The Dynamics of Living Matter

* Biitschli, Untersuchungcn uber tnicroscopische Schaume und das Protoplasma, Leipzig, 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.*

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- 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 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.

§ 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. 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

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.

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-

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. 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

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 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.

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.

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

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. 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 : — CONCENTRATION OF THE NACL SOLUTION HOURS IN PER CENT OF ITS ORIGINAL WEIGHT

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.) 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.