The Dynamics of Living Matter

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

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.

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

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, 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. 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.

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

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 : — 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

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. 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 —

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. 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 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. 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-

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. 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. 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

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