however, possible, as Freundlich * has already mentioned, that the charges of the col- loidal particles are due to the electrolytic dissociation of the latter. It had generally been noticed that the colloids of an acid character are negatively charged when in solution, while colloids of an alkaline char- acter are positively charged. This is exactly what should be expected if the charges of the colloidal particles in solution are due to electro- lytic dissociation. If the colloid is an acid, it will dissociate into one or more positively charged hydrogen-ions and a negatively charged colloid-ion; if the colloid is an alkali, it will dissociate into one or more negatively charged hydroxyl-ions and a positively charged colloid-ion.
Hardy f has shown that dialyzed white of egg (from the white of a hen's egg) is electro-positive when a trace of acid is added, while a trace of alkali makes it electro-negative. He believes that in the for- mer case, the hydrogen-ions are caught in the meshes of the colloidal particles of the white of egg and carry the latter with them when they migrate in an electrical field. When alkali is added, the hydroxyl- ions are caught in the meshes of the particles and drag the latter with them in an electrical field. I have called attention + to the fact that Hardy's observations allow of a different interpretation, namely, that they may be due to the electrolytic dissociation of the white of egg. The proteins have an amphoteric character, i.e. they are able to give off HO-ions, as well as H-ions, to the surrounding solution. If we add a trace of acid to the solution of dialyzed white of egg, the degree of dissociation of the acid part of the molecule is diminished, and it will dissociate chiefly into HO-ions and a colloid cation, and the latter will migrate in an electrical field to the cathode. If a trace of alkali, how- ever, is added to the surrounding solution, for the same reason, the white of egg will be prevented from sending as many HO-ions into solution as H-ions, and the molecule will dissociate mainly into H-ions and a colloid anion. Hence, the addition of a trace of acid will give
* Freundlich, Zeitseh. fur physikal. Chemie, Vol. 44, 1903.
t Hardy, Proceedings of the Royal Society, Vol. 66, p no, 1901.
j Loeb, University of California Publications, Vol. i,p. 149, 1904.
36 DYNAMICS OF LIVING MATTER
the colloidal particles a more positive charge, while a trace of alkali will give them a more negative charge.
Hardy was the first to call attention to the fact that the electrical charge of the colloidal particles - - or, in his opinion, the difference of potential between the particles and the surrounding solution --is a prerequisite for the stability of many colloidal solutions.* If in these solutions the charges are removed from the particles, a precipitation occurs through the clumping together of the small colloidal particles to larger aggregates and the falling of these aggregates. Hardy proved this in two ways: first, by carefully neutralizing acid white of egg with NaHO, until the particles no longer migrated with the positive or negative electric current. As soon as this occurred, a slight mechanical agitation of the particles was sufficient to produce a precipitation of the white of egg. The second proof consisted in show- ing that when a constant current is sent through the solution, the par- ticles that are carried to the electrode are precipitated. At the pole the particles lose their charge and become isoelectric with the surround- ing water. It is, however, not impossible that acid or alkaline white of egg is soluble, while the neutral white of egg is insoluble, or less soluble, in water.
It had been known for a long time that water which was rendered opaque through a suspension of small particles could be made clear if salts were added to the suspension. A similar experience had been made in connection with the precipitation of colloidal particles. It was further known that the precipitating power of various electro- lytes is a function of only one of the two ions, - - mostly the cation, — and that it increases with the valency of the active kind of ions. The fact that Freundlich found in experiments with a sol of arsenic sulphide, whose particles have a negative charge, that the precipitating force of salts with a bivalent cation was about seventy times as large as that of salts with a univalent cation, while salts with a trivalent metal pos- sessed a precipitating force five hundred times as large as that of a uni- valent cation, may serve as an example. Hardy added the important fact that in the case of sols with negatively charged particles, the pre- cipitation is due to the cations; while in the case of positive colloids the precipitation is caused by the anions of the precipitating salt. Hardy states that the precipitating power of an ion is an exponential function of its valency.f Freundlich, however, has shown clearly that where the cation of a salt causes the precipitation, the anion is not without some effect It seems quite possible that the facts found by Hardy indicate a purely chemical action of the precipitating salt.
* Hardy, lot. cit. f Hardy, Jour, of Physiology, Vol. 24, p. 288, 1899.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 37
A negatively charged colloid can form salts with metals; and it is in harmony with the general facts concerning the solubility of salts, that the latter decreases with the valency of the ion with which the colloid combines. Hardy, Bredig, and many other authors believe, however, that in this case the ions act only through their electric charges. Hardy has recently found that electro-negative globulin solutions are rapidly precipitated by the positive electrons sent out by radium.* It is pos- sible that the radiation, in this case, causes a precipitation only in- directly, while its direct action is a chemical change in the globulin solution.
If we accept the view of Hardy and Bredig, that we are dealing in the action of sols with an effect of an electrical charge of the ions, we shall do well to adopt Bredig's f explanation of this effect. The sur- face tension at the limit of two media reaches a maximum, when the difference of potential between the two media becomes a minimum. This is due to the fact that the electrical charges are antagonistic to the surface tension. The higher the surface tension between colloidal particle and surrounding liquid, the easier will the slightest agitation cause a clumping of the smaller particles into larger aggregates. Those who hold this view have thus far not yet shown how it happens that the valency of an ion has so great an effect upon the precipitation of the colloidal particles, although each precipitating salt carries equal quantities of positive and negative charges into the solution.
Not all the colloidal solutions show cataphoresis. Hardy men- tions that globulins which are held in solution by salts do not migrate when a constant current passes through them.
Life depends upon the existence of these colloidal solutions in the cells. All agencies which bring about a general gelation, bring life to a standstill; and such a standstill is permanent in case irreversible gels are formed, such as originate if proteins are heated. The liquid proteins of our body coagulate at a comparatively low temperature, and this is the reason that at a temperature of about 45° the cells of our body die very rapidly. The heavy metals also transform the proteins of our body into irreversible gels, and this may be a reason why tliey are so poisonous. There are, however, conditions in which the transformation of sols into gels does not lead to death, but to the formation of important morphological structures, e.g. Traube's membranes of precipitation. The astrospheres also originate, accord- ing to the botanist, Alfred Fischer, through a process of coagulation. It is, moreover, possible that a series of manifestations of life in cell-
* Hardy, Jour, of Physiology, Vol. 29, p. xxx, 1903. f Bredig, Anorganische Fertnente, Leipzig, 1901.
38 DYNAMICS OF LIVING MATTER
division and protoplasmic motion, rhythmic contractions, etc., depend upon alternating gelations and liquefactions. It is, however, useless to discuss such possibilities until more definite proofs of their real ex- istence have been furnished. Such proofs thus far exist only in regard to membranes of precipitation.
4. THE FORMATION OF SURFACE FILMS AND TRAUBE'S MEMBRANES
OF PRECIPITATION
It is a general rule that every free cell is surrounded by a solid film. The pseudopodia of many Infusorians could not exist were they entirely liquid. Liquid circular cylinders begin to fall apart into droplets as soon as their height becomes greater than the periphery of their base. The length of the pseudopodia of rhizopods is, however, very often a multiple of their circumference. As the interior of the pseudopodia shows phenomena of streaming, the solid part of the pseudopodia can only be at their surface. Such solid surface films may be exceedingly thin, according to Quincke's observations.
Ramsden has recently shown why masses of protoplasm must form solid films at their surface. He had formerly observed that the white of a hen's egg can be caused to coagulate by mere mechanical agita- tion.* The explanation of this fact was subsequently found in the fur- ther observation, that without any evaporation at the free surface of the protein solutions, solid or extremely viscous films are formed very rapidly. f If such solid particles be removed from the surface, i.e. by mechanical agitation, new particles will come to the surface and form membranes. This is in harmony with what was stated earlier in regard to the gathering of contaminating particles at the surface between two media.
What has been said here with reference to the formation of solid films at the surface of free cells, may also hold with regard to the for- mation of solid films at the surface of nuclei.
Traube has shown that where two liquid colloids come in con- tact, solid membranes may be formed. He investigated the mechanism of the formation of the membranes of plant cells and was led to the conclusion that the formation of these membranes, and the peculiarity of the cell to grow, depend upon a simple physical process. Certain colloids form a precipitate where they come in contact with each other, and this precipitate is impermeable for either colloid. The precipitate must therefore assume the shape of a thin film, which prevents the
* Ramsden, Archiv ftir Anatomic und Physiologic, Physiologische Abteilung, p. 517, 1894. t Ramsden, Zeitsch. fur physik. Chemie, Vol. 47, p. 336, 1904.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 39
further action of the two colloids upon each other. He called this film the membrane of precipitation.* The following example may be quoted : "A solution of a certain gelatine -- /3-gelatine -- was prepared, and a drop taken out of this solution with a glass rod. The drop remained hanging at the end of the rod, and was exposed to the air for several hours; it was then dipped in a 5 per cent solution of tannic acid. In about ten minutes a thin iridescent solid film formed at the surface of the drop. The /3-gelatine and the tannic acid had formed a membrane of precipitation at the common surface, which was impermeable for both colloids and thus prevented any further reaction between the two." But it is not necessary for the formation of membranes of precipitation that two colloids act upon each other. A crystalloid and a colloid may form a membrane of precipitation, as is the case when a drop of tannic acid is dipped into a neutral solution of lead acetate. Two crystal- loids can also form such membranes if they only form an amorphous precipitate which is impermeable for both crystalloids, e.g. ferrocyanide of potassium and ferric chloride. While these membranes are imper- meable for certain substances, they are not so for others; and Traube recognized the fundamental importance of this fact for life phenomena, "The cell membrane f makes a diminutive chemical factory of the con- tents of this cell by shutting it off from its surroundings, and enables each cell to lead a specifically different life from the neighboring cells." The substances which can permeate the membranes of precipitation vary according to the nature of the latter. All of them allow water to pass through; while they do not allow sugar or salts to pass through at all or not equally well. Traube pointed out that this semipermea- bility also explains the mechanism of cell growth. When the drop of /3-gelatine (or any other substance used for the experiment) had a greater concentration than the solution into which it was dipped, the drop began to grow in size as soon as the membrane of precipitation was formed. Traube thus became the originator of the modern theory of the growth of cells, which assumes that the growth is caused by the cell absorbing water in consequence of its osmotic pressure being higher than that of the surrounding solution.
Traube was inclined to explain the semipermeability of his artifi- cial membranes on the basis of the assumption that they possess very small pores or interstices which allowed only small molecules, such as water, to permeate; while the larger molecules, such as salts, could not pass through them. This assumption was no longer tenable after
* M. Traube, Rdcherfs und Du Bois Reymond's Archiv, 1867. Gesammdte Abhand- htngen, p. 213, Berlin, 1899.
t We should now say, the surface film of protoplasm.
40 DYNAMICS OF LIVING MATTER
Overton found that the alcohols, even those having large molecules, could pass into the cells much more readily than the salts with smaller molecules. Nernst had given another theory of semipermeability which is generally accepted; namely, that the substances which go through the semipermeable walls must first be dissolved in this mem- brane, and that therefore such substances must be absorbed most rapidly by these cells as are most soluble in the cell walls, or surface films of the cells. Overton * found that plant and animal cells which show the properties of semipermeability are generally most permeable for those substances which are most soluble in oil or fat, e.g. alcohol, ether, chloroform. He accepts Nernst's theory and draws the conclu- sion that the cells, or the protoplasm of the cells, are surrounded by a film of a fatty substance, such as lecithin or cholesterin, and that these substances give protoplasm the quality of semipermeability.
A similar conclusion had already been drawn by Quincke, who had noticed that protoplasm assumes a spherical shape when squeezed out of its cell into a watery liquid. This, he said, was only intelligible when protoplasm is surrounded by a film of oil or fat.f Quincke also pointed out that such films of oil must show the phenomenon of semi- permeability.
Hans Meyer and Overton J have noticed independently of each other that all narcotics have one property in common; namely, a comparatively great solubility in fat, or lipoids like lecithin or choles- terin. The special importance of this lies in the fact that the narcotic effect of a substance increases, on the whole, with the degree of its solubility in fat. They are inclined to believe that the chemical nature of the narcotic is otherwise of no or only minor importance, as they find that chemically inactive bodies may be very powerful narcotics, if only their solubility in oil is comparatively high. It seems to me, however, that in view of the presence of so many enzymes in our cells, substances may be very active in our body, which in the absence of such enzymes may appear rather inert. This does not, however, contradict the fact that the solubility of narcotics in fat plays a role in the absorp- tion of narcotics. Those cells in our body which are richest in lipoids, namely, the ganglionic cells, also feel first the effects of narcotics.
Meyer and Overton assume that the narcotics, such as alcohol, ether, etc., act merely by altering the physical properties of the cells in whose lipoids they dissolve. The fact that anaesthetics like ether and chloroform dissolve fat was utilized for an explanation of their
* Overton, Vierteljahreschrift der naturforschenden Gesellsch. in Zurich, Vol. 44, p. 88, 1899. (The original was not accessible to me.)
t Quincke, Sitzuugsberichte der Berliner Akademie der Wisscnschaften, p. 791, 1888. J Overton, Studien uber die Narcose, Jena, 1901.
GENERAL PHYSICAL CONSTITUTION' OF LIVING MATTER 41
physiological action immediately after the discovery of this action. At that time the various authors, e.g. von Biebra and Harless, ex- plained the action of ether and chloroform on the assumption that these substances caused the fat to leave the cells by dissolving it. This does not seem to harmonize with the fact that a person so soon recovers from the effects of a narcosis. Overton showed, moreover, that ciliary cells, when narcotized in water by ether or chloroform, may resume their activity when brought back into pure water. This would not be pos- sible if the narcotic effect of ether or chloroform had been due to the diffusing of fats from the cell; but the fact that a person can recover from the action of narcotics does not prove that their action is a purely physical one. A person who becomes unconscious from the lack of oxygen may also recover, if oxygen is admitted again, soon enough, and yet no one would conclude from this that the action of oxygen is purely physical. The rapidity of the absorption of narcotics may be due to their solubility in oil, and yet the effect they produce may be due to something entirely different.
5. OSMOTIC PRESSURE AND THE EXCHANGE OF LIQUIDS BETWEEN THE CELLS AND THE SURROUNDING LIQUID
The observations of Traube, Quincke, Ramsden, and Overton have given us some hints as to the nature of the surface films which surround protoplasm. Their importance lies in the fact that the con- tents of the cells are chiefly liquid, and that an exchange of dissolved substances occurs steadily between these substances and their sur- roundings. Animal cells are surrounded by a liquid which resembles sea water in its constitution, though its osmotic pressure is in land and fresh-water animals, and in some marine animals, less than that of sea water. The main force for the exchange of dissolved substances between the cells and the surrounding solution is the osmotic pressure. Inasmuch as the cells take up the salts, proteins, fats, and carbohy- drates that are dissolved in the blood, we cannot accept Overton's view that only water and those substances which are soluble in fat pass through the membranes, and that salts generally cannot pass through. We hold that the cell walls are not impermeable to salts, and that there is only a difference in the rate of diffusion of the various substances, many salts diffusing only very slowly into the protoplasm. The con- sequence is that for short experiments the cells act as if they were im- permeable for salts and permeable for water only. When cells are put into salt or sugar solutions, whose osmotic pressure is higher than that of the liquid of the cells, the cell loses water; and in the case of
42 DYNAMICS OF LIVING MATTER
most plant cells a condition ultimately arises in which the protoplasm becomes separated from the cellulose wall, the so-called plasmolysis. Nevertheless it cannot be said that plant cells are impermeable for salts, inasmuch as the building up of the living matter of the plant depends upon the diffusion of certain salts from the soil into the plant, e.g. nitrates, phosphates, sulphates, potassium salts, etc. For the animal cell this can be demonstrated still more strikingly. The com- mon striped muscle of the heart loses its excitability rather rapidly when put into a physiological salt solution to which a certain (but not too small) amount of KC1 is added; but if the muscle is taken out in time and put back into a pure NaCl solution, its excitability returns. The velocity with which the inhibiting effect of the potassium salts upon the irritability occurs, depends upon the concentration of the potassium salts in this solution; it is therefore certain that the potas- sium salts diffuse comparatively rapidly into the muscle and out of it. The same can be shown for Na, Ca, and many other, if not all salts. When the muscle is put into an isotonic solution of any sodium salt, rhythmical contractions begin, and the sooner the higher the concen- tration of the sodium salts. The same is true for solutions of barium salts. The velocity with which the sodium salts produce these twitch- ings varies with the nature of the anion of the salt. If to the solution of the sodium salt a small but definite quantity of a calcium salt be added, these contractions are suppressed. These facts are only con- ceivable if we assume that muscle cells are permeable for Na, Ca, and Ba salts, or ions. They must, however, be permeable for other salts also, e.g. Li, Cs, and Rd salts, as they begin to twitch in these solu- tions.* The more toxic salts, e.g. those of the heavy metals, must also be able to diffuse into the cells, as otherwise they could not be so toxic.
The salts diffuse more slowly into the muscle than water. If mus- cles be put into salt solutions of various concentrations, it will be ob- served that during the first hour or hours, the muscle absorbs water and swells in hypotonic solutions, while it loses water in hypertonic solutions. This phenomenon is, in wide limits, independent of the nature of the salt in solution. f If the muscle remains longer in the solu- tion, however, the influence of the osmotic pressure diminishes, and the specific effects of the salt appear. I found that in a 0.7 per cent solu- tion of NaCl, or an equivalent solution of NaBr or Nal, a muscle does not materially change its weight during eighteen hours. If there is
* Loeb, Festschrift fur Professor Pick, 1899. Pfluger's Archiv, Vol. 91, p. 248, 1902. See also the numerous papers of Ringer in this field of investigation.
f Loeb, Fftiiger's Archiv, Vol. 69, p. I, 1897; and E. Cooke, Jour, of Physiology, Vol. 23, p. 137, 1898.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 43
an increase in weight, as a rule, it does not exceed 7 per cent of the initial weight of the muscle. In the corresponding equimolecular solutions of potassium salts, the muscle increases its weight in the same time 40 per cent or more, while in an equimolecular solution of CaCl2, it may lose water, sometimes as much as 20 per cent.* In all these solutions the weight of the muscle changed but immaterially during the first hour. These facts become intelligible on the assump- tion that the salts diffuse into the muscle, although with less rapidity than the water. As far as the specific action of K, Na, and Ca salts upon the absorption of water by the muscle is concerned, it is some- what analogous to the behavior of various soaps. Potassium soaps are extremely hygroscopic, and absorb water in such quantities as to make them liquid, while calcium soaps absorb but little water; and sodium soaps occupy a position between these two. Soaps contain water in a form in which it can be squeezed out by a slight pressure. The same is also true for some, perhaps most of the water absorbed by muscles, and this holds also, according to Van Bemmelen and Hardy, for the water which is contained in irreversible gels, such as coagulated white of egg or a gel of silicic acid. Such gels evidently contain the water in capillary spaces. Evidently the Na-, K-, Ca-ions ultimately bring about a coagulation in the muscles ; but the structure and size or other physical properties of the interstices in the coagulated material change with the nature of the metal which brings about the coagulation. This is further corroborated by putting the muscle into solutions of a salt, e.g. NaCl, of various concentrations. During the first hour or so the volume of the muscle changes, as one would expect if the muscle were permeable for water, but impermeable or little permeable for salts; but after a longer period a paradoxical result is obtained. In solutions of higher osmotic pressure than the muscle, the latter increases in volume and weight, and within certain limits, the more so the higher the concentration of the solution, as the following table shows : —
_, _T „ ,, INCREASE IN WEIGHT OF MUSCLE IN TWENTY-FOUR
CONCENTRATION OF THE NACL SOLUTION HOURS IN PER CENT OF ITS ORIGINAL WEIGHT
1-05% + 0.7%
1-4% + 6.7%
1-75% +13%
2-1% +17-7%
2.45% +19%
2.8% +23.8%
This experiment might at first suggest that the osmotic pressure is not the force active in this case ; but this is not true. The osmotic pressure
* Loeb, Pfliiger's Archiv, Vol. 75, p. 303, 1899. (A dead muscle absorbs no water in a physiological salt solution, thus showing that the above-mentioned effects of K or Ca can- not be attributed merely to the death of the muscle.)
44 DYNAMICS OF LIVING MATTER
is the active force, but through the slow but gradual entrance of NaCl into the muscle, and possibly the loss of water and salts, on the part of the muscle, new conditions for the absorption of water are created which correspond with Van Bemmelen's observations on gels.
If a little acid be added to a 0.7 per cent solution of NaCl (which is about isosmotic with the gastrocnemius of a frog), the gastrocnemius will absorb considerable quantities of water from such a solution. The quantity of water absorbed increases with the quantity of acid used. For inorganic acids it can be shown that the effect is chiefly determined by the hydrogen-ions, and not by the anions. Organic acids, however, act considerably stronger than should be expected, if the effect were purely due to the free hydrogen-ions.* The cause of this anomaly is not yet known. If, however, acids are added to a hypertonic solution of NaCl, the effect is the reverse; the acid diminishes the amount of water absorbed by the muscle. f Alkalis increase the absorption of water under all circumstances. In these cases the acids and alkalis act probably through their combination with the proteids, whereby the conditions for the absorption and giving off of water are changed. It is therefore obvious that other forces than the mere osmotic pressure play a r61e in the absorption of liquids by tissues.
The same seems to be true for the reverse process; namely, the secretion of liquids from the cells. In these cases work is often done against the osmotic potential. It is evident that another force must be at work besides the mere osmotic pressure. The fact discov- ered by MacCallum, that the same salts which increase the peristaltic motion of the intestine also increase the secretory action of the glands of the intestine,! seems to indicate that this force may be of the nature of the contractile forces. The same salts which increase the secretion of liquid from the blood into the intestine also increase the secretory action of the kidneys.
Hober § has recently called attention to another possibility. Ham- burger had found that acids, e.g. CO2, increased the permeability of red blood corpuscles for certain anions. Hober has shown that such an increased permeability for anions must lead to a difference of potential between the inner and outer surface of the semipermeable elements, the inner surface assuming a positive charge. It is possible that such differences of potential, in case they lead to an electric current, may
* Loeb, Pflitger's Archiv, Vol. 69, p. I, 1897 '> Vol. 71, p. 457, 1898. f Loeb, Pfluger's Archiv, Vol. 75, p. 303, 1899.
t J. B. MacCallum, University of California Publications, Vol. I, p. 5, 1903; and pp. 81 and 125, 1904. Pfluger's Archiv, Vol. 104, 1904.
§ R. Hober, Pfluger's Archiv, Vol. 102, p. 196, 1904.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 45
bring about a cataphoresis of the water or the particles dissolved in it, into or out of the cell.
But in view of the observations of MacCallum I am more inclined to believe that contractile phenomena inside the cell furnish at least part of the energy of secretion and absorption in those cases where the osmotic forces alone cannot explain these phenomena. To illustrate what possible form these forces may assume, I may point out the rhythmical squeezing out of the liquid contents of the vacuole in Infusorians. Here the work of secretion is obviously done by protoplasmic contraction, and not by osmotic pressure. It is quite possible that, mutatis mutandis, something similar may occur in all cells, although this is only a surmise.
6. FURTHER LIMITATIONS OF TRAUBE'S THEORY OF SEMIPERMEABILITY
Traube's idea that all living cells are surrounded by a membrane which is absolutely permeable for water, does not seem correct for a number of marine animals. Fundulus heteroclitus, a marine fish, lives and develops exclusively in sea-water, i.e. in a solution whose osmotic pressure is, roughly estimated, like that of a half-grammolecular
/f}'l\
( -- } solution of NaCl. I have found that this fish as well as its eggs
can be put permanently into distilled water without the least injury. No swelling of the eggs or the tissues occurs under these conditions.* It may also be put into sea water whose osmotic pressure has been increased by the addition of a certain percentage of NaCl without perceptible shrinkage. This shows that water does not diffuse rapidly through the skin of the animal or the membrane of the egg. It cannot be stated, however, that it does not diffuse at all, since it is possible that a slight diffusion of water into the cells may be compensated by an increased secretion of water from the cells. In addition, the egg and animal must be but slighly permeable for salts, as otherwise the salts would diffuse from the blood and the tissues of the animal into the distilled water, and this would cause the death of the animal. The skin and the egg cannot be said to be absolutely impermeable, since gases like O and CO2 diffuse into the eggs, and since the latter rapidly dry out and die when taken out of the water and exposed to dry air. More- over, K and other toxic salts are able to diffuse slowly into the egg, as can be shown by the fact that if potassium salts are added to sea water, the heart of the embryo soon stops beating.
* Loeb, Ffliiger's Archiv, Vol. 55, p. 530, 1893. Am. Jour. Physiology, Vol. 3, pp. 327 and 383, 1900 ; and Vol. 6, p. 411, 1902.
46 DYNAMICS OF LIVING MATTER
The behavior of Fundulus to distilled water is not the rule for marine animals, as most of them when subjected to it die rapidly. I have recently made a series of experiments with a marine Crustacean Gam- marus, of the Bay of San Francisco.* The osmotic pressure of the sea-water of the bay varies at different times of the year between that
yyi
of about an -- and a f m NaCl solution. When Gammarus is brought
4 suddenly from bay water into distilled water, its respiratory motions
stop, as a rule, in about half an hour. This standstill becomes per- manent, unless they are put back into sea water within a short time (about ten minutes). If, however, Gammarus be put into a cane
m sugar, dextrose, or lactose solution of any concentration from — to
f m upward, they die just as rapidly, if not more so, than in distilled water. The same is true when the animals are put into a pure NaCl solution isosmotic with the sea water. They die still more rapidly when put into distilled water, to which all the other salts found in the sea water are added, with the exception of NaCl, and in the concentra- tion in which those salts occur in sea water. If they are put, however, into a solution of NaCl, KC1, and CaCl2, in that proportion in which these salts occur in the sea water, the animals may live as long as forty- eight hours; and if some MgCl2 is added to this solution, the animals may live as long as in sea water. If we prepare solutions composed of only two of the salts contained in the sea water; namely, NaCl + KC1, or NaCl + CaCl2, or NaCl + MgCl2, the Gammarus lives only a few hours. These experiments prove that the medium surrounding the Gammarus must not only have a definite osmotic pressure, but that this pressure must be supplied by specific salts. Perhaps the follow- ing data may explain, in part at least, why this lack of specific salts leads to the death of the animal.
7. THE ANTAGONISTIC EFFECTS OF SALTS
When the eggs of Fundulus are put immediately after fertilization into a pure solution of NaCl, whose concentration roughly equals that
m
in which this salt is contained in the ocean, — , or f m, no egg is
2
able to form an embryo. The eggs begin to segment and may go as far as the 64-cell stage, but after this they die. But if to the NaCl solution a small but definite amount of a bivalent metal (with the exception of the most poisonous ones, like Hg), is added, just as
* Loeb, Pfluger's Archiv, Vol. 97, p. 394, 1903 ; Vol. 101, p. 340, 1904.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER
47
many eggs form embryos as in normal sea water.* It is a striking fact that not only the salts of such bivalent metals which occur in the sea water or the body, e.g. Ca and Mg, render the pure NaCl solution harmless ; but also such salts as do not occur in the body, or are posi- tively poisonous, e.g. Sr, Ba, Co, Zn, Pb, and others. An example will illustrate these antagonistic effects between the salts of the univalent and bivalent metals.
PERCENTAGE OF THE FUNDULUS EGGS WHICH FORM AN EMBRYO IN THIS SOLUTION
0%
3% 3%
20%
75%
NATURE OF THE SOLUTION ioo c.c. f m NaCl
ioo c.c. \ m NaCl + \ c.c. — CaSO4
64
ioo c.c. f m NaCl + i c.c. — CaSO4
64
ioo c.c. \ m NaCl + 2 c.c. — CaSO4
64
ioo c.c. f m NaCl + 4 c.c. — CaSO4
64
ioo c.c. f m NaCl + 8 c.c. ~ CaSO4 70%
64
It is remarkable how small a quantity of calcium suffices to render the NaCl solution harmless. The anion has nothing to do with this effect of the calcium salt, as the result remained the same when any other soluble calcium salt was used, e.g. Ca(NO3)2 or CaCl2. The results also remained the same when in the place of the Ca salts, Sr, Ba, Co, Zn, or Pb salts were used, and even the quantities of the salt required to make the NaCl solution harmless were about the same for all the salts. I think it is one of the most striking facts known in toxicology that a pure solution of NaCl of that concentration in which this animal lives is poisonous, while this solution can be rendered less harmful or harmless by adding so poisonous a substance as Ba, Co, Zn, Pb, etc.
If the eggs of Fundulus are raised in a solution of a salt with another univalent cation than Na, e.g. K, Li, or NH4, we find that beginning with a certain concentration a solution of each of these salts becomes a poison for the eggs of Fundulus, that is to say, does not allow any egg to form an embryo. If, at that concentration of one of these salts, a small but definite amount of a salt with a bivalent cation is added, the eggs form embryos and they are able to develop. Trivalent cations, like Al and Cr, were also able to render the toxic concentrations of salts with a univalent metal less harmful. The antitoxic effect of a tetravalent cation, Th, however, was found to be only slight.
* Loeb, Pfluger'ls Archiv, Vol. 88, p. 68, 1901. Am. Jour. Physiology, Vol. 3, p. 327, 1900 ; Vol. 6, p. 411, 1902. Loeb und Gies, Pfluger's Archiv, Vol. 93, p. 246, 1902. Loeb, Pfluger's Archiv, Vol. 107, p. 252, 1905.
48 DYNAMICS OF LIVING MATTER
While a small quantity of a salt with a bivalent metal thus suffices to render a solution of a salt with a univalent cation harmless, it was found that it was not possible to produce similar antitoxic effects through the addition of a salt with an anion of higher valency. If sodium sulphate,
4W
sodium citrate, etc., was added to a — NaCl solution, the latter continued
2
to remain toxic for the Fundulus egg.
It is remarkable that not only the solutions of a salt with a univalent cation, like NaCl, can be rendered harmless by a salt with a bivalent cation, e.g. ZnSO4, but that also the reverse is true; namely, that a toxic solution of ZnSO4 can be rendered harmless by a solution of NaCl, provided the concentration of the ZnSO4 is not too high. In 100 c.c. of a f m NaCl solution no Fundulus egg forms an embryo.
Wl
When from 2 to 8 c.c. — solution of ZnSCX are added to this NaCl
64
solution, just as many eggs form embryos as in sea water or distilled
Ml
water. If, however, from 4 to 8 c.c. of a — ZnSO4 solution are added
to 100 c.c. distilled water, not a single egg is able to form an embryo, although in pure distilled water these eggs live and develop as well as in sea water. The Zn-ions are therefore not only able to prevent the toxic effects of a pure NaCl solution, but the NaCl of this solution also prevents, in this case, the toxic effects of the Zn-ions.
The quantitative relations are of some interest. About 4 c.c. —
solution of a salt with a bivalent metal are required to render 100 c.c. of a f m NaCl solution harmless. We may therefore say that for this concentration of NaCl one ion of the bivalent metal suffices to render 1000 molecules of the salt with the univalent metal harmless. When a
AM
NaCl solution of a lower concentration, namely, f m or - is used,
less salt with a bivalent metal is required for the antitoxic effect than in the case of a f m solution. If we use a NaCl solution with a concen-
7^Z
tration of or below — , it is no longer harmful for the eggs of Fundulus.
4 If we use stronger solutions than f w, we soon reach a limit where the
addition of a salt with a bivalent metal no longer renders the solution harmless. It is possible that, at this limit, the loss of water on the part of the egg acts harmfully, and this effect, of course, cannot be antagonized by the addition of another salt. If we try to determine how much NaCl is needed in order to render a solution of ZnSO4 harm- less, we find that a comparatively large amount of NaCl is required for
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 49
AM
this purpose. In order to prevent the poisonous effect of a - - ZnSO4
solution, so much NaCl had to be added that the concentration of the
m
NaCl in the solution was about -^-. About 50 molecules of NaCl were
o
therefore required to render one molecule of ZnSO4 harmless. The fact that the antitoxic effects of the salts with bivalent cations are so much greater than those of the salts with univalent cations is possibly respon- sible for the fact that I did not succeed in rendering a f m NaCl solution harmless through the addition of a salt with a univalent cation. The concentration of the solution would become so high that this might be sufficient to kill the eggs. The salts of certain metals are especially toxic, it being impossible to use those like Cu or Hg lor antitoxic effects, as they cause coagulation of the contents of the egg in smaller concen- trations than are required for the antitoxic effects of such a solution. The development of the egg of Fundulus requires at summer tem- perature from about twelve to twenty-four days. If we use a Ca salt
wz
to render a -- or f m NaCl solution harmless, an embryo can be formed,
and it may hatch, but will then die; if, however, a Zn or Ba salt be used for this purpose, an embryo is formed, and it may develop for a number of days quite normally ; but it dies before its development is complete. If we allow the egg to complete its development in distilled water or sea water, and put the larva, after it has hatched, into a mixture of
wi
100 c.c. - - NaCl, and a small amount of a Ba, or Co, or Zn salt, the 2
embryo dies even more quickly than if put into the pure NaCl solution. These facts indicate that for this fish the ZnSO4 remains toxic even in the presence of the NaCl, and that these two salts are only antago- nistic as long as the fish is surrounded by the egg membrane. This sug- gests the idea that the antagonism between these two salts is due only to the fact that they retard each other's rapidity of diffusion into the egg.*
«»
If the egg is put immediately after fertilization into a - - NaCl solution,
very soon so much NaCl diffuses into the egg that it poisons the fish. The same is true if a small amount of ZnSO4 is put into distilled water. But if both salts are put together into the distilled water, neither the NaCl nor the ZnSO4 can diffuse as rapidly into the egg, and the germ lives long enough to form an embryo. In a few days, however, death occurs, showing that the diffusion of the ZnSO4 was not prevented, but only retarded. Another fact corroborates the idea that it is only
* Loeb, Pfliiger's Archiv, Vol. 107, p. 252, 1905.
50 DYNAMICS OF LIVING MATTER
the rate of diffusion of salts through the membrane which is retarded in
4M
this case. A pure NaCl of the concentration - - or f m only prevents
j£
the formation of an embryo when the egg is put into the solution imme- diately after fertilization. If, however, the egg is put for the first twenty- four hours after fertilization into normal sea water and then into the pure
tn
NaCl solution of the above-mentioned concentration, the — or f m
2
NaCl solution is not so toxic. In all probability the membrane of the egg or the cells becomes more hardened or less permeable during the first twenty-four hours.
The antagonistic effects between two salts with a bivalent cation are not so general, yet I found that \ c.c. of a -f$ m SrCl2 solution diminished somewhat the toxicity of a T5g m solution of MgCl2.
While in the case of the membrane of the newly laid Fundulus egg, the addition of a trace of a salt with a bivalent cation sufficed to anni- hilate or diminish the toxic effect of a pure NaCl solution, we never find such simple relations for the Fundulus after it is hatched or for any living cell that is exposed directly to the solution without the inter- ference of a dead membrane, like the one which surrounds the fish egg. For such directly exposed living tissues or animals, it is a rule that a pure NaCl solution of sufficient concentration requires, besides the CaCL,, a trace of KC1, in order to become harmless, as was shown in the above-mentioned case of marine Gammarus. Besides, it is not possible to substitute in that case for the Ca any bivalent cation; only Sr can serve as a substitute for Ca in these cases. These limitations become intelligible on the assumption that the surrounding salts diffuse slowly into the cells. As long as this diffusion is so slow that the secretory activity of the cells or glands of an animal may remove them as fast as they enter, the cell or the animal may live in such a solution. I consider
/yt,i
this the reason why a Fundulus may live in a — NaCl solution, while it
o
"YVI
cannot live in a - • NaCl solution. A second condition for the main- 2
tenance of life is, according to this hypothesis, the continuation of the action of the secretory mechanism. If the latter depends on the con- tractile power of the protoplasm, as I believe it does, we can understand that, in order to make a NaCl solution harmless, not only Ca but also K are required. We shall see in a later lecture that apparently Na, Ca, and K are required for the contractile phenomena of protoplasm. Hober and Gordon* have pointed out the existence of an antago-
* Hober und Gordon, Hofmeisttr's Beitr'dge zur chemischen Physiologic und Pathologic, Vol. 5, p. 432, 1904.
GENERAL PHYSICAL CONSTITUTION OF LIVING MATTER 51
nism between the precipitating effects of salts of univalent and bivalent metals, which Linder and Picton had already found. If arsenic sulphide is precipitated with a mixture of two salts with a univalent cation, or of two salts with a bivalent cation, the effects of the two salts are added to each other. If a mixture of a salt with a univalent cation and a salt with a bivalent cation is used, however, for the precipitation, the result is an inhibition instead of a summation of the effects. Hober and Gordon have repeated and confirmed this observation. In addition, they have found that, just as in my own experiments, the valency of the anion plays no role. I am not able to state whether this explains the observations made on Fundulus.
It is rather remarkable that many authors have found distilled water to be poisonous for fresh-water animals. Locke showed that some authors had been deceived by the fact that their distilled water contained traces of copper salts, owing to the fact that the water had been distilled in copper vessels. But Bullot* found that for fresh-water Gammarus distilled water is toxic even if distilled with all necessary precautions in Jena glass or quartz or platinum vessels, and if care is taken that it is free from ammonia. He found that if a trace of NaCl is added to the distilled water (so that the concentration of the latter was 0.00008 N) fresh-water Gammarus could live indefinitely in the distilled water. The presence of a trace of NaCl in the distilled water possibly preserves the membrane better, or maintains better the secretory activity of the cells so that the animal can be freed from the excess of water which diffuses into it.
Dr. Wolfgang Ostwaldf investigated the duration of life of the same fresh-water Gammarus in solutions of higher concentration. He found that these animals live longer in a mixture of one hundred mole- cules NaCl, two molecules KC1, and two molecules CaCl2, than in a pure sugar or NaCl solution of the same concentration. This is in harmony with the assumption that the absorption as well as the secretive action of the cells requires the presence of Na, Ca, and K in definite proportions, as we shall see more fully later.
In connection with these experiments I made an observation which possibly may become of some use in the study of the phenomena of adap- tation. When marine Gammarus is put into sea water, which has been diluted with various quantities of distilled water, one notices that, with increasing dilution of the sea water, the duration of life of the Gammarus at first diminishes but little ; that, however, at a certain degree of dilu- tion (about ten times that of the normal sea water) the duration of life
* Bullot, University of California Publications, Physiology, Vol. I, p. 199, 1904. t W. Ostwald, Pft "tiger's Archiv, Vol. 106, p. 568, 1905.
52 DYNAMICS OF LIVING MATTER
decreases quite suddenly. It is obvious that the discontinuity in the curve of duration of life means that here a new condition, or a group of new conditions, enters which before that time were not noticeable. What are these conditions? Experiments which I have recently made on the eggs of sea urchins showed that up to a certain degree the dilution of the sea water with fresh water killed the eggs only slowly, but that beyond a certain degree of dilution death was rather sudden. This sudden death was due to a process of cytolysis in which the eggs were transformed into "shadows." I am inclined to believe that some- thing similar occurs in certain cells of marine Gammarus and of marine animals in general, when the dilution of the sea water falls below a certain limit.
This idea receives some support from the fact that Wolfgang Ostwald found that a rise in the concentration of the sea water above a certain limit also caused a sudden decline in the vitality curve of fresh-water Gammarus. If the concentration of the sea water be raised above a certain point, the eggs of the sea urchin also undergo cytolysis.*
* Loeb, Pflugtr's Archiv, Vol. 103, p. 257, 1904.
LECTURE IV ON SOME PHYSICAL MANIFESTATIONS OF LIFE
i.f HYPOTHESES OF MUSCULAR CONTRACTION
THE phenomena which allow us to discriminate between dead and living matter are physical processes, e.g. in higher animals, the contraction of the heart, the respiratory and other muscular motions. If the chemi- cal processes in living matter and the physical changes they bring about in the colloids were entirely known, the physical manifestations of life would also be clear to us. The periodic character of many of the mani- festations of life suggests the idea that these processes occur in several phases which are probably connected, partially at least, in a catenary way, so that the preceding process has effects which cause the subse- quent phase of the process.
These catenary mechanisms are for the most part still unknown. Inasmuch as the number of possible changes in the condition of colloids seems limited, the impression might be gathered that by a guess the whole secret of the physical manifestations of life might be unraveled. Such surmises find their way occasionally into print. As a rule, those who are familiar with the specific case for which the guess is made are not helped by it. It is not worth while to devote any time to the point- ing out of the futility if not open absurdity of most of these attempts.
The origin of animal heat from chemical energy offers no further mystery. We know that a kilo of sugar yields about four thousand calories of heat, if burned in the laboratory, and that it gives the same heat if oxidized in the body. In our modern theory of nutrition, the heat value of the various kinds of food is justly used as the basis for the calculation of their nutritive value. The times are gone when physi- cians and biologists dared to raise the objection — as they did against Robert Mayer — that our body inherits its heat.
As far as the transformation of chemical energy into mechanical energy in the muscle is concerned, Robert Mayer and Helmholtz con- sidered the muscle as a thermodynamical machine. They assumed that in the muscle the heat produced by chemical processes is partly
53
54 DYNAMICS OF LIVING MATTER
transformed into mechanical energy; but they refrained from stating how this transformation occurs. Engelmann tried to fill this gap.* Striped muscle consists of alternating stripes of optically isotropic and anisotropic substance. Engelmann observed that in the contraction of the muscle the anisotropic substance increases in volume, while the isotropic substance decreases. As the total volume of the muscle does not change during the contraction, Engelmann concluded that part of the liquid of the isojtropic substance diffused into the anisotropic during contraction. He showed by experiments on violin strings (made of catgut) that such a process of absorption can be produced by heat. The violin strings show the same double refraction as the aniso- tropic stripes in the muscle. When a violin string is suspended in water, and the latter suddenly heated, the string contracts, and is able to lift a weight in this contraction. This shortening of the string is caused by an absorption of water by the string; and this imbibition is caused by the increase in temperature. Like the contracting muscle, the violin string, in this case, becomes shorter and thicker. The process is reversible as the string elongates again upon cooling.
In the case of the muscle, Engelmann assumes that through the stimulus which causes muscular contraction, heat is produced (through the oxidation of carbohydrates); and that the increase in temperature causes the anisotropic substance to absorb water from the isotropic substance. This causes the change of form in the muscle - - the thicken- ing and shortening - - by which it is able to lift a weight.
For such a shortening of the violin string through heating, an increase of about 10° in the temperature of the water is necessary, while the temperature of a frog's muscle during a single contraction increases only by 0.001°. Engelmann points out, however, that the increase in the temperature of the whole muscle does not indicate the rise in tem- perature in individual spots in the muscle, which may be considerably higher. The foci of combustion heat the whole mass of the muscle, and we measure only the latter increase of temperature, which may of course be quite small. Provided we grant this, it is necessary to assume that the heat is sufficiently rapidly dissipated by conduction to allow the rapid succession of relaxation and contraction of the muscle in tetanus. We know that the muscles can contract and relax many times a second, e.g. the muscles of the wings of insects contract and relax more than a hundred times a second. I do not believe that the process of dissipation of heat in liquids is rapid enough to make Engel- mann's hypothesis probable, or even possible. It is, however, conceiv- able that with a slight modification his hypothesis may be rendered free
* Engelmann, Ueber den Ur sprung der Muskclkraft, Leipzig, 1893.
ON SOME PHYSICAL MANIFESTATIONS OF LIFE 55
from objections such as we have mentioned ; namely, by assuming that chemical, not thermal conditions determine the absorbtion of fluid by the anisotropic substance. The action of the nerve upon the muscle might consist in facilitating a chemical change which increases the absorption of water by the anisotropic substance of the muscle.
2. QUINCKE'S THEORY OF PROTOPLASMIC MOTION
The thermodynamic conception of muscular contraction has been abandoned by many authors, and the surface energy has been con- sidered in its stead as the cause of muscular contraction and work. D'Arsonval, Imbert, and more recently Bernstein, have tried to offer a hypothesis of this kind. We shall understand these hypotheses better if we first consider Quincke's theory of protoplasmic motion.*
When a drop of oil is put on the surface of water which is in contact with air, the oil spreads in an extremely thin layer at the limit between water and air. This process continues until a film of oil exists between water and air. The conditions for the spreading of the oil on the sur- face of the water are as follows: the particle of oil O at the left end of the oil drop (Fig. 7) is under the influence of three surface tensions
which pull at it in three different - v _~^rr^ _~ WAITER
directions, OA, OB, and OC, and with different force. One is the
surface tension between air and water, which tends to pull the particle from O in the direction OA. The second is the surface tension OB at the limit of oil and air, which tends to pull the particle O in the direction of the tangent OB from O. The third force is the surface tension at the limit of oil and water, which tends to pull the particle in the direction of the tangent OC from O. The surface tension at the limit of water and air is greater than the sum of the surface tensions at the limit between oil and air, and oil and water. The surface tension between air and water is 8.25 mg., between oil and air 3.76 mg., and between oil and water 2.73 mg. The particle O will therefore be pulled toward the left ; and the same will happen with the next particle of oil, until the surface water air is substituted by the surface oil air.
These phenomena of spreading are accompanied by motions in the neighboring particles of liquid. If oil spreads at the surface of water,
* Quincke, Sitzungsbtrichtc der Berliner Akadtmic der Wissensch., p. 791, 1888.
56 DYNAMICS OF LIVING MATTER
the moving oil will, through friction, set the adjoining particles of water also in motion. The superficial layers of water, therefore, will move away from the center of spreading, and water will move toward the
center from the interior, and from below. The arrows in Fig. 8 represent the currents in the water caused by the spreading of the oil. WATER Quincke holds that such
FlG 8 phenomena of spreading are
the cause of all protoplasmic
streaming. Such a streaming occurs constantly in the cells of Chara or Nitella. Quincke gives the following explanation for this process : all protoplasm contains oil or fat, and the surface layer of each cell must, therefore, be surrounded by a film of oil or fat. The oil will form through hydrolysis traces of fatty acid. The protoplasm contains substances which form soaps with the fatty acid. A soap solution must spread at the limit between oil and water, as the surface tension between oil and water is greater than the sums of surface tension between oil and soap solution, and between water and soap solution (which is zero). When the soap solution spreads, it must pull with it the adjacent particles of protoplasm. In this phenomenon of spreading, new particles of the surface of oil come in contact with protoplasm, new soap is formed, and the process is repeated. These phenomena of spreading, which constantly repeat themselves, furnish the energy for the constant streaming of protoplasm. The protoplasmic streaming occurs in the case of Chara or Nitella in one direction only. Quincke believes that this is due to an asymmetry in the structure of the cell, which renders the resistance to the streaming greater in one direction than in the opposite direction. Hence, the streaming occurs in one direction only; namely, that of least resistance.
The motion of an Amoeba can be imitated by bringing a drop of olive oil which contains a trace of fatty acid upon a one half to two per cent solution of Na2CO3. The oil, in this case, forms at its surface a film of solid soap. As soon as this dissolves at one spot, soap solution must spread at the surface of water and oil, and the moving soap solution must set also the neighboring layer of oil into motion. In the oil drop, therefore, two movements must occur, one at the periphery, which is directed away from, and one in the center, which is directed toward, the center of spreading. The arrows in Fig. 9 indicate these streams. The particles that flow from the interior toward the periphery produce a bulging out, and this is the analogue of the formation of a pseudo-
ON SOME PHYSICAL MANIFESTATIONS OF LIFE
57
SOAP
FIG. 9. — AFTER BUTSCHLI.
podium. According to Berthold,* the phenomena of streaming in the interior of an Amceba in the process of the formation of a pseudo- podium are such as to agree with the ideas of Quincke. Biitschli has come to the same conclusion. It seems to me, however, that if it is true that the Amceba is covered with a solid surface film, one condition for the formation of a pseudopodium must be a local liquefaction of protoplasm. In consequence of such a liquefaction, new protoplasm must flow out, which, subsequently, will form a new solid film at its surface. This may again be liquefied, and a new streaming may occur, etc. Such liquefactions can be caused by lack of oxygen, as we saw in a previous lecture; but they may also be caused by other chemical changes. I am inclined to believe that phenomena of liquefaction play at least some role in these processes of protoplasmic motion.
Imbertf published several years ago a hypothesis concerning the contraction of smooth muscle fibers, which assumes that the "stimulus" which causes the contraction of smooth muscles produces an increase in the surface tension between the longitudinal fibrils and the surround- ing liquid of the muscle cell. These fibrils are long and thin cylinders ; every increase in surface tension must have a tendency to make these fibrils more spherical, i.e. thicker and shorter. Such a change of form occurs indeed during contraction, but it is difficult to understand why the fibrils do not assume this form under the influence of surface tension alone, without stimulation. To meet this difficulty, Imbert assumes that smooth muscle fibers cannot contract unless they are stretched passively. He presupposes that their arrangement in the body is such that this prerequisite is generally fulfilled.
Bernstein has tried to explain away some of the weak spots in this hypothesis. t The surface energy at the limit between two media is equal to the product of surface tension into the surface. The work which surface tension can do is measured by the product of the decrease in surface, times the surface tension. From this it follows that the sur- face energy can do considerable work only, when the decrease in surface
* Berthold, Studien uber die Protoplasmamechanik, Leipzig, 1 886. t Imbert, Archives de physiol., 5th series, Vol. 9, p. 289, 1897. J Bernstein, Pfluger's Archiv, Vol. 85, p. 271, 1901.
58 DYNAMICS OF LIVING MATTER
is large. From this Bernstein justly argues that Imbert's assumption is incorrect, inasmuch as the area required for the work the muscle really does, must be much larger than that between the fibrils and the neighboring liquid. Bernstein therefore assumes that the fibril consists of a row of quite small ellipsoids, whose long axis is in the direction of the fibrils, and whose form is determined by elastic forces. An increase in the surface tension must make these ellipsoid elements more spherical, and thus the fibril becomes shorter and thicker.
How can the nerve impulse or an artificial stimulation of the muscle increase the surface tension? There are several possibilities. Sub- stances might be formed in this case which increase the surface tension at the limit between Bernstein's hypothetical ellipsoids and the surround- ing liquid. Another possibility might be that, through the process of innervation or stimulation, an existing difference of electrical potential between the ellipsoids and the surrounding liquid might be diminished. D'Arsonval explains the efficiency of electrical stimuli in this way.* Hermann has offered another hypothesis ; namely, that the contrac- tion is a process of coagulation, and the relaxation, a process of lique- faction.f He was led to this idea by the fact that the change in form which the muscle undergoes in the case of rigor mortis is similar to that in contraction, and that moreover a number of other features are common to both. Although all these hypotheses concerning muscular contraction have been known for a number of years, none has led to a new discovery. The reason lies possibly in the fact that one or more links in the catenary series of processes which underlie muscular con- traction have been ignored in these hypotheses. It is well known that a muscle gains in mass through " contractions, and that it undergoes atrophy when it remains at rest. This fact indicates, in my opinion, very clearly that phenomena or reactions which directly or indirectly lead to growth form a part in the process of muscular contraction. I consider it quite possible that no hypothesis concerning muscular contraction will prove fertile until this relation between activity and growth of the muscle is recognized.
3. CONCERNING THE THEORY OF CELL DIVISION
Were scientists with a purely physical training to be asked to give a hypothesis concerning cell division, I believe that their hypothesis would not take into consideration the phenomena of growth. Nevertheless, these phenomena form an obvious link in the catenary series of processes
* D'Arsonval, Archives de physiol., 5th series, Vol. I, p. 460, 1889. t Hermann, Handbuch der Physiologic, Vol. i, Part I, p. 332.
ON SOME PHYSICAL MANIFESTATIONS OF LIFE 59
which result in the division of the nucleus and the cell. It had been tacitly recognized by botanists that the growth of a cell precedes its division and is possibly the cause of the division. The botanist J. Sachs was the first to definitely state that in each species the ultimate size of a cell is a constant for each organ, and that two individuals of the same species but of different size differ in regard to the number, but not in regard to the size of their cells.* Amelung, a pupil of Sachs, determined the correctness of Sachs's theory by actual counts. Sachs, in addition, recognized that wherever there were large masses of protoplasm, e.g. in Siphonese and other cceloblasts, many nuclei were scattered throughout the protoplasm. He inferred from this that "each nucleus is only able to gather around itself and control a limited mass of protoplasm." f He points out that in the case of the animal egg the reserve material - fat granules, proteins, and carbohydrates — are partly transformed into the chromatin substances of the nuclei, and that the cell division of the egg results in the cells reaching that final size in which each nucleus has gathered around itself that mass of protoplasm which it is able to control. Morgan J and Driesch§ tested and confirmed the idea of Sachs for the eggs of Echinoderms. Driesch produced artificially larvae of sea urchins of one eighth, one fourth, and one half their normal size by isolating a single cleavage cell in one of the first stages of seg- mentation of the fertilized sea-urchin egg. He counted in each of the dwarf gastrulse resulting from these partial eggs the number of mesen- chyme cells and found that the larvae from a ^ blastomere possessed only £, those from a \ blastomere only ^, and those from a | blasto- mere only •§• of the number of cells which a normal larva developing from a whole egg possessed. Moreover, he could show that when two eggs were caused to fuse so as to produce a single larva of double size, the gastrulas of such larvae had twice the number of mesenchyme cells. Driesch drew from his observations the conclusion that each morphogenetic process in an egg reaches its natural end when the cells formed in the process have reached their final size.
Gerassimowll found that by exposing dividing cells of Spirogyra to a low temperature the division became irregular, and it happened that the nuclear material instead of being divided between the two
* T- v. Sachs, " Physiologische Notizen," VI, Flora, 1893. t Sachs, " Phvsiologische Notizen," IX, p. 425, Flora, 1895.
mechanik
Vol
XVI, 1 903.
§ Driesch, "Von der Beendigung morphogener Elementarprocesse," Arch, fur Enhvicke- Inng'smechanik, Vol. VI, 1898. "Die isolirten Blastomeren des Echinidenkeims," ibid., Vol. X, 1900.
|| Gerassimow, Zeitsch.fur allgemeine Physiologic, Vol. I, p. 22O, 1902.
60 DYNAMICS OF LIVING MATTER
masses of protoplasm remained in one of the two daughter cells ; some- times all the chromosomes were united into one nucleus and sometimes he obtained two nuclei. He found that cells with an increased mass of chromatin only began to divide after their protoplasm had reached a much greater mass than that found in the normal cells with half the mass of the chromosomes. This fact seems to indicate that cell division is determined by the ratio of the mass of the chromosomes to that of the protoplasm. If the mass of the chromosomes in a cell is increased, the tendency for cell division does not develop until the mass of protoplasm is increased also. It is the merit of Boveri to have found the law which governs this condition for cell division.* He compared the process of segmentation in normally fertilized fragments of sea-urchin eggs, which contained the normal number of chromo- somes, with that of enucleated fragments which contained only the sperm nucleus, and whose mass of chromatin was only one half of that of the normal fertilized egg. In order to understand his results, the reader's attention should be called to the following fact: in the process of cell division each daughter nucleus of the egg contains just as many chromosomes as the mother nucleus, but the mass of chromatin (and possibly the other constituents of the nucleus) of each chromosome in the daughter nucleus is only one half of that of the corresponding chromosome of the mother nucleus. The next phase -- the resting stage between two divisions — consists in the growth of the chromosomes of the two daughter nuclei until they have reached the mass of the original chromosomes and then a new nuclear and cell division begins. The material for the growth of the chromosomes is furnished by the protoplasm and according to the above-quoted idea of Sachs by the reserve material included in the protoplasm and not by the "living" part of the latter itself. It is, however, questionable whether this latter discrimination has any real basis. In this way the process of cell division in the egg consists in the gradual transformation of proto- plasmic into chromatin material of the nucleus until a definite ratio between the mass of the chromosomes and the protoplasm is reached. When this is established, no new cell divisions are possible until the mass of the protoplasm is increased again through the absorption of food stuffs on the part of the cell. The process of the transformation of protoplasm into chromatin is necessarily rendered discontinuous through the fact that the chromosomes cannot grow indefinitely, but that growth will stop as soon as they have reached a certain size, and this fact leads apparently to the process of nuclear divisions. I
* Boveri, Zellen-Studien, Heft 5. Ueber die Abhangigkeit der Kertigrosse und Zellen- zahl der Seeigel-Larven -von der Chromosomenzahl der Ausgangszellen, Jena, 1905.
ON SOME PHYSICAL MANIFESTATIONS OF LIFE 6 1
have thought of the possibility that the continuation of the synthetical process which leads to the formation of nuclear material from certain constituents of the protoplasm gives rise to the formation of astrospheres as soon as the maximal growth of the chromosomes is reached. But" this does not need to enter into our consideration for the present.
Since each daughter nucleus of a dividing blastomere has the same number of chromosomes as the original nucleus of the egg, it is clear that in a normally fertilized egg each nucleus has twice the mass of chromosomes as the nucleus of a merogonic egg, i.e. an enucleated frag- ment of protoplasm which has only the sperm nucleus. Boveri has not only ascertained this fact but he has also ascertained the further fact that the final size of the cells after the cell divisions have come to a standstill is always in proportion to the original mass of the chromatin contained in the egg ; the cells of the merogonic embryo, e.g. the mesen- chyme cells, are only half the size of the same cells in the normally fertilized embryo. Driesch has just furnished a further proof of Boveri's law, that the final ratio of the mass of the chromatin substance in a nucleus to the mass of protoplasm is a constant in a given species. He compared the size of the mesenchyme cells in a sea-urchin embryo produced by artificial parthenogenesis with those of a normally fertilized egg and found them half of the size of the latter. When the fertilized eggs and the parthenogenetic eggs are equal in size from the start, - which is practically the case if eggs of the same female are used, --the process of the formation of mesenchyme cells comes to a standstill in the normally fertilized eggs when the number of mesenchyme cells is half as large as the final number of mesenchyme cells found in the parthenogenetic egg.* As a matter of fact, Boveri's results as well as those of Driesch were obtained by counting the cells formed by eggs of equal size and not by only measuring the size of the cells. It is most remarkable that certain apparent exceptions to Boveri's law which Driesch has actually found have been predicted by Boveri.
The fact that the process of cell division comes to a standstill when the ratio of the mass of the chromosomes in the nuclei of an egg or an organ to that of the surrounding protoplasm reaches a certain limit, suggests in my opinion the possibility that this ratio is determined by the laws of mass action and chemical equilibrium. If this is correct, the synthesis of nuclein compounds from the protoplasmic constituents must be a reversible process. This suggestion would gain in probability if it could be shown that a reduction of size in protoplasm in the case of starvation is also followed or accompanied by a reduction in the size of the nuclei.
* Driesch, Archiv Jiir Entwickehtngsmeckanik, Vol. 19, p. 648, 1905.
62 DYNAMICS OF LIVING MATTER
The fact that the cell division is as a rule preceded by a synthetical process explains possibly the fact mentioned in the second lecture that the phenomena of cell division in a fertilized egg come soon if not immediately to a standstill when the atmospheric oxygen is with- drawn from the egg. We have mentioned Schmiedeberg's view in regard to the role of oxygen in synthetical processes. But even if this view were not correct, we can understand that lack of oxygen might indirectly interfere with the synthesis of the nuclein compounds.
E. P. Lyon has shown that the chemical conditions and processes in the cell differ in the various phases of cell division. He found that during different stages of cell division the egg of the sea urchin shows a different resistance to the effects of HCN, and this difference repeats itself during each of the successive segmentations. More recently he has added the important fact that the production of CO2 on the part of the egg of the sea urchin also undergoes periodic variations during segmentation.
If we now turn to the physical side of the phenomena of cell division, we shall meet almost the same uncertainty which confronted us in the case of muscular contraction. We shall therefore confine ourselves to the enumeration of a few facts, with occasional hints for possible further work.
When we watch the process of cell division in an egg, we can dis- criminate at least three distinct phases of this process: first, the ap- pearance of systems of radiation — the so-called astrospheres — in the protoplasm of the cell. The second phase is the disappearance (liquefac- tion?) of the nuclear wall, and the division and migration of certain constituents of the nucleus, namely, the chromosomes toward the centers of the astrospheres, and the formation of two new nuclei. The third phase consists in the separation of the protoplasm into two pieces in a plane, which, from the position of the astrospheres, as a rule, can be predicted. This latter separation is the process of cell division proper.
If newly fertilized eggs of the sea urchin are put into sea water, whose osmotic pressure has been adequately raised by the addition of some salt, e.g. NaCl, or sugar, no segmentation occurs as long as the eggs remain in this solution. If they are brought back from this solu- tion into normal sea water, they will segment, provided they have not been left too many hours in the hypertonic sea water. There is, how- ever, a characteristic difference between this segmentation and the normal segmentation. If the eggs are brought back into normal sea water after two hours, they do not divide, as a rule, first into two, and then into four cells, but into three or four cells simultaneously. If they are left for three or four hours in the hypertonic solution, and then
ON SOME PHYSICAL MANIFESTATIONS OF LIFE 63
brought back into normal sea water, they break apart into from six to sixteen cells simultaneously in about ten or twenty minutes after being put back into normal sea water. If they remain for five or six hours in the hypertonic solution, many eggs suffer. If they do not suffer they soon break up, when put into normal sea water, into a still larger number of cells than if they remained only for three or four hours in the hypertonic sea water. I have seen such eggs divide simultaneously into about forty cells, or more, in from ten to twenty minutes after being put back into normal sea water. These phenomena of segmen- tation are accompanied by violent phenomena of streaming or proto- plasmic motion at the surface of the egg. From these facts I concluded that while the hypertonic sea water inhibits the cell division, it allows the division of the nucleus, which precedes the segmentation of the protoplasm.* W. W. Normanf undertook a histological examination of the eggs under these conditions. He found that if the concentra- tion of the sea water be adequately, but not excessively, raised through the addition of a definite amount of NaCl, KC1, or MgCl2, the nuclei of the eggs divide in the hypertonic sea water karyokinetically, into two, four, and eight successively, while no cell division occurs. When such an egg with eight nuclei is put back into normal sea water, it divides as a rule into more than eight cells simultaneously. If a slightly too high concentration is used, the distribution of the nuclei in the egg does not become so regular; if the concentration is still a little higher, an excessive number of astrospheres is formed, as Morgan and Norman found. In this case, the nuclear material is often not scattered in the egg, although the nucleus seems to be broken into smaller fragments, for if brought back into normal sea water such eggs break up rapidly into a larger number of cells. R. Hertwig had already observed the formation of astrospheres in the unfertilized eggs of the sea urchin when he added a little sulphate of quinine to the sea water, and Morgan applied the method used by myself and Norman to the unfertilized egg and found an excessive number of astrospheres, just as Norman had observed in the fertilized egg.J
It is obvious from these and other experiments not mentioned here that the loss of water on the part of the fertilized egg ultimately retards all the phases of nuclear and cell division, but not all quantitatively alike. It seems that the chemical process of transformation of protoplasmic into chromatin material is less interfered with than the cell division proper. This follows from the fact that the chromosomes may divide without
* Loeb, Jour, of Morphology, Vol. 7, p. 253, 1892.
f W. W. Norman, Archiv fiir Entivickehingsmcchanik, Vol. 3, p. 106, 1896.
J Morgan, Archiv fiir Entwicktlungsmechanik, Vol. 8, p. 448, 1899.
64 DYNAMICS OF LIVING MATTER
cell division. Under normal conditions, the growth of the chromo somes is followed by a formation of astrospheres and division of the nuclei, and this in turn is followed by a cell division. The loss of water which the egg undergoes in the hypertonic sea water seems to inter- fere mostly with the cell division. It is possible that the viscosity of the protoplasm is increased by the loss of water, and that this condi- tion interferes somewhat with the migration of chromosomes after they have divided and still more with the segmentation of the protoplasm. The growth of the chromosomes and the subsequent formation of as- trospheres seem, however, to continue for some time in the hypertonic sea water.
O. and R. Hertwig (as well as Roux) have noticed that as a rule the plane of division of a non-spherical cell is at right angles with the direction of the greatest diameter, or extension of the cell. Driesch has given a nice experimental proof for this rule. If the newly fer- tilized egg of the sea urchin be gently pressed under a cover glass, so that it is slightly flattened, the plane of division is at right angles to the slide. The position of the plane of cleavage is determined by the position of the nuclear spindle, and the latter depends upon the position of the centrosomes or astrospheres. The question hence arises, How does it happen that in most cases the common diameter of the two astrospheres coincides with the longest diameter of a cell? This posi- tion of the astrospheres or centrosomes becomes comprehensible on the assumption that these organs not only repel each other, but are also repelled by the external surface of the nuclei and the inner surface of the cell limit. The forces involved in this repulsion must be forces such as occur in liquids, as the contents of the egg of the sea urchin is mainly liquid. It must, moreover, be taken into consideration that the space in which the process of cell division occurs, is generally of microscopic and always of capillary dimensions. It is therefore quite possible that the repelling forces in this case are capillary forces. There is, however, another fact to be considered ; namely, that in the process of cell division the egg of some animals becomes elliptic, with its long axis falling in the direction of the common diameter of both astro- spheres. This has given rise to the idea that the spindle or the astro- spheres elongated the egg. I have often noticed - - as others have undoubtedly done before me — an elongation of the egg of the sea urchin in the direction of the spindle, but this always occurred imme- diately before the cell division. It gives easily the impression as if contractile forces were active in radial directions in the astrospheres and that these forces had something to do with the process of cell divi- sion. Certain deviations from Hertwig's law may be only apparent.
OAT SOME PHYSICAL MANIFESTATIONS OF LIFE 65
Such exceptions occur in epithelial cells, but it is quite possible, that not the whole cell but only one part of it is in this case the seat of the processes which cause the orientation of the astrospheres or the cen- trosomes.
R. Lillie has expressed the idea that electrical forces play a role here. - Were this idea correct, it should be an easy matter to control the orientation of the plane of cleavage in the cell by means of a gal- vanic current; such is, however, not the case, at least as far as our present experience goes. If hydrodynamic and "contractile" forces are responsible for the orientation of the astrospheres or centrosomes in the cell, it should be expected that these latter organs are solid or at least more viscous than the rest of the liquids of the cell. If the centro- somes are fixed organs of the cells, and multiply by division, they must naturally be solid or at least possess a solid surface. Alfred Fischer assumed that the formation of astrospheres depended upon a process of coagulation. This has not yet been proved, although this author has imitated the well-known figures of astrospheres in coagulated proteins.
If the process of nuclear division in transparent cells, e.g. egg cells, is observed, the impression is easily gathered that the astrospheres cause a liquefaction of the nuclear membrane and an emulsification of certain constituents of the nucleus. If this observation be correct, the phenomena of spreading and the phenomena of streaming con- nected with such a process might be the forces which carry the chro- mosomes of the nucleus toward the center of the astrospheres. This assumption is in harmony with the fact that the withdrawal of water from the egg cell diminishes the velocity of the nuclear division,* inas- much as the loss of water may easily increase the viscosity of protoplasm, and thus diminish the velocity of the process of streaming, finally ren- dering it entirely impossible.
The phenomena of streaming can be demonstrated most beauti- fully in the experiment described above in which eggs of the sea urchin were put into hypertonic sea water, whose concentration was just adequate to prevent the cell division, without preventing the nuclear division. When such eggs are put back into normal sea water after about three hours, the most powerful phenomena of streaming may be witnessed, resulting in the formation of knobs. The streaming seems to occur around the chromosomes or fragments of nuclear mate- rial as a center. Afterwards, each such knob, or projection, formed by the streaming becomes a separate cell.f
* Loeb, Am. Jour, of Morphology, Vol. 7, p. 253, 1892.
t This amceboid, character of cell division had been observed and described before by O. and R. Hertwig and called " Knospenfitrchung"
F
66 DYNAMICS OF LIVING MATTER
In my earlier experiments on artificial parthenogenesis, I frequently had opportunity to observe cell divisions of a character which made it clear that phenomena of streaming underlie cell division, at least, in these cases. Figures 10-13 give an illustration of such a case. The egg had been treated with hypertonic sea water, and when put back
FIG. 10. FIG. ii. FIG. 12. FIG. 13.
into normal sea water divided as represented in these drawings. The division began (as was frequently the case) on one side (Fig. 10), and the protoplasm then flowed in the direction of the two arrows (Fig. u) in opposite directions toward the two nuclei. The connecting piece becomes empty of protoplasm and only the pigmented solid surface film is left (Fig. 12), and finally this also disappears (Fig. 13). It is, however, possible that contractile forces acting in a radial direction in an astrosphere might bring about similar results.
The process of the cell division proper seems to consist also of several phases. A reduction of volume seems to occur in this process, inasmuch as the combined volume of the two daughter cells appears immediately after the division, smaller than the volume of the mother cell. This diminution of volume may be due to a loss of water, or watery liquid, on the part of the cell. There may also be a process of gelation on the part of certain constituents of the cell, e.g. the nucleus, which at this stage appears to form a solid mass, or possesses at its surface a solid wall.
4. THE ORIGIN OF RADIANT ENERGY IN LIVING ORGANISMS
The first investigation of animal phosphorescence that was of any consequence goes back to Faraday, who showed that the phosphores- cent part of a glowworm continues to send out light if it be made into a pulp. This observation speaks against the view of Kolliker and Pfliiger that the phosphorescence of animals is a function of "living" matter, and even, in certain cases, under the control of the nervous system. They were led to their view by the observation that "stimu- lation" could call forth the process of phosphorescence, while poisons and high temperatures caused it to disappear. From this Pfliiger *
* Pfliigcr's Archiv, Vol. 10, p. 251, 1875.
ON SOME PHYSICAL MANIFESTATIONS OF LIFE 6/
drew the conclusion that phosphorescent matter is irritable, and "irrita- bility" is considered a sign of life. We must not, however, overlook the possibility that stimulation of an animal may produce the process of phosphorescence indirectly, e.g. by causing motions on the part of the animal which bring the phosphorescent matter into contact with oxygen. Giesebrecht * has furnished an absolute proof for the fact that phosphorescence may be produced in animals by non-living mate- rial. He found that certain pelagic copepods, e.g. Pleuromma gracile and Leuckartia flaviensis show phosphorescence, and that this phe- nomenon is confined to definite points of their body, which correspond to the ducts of certain glands of the skin of the animals. These glands secrete drops of a greenish yellow substance. As long as the animals lie quiet there is no phosphorescence visible, but they show this phenom- enon when pressed or heated, or if brought in contact with ammonia, alcohol, or glycerine. This might easily be interpreted as signifying that the phosphorescence of these animals is a phenomenon, which is produced by the stimulation of the animal. Giesebrecht found, how- ever, that the phosphorescence occurs only when the secretion of the glands is brought to the surface of the animal, and comes in contact with the sea water. He proved, moreover, that the secretion even re- tains its power of phosphorescing after the death of the animal. Dead copepods, which had been preserved in a dry condition for three weeks, still showed the phosphorescence at the opening of the glands, whenever they were put into water. The above-mentioned "stimuli" caused the phosphorescence only indirectly, by causing the squeezing out of the secretion of the glands from the duct.
How the contact of the secretion with water can cause the phos- phorescence is not yet clear. Radziszewski f has found that a number of organic compounds show phosphorescence at a comparatively low temperature, e.g. 10° C., when they come in contact with atmos- pheric oxygen, and the reaction is alkaline. Among these substances are the soaps of oleic acid, a number of alcohols, etc. This author assumes that the phosphorescence of animals is caused in the same way. Traces of the phosphorescent substances and of oxygen suffice for the production of the phenomenon. We can readily understand that mo- tions of an animal are favorable for the production of phosphorescence, as they tend to bring the oxygen (e.g. in the tracheae of insects) in con- tact with new particles of the phosphorescent substance. Giesebrecht questions the importance of oxygen for this process, inasmuch as in
* Giesebrecht, Mittheilungcn aits der zoologischen Station zii Neapel, Vol. 2, p. 648, 1895.
t Radziszewski, Liebig's Annalen der Chemie, 1880.
68
DYNAMICS OF LIVING MATTER
his experiments the animals also showed phosphorescence in boiled water; but as very little oxygen suffices for the phenomenon, it is possible that in Giesebrecht's experiments sufficient oxygen was present for the pro- cess. The experimenters agree, in general, that free oxygen is neces- sary for phosphorescence. It is possible, however, that the conditions for phosphorescence may vary with the nature of the substance.
5. ELECTRICAL PHENOMENA IN LIVING ORGANISMS
When Galvani noticed that the muscles of the leg of a frog twitch when touched with two metals, he believed that this phenomenon indicated the production of electricity in living organisms. Volta subsequently showed that the nerve-muscle preparation only acts as a sensitive rheoscope. Thus a misunderstood biological observation became the germ for the development of electrochemistry. It was found afterward that living organisms produce indeed some electrical energy, but in spite of the most diligent search nobody has yet been able to prove that the electrical energy thus produced plays any role in an essential life phenomenon, although this may be the case.
The liquids of the body must be the cause of the differences of potential in the tissues as only the electrolytes dissolved in these liquids are capable of producing differences of potential. The most common instances of the production of a difference of electrical potential are
the cases of an active or dying nerve or muscle. When an element of a nerve is active or injured, and one electrode of a galvanometer is applied to the active or injured spot (Fig. 14), an- other to the neighboring resting, or normal element of the nerve or muscle, a cur- rent of positive electricity travels through the galvanometer from the resting, or normal, to the active, or injured element of the nerve or muscle. The activity of the muscle, or its injury, is accompanied by a production of acid, i.e. carbonic, and possibly, lactic acid. According to Waller CO2 is also produced in the active nerve.* I concluded from this that these currents might be due to the formation of acid. The H-ions have a much greater velocity of migration than any anion, and hence,
* Waller, Lectures on Physiology, I. On Animal Electricity, London, 1897.
|
+ |
4- |
|
|
Active or Resting or Injured. Normal |
FIG. 14.
ON SOME PHYSICAL MANIFESTATIONS OF LIFE 69
if in an active or injured- element of the nerve or muscle acid is formed, the hydrogen-ions must migrate faster into the neighboring tissue than the anions. Consequently, the active element will have an excess of free negatively charged ions, while the neighboring resting elements will assume a positive charge (Fig. 14). I published this explanation of the origin of currents of action in a preliminary way in an address at the Naturalists' meeting in 1897.* Oker-Blom-f has since expressed a similar view in regard to the current of demarcation ; and he mentions that Tschagovetz has published a similar view in a Russian journal.
Ostwald J has pointed out that the semipermeable membranes may possibly be permeable for only one class of ions, positive or negative, and "not only the currents in muscles and nerves, but also the myste- rious effects of electrical fishes might find their explanation by such a property of the semipermeable membranes." Bernstein § and Briin- ings || have recently adopted this view. Bernstein pointed out that, in order to explain the above-mentioned current of action, a specific permeability of the semipermeable membranes for cations must be assumed. It is hardly possible that differences in electrical poten- tial can arise in any other way in the tissues than by a separation of anions and cations of the electrolytes dissolved in the tissues. As the difference in the rate of diffusion for different ions always exists, especially when acids or alkalies are formed, and as a difference of the permeability of the protoplasm for oppositely charged ions may also easily exist or arise, it is not difficult to understand that so many life phenomena are accompanied by electrical changes and currents, e.g. when light falls upon the retina, or when glands secrete.
Plants also show such currents, especially such plants, as are dis- tinguished by a comparatively quick conduction of stimuli, e.g. Mimosa or Drosera. This lends, perhaps, support to the idea expressed by Hermann that the current of action is the cause, or means, of the propa- gation of the nerve impulse. When a nerve or muscle is stimulated, the stimulated spot becomes negatively electrical as compared with the neighboring resting spot. In the next element of time this latter spot becomes the seat of activity, and now becomes negative toward the more distantly situated piece of nerve, etc. A region or wave of negative potential is thus propagated from the original seat of stimu- lation in both directions, through the nerve. Bernstein has found that this negative wave is propagated with the same velocity as the
* Loeh, Science, N. S., Vol. 7, p. 154, 1898. t Oker-Blom, Pfiuger's Archiv, Vol. 84, p. 191, 1901. j Ostwald, Zeitsch. fur physik. Chemie, Vol. 6, 1890. § Bernstein, Pfliiger's Archiv, Vol. 92, p. 521, 1901. || Briinings, Pftilger1 s Archiv, Vol. 100, p. 367, 1903.
70 DYNAMICS OF LIVING MATTER
nerve impulse, so that there exists a possibility that this difference of potential which originates upon stimulation is the cause, or the means, of propagation for the nerve impulse. Hermann has given a more detailed sketch of such an assumption.* The axis cylinder of the nerve is surrounded by a liquid conductor of electricity, i.e. a solution of electrolytes. If a certain element A of the axis cylinder be stimulated, it will assume a negative charge, while the neighboring parts B assume a positive charge. This leads to the formation of a microscopic cur- rent from B through the liquid conductor to A. This current may be considered as a stimulating current for the axis cylinder with an anode at A and a cathode at B. We shall see in a later lecture that if a current be made, the stimulation occurs at the cathode, while the anode is put into a condition of diminished irritability. Therefore, the region A now returns to a condition of rest, while B becomes active. Then the same process is repeated for B and its neighboring element, etc. Waller has been able to determine the beginning of life in the hen's egg and in seeds of plants by galvanometric tests; he has also deter- mined the cessation of life in the same manner. f These facts may serve as a further indication that all life phenomena are accompanied by electrical phenomena. We shall see later that salts play a great role in life phenomena ; and it is obvious that if changes in the nature and number of ions in a solution accompany life phenomena, electrical currents must also be a necessary consequence.
* Hermann, Handbuch der Physiologie, Vol. 2, 1st part, p. 193, 1879. t Waller, C. R. de r Academic des Sciences, Vol. 131, pp. 485 and 1173, 1900. Proceed- ings of the Royal Society, Vol. 68, p. 79, 1901.
LECTURE V
THE ROLE OF ELECTROLYTES IN THE FORMATION AND PRES- ERVATION OF LIVING MATTER
i. ON THE SPECIFIC DIFFERENCE BETWEEN THE NUTRITIVE SOLU- TIONS FOR PLANTS AND ANIMALS
THE green plants are the factories in which the material for the nutrition for animals and fungi is prepared. The green plant, how- ever, manufactures also, as long as it grows, its own living matter out of the electrolytes of the soil and the CO2 of the air. The CO2 is util- ized for the formation of carbohydrates and probably fats; the salts of ammonia, nitrates, phosphates, and sulphates are used for the building lip of nitrogenous compounds. One of the nutritive solutions * which is most commonly used for phanerogamic plants is as follows : —
4 g. Ca(N03)2 i g. KNO3 i g. MgS04 + 7 H20 i g. KH2P04 0.5 g. KC1
The whole is dissolved in from 3 1. to 7 1. of water. A few drops of ferric chloride are added to this solution. This solution may vary within certain limits. It contains besides the anions CO3, NO3, SO4, and PO4, which are necessary for the synthesis of the essential compounds of the plant, the cations K, Ca, Mg, which do not seem equally necessary for the synthesis of living matter. In addition to these, free oxygen is absolutely necessary for the formation of living matter in green plants.
For the fungi the nutritive solutions are similarly constituted, with this difference only, that they cannot make carbohydrates of CO2 ; and they are therefore compelled to get their sugar from plants or animals. If raised in a solution containing sugar or certain organic acids (e.g. acetic, tartaric acids) and certain salts, they can also make
* Knop's Solution. See Pfeffer, PflanzenpJtysiologie, 2d edition, Vol. I, p. 413, 1897.
71
72 DYNAMICS OF LIVING MATTER
all the constituents of living matter, e.g. fats, proteins, nucleins. Raulin, a pupil of Pasteur, has investigated with unparalleled thor- oughness the optimal nutritive solutions for a fungus, Aspergillus niger. Raulin * determined which nutritive solution gave the great- est development of living matter from a given quantity of spores, and found that it possessed the following composition : —
Water 1500 g.
Cane sugar 70 g.
Tartaric acid 4 g.
(NH4)3PO4 0.60 g.
K2CO3 ......... 0.60 g.
MgCO3 0.40 g.
(NH4)2 SO4 0.25 g.
ZnSO4 0.07 g.
FeSO4 ......... 0.07 g.
K2SiO3 ......... 0.07 g.
Of course, to this list must be added atmospheric oxygen.
Part of the free acid in Raulin's solution is neutralized by the HO- ions due to the presence of (NH4)3PO4 and K2CO3 in the solution. The sugar, fatty acid, ammonia, SO4, and PO4 are used for the build- ing up of living matter ; but it is not clear what the role of K, Mg, Zn, and Fe is. It is remarkable that Ca is not required, and it seems to be a general fact that Ca is not of great importance for the fungi, while it is of great importance for animals, and apparently also for the higher plants. But what is the role of the cations?
It has been noticed that the living tissues of plants, as well as of animals, possess a selective power for certain salts, especially for K-salts. Although in fresh-water streams the concentration of K-salts is often very low, the plants which live in it are capable of storing up a com- paratively large amount in their tissues. The muscle of animals shows the same phenomenon, inasmuch as it contains a much higher per- centage of potassium than the blood. This "selective power" admits of only one explanation; namely, that the potassium is used for the building up of more complex compounds in which the K cannot be dissociated as a free ion. If a tissue utilizes one kind of metals in this way, e.g. K, while another metal, e.g. Na, is chiefly used for the forma- tion of dissociable compounds with Na as a free ion, the consequence will be that the ashes of a tissue contain K and Na in altogether differ- ent proportions from what they are contained in the surrounding solu-
* See Duclaux, Traite de microbiologie, Vol. I, p. 176, li
ELECTROLYTES IN LIVING MATTER
73
tion. I think we may take it for granted that, at least, K forms a nondissociable constituent of the protoplasm of a number of tissues of animals and plants, and that it therefore may be considered a building stone for living matter in the same sense as the above-mentioned anions.
This fact explains the so-called oligodynamic effects. This term was applied to the fact that certain heavy metals like Cu, or, as I believe, traces of their salts, can produce a toxic effect. Obviously the Cu-ions form upon their entrance into the cell undissociable or practically undissociable compounds with protein substances, and thus their con- centration is kept lower in the cell than in the surrounding solution. The consequence is that in due length of time enough Cu may diffuse into the cell to act toxically. There is therefore no reason why we should continue to set aside the oligodynamic effects as a distinct group of phenomena in biology. It is quite possible that an ion may be utilized in two ways by a tissue; namely, for the synthesis of mole- cules from which it can no longer be dissociated as an ion, and in the form of salts, — possibly ion-proteids, — where the metal can disso- ciate as an ion.
What is true for the K may also be true for the Mg, but it can scarcely be so for the Zn in Raulin's solution, although, curiously enough, it is not so much less important for Aspergillus than K. Raulin found that if he allowed spores to develop on the above-mentioned solution, which contained all fourteen constituents, except K, the crop was only one twenty-fifth of the dry weight of that which he got when he added K. When the trace of Zn contained in that solution was omitted, the dry weight of the crop was only one tenth of that which he obtained when Zn was added. Raulin has made similar determinations for all the constituents of his nutritive solution. The figure following each sub- stance in the table below expresses how many times greater the dry weight of the crop was with the addition of the substance than without it.
NH
Mg
K
S04
Zn
Fe
SiO,
153 182
9i
25
25 10
2.7 1.4
It was to be expected that the omission of NH4 from the solution would reduce the crop considerably (to T^-g of its weight), inasmuch as it
74 DYNAMICS OF LIVING MATTER
furnishes the material for the manufacture of the proteins. The PO4 is needed for the nucleins, and it is probable that the Mg is necessary for the building up of definite important compounds; but the Zn is no part of any compound of the plant. It is therefore obvious that the nutritive solution for a plant not only contains substances which are of importance for the building up of its living matter, but also sub- stances which do not enter into these compounds and are yet of im- portance. I am inclined to believe that the explanation of the latter facts takes us back to the antagonistic salt effects discussed in the previous lecture.
If we compare the nutritive solutions for animals with those of plants, we find in general that PO4-, NH4-, NO3-ions, which are of such im- portance for plants, are either of no importance for animals, or are directly poisonous, e.g. the NH4-ions. Inasmuch as the animals get all their proteins and carbohydrates directly or indirectly from plants, it is to be expected that they do not depend upon the CO2 of the air or the NH4, NO3, or PO4 of the soil. We meet, however, with another striking difference between animals and plants, which was not to be expected a priori; namely, the fact that Na, which appears neither in Knop's solution for Phanerogams nor in Raulin's solution, is one of the most, if not the most, important constituent of a nutritive solu- tion for animals. Next in importance for animals is Ca, which does not appear in Raulin's solution, although it seems to be important for phanerogamic plants.
We have already seen that the majority of marine animals, e.g. marine Gammarus, can only live in solutions which contain certain salts, NaCl, CaCl2, KC1, and MgCl2 in definite proportions. The lack of Mg is not so fatal as the lack of one of the other three metals. One anion is sufficient; namely, Cl. Without Na, K, or Ca the animal lives at the utmost but a couple of hours, as a rule a much shorter time; while in a mixture of NaCl, KC1, and CaCl2 it may live as long as two days, and still longer upon the addition of MgCl2.
Similar results were obtained in experiments on the substances which Tubularians need for regeneration and growth. These Hy- droids can only live in a solution which contains NaCl, KC1, CaCl2, and MgCl2. If one of these salts is lacking, no polyp can be regener- ated. In order to allow the polyp to grow, a substance must be added which keeps the reaction of the solution neutral; namely, NaHCO3*
The conditions for the development of the eggs of the sea urchin,
* Loeb, Pfluger's Archiv, Vol. 101, p. 340, 1904.
ELECTROLYTES IN LIVING MATTER 75
Strongylocentrotus purpuratus, are similar.* NaCl, KC1, and CaCl2 are necessary: as without one of these salts no segmentation is pos- sible. For the complete development Mg and SO4 are also required, but these latter two constituents do not possess the same degree of importance as Ca, Na, or K. In addition, a substance is needed which keeps the solution neutral, e.g. NaHCO3. Other constituents of the sea water, such as PO4, Fe, are not required. This latter statement disagrees with the conclusions of Herbst. f
The same is true for Medusae : they will keep alive in solutions of NaCl, KC1, CaCL,, MgCl2, in the proportion and concentration in which these solutions occur in the sea water. In addition, a substance is required which keeps the sea water neutral, e.g. NaHCO3. I think these examples may suffice as the proof of the fact that for marine animals NaCl and CaCL, and KC1 are essential for the maintenance of life. It is questionable whether the substances which growing animals require for the manufacture of living matter are taken from the surrounding solution. Were this the case, only traces of any of these salts should be sufficient, while in reality the proportion of Na, K, and Ca can vary only within certain limits in the solution.
The tissues of marine animals seem to require a solution of the same character. Dr. Rogers has, at my suggestion, determined in which solution the heart of a marine crab beats longest. He found that sea water is an excellent "nutritive" solution for the heart, and that the same is true for a van't Hoff solution; namely, a solution of 100 molecules NaCl, 2.2 KC1, 2 CaCl2, 7.8 MgCl2, 3.8 MgSO4. To this should be added a trace of NaHCO3. The action of sea water becomes better if a little CaCL, is added, possibly on account of a slight antago- nistic effect between Ca and Mg.
It is remarkable that the tissues of fresh-water and land animals, e.g. the frog, the tortoise, and apparently the mammals, live longest in a solution which has the same constitution as the sea water, and differs from the latter only in its concentration. The optimal concen- tration of the solutions for frogs and land animals is about that of a
Wl
•Q- solution of NaCl. In order to keep the isolated heart of cold-blooded
o
animals alive, Ringer has recommended the following solution : — J
* Loeb, Pfliiger's Arehiv, Vol. 103, p. 503, 1904.
t Herbst, Arehiv fitr Entivickehitigsmechanik, Vol. 5, p. 649, 1897, and numerous other papers on the same subject.
Herbst did not recognize the antagonistic effects of salts, and so concluded that if the elimination of one of the constituents of the sea water was injurious, this proved the neces- sity of the omitted substance for the animal. The above-mentioned observations on Fundu- lus show in m.y opinion the fallacy of this conclusion.
t Quoted after Rusch, P/Higer's Arehiv, Vol. 73, p. 535, 1898.
76 DYNAMICS OF LIVING MATTER
Water 1000 g.
NaCl ......... 6 g.
KC1 0.075 g-
CaCl2 ......... o.i g.
NaHCO3 o.i g.
Rusch has applied this solution with success for the isolated heart of warm-blooded animals, with the difference only that he added 8 g. instead of 6 g. of NaCl to 1000 g. of water.
Locke * recommends the following solution for the isolated heart of the rabbit: —
Water 1000 g.
NaCl 9.10 g.
KC1 ......... 0.2 g.
CaCL, ......... 0.2 g.
NaHCO3 o.i g.
The solution is said to be more effective if a little dextrose is added,
«•£• i g-
Otherwise Locke's solution differs from Ringer's by a somewhat
higher amount of KC1 and CaCl2. His figures for these latter salts are approximately those which Abderhalden found for the concen- tration of these salts in the serum of rabbits; namely, 0.024 per cent CaCl2 and 0.042 per cent KCl.f
If we express the percentage solutions of Ringer, Locke, or Abder- halden, in the values of grammolecular solutions, we find that it is approximately 100 molecules NaCl to 2 molecules of CaCl2. This is practically the proportion in which these salts exist in the sea water, and in which marine animals live longest. This proportion may vary a little for marine animals, and the same is true for the solutions in which the tissues of animals live best, as a comparison of the figures of Ringer and Locke shows.
An observation mentioned already in a former lecture shows con- clusively that the mixture of 100 NaCl, 2 KC1, and 2 CaCl2 cannot be considered as a nutritive solution for animals, but must play a different role. Fundulus lives just as well in distilled water as in sea water. This fact proves that these animals do not depend for their nutrition
* Locke, Centralblatt fur Physiologie, Vol. 14, p. 670, 1901.
t The fact that Locke also mentions sugar as one of the necessary constituents of his solution indicates that he considers the other constituents also as nutritive material. This would, however, be wrong. From my own experiments I do not think that the addition of sugar is of any value.
ELECTROLYTES IN LIVING MATTER 77
and development upon the salts dissolved in the sea water. If the young fish, however, are put into a pure solution of NaCl of the concen-
(tyyi \ -). the animal 2/
dies in less than twelve hours. If CaCl2 is added, the animal does not live more than twenty-four hours. If it is desired to keep animals alive permanently, - - my experiments lasted for ten days, - - not only 2 CaCl2 but also 2 KC1 must be added to 100 NaCl. This is exactly the solution which is generally considered as a nutritive solution for animals.* I believe that these facts show that we must discriminate between nutritive and protective solutions. Ringer's solution, as well as the sea water, are primarily protective and not nutritive solutions. What is meant by this becomes clear if we remember what was said concern-
*>yi
ing the antagonistic effects of salt. A - - solution of NaCl, as well as a
Wl
'- solution of ZnSO4, alone are each poisonous for the eggs of Fundulus. °4
If mixed, the solution becomes considerably less poisonous. It is prob- able that these two salts if together in solution materially diminish their rate of diffusion into the tissues. It follows from these experi- ments that the role of the Ca and Mg in the sea water, as well as in a Ringer's solution, consists partly in antagonizing the effects which would be produced by the NaCl were it alone in solution. The experiments with Fundulus suggest that in this case the presence of the Ca and Mg in the sea water diminishes the rapidity of diffusion of the NaCl into the tissues. It is possible that the Zn acts in some protective way in the case of Raulin's solution, although in regard to this it is not possible to make a definite statement.
But there remains the other fact that K is also needed. The life of Gammarus and many other marine animals is not essentially pro- longed by the addition of CaCl2 or MgCL, or both to the NaCl solution, but is materially prolonged by the addition of CaCl2 and KC1 to the NaCl solutions. Besides we cannot, in general, substitute any other bivalent metal (with the exception of Sr) for Ca; nor can we sub- stitute any other univalent cations for Na and K. This indicates that the metals Na, Ca, K, and Mg play a role in life phenomena other than that of serving for the synthesis of living matter, and also other than that of merely regulating the velocity of diffusion. We have already alluded to the possibility of their necessity for phenomena of secretion. We are inclined to believe that what is generally called irritability and contractility is due to the influence of these ions.
* Loeb, Pfluger's Archiv, Vol. 55, p. 530, 1893.
78 DYNAMICS OF LIVING MATTER
2. CONCERNING A THEORY OF IRRITABILITY AND THE ROLE OF NA,
K, AND CA FOR ANIMAL LIFE
In 1899 I outlined a general theory of irritability which may be briefly summarized in the following sentences which I quote from a former paper: "The salts, or electrolytes in general, do not exist in living tissues as such exclusively, but are partly in combination with proteids (or fatty acids). The salts or electrolytes do not enter into this combination as a whole, but through their ions. The great importance of these ion-proteid compounds (or soaps) lies in the fact that, by the substitution of one ion for another, the physical properties of the proteid compounds change (e.g. their surface tension, their power to absorb water, or their viscosity or state of matter). We thus possess in these ion-proteid or soap compounds essential constituents of living matter, which can be modified at desire, and hence enable us to vary and control the life phenomena themselves." *
Life phenomena, and especially irritability, depend "on the pres- ence in the tissues of a number of various metal proteids, or soaps (Na, Ca, K, and Mg), in definite proportions."
I first applied this conception to a phenomenon which had hitherto been observed only occasionally ; namely, rhythmical contraction of the muscles of the skeleton.f I found that such rhythmical contractions occur only in solutions of electrolytes, i.e. in compounds which are capable of ionization. In solutions of nonconductors (urea, various sugars, and glycerine), these rhythmical contractions are entirely or practically impossible. Only in certain, not in all, salt solutions are such rhythmical contractions possible. All the solutions of Na-salts are able to produce them, but in a 0.7 per cent NaCl solution, contrac- tions begin later, and are less powerful, than in an equimolccular NaBr solution. The experiments on the rhythmical contractions of the muscles of the skeleton led to some other data concerning the effects of those salts. Solutions of Na-salts produce rhythmical contractions only if the muscle cells contain Ca-ions in sufficient numbers. As soon as there is a lack of Ca-ions in the tissues, the Na-ions are no longer able to cause rhythmical contractions. On the other hand, if we add Ca-salts in sufficient quantity to the NaCl solution, it will no longer cause rhythmical contraction in a fresh muscle of the frog. It there-
* Loeb, Am. Jour. Physiology, Vol. 3, p. 337, 1900.
t Loeb, Festschrift fur Professor Pick, Wiirzburg, 1899.
The idea of the general existence of such ion-proteid compounds was developed inde- pendently by Pauli and myself in 1899. Loeb, Pfliigeijs Archiv, Vol. 75, p. 303, 1899 ; and Festschrift fur Pick, 1899 ; and W. Pauli, Wiener akaJemischer Anzeiger, October 12, 1899 ; and Ueber physikalisch-chemische Methoden und Probletne in der Medizin, Wien, 1900.
ELECTROLYTES IN LIVING MATTER 79
fore looks as if the presence of a certain quantity of Na-ions caused contractions ; but if the quantity of the Na-ions becomes too great in proportion to the Ca-ions, the muscle loses its irritability. On the other hand, if there are too many Ca-ions present, the rhythmical con- tractions become also impossible. The quotient of the concentration
£
of the Na-ions over the concentration of the Ca-ions, — — a, becomes
Cca
therefore of importance for phenomena of irritability. We shall see later that Mg acts very much like Ca in this respect.
It is hardly necessary to mention that this suggested the possibility that muscular contraction, in general, is due to a substitution of Na for Ca, or vice -versa, in certain compounds (proteins or soaps) in the muscle. Every substance or agency will act as a stimulant which brings about such a change of the metals in these compounds in the muscle.
It may be added that all the salts of univalent metals act like the Na-salts, inasmuch as they cause rhythmical contractions when the