Principles of General Physiology

Now these two points of view are not to be regarded as antagonistic or mutually exclusive. As Perrin remarks (1905, p. 110), with respect to the fact that change of electrification is accompanied by change in the composition of the double layer and, therefore, in the composition of the colloid as given by chemical analysis, " physical and chemical variations are here two aspects of one and the same phenomenon." On the other hand, there are certain physical properties not necessarily involved in the chemical description of proteins, which must play a part in their behaviour. Since they do not diffuse through parchment paper, we know that they are large enough to exhibit the properties of matter in mass, the most characteristic being those connected with the possession of surface. This involves electrical relations differing from those of simple electrolytes, and so forth.

When we find a book, " The Physical Chemistry of the Proteins," by J. Brailsford Robertson, 1912, which professes to treat the whole subject without reference to any of the conceptions which the modern development of the theory of the colloidal state has introduced, wicannot but agree with the reviewer (W. O.) in Zeitsch. f. physik. Chemie, 81, 508, whose remarks will serve to call attention to the facts to be taken into consideration in an adequate treatment. "But in so far as the colloidal, that is nonhomogeneous, charm t< i of protein compounds has been proved experimentally beyond all douot, it appears to me (the reviewer) that, in the intentional laying aside of this fact, an error of method is committed, an error which brings with it considerable danger of laying more weight on a particular interpretation of facts, in themselves correctly observed, than is desirable in the interests of science. This danger is all the more serious when the author is one who is able to manipulate, with considerable skill and corresponding predilection, complex mathematical formulae, and by that means is able to introduce as many variables into the theoretical treatment of his problems as satisfactory agreement with experimental results requires. Owing to the complex and changeable nature of the substances in question, a completely exact agreement between measurement and calculation can never be expected, and, for this reason, the widest possibilities for theoretical presentation offer themselves."

With reference to the two theories, the purely electro-chemical and the colloidal, where the conditions at finite boundary surfaces are taken into account; the same reviewer says, " The two are, in point of fact, nowhere and never in opposition, but are merely different stages in the complete analysis of the physico-chemical phenomena." An illustration due to A. W. Stewart (Chemical World, 2, 53) will serve to show the misleading effect of a one-sided consideration. "Suppose that we gave two specimens, a diamond and some graphite, to be examined by purely chemical methods on the one hand, and by purely physical methods on the other. The chemist, relying on his analysis, would declare them to be identical, consisting, as they do, of carbon. The physicist, on the other hand, from an examination of colour, density, form, etc., would pronounce them to be different from one another. Both would be right, but each in possession of half the truth only." If we wanted to cut glass, it would not be of any help to be told that the chemical composition of diamond and graphite is the same. On the other hand, suppose that we wanted to prepare carbon dioxide by burning in oxygen, either would serve, although we should hardly choose the diamond for the purpose. Should the electronic theory of the constitution of atoms be correct, reconciliation between the chemical and physical points of view will presumably be found in molecular physics.

In order to indicate the kind of problems which confront us in the study of proteins, the work of Hopkins and Savory (1911, p. 249) on Bence-Jones' protein, may be referred to. This substance is found in the urine in certain disorders of metabolism, and is characterised by its peculiar behaviour on heating. In the absence of salts, it coagulates on heating to about 50°, and becomes an irreversible or suspensoid colloid. If neutral salts are present, the coagulum is redissolved on boiling, owing, perhaps, to the formation of a chemical compound with salts, although, as we shall see later, this interpretation is rather questionable. If the suspensoid particles are given a positive or negative charge by traces of acid or alkali, the precipitating effect of electrolytes comes into play, and the anion or cation becomes prepotent, according to Hardy's rule. We have then a complex state of antagonistic effects. There are two relations to be taken into account, one between the salt as a whole and the protein molecule, perhaps a chemical one, although the lyotropic effects described in the preceding section must not be forgotten, the other relation, a physico-chemical, colloidal or electrical one between the particle (qua particle) and the ions of the salt as carriers of electric charges. In no other way can the experimental facts be satisfactorily explained.

A few words are requisite at this stage as to the chemical nature of proteins, so far as to make intelligible the way in which it intervenes in their colloidal reactions. A more complete account will be found in Chapter IX. It has been shown, mainly by the work of Emil Fischer ("Collected Papers," 1906), that these substances are formed by the condensation of a number of molecules of various amino-acids. Now the amino-acids are characterised by the presence of one or more NH2 groups, giving them basic properties, and one or more carboxyl groups, giving them acidic properties. Alanine, or amino-propionic acid, is

They belong, therefore, to the class of electrolytes called by Bredig (1899) amphoteric, behaving towards strong bases as acids, and towards strong acids as bases. When the COOH and NH2 groups are equal in number, as in alanine, the amino-acid is very nearly equally strong as a base and an acid, and is therefore practically neutral in reaction, actually very faintly acid. If the NH9 groups are in excess, as in the diamino-monocarboxylic acid, lysine, the substance becomes a fairly strong base ; while, if the carboxyl groups are in excess, as in the monoamino-dicarboxylic acid, aspartic acid, we have a fairly strong acid. These acids are capable of combining together by the COOH of one uniting with the NH2 of another, with elimination of water, thus : —

There are always some NH2 and some COOH groups left uncombined, and according to the relative number of these, the resulting protein or polypeptide will have the properties either of a base, a neutral substance, or an acid. This brief sketch will suffice for our present purpose, although it must be remembered that some of the constituent amino-acids are complex compounds containing aromatic, pyrrol, iminazol, etc., groups. When combined with base or acid, say sodium or hydrochloric acid, an amino-acid forms a salt, thus alanine becomes sodium amino-propionate or alanine hydrochloride respectively : —

Similarly, the free NH2 and COOH groups of the protein can react with acid or base to form a salt. Now, like all salts, these salts of proteins are electrolytically dissociated in solution, the sodium salt of globulin, for example, partially dissociates into Na- and a large organic anion, which has the properties of the colloidal state. The hydrochloride dissociates into 01' and a large colloidal organic cation. We see thus how, by direct chemical means, we can obtain the same protein with a negative or a positive charge. It appears, also, that these colloidal ions are very ready to form aggregates, as the simple, insoluble, inorganic salts, such as

arsenious .sulphide, do. It remains as yet uncertain whether many of the complex proteins, occurring naturally, are not aggregates of various simpler ones, united by means not strictly chemical. As remarked above, proteins may be either weak aoids or weak bases. In t In- former case, owing to the preponderance in number of H- ions given off, the colloidal ion is left negative, just as silicic acid is ; in the latter case, the protein particle will be positive, as it gives off rapidly moving OH' ions. If the protein is an aggregate, it is clear that dissociation will occur in the case of those molecules on the surface only, and the colloidal ion will be more bulky than if the protein is in single molecules.

It is of interest to record the fact that crystals of leucine, a simple amino -arid, suspended in their own saturated solution, have a small negative charge, as would be expected from the slightly more acidic than basic character of leucine itself. When a solution of leucine is made acid, there is a deposition of the solute on the cathode, when a current is passed through the solution, and vice versa when made alkaline. Thus it behaves in the same way as a protein under the same conditions.

On addition of small amounts of a strong acid to a solution of a very weak acid, by the law of mass action the dissociation of the weak acid is practically abolished, so that its molecules are almost entirely present as neutral uncharged elements. The same thing happens when a strong acid is added to a protein solution. But owing to the amino acid nature of the latter, the effect in question is replaced by formation of a salt when the quantity of acid added is increased : the acid then combines with the basic groups of the amino-acid. At a particular concentration of acid, therefore, the protein exists with a maximum of electricallyneutral molecules. This is the isoelectric point, which varies with different proteins, according to the degree of their acidic properties.

Now it is found experimentally that the lyophile character varies greatly according to the presence or absence of the electric charge, i.e., whether the protein is in the form of an ion or otherwise (Pauli, 1912, p. '226). The increase of hydration implied in this, goes with increase of properties such as viscosity, imbibition, solubility, osmotic pressure, difficulty of coagulation by alcohol and heat, surface tension, and rotation of polarised light. The importance of the distribution of the solvent between the phases of a colloidal system has been emphasised by Hatschek, as already mentioned, and we see now how the effect of acid and of alkali in increasing the water content of the dispersed phase may be explained by the production of protein ions. As will be shown in more detail in Chapter VIII., ions are usually associated with a considerable number of molecules of the solvent.

The action of neutral salts is not so simple. The example of the Bence-Jones' protein, given previously, points to a double action, if not a triple one. The effect of salts in large concentration in removing water from the internal phase, and thus producing what is known as "salting out," has been described under the head of emulsoids above (page 97). The precipitating power of salts follows the " Hof meister series" and it is important to note that certain properties of water, such as surface tension, viscosity, compressibility, are affected in the same order, so that it is to be supposed that the action of salts in removing water is exerted rather on the water itself than on the protein (Pauli, 1912, p. 238).

The same series was found by Rothmund, independentlv (%<tt*ch. /". />/< //*//.•. ('In in., 33, 401), to apply to the case of the solubility of phenylthiocarbamide. Schryver (1910) brings the phenomena into relationship with the effect of the salts on the surface tension of water. Whether there is actual chemical union between proteins and neutral salts is a matter of some dispute. It has been suggested that reaction may occur in such a way that both potassium and chlorine, for example, may join on to the nitrogen of the NH2 group, as H and Cl do. Another possibility is that the K may unite with COOH in the usual way, while the Cl joins to the NH.,, with the aid of the H displaced from COOH. These suggestions do not seem very probable from the chemical point of view, although, of course, not impossible. Until recently, no direct evidence had been brought forward in favour of combinations of a chemical kind, but Pfeiffer and Modelski (1912) state that they have obtained crystalline

salts of glycocoll with calcium chloride and lithium chloride, although none could be obtained with potassium chloride. A fact which also makes the evidence rather uncertain is that, in order to maintain constant composition in successive recrystallisation, it was necessary to add dilute acetic acid. If recrystallised from water, no constant composition was shown. I have repeated some of these experiments, but have been unable to get crystals of constant composition, even using acetic acid, and have been compelled to conclude that the preparations of Pfeiffer and Modelski consisted of mixed crystals, although it is difficult to account for their results being in accordance with these required by the chemical formula?. I found that, unless the solutions were very highly concentrated, the pure amino-acid, both in the case of glycocoll and of leucine, crystallised out first and that it was not till the mixture was evaporated nearly to dryness, or by the addition of alcohol, that the neutral salt came down also. Further evidence on this point is therefore necessary.

On the other hand, there is certain evidence that salts are adsorbed by proteins. Such effects as those on the temperature of coagulation are found to be expressed by a formula similar to that of adsorption, and not by stoichiornetrical relations. The amount of salt attached to the protein particle is found to be in certain proportion to that free in the external phase. Pauli (1912, p. 231) also points out that there is evidence that a surface action is in question, in that the viscosity of protein solutions is always lowered by the addition of a salt. This implies that the surface of contact is changed from one between albumin and water to one between salt and water.

If salts are adsorbed by protein, it is to be expected that, in the case of comparatively insoluble salts, a considerable difference would be found in their apparent solubility in water and in protein solutions. This has been found to be the case, by Pauli and Samec (1909, p. 241), for calcium sulphate, phosphate and carbonate,, silicic acid and uric acid. The amount of very soluble salts adsorbed would be too small a percentage of the total solubility to be detected.

Some experiments made by myself (1906, p. 182) on the rate of removal of salts from gelatine by. water, also point to the adsorption nature of the union, as also other experiments on the taking up of salts. A further fact in connection with the question before us is that measurements of electrical conductivity show no effects of neutral salts similar to those which are so obvious where we know that true chemical reaction takes place, viz., with strong acids and alkalies.

The experiments of Bugarsky and Liebermann (1898, pp. 68, 72) show that no combination occurs between proteins and neutral salts, whereas it does between proteins and strong acids or alkalies. (See also page 221 below.) Chemical combination between neutral salts and proteins is, then, very doubtful and cannot be used in explanation of observed facts unless directly proved to take place. The fact that the effect of anions on the imbibition of water by starch and by albumin is identical, as shown by Samec (1911, p. 154), is difficult to understand on the assumption of a chemical union.

That there are relationships of an electrical nature between proteins and ions, apart from effects on the solvent or chemical reactions, is shown especially by the work of Mines (1912, p. 217) with regard to the action of various ions on emulsoids, inclusive of proteins. There is, in fact, an .action similar to that on suspensoids or hydrophobe colloids. This latter we have already seen to be due to electrical charges as such and it is natural to look for similar effects in the case of proteins. Perhaps the most striking evidence in this connection is the behaviour of the heart muscle, which will be better discussed in Chapter VII., under the action of electrolytes in general. For the present, we may note that the heart of the dogfish is 10,000 times more sensitive to various simple trivalent ions than to the bivalent ion Mg- (p. 216). This extraordinary disparity between the effects of two ions, not very different chemically, but whose electric charges are as 3 to 2, shows distinctly that electrolytes have an effect on proteins or other emulsoids in addition to the possible formation of salts, and that this effect is in relation to their electric charges. This again being due to the adsorption by a surface of ions of opposite charge to its own, will clearly depend on the sign and amount of the charge of the protein particle; in water this charge is small as a rule, but in acid or alkali, by the increased production of colloidal ions, the charge will be

greater, and, as we have seen in the case of the Bence-Jones' protein, the action of salts as ions on particles becomes more marked. Silk is a protein and forms a convenient means of testing some of the relations of these substances to electrolytes. In pure water, it has a slight negative charge, due, no doubt, to its acidic function exceeding its basic one. As an electro-negative colloid, it is especially sensitive to the action of cations, so far as concerns all properties depending on its charge. I have recently tested its behaviour towards a colloidal acid, that of Congo-red, with which it, as a potential base, is capable of forming a salt of the usual red colour, being dyed, in fact. Now ooth the silk and the colloidal acid are negative, so that very little adsorption takes place, unless we reverse the sign of the charge on the silk by the addition of cations. Calcium sulphate, of very low concentration, was used for the purpose. The effect of the elrrtnilvtr was the same as in the case of filter paper as described on page 58 above. The dye was adsorbed by the silk, which was thus dyed blue, the colour of the free acid, like the adsorption compound of thorium hydroxide with the same acid. On heating, chemical reaction occurred, with the formation of a red salt of silk protein. The interest of this experiment is that it shows the intervention of electric forces in addition to the purely chemical ones. Leucine, suspended in its saturated solution, also forms a blue adsorption compound with the Congo-red acid, which becomes a red salt on warming.

The natural proteins are, as we have seen, comparatively insensitive to the action of neutral salts. Certain of them, however, known as albumins and globulins, are capable of a change, called " denaturation," by which they approximate to the suspensoid class, in so far as becoming more sensitive to the action of salts, although their high viscosity and low surface tension shows them to be also hydrophile. A familiar instance of "denaturation" is the effect of boiling water on white of egg. What precisely happens, is as yet unknown, although the work of Hardy (1899, i. p. 182) and of Chick and Martin (1912) has thrown much light on the process. Hardy showed that in the coagulation of egg-white by heat there are two distinct stages : (1) denaturation, by which the protein becomes precipitable by salts, according to the same law of valency as the inorganic suspensoids, and (2) the agglutination of the denaturated particles by electrolytes, if present.

As we saw in the case of blood corpuscles acted on by cerium salt, if the concentration of the Ce-<> ions be large, the sign of the charge is reversed on all the corpuscles together, and redispersion takes place. Similarly, dispersion of protein particles by salts can occur. When all particles are equally charged, although of an opposite sign to their original one, mutual repulsion ensues, while dispersion is also assisted by the lowering of surface tension which is the result of the increased charge. Chick and Martin (1912, p. 293) call attention to the relation of the facility with which weak acid or alkali causes redispersion of the heat coagulum of a protein to the nature of the aggregated precipitate. In a loosely agglutinated mass, each particle is sufficiently distinct to carry its own charge, whereas when the particles are closely packed without interspaces, the charge will be on the surface of the mass as a whole. A small charge will readily produce breaking up in the former case, but can only affect the most superficial particles in the latter. For further information the reader is referred to the papers of the investigators named.

Chick and Martin (1913) have also devoted a detailed investigation to the phenomena of "salting out," which should be consulted. We may note that the effect of hydrogen ion concentration shows that electrical charge plays a considerable part, as does also the effect of the valency of the precipitating ion. From the preceding short account of the colloidal nature of proteins, it will be obvious that the phenomena presented by them are of much complexity, and are not yet altogether clear. Owing to their great variety in chemical constitution and the corresponding variety in their behaviour, it becomes almost a necessity to devote a special study to each one. There can be no doubt that their manifold capabilities of change in state make them very important in physiological processes. The effect of electrolytes, and especially of H' and OH' ions, on this state may be emphasised. Adsorption of salts, especially of those which are comparatively insoluble, is also to be remembered. Since the degree of adsorption is proportional to the surface, it will be seen how, by alterations of state of aggregation, electric charge, or surface tension,

adsorbed, and therefore inactive, substances may be set free to manifest their activity, or "mobilised," to use a frequent form of expression. The proteins of blood plasma do not appear to serve as food to the tissue cells. Quagliariello (1912, p. 174) showed that, when injected into the blood vessels, they are only utilised with extreme slowness. It appears that their chief value is due to their properties as colloids. When a solution of an electro-positive colloid, such as ferric hydroxide, is added to one of an electro-negative colloid, such as arsenious sulphide, if the proportion of the two is such that the charges will mutually annul each other, both colloids are precipitated as a complex, and the solution is left free from both. This phenomenon has been investigated especially by Biltz (1904). The precipitate will, in such a case, have no charge. If excess of either colloid is present, only partial precipitation will occur, and both colloids will be present in the precipitate and in the liquid above, although in different proportion in the two. In other words, we have an adsorption compound formed, whose composition depends on the relative concentration of its components in the solution, and whose electric charge has the sign of that colloid which is in excess. The fact that only partial precipitation or mere aggregation takes place when either colloid is in excess is sometimes put in the form that the precipitate is soluble in excess of either colloid.

In such a comparatively simple system we see already conditions of much complexity, and when, in addition, emulsoid colloids, or proteins, are present, the possibilities are still more manifold. The triple adsorption compounds of Raehlmann (1906) have already been described (page 65), and one or two examples of other complex systems may be instructive. The fact that filter papers take up a greatly increased amount of Congo-red when its negative charge is reduced or reversed by cations, such as Ca- •, has been previously referred to. Now, if gelatine be added, this effect is practically abolished, because the gelatine coats the paper with an emulsoid, itself insensitive to Ca1 • ions. Egg-white behaves in the same way as gelatine, if in neutral solution ; but, if made acid (i.e., electro-positive), it increases the action of Ca- • ions, and if alkaline, it diminishes their action, as in neutral solution (see Bayliss, 1906, p. 201).

The following experiment of Larguier des Bancels (1908, p. 198) is of interest in showing how an effect varies according to concentration : 2 c.c. of a dilute (0'125 per cent.) solution of aniline blue is totally precipitated by 5 drops of a certain ferric hydroxide preparation. If 5 drops of saturated ammonium sulphate be added in addition, only partial precipitation occurs ; the solution is left deep blue. But if 40 drops of the ammonium sulphate be added, the precipitate is again nearly total.

As cases where we have to deal with complex mixtures of interacting colloids, we may mention : the coagulation of the blood, and the innumerable phenomena connected with haemolysins, immunity, and anaphylaxis, together with intracellular processes in general. An elaborate system of names has been introduced, especially in connection with haemolysins and the coagulation of the blood, names which imply definite chemical individuals. From the complexity of the results to be obtained in colloidal reactions, from a very few distinct chemical substances, it seems more than probable that, as soon as sufficient knowledge is obtained of the nature of the phenomena in the systems referred to, the necessity for most of these names will be found to have disappeared. At present we find an investigator content to refer an experimental result to, say, "deviation of complement," apparently unaware that he is merely translating into a classical language what he has previously described in his mother-tongue. This particular case appears to be merely one of adsorption, a general phenomenon explicable on such fundamental laws as the principle of Clausius and Carnot. This question of terminology will be of necessity mentioned again (pages 307 and 328 below).

The papers of Gengou (1908) will be found very instructive in connection with the subject of the present section. Certain processes have incidentally been given in the previous pages. As a general statement, it may be said that the object to be attained is the formation of excessively minute particles of the substance which it is desired to obtain in the colloidal state. In the case of the suspensoid or irreversible class, it will be plain that we cannot take a portion of the dry solid and dissolve it in water in the way usually done in preparing solutions. This can be done with emulsoids in most cases, especially with the proteins ; so that, if a preparation of colloidal silver, for example, is desired in the dry state, such that it can be made into a solution by mere addition of water, the method adopted is to coat the particles with some protein. This is done by adding such a protein to a solution of the suspensoid and then evaporating to dryness. The commercial "collargol" is such a preparation of colloidal silver.

In the case of the suspensoids, in order to prepare their solutions, the particles themselves must, as a rule, be formed in the liquid which is to be the medium of dispersion. Arsenious sulphide is formed by passing hydrogen sulphide through a solution of arsenious acid and sols of various metals by disintegration with the electric arc (Bredig) or spark (Svedberg) in the water or other liquid. In order that such sols shall be permanent, foreign electrolytes must be removed as far as possible.