AMPHOTERIC COLLOIDS : V. THE INFLUENCE OF THE VALENCY OF ANIONS UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. When we plot the values of osmotic pressure, swelling, and viscosity of gelatin solutions as ordinates over the pH as abscissæ, practically identical curves are obtained for the effect of monobasic acids (HCl, HBr, HNO₃, and acetic acid) on these properties. 2. The curves obtained for the effect of H₂SO₄ on gelatin are much lower than those obtained for the effect of monobasic acids, the ratio of maximal osmotic pressures of a 1 per cent solution of gelatin sulfate and gelatin bromide being about 3:8. The same ratio had been found for the ratio of maximal osmotic pressures of calcium and sodium gelatinate. 3. The curves representing the influence of other dibasic and tribasic acids, viz. oxalic, tartaric, succinic, citric, and phosphoric, upon gelatin are almost identical with those representing the effect of monobasic acids. 4. The facts mentioned under (2) and (3) permit us to decide between a purely chemical and a colloidal explanation of the influence of acids on the physical properties of gelatin. In the former case we should be able to prove, first, that twice as many molecules of HBr as of H₂SO₄ combine with a given mass of gelatin; and, second, that the same number of molecules of phosphoric, citric, oxalic, tartaric, and succinic acids as of HNO₃ or HCl combine with the same mass of gelatin. It is shown in the present paper that this is actually the case. 5. It is shown that gelatin sulfate and gelatin bromide solutions of the same pH have practically the same conductivity. This disproves the assumption of colloid chemists that the difference in the effect of bromides and sulfates on the physical properties of gelatin is due to a different ionizing and hydratating effect of the two acids upon the protein molecule.     

AMPHOTERIC COLLOIDS : III. CHEMICAL BASIS OF THE INFLUENCE OF ACID UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. The method of removing the excess of hydrobromic acid after it has had a chance to react chemically with gelatin has permitted us to measure the amount of Br in combination with the gelatin. It is shown that the curves representing the amount of bromine bound by the gelatin are approximately parallel with the curves for the osmotic pressure, the viscosity, and swelling of the gelatin solution. This proves that the curves for osmotic pressure are an unequivocal function of the number of gelatin bromide molecules formed under the influence of the acid. The cc. of 0.01 N Br in combination with 0.25 gm, of gelatin we call the bromine number. 2. The explanation of this influence of the acid on the physical properties of gelatin is based on the fact that gelatin is an amphoteric electrolyte, which at its isoelectric point is but sparingly soluble in water, while its transformation into a salt with a univalent anion like gelatin Br makes it soluble. The curve for the bromine number thus becomes at the same time the numerical expression for the number of gelatin molecules rendered soluble, and hence the curve for osmotic pressure must of necessity be parallel to the curve for the bromine number. 3. Volumetric analysis shows that gelatin treated previously with HBr is free from Br at the isoelectric point as well as on the more alkaline side from the isoelectric point (pH ≧ 4.7) of gelatin. This is in harmony with the fact that gelatin (like any other amphoteric electrolyte) can dissociate on the alkaline side of its isoelectric point only as an anion. On the more acid side from the isoelectric point gelatin is found to be in combination with Br and the Br number rises with the pH. 4. When we titrate gelatin, treated previously with HBr but possessing a pH = 4,7, with NaOH we find that 25 cc. of a 1 per cent solution of isoelectric gelatin require about 5.25 to 5.5 cc. of 0.01 N NaOH for neutralization (with phenolphthalein as an indicator). This value which was found invariably is therefore a constant which we designate as "NaOH (isoelectric)." When we titrate 0.25 gm. of gelatin previously treated with HBr but possessing a pH < 4.7 more than 5.5 cc. of 0.01 N NaOH are required for neutralization. We will designate this value of NaOH as "(NaOH)_n," where n represents the value of pH. If we designate the bromine number for the same pH as "Br_n" then we can show that the following equation is generally true: (NaOH)_n = NaOH (isoelectric) + Br_n. In other words, titration with NaOH of gelatin (previously treated with HBr) and being on the acid side of its isoelectric point results in the neutralization of the pure gelatin (NaOH isoelectric) with NaOH and besides in the neutralization of the HBr in combination with the gelatin. This HBr is set free as soon as through the addition of the NaOH the pH of the gelatin solution becomes equal to 4.7. 5. A comparison between the pH values and the bromine numbers found shows that over 90 per cent of the bromine or HBr found was in our experiments in combination with the gelatin.     

AMPHOTERIC COLLOIDS : I. CHEMICAL INFLUENCE OF THE HYDROGEN ION CONCENTRATION.

1. It has been shown in this paper that while non-ionized gelatin may exist in gelatin solutions on both sides of the isoelectric point (which lies for gelatin at a hydrogen ion concentration of C_H = 2.10^–5 or pH = 4.7), gelatin, when it ionizes, can only exist as an anion on the less acid side of its isoelectric point (pH > 4.7), as a cation only on the more acid side of its isoelectric point (pH < 4.7). At the isoelectric point gelatin can dissociate practically neither as anion nor as cation. 2. When gelatin has been transformed into sodium gelatinate by treating it for some time with M/32 NaOH, and when it is subsequently treated with HCl, the gelatin shows on the more acid side of the isoelectric point effects of the acid treatment only; while the effects of the alkali treatment disappear completely, showing that the negative gelatin ions formed by the previous treatment with alkali can no longer exist in a solution with a pH < 4.7. When gelatin is first treated with acid and afterwards with alkali on the alkaline side of the isoelectric point only the effects of the alkali treatment are noticeable. 3. On the acid side of the isoelectric point amphoteric electrolytes can only combine with the anions of neutral salts, on the less acid side of their isoelectric point only with cations; and at the isoelectric point neither with the anion nor cation of a neutral salt. This harmonizes with the statement made in the first paragraph, and the experimental results on the effect of neutral salts on gelatin published in the writer''s previous papers. 4. The reason for this influence of the hydrogen ion concentration on the stability of the two forms of ionization possible for an amphoteric electrolyte is at present unknown. We might think of the possibility of changes in the configuration or constitution of the gelatin molecule whereby ionized gelatin can exist only as an anion on the alkaline side and as a cation on the acid side of its isoelectric point. 5. The literature of colloid chemistry contains numerous statements which if true would mean that the anions of neutral salts act on gelatin on the alkaline side of the isoelectric point, e.g. the alleged effect of the Hofmeister series of anions on the swelling and osmotic pressure of common gelatin in neutral solutions, and the statement that both ions of a neutral salt influence a protein simultaneously. The writer has shown in previous publications that these statements are contrary to fact and based on erroneous methods of work. Our present paper shows that these claims of colloid chemists are also theoretically impossible. 6. In addition to other physical properties the conductivity of gelatin previously treated with acids has been investigated and plotted, and it was found that this conductivity is a minimum in the region of the isoelectric point, thus confirming the conclusion that gelatin can apparently not exist in ionized condition at that point. The conductivity rises on either side of the isoelectric point, but not symmetrically for reasons given in the paper. It is shown that the curves for osmotic pressure, viscosity, swelling, and alcohol number run parallel to the curve of the conductivity of gelatin when the gelatin has been treated with acid, supporting the view that these physical properties are in this case mainly or exclusively a function of the degree of ionization of the gelatin or gelatin salt formed. It is pointed out, however, that certain constitutional factors, e.g. the valency of the ion in combination with the gelatin, may alter the physical properties of the gelatin (osmotic pressure, etc.) without apparently altering its conductivity. This point is still under investigation and will be further discussed in a following publication. 7. It is shown that the isoelectric point of an amphoteric electrolyte is not only a point where the physical properties of an ampholyte experience a sharp drop and become a minimum, but that it is also a turning point for the mode of chemical reactions of the ampholyte. It may turn out that this chemical influence of the isoelectric point upon life phenomena overshadows its physical influence. 8. These experiments suggest that the theory of amphoteric colloids is in its general features identical with the theory of inorganic hydroxides (e.g. aluminum hydroxide), whose behavior is adequately understood on the basis of the laws of general chemistry.     

INFLUENCE OF THE CONCENTRATION OF ELECTROLYTES ON SOME PHYSICAL PROPERTIES OF COLLOIDS AND OF CRYSTALLOIDS

1. When a 1 per cent solution of a metal gelatinate, e.g. Na gelatinate, of pH = 8.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate which can be measured by the rise of the level of the liquid in a manometer. When to such a solution alkali or neutral salt is added the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. This depressing effect of the addition of alkali and neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. 2. When a neutral M/256 solution of a salt with monovalent cation (e.g. Na₂SO₄ or K₄Fe(CN)₆, etc.) is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain initial rate. When to such a solution alkali or neutral salt is added, the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. The depressing effect of the addition of alkali or neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. The membranes used in these experiments were not treated with gelatin. 3. It can be shown that water diffuses through the collodion membrane in the form of positively charged particles under the conditions mentioned in (1) and (2). In the case of diffusion of water into a neutral solution of a salt with monovalent or bivalent cation the effect of the addition of electrolyte on the rate of diffusion can be explained on the basis of the influence of the ions on the electrification and the rate of diffusion of electrified particles of water. Since the influence of the addition of electrolyte seems to be the same in the case of solutions of metal gelatinate, the question arises whether this influence of the addition of electrolyte cannot also be explained in the same way, and, if this be true, the further question can be raised whether this depressing effect necessarily depends upon the colloidal character of the gelatin solution, or whether we are not dealing in both cases with the same property of matter; namely, the influence of ions on the electrification and rate of diffusion of water through a membrane. 4. It can be shown that the curve representing the influence of the concentration of electrolyte on the initial rate of diffusion of water from solvent into the solution through the membrane is similar to the curve representing the permanent osmotic pressure of the gelatin solution. The question which has been raised in (3) should then apply also to the influence of the concentration of ions upon the osmotic pressure and perhaps other physical properties of gelatin which depend in a similar way upon the concentration of electrolyte added; e.g., swelling. 5. When a 1 per cent solution of a gelatin-acid salt, e.g. gelatin chloride, of pH 3.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate. When to such a solution acid or neutral salt is added—taking care in the latter case that the pH is not altered—the initial rate with which water will diffuse into the solution is diminished and the more so the more acid or salt is added. Water diffuses into a gelatin chloride solution through a collodion membrane in the form of negatively charged particles. 6. When we replace the gelatin-acid salt by a crystalloidal salt, which causes the water to diffuse through the collodion membrane in the form of negatively charged particles, e.g. M/512 Al₂Cl₆, we find that the addition of acid or of neutral salt will diminish the initial rate with which water diffuses into the M/512 solution of Al₂Cl₆, in a similar way as it does in the case of a solution of a gelatin-acid salt.     

AMPHOTERIC COLLOIDS : II. VOLUMETRIC ANALYSIS OF ION-PROTEIN COMPOUNDS; THE SIGNIFICANCE OF THE ISOELECTRIC POINT FOR THE PURIFICATION OF AMPHOTERIC COLLOIDS.

1. It is shown by volumetric analysis that on the alkaline side from its isoelectric point gelatin combines with cations only, but not with anions; that on the more acid side from its isoelectric point it combines only with anions but not with cations; and that at the isoelectric point, pH = 4.7, it combines with neither anion nor cation. This confirms our statement made in a previous paper that gelatin can exist only as an anion on the alkaline side from its isoelectric point and only as a cation on the more acid side of its isoelectric point, and practically as neither anion nor cation at the isoelectric point. 2. Since at the isoelectric point gelatin (and probably amphoteric colloids generally) must give off any ion with which it was combined, the simplest method of obtaining amphoteric colloids approximately free from ionogenic impurities would seem to consist in bringing them to the hydrogen ion concentration characteristic of their isoelectric point (i.e., at which they migrate neither to the cathode nor anode of an electric field). 3. It is shown by volumetric analysis that when gelatin is in combination with a monovalent ion (Ag, Br, CNS), the curve representing the amount of ion-gelatin formed is approximately parallel to the curve for swelling, osmotic pressure, and viscosity. This fact proves that the influence of ions upon these properties is determined by the chemical or stoichiometrical and not by the "colloidal" condition of gelatin. 4. The sharp drop of these curves at the isoelectric point finds its explanation in an equal drop of the water solubility of pure gelatin, which is proved by the formation of a precipitate. It is not yet possible to state whether this drop of the solubility is merely due to lack of ionization of the gelatin or also to the formation of an insoluble tautomeric or polymeric compound of gelatin at the isoelectric point. 5. On account of this sudden drop slight changes in the hydrogen ion concentration have a considerably greater chemical and physical effect in the region of the isoelectric point than at some distance from this point. This fact may be of biological significance since a number of amphoteric colloids in the body seem to have their isoelectric point inside the range of the normal variation of the hydrogen ion concentration of blood, lymph, or cell sap. 6. Our experiments show that while a slight change in the hydrogen ion concentration increases the water solubility of gelatin near the isoelectric point, no increase in the solubility can be produced by treating gelatin at the isoelectric point with any other kind of monovalent or polyvalent ion; a fact apparently not in harmony with the adsorption theory of colloids, but in harmony with a chemical conception of proteins.     

THE AMPHOTERIC PROPERTIES OF SOME AMINO-ACIDS AND PEPTIDES

The titration curves of solutions of glycine, alanine, α-ammo-butyric acid, leucine, glycyl-glycine, alanyl-glycine, alanyl-alanine, acetone, acetamide, urea, acetic acid, and aceturic acid were determined and some of the relations as dependent upon the chemical structures discussed. The isoelectric points of some of the amphoteric electrolytes were found experimentally. The definition of isoelectric point, its theoretical significance, and method of calculation were considered in some detail.     

THE EFFECT OF VALENCY OF CATIONS AND ANIONS ON NEGATIVELY AND POSITIVELY CHARGED RED BLOOD CELLS

1. Under comparable conditions, valency effect may be demonstrated with a suspension of red blood cells and the cations and anions of salts. 2. The valency of the cation determines the degree of the effect on negatively charged cells, the valency of the anion, the effect on positively charged cells. 3. Anomalies in valency effects with different salts and red cell suspensions are in part due to variations in H ion concentration, depending on the degree of hydrolysis of the salt.     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : II. THE INFLUENCE OF SALTS.

It is shown by the older experiments by Loeb and by the experiments reported in this paper that the effect of salts on the membrane potentials, osmotic pressure, swelling of gelatin chloride, and that type of viscosity which is due to the swelling of protein particles, depends only on the valency but not on the chemical nature of the anion of the salt, and that the cation of the salt has no effect on these properties, if the pH of the protein solution or protein gel is not altered by the salt. The so called Hofmeister series of salt effects on these four properties are purely fictitious and due to the failure of the former authors to measure the hydrogen ion concentration of their protein solutions or gels and to compare the effects of salts at the same pH of the protein solution or the protein gel. These results confirm the older experiments of Loeb and together they furnish a further proof for the correctness of the idea that the influence of electrolytes on the four properties of proteins is determined by membrane equilibria. Such properties of proteins which do not depend on membrane equilibria, such as solubility or cohesion, may be affected not only by the valency but also by the chemical nature of the ions of a salt.     

THE INFLUENCE OF ALCOHOL UPON THE GROWTH OF SEEDLINGS

In this paper it is shown that if the dry seeds of the cantaloupe (Cucumis melo) are soaked for 3 hours in solutions of ethyl alcohol of concentration ranging from 2 to 16 per cent by volume, and then germinated and grown in distilled water in the dark, the total growth attained is greater by amounts ranging from 9 to 35 per cent than is that made by seeds treated in every way identically except that they are initially soaked in distilled water instead of alcohol. It is shown that this result is not due simply to differences in osmotic pressure in the different alcohol solutions. It is probably due to a simple selective action of the alcohol which eliminates the constitutionally weak and defective seeds.     

THE EFFECT OF RENNIN UPON CASEIN : II. FURTHER CONSIDERATION OF THE PROPERTIES OF PARACASEIN.

The properties of the paracasein and casein preparations studied are compared in Table VI. See PDF for Structure I. Casein retains its characteristic solubility in NaOH: (1) after being exposed to a high degree of alkalinity during its preparation, (2) when recovered from partially hydrolyzed solutions in NaOH, and (3) after being kept for a prolonged time at the isoelectric point at 5°C. II. It follows from I, that: (1) paracasein is not identical to casein modified by an excess of alkali, and that (2) this protein was not produced from casein by a partial hydrolysis of the latter in presence of NaOH.