Protoplasmic Action and Nervous Action

Emulsions and foam structures are essentially similar systems, with the difference (as usually defined) that in a foam the disperse or discontinuous phase is a gas, in an emulsion a liquid. Jellies or gels also resemble these systems in constitution in many cases. It is now known that various different types of gel structure exist; many jellies, however, are essentially dense emulsions; thus stiff* foams of air with a soap solution (or solutions of albumin, saponin, or other surface-active colloidal substances) have many of the characters of solids or semisolids; i.e., permanence of form, elasticity, high viscosity; and all transitions between liquid and solid systems of the emulsion type are known — a fact familiar to anyone who makes a lather of soap solution. The same is true of an emulsion of one liquid in another; thus an emulsion of oil in a soap solution may be made

^For a general review cf. Bancroft's series of articles on '"The Theory of Emulsification " in Journal of Physical Chemistry, XVI-XIX (191 2-15); also the recent book of Clayton, The Theory of Emulsions and Emulsification. so concentrated — with more than 90 per cent of oil as a disperse phase in some of the emulsions prepared by Pickering^ — that the whole mass has a jelly-like consistency. The differences of opinion as to whether living protoplasm belongs to the ''sol" or ''gel" type are thus seen to be unimportant, since transitions between these states are continuous, and in fact many forms of protoplasm exhibit a liquid consistency at one stage (or under certain conditions) and a solid consistency at another.^

In a foam of air and soap solution the individual bubbles do not coalesce, although the intervening films may be extremely thin; evidently the structural stability of the system as a whole is determined by the properties of the films. If we break down these films, mechanically or otherwise, the whole foam structure collapses. The stability of emulsions of oil in aqueous media is similarly conditioned; in this case the coalescence of the separate oil droplets is prevented by interfacial films of soap or other material, and such an emulsion can be also destroyed (de-emulsified) by altering the material composing the films, e.g., by adding strong acid if soap is the emulsifying material. Such facts lead to the general question of the conditions determining the stability of a foam structure or emulsion.

Two chief conditions for the persistence of an air foam are that the layer of solution separating the adjacent bubbles should have (i) a low surface-tension and (2) a high viscosity. A low surface-tension is favorable because the tangentially acting forces tending * Cf . Bayliss' recent paper in Proceedings of the Royal Society, B, to rupture the films or laraellae are then small; i.e., the natural tendency of the film material to minimize its surface, or "draw together," is slight. But this condition is not alone sufficient, as seen in the fact that pure liquids of low surface-tension against air, like ether, benzol, alcohol, etc., do not give permanent foams, any more than does water. It is well known that mixtures of alcohol and water foam more readily than either liquid alone ; and this is especially true of mixtures of water and a second liquid of great surface-activity and high viscosity, such as amyl alcohol; hence the important generalization that pure liquids do not foam — do not form permanent disperse systems with air. Nor do mixtures of two pure, mutually immiscible liquids readily form permanent emulsions. Typically the presence of a third substance is necessary, and it is important that this third substance should be of such a kind as to lower the surface-tension at the boundary between the phases, and also to impart to the surface layer a relatively high viscosity or resistance to displacement. Under some conditions this viscosity may be sufficient to impart to the interfacial layer the properties of a solid film. In general, a third substance is effective as an emulsifying agent in proportion to its power of forming at the boundary a film having these properties of low surfacetension and high viscosity. Most substances which *form stable emulsions of oil in water (soap, proteins, gums) are of this kind. The interfacial films or lamellae then resist disruption and the disperse droplets are prevented from fusing. If the film is considered as a phase, most emulsions would be classed as three-phase systems (triphasic).

Another condition favoring stability in an emulsion is a small diameter in the disperse droplets. The disperse particles in suspensions and emulsions are electrically charged, and if they are sufficiently minute the forces due to their mutual electrostatic repulsion may be sufficient to prevent contact and fusion/ We must therefore qualify the statement that at least three components are necessary in a permanent emulsionsystem by the proviso that the subdivision be not excessively minute. This factor, however, is of minor importance in most emulsion systems, and probably is not of great importance from a biological point of view. It is worthy of note, however, that in some cases mutual electrostatic repulsion appears to play a part in determining the distribution of colloidal particles, droplets, or other minute freely mobile particles in cells; e.g., the distribution of the chromatin in the equatorial plates and spiremes of mitotic figures shows evidence of this factor.^

An emulsion, being a system of disperse charged particles, resembles in this respect any colloidal suspension; hence electrolytes influence the stabiHty of emulsions, because of the influence of the ions on the interfacial potentials, just as they influence the stabihty of other colloidal systems.^ Generally speaking, any mechanical, chemical, or electrical conditions which alter the surface lamellae affect the stability and other* 3 For the action of electrolytes on the stability of emulsions cf , Powis, Z. physik. Chem., LXXXIX (1914-15), 186. Cf. Northrop and De Kruif, /. Gen. Physiol., IV (1921-22), 639, for an account of the analogous action of electrolytes in the agglutination of bacteria.

properties of an emulsion system. Emulsions of oil in alkaline water or soap solution are destroyed by adding strong acid (HCl) which breaks down the soap films. Similarly a foam structure may be destroyed mechanically or by adding a surface-active substance of low viscosity; thus a few drops of ether destroy a beer-foam, a fact explained by Quincke as due to the displacement of the material composing the surface lamellae.^ Similarly a saponin solution to which sufficient alcohol is added does not form a permanent foam; the addition of isobutyric acid to a saponin solution also prevents foaming, but if alkali is added to neutralize the acid and form the surface-inactive salt, foaming results.^ Many other facts of a similar kind are well known. The conditions of de-emulsification ('' cracking" of emulsions) deserve careful study by biologists, for changes of this kind are almost certainly concerned in many forms of protoplasmic activity; e.g., secretion and the processes of activation, stimulation, and cytolysis.

In general, therefore, we may define the chief condition of stability in emulsion systems as the presence of material, differing from that composing the two chief phases, in the form of thin continuous layers or films deposited or adsorbed at the boundary surfaces. The thickness of these films may be extremely slight; when a material is surface-active and is free to spread over the surface separating the phases, conditions of equilibrium may not be reached until the layer is only one or two molecules thick. ^ Such a film, however, is

capable of holding one liquid finely dispersed in another in a permanent state of emulsion. We may infer that in at least some forms of protoplasmic emulsion-structure the interfacial films are of molecular thickness, a consideration of much importance in relation to the properties of irritability and transmissivity (or propagation of excitation-states), as will be seen below. It will be clear from the above that in the formation of emulsions — and hence of living protoplasm as a system based upon the emulsion type of structure — the conditions determining the formation of interfacial films are of primary importance. Adsorption, the process by which material collects or concentrates at boundary surfaces, is thus a fundamental factor in the formation and behavior of emulsion systems and of colloidal systems in general. The physics and chemistry of adsorption processes have recently been discussed fully in several excellent textbooks,^ so that it is unnecessary here to give any detailed account. One general fact, however, which may be emphasized as especially important from the physiological point of view, is that adsorbed substances are typically more subject to chemical change than substances uniformly distributed in a solution. Both the increase of concentration and the presence of surface factors are concerned in the increase of reactivity, the catalytic action of many finely divided materials (charcoal, platinum, etc.) is usually referred to the increased concentration of the chemically altered material

^ Hober, Physikalische Chemie der Zelle und der Gewehe (1914); Freundlich, Kapillar chemie, Leipzig (1909); Bayliss, Principles of General Physiology; Bancroft, Applied Colloid Chemistry. at the surface of the catalytic agent, but it appears probable that other factors (electrical) also enter in many cases of adsorption-catalysis (see below). In considering the case of protoplasmic systems, we may regard adsorption as of importance in two chief respects: (i) as an essential condition in the determination of structure (through the formation of the adsorption-films of the protoplasmic emulsion and in membrane structure in general), and (2) as a main factor determining the character and velocity of the chemical reactions; i.e., as influencing or controlling cell-metabolism.

It is well known that the adsorption of dissolved substances of low molecular weight is, as a rule, a strictly reversible process, with the equilibrium conditions defined by the formula xjm = kcn , where x is the quantity adsorbed, m the mass of the adsorbent, c the concentration of the substances in solution, and k and n constants. On the other hand, in the case of colloidal substances or other substances of high molecular weight, adsorption frequently leads to a change of properties, the substances becoming converted into relatively insoluble or resistant varieties^ (possibly polymerized). In such cases the process may be difficultly reversible or irreversible, a fact of much interest as bearing on the question of the conditions under which the more permanent portion of the protoplasmic substratum is formed. In general, organic growth appears to depend on the deposition of relatively stable or persistent structural elements or material in apposition to other elements or material of

^ Cf. Hober, op, cit., p. 220, for instances of anomalous or irreversible adsorption. the same kind, a process suggesting an irreversible type of adsorption. A few examples of irreversible adsorption may be cited for illustration. Freundlich and Losev^ found that a solution of the dye, crystal violet, was completely decolorized by animal charcoal, and that washing would not give back the dye. This may mean that the concentration of the dye in solution at equilibrium is indefinitely small; but more probably it points to the formation of an insoluble modification as the result of adsorption. Proteins like egg-albumin when adsorbed at the surface of drops of chloroform, form thin highly insoluble and resistant pellicles; i.e., the protein undergoes the change usually described as "denaturation." Various other cases of anomalous adsorption are probably to be referred to conditions of a similar kind; the adsorbed material apparently undergoes some chemical modification.

A further fact of fundamental biological interest is that the adsorbent action of many materials has a certain specificity or selective character; i.e., the action varies from adsorbent to adsorbent independently of the latter's state of subdivision. This phenomenon has apparently the same ultimate basis as have the specific chemical affinities between substances; in fact, the distinction between adsorption and the formation of true chemical compounds is now very generally recognized as ill defined.^ The phenomena of cohesion, adhesion, and capillarity are closely related to adsorption; thus water wets (is adsorbed by) certain solid surfaces,

but not others. It is well known that the interfacial tension between an adsorbing surface and a solution of an adsorbable substance is a direct function of the degree of adsorption of the latter. When the adsorbed substances are of low molecular weight — e.g., in homologous series of alcohols, organic acids, or similar compounds — it is usually found that the order of relative adsorption is not altered by altering the adsorbent, although the degree of adsorption may vary widely with the different adsorbents. With more complex molecules, however, relations of an apparently arbitrary or specific kind often enter, and presumably the relations between the molecular structure or configuration of the adsorbing surface and that of the adsorbed substance then become important. Specific adsorptions, like specific chemical combinations (between enzyme and substrate, or antigen and anti-body) are thus probably largely dependent on similarities of chemical configuration. Hence a good adsorbent for one substance may be a poor one for another.^ Freundlich cites various instances illustrating the differences between the adsorbent powers of different materials for the same substance.^ Thus charcoal adsorbs crystal violet 20 times as effectively as silk, and 156 times as effectively as cotton wool. He found that different adsorbents usually showed the same order of relative adsorption for the dyes used; with four solid adsorbents the general order of adsorbent action was charcoal>wool> silk > cotton, but the ratios of the adsorption constants varied with different dyes.

^ Cf. Bayliss, op. cit., p. 60, for instances of specific adsorption; also Bancroft's Applied Colloid Chemistry, p. 3. Wohler and Plliddemann^ found that iron oxide adsorbed 10 times as much benzoic acid as acetic acid; chromic oxide adsorbed both about equally; while platinum sponge adsorbed more acetic than benzoic, but both shghtly. According to Freundlich, gelatine adsorbs sugar only after having been treated with formaldehyde.'' The influence of the specific molecular structure of the adsorbent on its selective adsorption is well shown in the investigations of Marc. A crystalline adsorbent, BaC03 (rhombic) adsorbs KNO3 (rhombic) but not, or slightly, NaN03 (hexagonal); CaC03 (hexagonal) adsorbs NaN03 but not KN03.^ These observations throw an interesting light on the phenomena of crystallization; it is well known that the specific molecular configuration of a substance determines the form in which it crystallizes, as originally shown by Pasteur's observations on the separation of laevo- and dextrotartrate in separate crystals in the crystallization of the optically inactive solution. Apparently the abstraction of molecules from solution and their deposition to form the regular solid structure or crystal are determined by conditions of the same kind as those determining selective adsorption. Adsorption of molecules at the surface of the crystal is a preliminary to the growth of the latter; this growth is evidently dependent on mutual apposition of molecules similar in configuration ^and dimensions and with their axes parallel."* In organic growth — another

4 Crystal growth, in fact, appears to afford the clearest cases of specificity in adsorption. type of specific growth and form-determination — similar processes are almost certainly concerned. The reversible character of many adsorption processes is a property of great biological importance and apparently one essential to certain physiological effects, such as narcosis, which are universal in living protoplasm. There is no doubt that this reversibility also plays an essential part in the normal chemical processes of protoplasm. The displacement of one adsorbed compound by another of greater surface-activity presupposes reversible adsorption, and various biological instances of this effect are known. Thus an adsorbed enzyme can be removed from an adsorbing surface by adding a more surfaceactive substance; e.g., rennin adsorbed by charcoal and added to milk will not coagulate the latter, but on the addition of saponin the rennin is set free and causes coagulation. A solution of rennin is inactivated by shaking with air, but not if saponin is present; the latter protects the enzyme by preventing adsorption at the air-water interface.'

Similar cases of inactivation by shaking are cited by Meltzer and Shaklee.^ Apparently only the adsorbed enzyme is inactivated; it has already been mentioned that changes of physical state frequently result from adsorption; Ramsden's observation that proteins can be coagulated by shaking with air is an instance of the same phenomenon.^ Hence the prevention of adsorption through the presence of another surface-active compound may be a factor in preventing physical and chemical alteration in living protoplasm. Preventive effects of this kind probably form a chief factor in the anaesthetic action of surface-active compounds, as well as in the protective action which they often exhibit (against salt action, haemolysis, or mechanical injury)/

In general, colloids of the suspensoid group are less readily adsorbed than those of the emulsoid group, in correspondence with the fact that the latter are usually surface-active, the former not; For the same reason the suspensoids do not usually act as emulsifying agents, while many emulsoids are highly effective in this regard. The distinction, however, is not absolute, since finely divided insoluble substances of various kinds may emulsify oils under certain conditions;^ what is essential is that the material should collect and form a continuous layer at the surface between the phases.

As a rule proteins are surface-active; their solutions have lower surface-tensions than pure water and they are readily adsorbed. The conditions are, however, complex; the degree of adsorption may vary with the same protein according to its state of subdivision (which varies with the salt content) or according to the H-ion concentration of the solution; the latter condition determines the proximity to the isoelectric point and hence ^Cf. Bancroft, loc. cit.; Journal of Physical Chemistry, XVI

the electrical properties of the particles, a factor which influences their adsorption.^ Observations on the relation between the concentration of proteins in solution and the degree of their adsorption give many abnormalities, probably referable chiefly to variations in the aggregation state.^ The particles cohere and form larger aggregates which condense at surfaces, forming films of modified protein. Such processes are largely irreversible, and chemical change probably also enters as a factor; the changes in the properties of enzymes, dyes, and other colloidal compounds in adsorption are probably to be thus explained. Many cases of abnormal and irreversible adsorption belong here; such abnormalities are especially characteristic of colloids of the emulsoid group. The surface-activity of proteins, and the readiness with which they form films, filaments, and other coherent structures, at the surfaces where they are adsorbed, are undoubtedly properties of great biological importance; and we may assume that in the deposition of proteins in a solid or semi-solid state to form the more permanent structural elements of protoplasm such processes play a chief part. As already indicated, it seems probable that specific adsorption, a process apparently based on the tendency of molecules of similar configuration to cohere or coalesce to form larger aggregates, is the fundamental factor underlying the specificity of growth processes.

As already pointed out, adsorption may furnish the conditions for many of the chemical reactions in cells; ^ Cf . Christiansen, cited by Pauli, Colloid Chemistry of Proteins, Philadelphia (1922), p. 89. i.e., material concentrated or condensed at the protoplasmic surfaces may in this manner first become capable of chemical interaction. In a recent paper^ Bayliss gives instances showing that in many reactions in polyphasic systems adsorption is the initial process which forms a necessary preliminary to the true chemical combination following.

We may conclude that the determination and control of chemical reactions by adsorption are universal in living protoplasm. The presence of colloidal complexes or ^'adsorption compounds" — e.g., compounds of lecithin with proteins, such as lecithin-vitellin (in the ethereal extract of egg yolk) and jecorin (dextrose and lecithin plus protein) — is frequent in organisms. Inorganic salts and ions are probably also largely present in a condition of adsorption;^ and the indications are that the action of the surface-active pharmacological compounds (especially the anaesthetics) is largely so determined. According to Loewe, the chief relation between lipoids and narcotic compounds is one of adsorption,^ although the relative solubilities of these compounds in the different protoplasmic phases (partition-coefficients) probably also enter as an important factor in narcotic action."*

The promotion of chemical action by adsorption is often called ''adsorption-catalysis." Well-known examples are the formation of H2SO4 from SO2 in the presence of platinum, the reduction of various compounds by hydrogen in the presence of platinum, and the oxidation of compounds by blood-charcoal (oxalic acid, etc.). The combination of tannin with leather is a good illustration of the determination of a chemical reaction by a previous adsorption; the tannin is first adsorbed, then it combines. The same condition is shown in the union of dyes with heat-denatured egg-white; the process is at first readily reversible (by acid), but not later, indicating that the first stage of the process is a close contact or adhesion, which is then followed by chemical combination.^ The toxin-antitoxin reactions and the opsonin reaction with leucocytes are further biological instances of a similar kind. According to Morgenroth, tetanus toxin is taken up or attached by living cells at 8°, but does not become active until 20°. In the action of enzymes adsorption processes play an important part, as already indicated.^

The relations of electrostatic attraction or repulsion between the charged surface of the adsorbent and the charge on the particles of the dissolved substance constitute a factor of decisive importance in many adsorption processes. For example, acid dyes (whose colloidal particles are negatively charged) are as a class more readily adsorbed by suspended alumina (with positive particles) than by kaolin, a silicate with negative particles, and vice versa. Color bases show the reverse behavior, being adsorbed by substances which form

negatively charged surfaces; e.g., silicates, carbon, and adsorbents of chemically acid character, but not by hydrates like alumina or other positive adsorbents.^ Suspensoids of heat-denatured albumin show the same behavior; when the particles are made positive by the addition of a little acid they become adsorbents for acid dyes, which also cause precipitation; while in the negative condition, i.e., on the alkaline side of the isoelectric point, they are precipitated by (and adsorb) basic but not acid dyes. The mutual precipitation of oppositely charged colloidal solutions when mixed in suitable proportions is an example of the same phenomenon.

The importance of the electrical factor in the general behavior of colloids is indicated by the remarkable changes in adsorptive and chemical properties which a given protein exhibits when the H-ion concentration of the solution passes from one side of the isoelectric point to the other. On the acid side precipitation is induced by the anions of an added electrolyte, on the alkaline side by the cations, as Hardy first showed in the case of heat-modified egg-albumin.^ Recently the relation between the charged condition of the protein aggregates and their chemical and other behavior has been investigated in much detail by Loeb,^ who has determined many striking correlations between the physical and the chemical properties of proteins at varying H-ion con-

^ Cf . Michaelis, Physikalische Chemie iind Medizln, edited by Koranyi and Richter, Leipzig, II (1908), 341. 3 For a summary of these important investigations cf . Loeb's recent book Proteins and the Theory of Colloidal Behavior, New York, 1922. centrations. As Pauli^ also has pointed out, many properties (viscosity, osmotic pressure, refractive index, precipitability by alcohol) pass through a minimum which is coincident with the isoelectric point.

In all colloids the electrical factor plays a special part in changes of aggregation state or dispersion. In precipitation by electrolytes adsorption processes enter; one or the other ion is adsorbed predominantly, i.e., the ion of opposite sign to the colloidal particle; accordingly this ion is the precipitant. In general the more readily adsorbed an electrolyte is, the more effective it is as a precipitating agent. Freundlich has shown this clearly for the salts of a number of organic bases. ^ The relative precipitating effectiveness of the several salts, with colloidal arsenious sulphide, is shown in the following table:

The order of precipitating concentrations is the reverse of the order of relative adsorption. Theoretically the two ions of an electrolyte should have different adsorption constants in relation to an adsorbing surface. The nature and quantity of the ions adsorbed will influence the electrical conditions at the interface, and, secondarily, all processes in which these conditions are a factor, such as colloidal stability, state of dispersion, cataphoresis and electrical endosmose, and catalytic action. The action of electrolytes on colloids shows many indications of adsorption effects; those ions which other evidence indicates are in general the most strongly adsorbed (H, OH, polyvalent cations) have a correspondingly marked influence on the colloidal state. The influence of adsorption is shown with especial distinctness in the action of salts on protein solutions, as Pauli's work has especially shown; the characteristic curves relating temperatures of heatcoagulation, melting points of gels, and precipitability by alcohol to concentration of salt (when the salt is present in excess of that required to form stoichiometric compounds) are clearly of the adsorption t>pe.^ The same is true for the influence of salts on the osmotic pressure of protein solutions.^

Apparently, in all processes where surface effects are concerned, the different salts of the same metal differ in their action according to the nature of their anions; according to Rontgen and Schneider^ the order of relative adsorption of anions is S04<Cl<Br and N03<I; this series corresponds to the characteristic lyotropic series of Hofmeister, which is shown not only in the above-cited work on proteins and in many of the physiological effects produced by salts^ but also in various purely physical phenomena involving surface factors