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.     

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.     

THE ISOELECTRIC POINT OF RED BLOOD CELLS AND ITS RELATION TO AGGLUTINATION

1. The movement of normal and sensitized red blood cells in the electric field is a function of the hydrogen ion concentration. The isoelectric point, at which no movement occurs, corresponds with pH 4.6. 2. On the alkaline side of the isoelectric point the charge carried is negative and increases with the alkalinity. On the acid side the charge is positive and increases with the acidity. 3. On the alkaline side at least the charge carried by sensitized cells is smaller and increases less rapidly with the alkalinity than the charge of normal cells. 4. Both normal and sensitized cells combine chemically with inorganic ions, and the isoelectric point is a turning point for this chemical behavior. On the acid side the cells combine with the hydrogen and chlorine ions, and in much larger amount than on the alkaline side; on the alkaline side the cells combine with a cation (Ba), and in larger amount than on the acid side. This behavior corresponds with that found by Loeb for gelatin. 5. The optimum for agglutination of normal cells is at pH 4.75, so that at this point the cells exist most nearly pure, or least combined with anion and cation. 6. The optimum for agglutination of sensitized cells is at pH 5.3. This point is probably connected with the optimum for flocculation of the immune serum body.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. I

1. This paper contains experiments on the influence of acids and alkalies on the osmotic pressure of solutions of crystalline egg albumin and of gelatin, and on the viscosity of solutions of gelatin. 2. It was found in all cases that there is no difference in the effects of HCl, HBr, HNO₃, acetic, mono-, di-, and trichloracetic, succinic, tartaric, citric, and phosphoric acids upon these physical properties when the solutions of the protein with these different acids have the same pH and the same concentration of originally isoelectric protein. 3. It was possible to show that in all the protein-acid salts named the anion in combination with the protein is monovalent. 4. The strong dibasic acid H₂SO₄ forms protein-acid salts with a divalent anion SO₄ and the solutions of protein sulfate have an osmotic pressure and a viscosity of only half or less than that of a protein chloride solution of the same pH and the same concentration of originally isoelectric protein. Oxalic acid behaves essentially like a weak dibasic acid though it seems that a small part of the acid combines with the protein in the form of divalent anions. 5. It was found that the osmotic pressure and viscosity of solutions of Li, Na, K, and NH₄ salts of a protein are the same at the same pH and the same concentration of originally isoelectric protein. 6. Ca(OH)₂ and Ba(OH)₂ form salts with proteins in which the cation is divalent and the osmotic pressure and viscosity of solutions of these two metal proteinates are only one-half or less than half of that of Na proteinate of the same pH and the same concentration of originally isoelectric gelatin. 7. These results exclude the possibility of expressing the effect of different acids and alkalies on the osmotic pressure of solutions of gelatin and egg albumin and on the viscosity of solutions of gelatin in the form of ion series. The different results of former workers were probably chiefly due to the fact that the effects of acids and alkalies on these proteins were compared for the same quantity of acid and alkali instead of for the same pH.     

ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND ANOMALOUS OSMOSIS

1. It has been shown in previous publications that when solutions of different concentrations of salts are separated by collodion-gelatin membranes from water, electrical forces participate in addition to osmotic forces in the transport of water from the side of the water to that of the solution. When the hydrogen ion concentration of the salt solution and of the water on the other side of the membrane is the same and if both are on the acid side of the isoelectric point of gelatin (e.g. pH 3.0), the electrical transport of water increases with the valency of the cation and inversely with the valency of the anion of the salt in solution. Moreover, the electrical transport of water increases at first with increasing concentration of the solution until a maximum is reached at a concentration of about M/32, when upon further increase of the concentration of the salt solution the transport diminishes until a concentration of about M/4 is reached, when a second rise begins, which is exclusively or preeminently the expression of osmotic forces and therefore needs no further discussion. 2. It is shown that the increase in the height of the transport curves with increase in the valency of the cation and inversely with the increase in the valency of the anion is due to the influence of the salt on the P.D. (E) across the membrane, the positive charge of the solution increasing in the same way with the valency of the ions mentioned. This effect on the P.D. increases with increasing concentration of the solution and is partly, if not essentially, the result of diffusion potentials. 3. The drop in the transport curves is, however, due to the influence of the salts on the P.D. (ε) between the liquid inside the pores of the gelatin membrane and the gelatin walls of the pores. According to the Donnan equilibrium the liquid inside the pores must be negatively charged at pH 3.0 and this charge is diminished the higher the concentration of the salt. Since the electrical transport is in proportion to the product of E x ε and since the augmenting action of the salt on E begins at lower concentrations than the depressing action on ε, it follows that the electrical transport of water must at first rise with increasing concentration of the salt and then drop. 4. If the Donnan equilibrium is the sole cause for the P.D. (ε) between solid gelatin and watery solution the transport of water through collodion-gelatin membranes from water to salt solution should be determined purely by osmotic forces when water, gelatin, and salt solution have the hydrogen ion concentration of the isoelectric point of gelatin (pH = 4.7). It is shown that this is practically the case when solutions of LiCl, NaCl, KCl, MgCl₂, CaCl₂, BaCl₂, Na₂SO₄, MgSO₄ are separated by collodion-gelatin membranes from water; that, however, when the salt has a trivalent (or tetravalent?) cation or a tetravalent anion a P.D. between solid isoelectric gelatin and water is produced in which the wall assumes the sign of charge of the polyvalent ion. 5. It is suggested that the salts with trivalent cation, e.g. Ce(NO₃)₃, form loose compounds with isoelectric gelatin which dissociate electrolytically into positively charged complex gelatin-Ce ions and negatively charged NO₃ ions, and that the salts of Na₄Fe(CN)₆ form loose compounds with isoelectric gelatin which dissociate electrolytically into negatively charged complex gelatin-Fe(CN)₆ ions and positively charged Na ions. The Donnan equilibrium resulting from this ionization would in that case be the cause of the charge of the membrane.     

SYNERESIS AND SWELLING OF GELATIN

1. When solid blocks of isoelectric gelatin are placed in cold distilled water or dilute buffer of pH 4.7, only those of a gelatin content of more than 10 per cent swell, while those of a lower gelatin content not only do not swell but actually lose water. 2. The final quantity of water lost by blocks of dilute gelatin is the same whether the block is immersed in a large volume of water or whether syneresis has been initiated in the gel through mechanical forces such as shaking, pressure, etc., even in the absence of any outside liquid, thus showing that syneresis is identical with the process of negative swelling of dilute gels when placed in cold water, and may be used as a convenient term for it. 3. Acid- or alkali-containing gels give rise to greater syneresis than isoelectric gels, after the acid or alkali has been removed by dialysis. 4. Salt-containing gels show greater syneresis than salt-free gels of the same pH, after the salt has been washed away. 5. The acid and alkali and also the salt effect on syneresis of gels disappears at a gelatin concentration above 8 per cent. 6. The striking similarity in the behavior of gels with respect to syneresis and of gelatin solutions with respect to viscosity suggests the probability that both are due to the same mechanism, namely the mechanism of hydration of the micellæ in gelatin by means of osmosis as brought about either by diffusible ions, as in the presence of acid or alkali, or by the soluble gelatin present in the micellæ. The greater the pressures that caused swelling of the micellæ while the gelatin was in the sol state, the greater is the loss of water from the gels when the pressures are removed. 7. A quantitative study of the loss of water by dilute gels of various gelatin content shows that the same laws which have been found by Northrop to hold for the swelling of gels of high concentrations apply also to the process of losing water by dilute gels, i.e. to the process of syneresis. The general behavior is well represented by the equations: See PDF for Equation and See PDF for Equation where P ₁ = osmotic pressure of the soluble gelatin in the gel, P ₂ = stress on the micellæ in the gelatin solution before setting, K_e = bulk modulus of elasticity, V_o = volume of water per gram of dry gelatin at setting and V_e = volume of water per gram of gelatin at equilibrium.     

CHANGES IN THE STABILITY AND POTENTIAL OF CELL SUSPENSIONS : II. THE POTENTIAL OF ERYTHROCYTES.

1. Human and sheep erythrocytes, when placed in 0.01 N buffer solutions at reactions more acid than pH 5.2, undergo a progressive change in potential, becoming less electronegative or more electropositive. This change usually occurs within 2 hours at ordinary room temperatures. It did not occur when rabbit erythrocytes were used. 2. This change is due primarily to the liberation of hemoglobin from some of the cells. 3. Hemoglobin, even in very low concentrations, markedly alters the potential of erythrocytes in the more acid reactions. This is due to a combination between the electropositive hemoglobin and the erythrocytes. The effect of the hemoglobin is most marked in the more acid solutions; it occurs only on the acid side of the isoelectric point of the hemoglobin. 4. The isoelectric point of erythrocytes in the absence of salt, or in the presence of salts having both ions monovalent, occurs at pH 4.7. This confirms the observations of Coulter (1920–21). Divalent anions shift the isoelectric point to the acid side. 5. The effect of salts on the potential of erythrocytes is due to the ions of the salts, and is analogous in every way to the effect of salts on albumin-coated collodion particles, as discussed by Loeb (1922–23).     

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.     

THE SWELLING OF GELATIN AND THE VOLUME OF SURROUNDING SOLUTION

The swelling of isoelectric gelatin added to various volumes of acid of different concentration at 5°C. has been determined. The swelling is determined only by the concentration of the supernatant solution at equilibrium and is independent of the volume of acid. Similar experiments with unpurified gelatin show that in this case, owing to the presence of neutral salts the swelling is a function of the volume as well as the concentration of acid. Both results are predicted by the Procter-Wilson-Loeb theory of the swelling of gelatin.     

FRACTIONATION OF GELATIN

1. It is possible to fractionate gelatin by means of reprecipitation at 23°C. of a salt-free solution of pH 4.7 into two fractions, one of which is soluble in water at any temperature, and a second one which does not dissolve in water even when heated to 80°C. 2. The proportion of the soluble fraction in gelatin is much greater than of the insoluble one. 3. The insoluble fraction of gelatin does not swell when mixed with water, but it does swell in the presence of acid and alkali which finally dissolve it. 4. Blocks of concentrated gel made by dissolving various mixtures of the soluble and insoluble fractions of gelatin in dilute NaOH swell differently when placed in large volumes of dilute buffer solution pH 4.7 at 5°C. The gel consisting of the insoluble material shows only a trace of swelling, while those containing a mixture of soluble and insoluble swell considerably. The swelling increases rapidly as the proportion of the soluble fraction increases. 5. A 5 per cent gel made up by dissolving the insoluble fraction of gelatin in dilute NaOH loses about 70 per cent of its weight when placed in dilute buffer pH 4.7 at 5°C. A similar gel made up of ordinary gelatin loses only about 20 per cent of its weight under the same conditions. 6. It was not found possible to resynthesize isoelectric gelatin from its components. 7. An insoluble substance similar in many respects to the one obtained by reprecipitation of gelatin is produce on partial hydrolysis of gelatin in dilute hydrochloric acid at 90°C.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : I. MEMBRANE POTENTIALS.

1. It is shown that a neutral salt depresses the potential difference which exists at the point of equilibrium between a gelatin chloride solution contained in a collodion bag and an outside aqueous solution (without gelatin). The depressing effect of a neutral salt on the P.D. is similar to the depression of the osmotic pressure of the gelatin chloride solution by the same salt. 2. It is shown that this depression of the P.D. by the salt can be calculated with a fair degree of accuracy on the basis of Nernst''s logarithmic formula on the assumption that the P.D. which exists at the point of equilibrium is due to the difference of the hydrogen ion concentration on the opposite sides of the membrane. 3. Since this difference of hydrogen ion concentration on both sides of the membrane is due to Donnan''s membrane equilibrium this latter equilibrium must be the cause of the P.D. 4. A definite P.D. exists also between a solid block of gelatin chloride and the surrounding aqueous solution at the point of equilibrium and this P.D. is depressed in a similar way as the swelling of the gelatin chloride by the addition of neutral salts. It is shown that the P.D. can be calculated from the difference in the hydrogen ion concentration inside and outside the block of gelatin at equilibrium. 5. The influence of the hydrogen ion concentration on the P.D. of a gelatin chloride solution is similar to that of the hydrogen ion concentration on the osmotic pressure, swelling, and viscosity of gelatin solutions, and the same is true for the influence of the valency of the anion with which the gelatin is in combination. It is shown that in all these cases the P.D. which exists at equilibrium can be calculated with a fair degree of accuracy from the difference of the pH inside and outside the gelatin solution on the basis of Nernst''s logarithmic formula by assuming that the difference in the concentration of hydrogen ions on both sides of the membrane determines the P.D. 6. The P.D. which exists at the boundary of a gelatin chloride solution and water at the point of equilibrium can also be calculated with a fair degree of accuracy by Nernst''s logarithmic formula from the value pCl outside minus pCl inside. This proves that the equation x² = y ( y + z) is the correct expression for the Donnan membrane equilibrium when solutions of protein-acid salts with monovalent anion are separated by a collodion membrane from water. In this equation x is the concentration of the H ion (and the monovalent anion) in the water, y the concentration of the H ion and the monovalent anion of the free acid in the gelatin solution, and z the concentration of the anion in combination with the protein. 7. The similarity between the variation of P.D. and the variation of the osmotic pressure, swelling, and viscosity of gelatin, and the fact that the Donnan equilibrium determines the variation in P.D. raise the question whether or not the variations of the osmotic pressure, swelling, and viscosity are also determined by the Donnan equilibrium.     

ON THE LOCATION OF THE FORCES WHICH DETERMINE THE ELECTRICAL DOUBLE LAYER BETWEEN COLLODION PARTICLES AND WATER

1. The cataphoretic P.D. of suspended particles is assumed to be due to an excess in the concentration of one kind of a pair of oppositely charged ions in the film of water enveloping the particles and this excess is generally ascribed to a preferential adsorption of this kind of ions by the particle. The term adsorption fails, however, to distinguish between the two kinds of forces which can bring about such an unequal distribution of ions between the enveloping film and the opposite film of the electrical double layer, namely, forces inherent in the water itself and forces inherent in the particle (e.g. chemical attraction between particle and adsorbed ions). 2. It had been shown in a preceding paper that collodion particles suspended in an aqueous solution of an ordinary electrolyte like NaCl, Na₂SO₄, Na₄Fe(CN)₆, CaCl₂, HCl, H₂SO₄, or NaOH are always negatively charged, and that the addition of these electrolytes increases the negative charge as long as their concentration is below M/1,000 until a certain maximal P.D. is reached. Hence no matter whether acid, alkali, or a neutral salt is added, the concentration of anions must always be greater in the film enveloping the collodion particles than in the opposite film of the electrical double layer, and the reverse is true for the concentration of cations. This might suggest that the collodion particles, on account of their chemical constitution, attract anions with a greater force than cations, but such an assumption is rendered difficult in view of the following facts. 3. Experiments with dyes show that at pH 5.8 collodion particles are stained by basic dyes (i.e. dye cations) but not by acid dyes (i.e. dye anions), and that solutions of basic dyes are at pH 5.8 more readily decolorized by particles of collodion than acid dyes. It is also shown in this paper that crystalline egg albumin, gelatin, and Witte''s peptone form durable films on collodion only when the protein exists in the form of a cation or when it is isoelectric, but not when it exists in the form of an anion (i.e. on the alkaline side of its isoelectric point). Hence if any ions of dyes or proteins are permanently bound at the surface of collodion particles through forces inherent in the collodion they are cations but not anions. The fact that isoelectric proteins form durable films on collodion particles suggests, that the forces responsible for this combination are not ionic. 4. It is shown that salts of dyes or proteins, the cations of which are capable of forming durable films on the surface of the collodion, influence the cataphoretic P.D. of the collodion particles in a way entirely different from that of any other salts inasmuch as surprisingly low concentrations of salts, the cation of which is a dye or a protein, render the negatively charged collodion particles positive. Crystalline egg albumin and gelatin have such an effect even in concentrations of 1/130,000 or 1/65,000 of 1 per cent, i.e. in a probable molar concentration of about 10^–9. 5. Salts in which the dye or protein is an anion have no such effect but act like salts of the type of NaCl or Na₂SO₄ on the cataphoretic P.D. of collodion particles. 6. Amino-acids do not form durable films on the surface of collodion particles at any pH and the salts of amino-acids influence their cataphoretic P.D. in the same way as NaCl but not in the same way as proteins or dyes, regardless of whether the amino-acid ion is a cation or an anion. 7. Ordinary salts like LaCl₃ also fail to form a durable film on the surface of collodion particles. 8. Until evidence to the contrary is furnished, these facts seem to suggest that the increase of the negative charge of the collodion particles caused by the addition of low concentrations of ordinary electrolytes is chiefly if not entirely due to forces inherent in the aqueous solution but to a less extent, if at all, due to an attraction of the anions of the electrolyte by forces inherent in the collodion particles.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : II. OSMOTIC PRESSURE.

1. It had been shown in previous publications that the osmotic pressure of a 1 per cent solution of a protein-acid salt varies in a characteristic way with the hydrogen ion concentration of the solution, the osmotic pressure having a minimum at the isoelectric point, rising steeply with a decrease in pH until a maximum is reached at pH of 3.4 or 3.5 (in the case of gelatin and crystalline egg albumin), this maximum being followed by a steep drop in the osmotic pressure with a further decrease in the pH of the gelatin or albumin solution. In this paper it is shown that (aside from two minor discrepancies) we can calculate this effect of the pH on the osmotic pressure of a protein-acid salt by assuming that the pH effect is due to that unequal distribution of crystalloidal ions (in particular free acid) on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 2. It had been shown in preceding papers that only the valency but not the nature of the ion (aside from its valency) with which a protein is in combination has any effect upon the osmotic pressure of the solution of the protein; and that the osmotic pressure of a gelatin-acid salt with a monovalent anion (e.g. Cl, NO₃, acetate, H₂PO₄, HC₂O₄, etc.) is about twice or perhaps a trifle more than twice as high as the osmotic pressure of gelatin sulfate where the anion is bivalent; assuming that the pH and gelatin concentrations of all the solutions are the same. It is shown in this paper that we can calculate with a fair degree of accuracy this valency effect on the assumption that it is due to the influence of the valency of the anion of a gelatin-acid salt on that relative distribution of the free acid on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 3. The curves of the observed values of the osmotic pressure show two constant minor deviations from the curves of the calculated osmotic pressure. One of these deviations consists in the fact that the values of the ascending branch of the calculated curves are lower than the corresponding values in the curves for the observed osmotic pressure, and the other deviation consists in the fact that the drop in the curves of calculated values occurs at a lower pH than the drop in the curves of the observed values.     

THE REVERSAL OF THE SIGN OF THE CHARGE OF MEMBRANES BY HYDROGEN IONS

1. It had been shown in previous papers that when a collodion membrane has been treated with a protein the membrane assumes a positive charge when the hydrogen ion concentration of the solution with which it is in contact exceeds a certain limit. It is pointed out in this paper that by treating the collodion membrane with a protein (e.g. oxyhemoglobin) a thin film of protein adheres to the membrane and that the positive charge of the membrane must therefore be localized in this protein film. 2. It is further shown in this paper that the hydrogen ion concentration, at which the reversal in the sign of the charge of a collodion membrane treated with a protein occurs, varies in the same sense as the isoelectric point of the protein, with which the membrane has been treated, and is always slightly higher than that of the isoelectric point of the protein used. 3. The critical hydrogen ion concentration required for the reversal seems to be, therefore, that concentration where enough of the protein lining of the membrane is converted into a protein-acid salt (e.g. gelatin nitrate) capable of ionizing into a positive protein ion (e.g. gelatin) and the anion of the acid used (e.g. NO₃).     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : I. THE ACTION OF ACIDS.

1. The action of a number of acids on four properties of gelatin (membrane potentials, osmotic pressure, swelling, and viscosity) was studied. The acids used can be divided into three groups; first, monobasic acids (HCl, HBr, HI, HNO₃, acetic, propionic, and lactic acids); second, strong dibasic acids (H₂SO₄ and sulfosalicylic acid) which dissociate as dibasic acids in the range of pH between 4.7 and 2.5; and third, weak dibasic and tribasic acids (succinic, tartaric, citric) which dissociate as monobasic acids at pH 3.0 or below and dissociate increasingly as dibasic acids, according to their strength, with pH increasing above 3.0. 2. If the influence of these acids on the four above mentioned properties of gelatin is plotted as ordinates over the pH of the gelatin solution or gelatin gel as abscissæ, it is found that all the acids have the same effect where the anion is monovalent; this is true for the seven monobasic acids at all pH and for the weak dibasic and tribasic acids at pH below 3.0. The two strong dibasic acids (the anion of which is divalent in the whole range of pH of these experiments) have a much smaller effect than the acids with monovalent anion. The weak dibasic and tribasic acids act, at pH above 3.0, like acids the anion of which is chiefly monovalent but which contain also divalent anions increasing with pH and with the strength of the acid. 3. These experiments prove that only the valency but not the other properties of the anion of an acid influences the four properties of gelatin mentioned, thus absolutely contradicting the Hofmeister anion series in this case which were due to the failure of the earlier experimenters to measure properly the pH of their protein solutions or gels and to compare the effects of acids at the same pH of the protein solution or protein gel after equilibrium was established. 4. It is shown that the validity of the valency rule and the non-validity of the Hofmeister anion series for the four properties of proteins mentioned are consequences of the fact that the influence of acids on the membrane potentials, osmotic pressure, swelling, and viscosity of gelatin is due to the Donnan equilibrium between protein solutions or gels and the surrounding aqueous solution. This equilibrium depends only on the valency but not on any other property of the anion of an acid. 5. That the valency rule is determined by the Donnan equilibrium is strikingly illustrated by the ratio of the membrane potentials for divalent and monovalent anions of acids. Loeb has shown that the Donnan equilibrium demands that this ratio should be 0.66 and the actual measurements agree with this postulate of the theory within the limits of accuracy of the measurements. 6. The valency rule can be expected to hold for only such properties of proteins as depend upon the Donnan equilibrium. Properties of proteins not depending on the Donnan equilibrium may be affected not only by the valency but also by the chemical nature of the anion of an acid.     

Non-enzymic Browning of Soy Sauce

Ion exchange resins have been used to separate soy sauce into three fractions of distinctly different composition: a cation fraction, a neutral fraction and an anion fraction. Almost all of the constituents responsible for browning were recovered in these three fractions.

Storage experiments show that when the three fractions were stored separately, only the cation fraction darkened considerably. When they were combined and stored, the color of the mixture increased at nearly the same rate as that of the original soy sauce. Neutral sugars are important constituents of the neutral fraction with respect to browning. The browning rate of a sugar-amino acid mixture (simulated soy sauce), was about 10% of soy sauce. The effect of the anion fraction (mainly caused by organic acids) and the ashed cation fraction on the over-all browning of soy sauce is calculated to be 1O~12% and 20%, respectively.

The sum of the contribution rate of the anion fraction, the neutral fraction, the amino acids and the ashed cation fraction in the browning of soy sauce was concluded to be approximately 40%. Compounds responcible for residual part of 60% should be considered to exist in the cation fraction. It was suggested that such compounds have strong reducing power and 0₂-uptaking ability.     

ELECTRIFICATION OF WATER AND OSMOTIC PRESSURE

1. Amphoteric electrolytes form salts with both acids and alkalies. It is shown for two amphoteric electrolytes, Al(OH)₃ and gelatin, that in the presence of an acid salt water diffuses through a collodion membrane into a solution of these substances as if its particles were negatively charged, while water diffuses into solutions of these electrolytes, when they exist as monovalent or bivalent metal salts, as if the particles of water were positively charged. The turning point for the sign of the electrification of water seems to be near or to coincide with the isoelectric point of these two ampholytes which is a hydrogen ion concentration of about 2 x 10^–5 N for gelatin and about 10^–7 for Al(OH)₃. 2. In conformity with the rules given in a preceding paper the apparently positively charged water diffuses with less rapidity through a collodion membrane into a solution of Ca and Ba gelatinate than into a solution of Li, Na, K, or NH₄ gelatinate of the same concentration of gelatin and of hydrogen ions. Apparently negatively charged water diffuses also with less rapidity through a collodion membrane into a solution of gelatin sulfate than into a solution of gelatin chloride or nitrate of the same concentration of gelatin and of hydrogen ions. 3. If we define osmotic pressure as that additional pressure upon the solution required to cause as many molecules of water to diffuse from solution to the pure water as diffuse simultaneously in the opposite direction through the membrane, it follows that the osmotic pressure cannot depend only on the concentration of the solute but must depend also on the electrostatic effects of the ions present and that the influence of ions on the osmotic pressure must be the same as that on the initial velocity of diffusion. This assumption was put to a test in experiments with gelatin salts for which a collodion membrane is strictly semipermeable and the tests confirmed the expectation.     

THE MECHANISM BY WHICH TRIVALENT AND TETRAVALENT IONS PRODUCE AN ELECTRICAL CHARGE ON ISOELECTRIC PROTEIN

1. Experiments on anomalous osmosis suggested that salts with trivalent cations, e.g. LaCl₃, caused isoelectric gelatin to be positively charged, and salts with tetravalent anions, e.g. Na₄Fe(CN)₆, caused isoelectric gelatin to be negatively charged. In this paper direct measurements of the P.D. between gels of isoelectric gelatin and an aqueous solution as well as between solutions of isoelectric gelatin in a collodion bag and an aqueous solution are published which show that this suggestion was correct. 2. Experiments on anomalous osmosis suggested that salts like MgCl₂, CaCl₂, NaCl, LiCl, or Na₂SO₄ produce no charge on isoelectric gelatin and it is shown in this paper that direct measurements of the P.D. support this suggestion. 3. The question arose as to the nature of the mechanism by which trivalent and tetravalent ions cause the charge of isoelectric proteins. It is shown that salts with such ions act on isoelectric gelatin in a way similar to that in which acids or alkalies act, inasmuch as in low concentrations the positive charge of isoelectric gelatin increases with the concentration of the LaCl₃ solution until a maximum is reached at a concentration of LaCl₃ of about M/8,000; from then on a further increase in the concentration of LaCl₃ diminishes the charge again. It is shown that the same is true for the action of Na₄Fe(CN)₆. From this it is inferred that the charge of the isoelectric gelatin under the influence of LaCl₃ and Na₄Fe(CN)₆ at the isoelectric point is due to an ionization of the isoelectric protein by the trivalent or tetravalent ions. 4. This ionization might be due to a change of the pH of the solution, but experiments are reported which show that in addition to this influence on pH, LaCl₃ causes an ionization of the protein in some other way, possibly by the formation of a complex cation, gelatin-La. Na₄Fe(CN)₆ might probably cause the formation of a complex anion of the type gelatin-Fe(CN)₆. Isoelectric gelatin seems not to form such compounds with Ca, Na, Cl, or SO₄. 5. Solutions of LaCl₃ and Na₄Fe(CN)₆ influence the osmotic pressure of solutions of isoelectric gelatin in a similar way as they influence the P.D., inasmuch as in lower concentrations they raise the osmotic pressure of the gelatin solution until a maximum is reached at a concentration of about M/2,048 LaCl₃ and M/4,096 Na₄Fe(CN)₆. A further increase of the concentration of the salt depresses the osmotic pressure again. NaCl, LiCl, MgCl₂, CaCl₂, and Na₂SO₄ do not act in this way. 6. Solutions of LaCl₃ have only a depressing effect on the P.D. and osmotic pressure of gelatin chloride solutions of pH 3.0 and this depressing effect is quantitatively identical with that of solutions of CaCl₂ and NaCl of the same concentration of Cl.     

THE ORIGIN OF THE ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND OF LIVING TISSUES

1. When a solution of a salt of gelatin or crystalline egg albumin is separated by a collodion membrane from a watery solution (free from protein) a potential difference is set up across the membrane in which the protein is positively charged in the case of protein-acid salts and in which the protein is negatively charged in the case of metal proteinates. The turning point is the isoelectric point of the protein. 2. Measurements of the pH of the (inside) protein solution and of the outside watery solution show that when equilibrium is established the value pH inside minus pH outside is positive in the case of protein-acid salts and negative in the case of metal proteinates. This is to be expected when the P.D. is caused by the establishment of a Donnan equilibrium, since in that case the pH should be lower outside than inside in the case of a protein-acid salt and should be higher outside than inside in the case of a metal proteinate. 3. At the isoelectric point where the electrical charge is zero the value of pH inside minus pH outside becomes also zero. 4. It is shown that a P.D. is established between suspended particles of powdered gelatin and the surrounding watery solution and that the sign of charge of the particles is positive when they contain gelatin-acid salts, while it is negative when the powdered particles contain metal gelatinate. At the isoelectric point the charge is zero. 5. Measurements of the pH inside the powdered particles and of the pH in the outside watery solution show that when equilibrium is established the value pH inside minus pH outside is positive when the powdered particles contain a gelatin-acid salt, while the value pH inside minus pH outside is negative when the powdered particles contain Na gelatinate. At the isoelectric point the value pH inside minus pH outside is zero. 6. The addition of neutral salts depresses the electrical charge of the powdered particles of protein-acid salts. It is shown that the addition of salts to a suspension of powdered particles of gelatin chloride also diminishes the value of pH inside minus pH outside. 7. The agreement between the values 58 (pH inside minus pH outside) and the P. D. observed by the Compton electrometer is not only qualitative but quantitative. This proves that the difference in the concentration of acid (or alkali, as the case may be) in the two phases is the only cause for the observed P.D. 8. The Donnan theory demands that the P.D. of a gelatin chloride solution should be 1½ times as great as the P.D. of a gelatin sulfate solution of the same pH and the same concentration (1 per cent) of originally isoelectric gelatin. This is found to be correct and it is also shown that the values of pH inside minus pH outside for the two solutions possess the ratio of 3:2. 9. All these measurements prove that the electrical charges of suspended particles of protein are determined exclusively by the Donnan equilibrium.     

ELECTROKINETIC PHENOMENA : X. ELECTRIC MOBILITY AND CHARGE OF PROTEINS IN ALCOHOL-WATER MIXTURES

1. The electrophoretic velocities of gelatin-, egg-albumin-, and gliadin-covered quartz particles in various alcohol-water solutions are, within the limits employed in usual experimental procedures, proportional to the field strength. 2. The electrophoretic mobilities of small, irregularly shaped quartz particles covered with an adsorbed film of protein in alcohol-water solutions are equal to the electroosmotic mobilities of the liquid past similarly coated flat surfaces. Hence the size and shape of such particles does not influence their mobilities, which depend entirely on the protein film. 3. The corrected mobility and hence presumably the charge of gelatin-covered quartz particles in solutions containing 35 per cent ethyl alcohol is proportional to the combining power of the gelatin; therefore the gelatin is adsorbed with the active groups oriented toward the liquid. The same is true in 60 per cent alcohol. 4. The charge calculated by means of the Debye-Henry approximation from the mobility of gelatin in solutions containing up to 35 per cent ethyl alcohol is, in the neighborhood of the isoelectric point, proportional to the combining power of the gelatin. Therefore the dielectric constant and the viscosity of the bulk of the medium may be used in the Debye-Henry approximation Q = 6 π η r v_m (1 + κ r) to predict changes in charge from mobility. 5. In the neighborhood of the isoelectric point gelatin is probably completely ionized in buffered ethyl alcohol-water mixtures up to 60 per cent alcohol. 6. In the presence of ethyl alcohol the isoelectric point of gelatin is shifted toward smaller hydrogen ion activities. This shift, like that caused by alcohol in the isoelectric points of certain amino acids, is approximately linearly related to the dielectric constant of the medium.