THE COLLOIDAL BEHAVIOR OF SERUM GLOBULIN

1. The globulin prepared from ox serum by dilution and precipitation with carbon dioxide has been found, by electrometric titration experiments, to behave like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of globulin chloride, phosphate, or acetate and a solution of the corresponding acid, free from protein, separated from the globulin by a collodion membrane, was found to be influenced by hydrogen ion concentration and salt concentration in the way predicted by Donnan''s theory of membrane equilibrium. In experiments with sodium globulinate and sodium hydroxide it was found that the potential difference could be similarly explained. 3. The osmotic pressure of such solutions could be qualitatively accounted for by the Donnan theory, but exhibited a discrepancy which is explicable by analogy with certain experiments of Loeb on gelatin. 4. The application of Loeb''s theory of colloidal behavior, which had previously been found to hold in the case of gelatin, casein, egg albumin, and edestin, has thus been extended to another protein, serum globulin.     

THE ELIMINATION OF DISCREPANCIES BETWEEN OBSERVED AND CALCULATED P.D. OF PROTEIN SOLUTIONS NEAR THE ISOELECTRIC POINT WITH THE AID OF BUFFER SOLUTIONS

1. It had been noticed in the previous experiments on the influence of the hydrogen ion concentration on the P.D. between protein solutions inside a collodion bag and aqueous solutions free from protein that the agreement between the observed values and the values calculated on the basis of Donnan''s theory was not satisfactory near the isoelectric point of the protein solution. It was suspected that this was due to the uncertainty in the measurements of the pH of the outside aqueous solution near the isoelectric point. This turned out to be correct, since it is shown in this paper that the discrepancy disappears when both the inside and outside solutions contain a buffer salt. 2. This removes the last discrepancy between the observed P.D. and the P. D. calculated on the basis of Donnan''s theory of P.D. between membrane equilibria, so that we can state that the P.D. between protein solutions inside collodion bags and outside aqueous solutions free from protein can be calculated from differences in the hydrogen ion concentration on the opposite sides of the membrane, in agreement with Donnan''s formula.     

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.     

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.     

THE COMBINATION OF SALTS AND PROTEINS. I

1. It has been found that the ratios of the total concentrations of Ca, Mg, K, Zn, inside and outside of gelatin particles do not agree with the ratios calculated according to Donnan''s theory from the hydrogen ion activity ratios. 2. E.M.F. measurements of Zn and Cl electrode potentials in such a system show, however, that the ion activity ratios are correct, so that the discrepancy must be due to a decrease in the ion concentration by the formation of complex ions with the protein. 3. This has been confirmed in the case of Zn by Zn potential measurements in ZnCl₂ solutions containing gelatin. It has been found that in 10 per cent gelatin containing 0.01 M ZnCl₂ about 60 per cent of the Zn⁺⁺ is combined with the gelatin. 4. If the activity ratios are correctly expressed by Donnan''s equation, then the amount of any ion combined with a protein can be determined without E.M.F. measurements by determining its distribution in a proper system. If the activity ratio of the hydrogen ion and the activity of the other ion in the aqueous solution are known, then the activity and hence the concentration of the ion in the protein solution can be calculated. The difference between this and the total molar concentration of the ion in the protein represents the amount combined with the protein. 5. It has been shown that in the case of Zn the values obtained in this way agree quite closely with those determined by direct E.M.F. measurements. 6. The combination with Zn is rapidly and completely reversible and hence is probably not a surface effect. 7. Since the protein combines more with Zn than with Cl, the addition of ZnCl₂ to isoelectric gelatin should give rise to an unequal ion distribution and hence to an increase in swelling, osmotic pressure, and viscosity. This has been found to be the case.     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : III. THE INFLUENCE OF SALTS ON OSMOTIC PRESSURE,MEMBRANE POTENTIALS,AND SWELLING OF SODIUM GELATINATE.

A detailed study was made on the influence of salts on those physicochemical properties of sodium gelatinate which are regulated by Donnan''s law of membrane equilibria; namely, osmotic pressure, membrane potentials, and swelling. It was found that the influence of salts on these properties in the case of sodium gelatinate obeys the same rules of valency as in the case of the influence of salts on gelatin chloride as discussed in a previous publication. The rules state that when a salt is added to an ionized protein, without causing a change in the hydrogen ion concentration of the protein, the general effect is a depression of the mentioned properties. The degree of depression depends not only on the concentration of the salt but on the electrical properties of the ions constituting the salt. Of the two or more oppositely charged ions of which a salt consists, only the valency of those ions which carry charges opposite to those carried by the protein ions affects the degree of depression which increases with the valency of the ions. It was also found that the phenomenon of swelling of gelatin becomes modified by solubility of the gelatin when salts are added in concentrations higher than N/4. Emphasis is laid on the point that the valency rule holds perfectly also in relation to swelling as long as the phenomenon is pure swelling which is the case when salt solutions of concentrations lower than N/4 are added to gelatin.     

MEMBRANE POTENTIALS AND CATAPHORETIC POTENTIALS OF PROTEINS

1. It has been shown in preceding publications that the membrane potentials of protein solutions or gels are determined by differences in the concentration of a common ion (e.g. hydrogen ion) inside a protein solution or protein gel and an outside aqueous solution free from protein, and that the membrane potentials can be calculated with a good degree of accuracy from Donnan''s equation for membrane equilibria. 2. On the basis of the theory of electrical double layers developed by Helmholtz, we are forced to assume that the cataphoretic potentials of protein particles are determined by a difference in the concentration of the two oppositely charged ions of the same electrolyte in the two strata of an electrical double layer surrounding the protein particle but situated entirely in the aqueous solution. 3. The membrane potentials of proteins agree with the cataphoretic potentials in that the sign of charge of the protein is negative on the alkaline side and positive on the acid side of the isoelectric point of the protein in both membrane potentials and cataphoretic potentials. The two types of potential of proteins disagree, especially in regard to the action of salts with trivalent and tetravalent ions on the sign of charge of the protein. While low concentrations of these salts bring about a reversal of the sign of the cataphoretic potentials of protein particles (at least in the neighborhood of the isoelectric point), the same salts can bring the membrane potentials of proteins only to zero, but call bring about no or practically no reversal of the sign of charge of the protein. Where salts seem to bring about a reversal in the membrane potential of protein solutions, the reversal is probably in reality always due to a change in the pH. 4. We may state, as a result of our experiments, that the cataphoretic migration and the cataphoretic P.D. of protein particles or of suspended particles coated with a protein are the result of two groups of forces; namely, first, forces inherent in the protein particles (these forces being linked with the membrane equilibrium between protein particles and the outside aqueous solution); and second, forces inherent entirely in the aqueous solution surrounding the protein particles. The forces inherent in the protein particles and linked with the membrane equilibrium prevail to such an extent over the forces inherent in the water, that the sense of the cataphoretic migration of protein particles is determined by the forces resulting from the membrane equilibrium.     

THE IONIZATION OF PROTEIN CHLORIDES

1. By the use of the silver-silver chloride electrode, measurements have been made of the chloride ion concentrations of 1 per cent solutions of five proteins, containing from 0.001 N to 0.1 N hydrochloric acid. The hydrogen ion concentrations of the same solutions have been measured by the use of the hydrogen electrode. 2. The measurements indicate that the chlorides of gelatin, egg albumin, casein, edestin, and serum globulin are highly ionized electrolytes, ionizing to yield chloride ion and a positive protein-hydrogen ion. Their ionization is therefore similar to that of ammonium chloride. 3. The results do not support the idea that a protein chloride does not yield chloride ion on dissociation. They are not in agreement with the idea that the depressing effect of an excess of HCl on the viscosity and other colloidal properties of a protein chloride solution is due to a repression of the ionization of the protein chloride. The results are, however, in complete accord with the theory of colloidal behavior advocated by Loeb.     

THE COMBINATION OF CERTAIN PROTEINS WITH HYDROCHLORIC ACID

Electromotive force measurements of cells without liquid junction, of the type Ag, AgCl, HCl + protein, H₂, have been made at 30°C. with the proteins gelatin, edestin, and casein in 0.1 M hydrochloric acid. The data are consistent with the assumptions of a constant combining capacity of each protein for hydrogen ion, no combination with chloride ion, and Failey''s principle of a linear variation of the logarithm of the mean activity coefficient of the acid with increasing protein concentration. The combining capacities for hydrogen ion so obtained are 13.4 x 10^–4 for edestin, 9.6 x 10^–4 for gelatin, and 8.0 x 10^–4 for casein, in equivalents of combined H⁺ per gm. of protein.     

THE EFFECT OF VARIOUS ACIDS ON THE DIGESTION OF PROTEINS BY PEPSIN

1. At equal hydrogen ion concentration the rate of pepsin digestion of gelatin, egg albumin, blood albumin, casein, and edestin is the same in solutions of hydrochloric, nitric, sulfuric, oxalic, citric, and phosphoric acids. Acetic acid diminishes the rate of digestion of all the proteins except gelatin. 2. There is no evidence of antagonistic salt action in the effect of acids on the pepsin digestion of proteins. 3. The state of aggregation of the protein, i.e. whether in solution or not, and the viscosity of the solution have no marked influence on the rate of digestion of the protein.     

THE ORIGIN OF THE POTENTIAL DIFFERENCES RESPONSIBLE FOR ANOMALOUS OSMOSIS

1. Collodion bags coated with gelatin on the inside were filled with a M/256 solution of neutral salt (e.g., NaCl, CaCl₂, CeCl₃, or Na₂SO₄) made up in various concentrations of HNO₃ (varying from N/50,000 to N/100). Each collodion bag was put into an HNO₃ solution of the same concentration as that inside the bag but containing no salt. In this case water diffuses from the outside solution (containing no salt) into the inside solution (containing the salt) with a relative initial velocity which can be expressed by the following rules: (a) Water diffuses into the salt solution as if the particles of water were negatively charged and as if they were attracted by the cation and repelled by the anion of the salt with a force increasing with the valency of the ion. (b) The initial rate of the diffusion of water is a minimum at the hydrogen ion concentration of about N/50,000 HCl (pH 4.7, which is the point at which gelatin is not ionized), rises with increasing hydrogen ion concentration until it reaches a maximum and then diminishes again with a further rise in the initial hydrogen ion concentration. 2. The potential differences between the salt solution and the outside solution (originally free from salt) were measured after the diffusion had been going on for 1 hour; and when these values were plotted as ordinates over the original pH as abscissae, the curves obtained were found to be similar to the osmotic rate curves. This confirms the view expressed by Girard) Bernstein, Bartell, and Freundlich that these cases of anomalous osmosis are in reality cases of electrical endosmose where the driving force is a P.D. between the opposite sides of the membrane. 3. The question arose as to the origin of these P. D. and it was found that the P.D. has apparently a double origin. Certain features of the P.D. curve, such as the rise and fall with varying pH, seem to be the consequence of a Donnan equilibrium which leads to some of the free HNO₃ being forced from the solution containing salt into the outside solution containing no (or less) salt. This difference of the concentration of HNO₃, on the opposite sides of the membrane leads to a P.D. which in conformity with Nernst''s theory of concentration cells should be equal to 58 x (pH inside minus pH outside) millivolts at 18°C. The curves of the values of (pH inside minus pH outside) when plotted as ordinates over the original pH as abscissae lead to curves resembling those for the P. D. in regard to location of minimum and maximum. 4. A second source of the P.D. seems to be diffusion potentials, which exist even if no membranes are present and which seem to be responsible for the fact that the rate of diffusion of negatively charged water into the salt solution increases with the valency of the cation and diminishes with the valency of the anion of the salt. 5. The experiments suggest the possibility that the establishment of a Donnan equilibrium between membrane and solution is one of the factors determining the Helmholtzian electrical double layer, at least in the conditions of our experiments.     

THE R?LE OF THE ACTIVITY COEFFICIENT OF THE HYDROGEN ION IN THE HYDROLYSIS OF GELATIN

1. The hydrolysis of gelatin at a constant hydrogen ion concentration follows the course of a monomolecular reaction for about one-third of the reaction. 2. If the hydrogen ion concentration is not kept constant the amount of hydrolysis in certain ranges of acidity is proportional to the square root of the time (Schütz''s rule). 3. The velocity of hydrolysis in strongly acid solution (pH less than 2.0) is directly proportional to the hydrogen ion concentration as determined by the hydrogen electrode i.e., the "activity;" it is not proportional to the hydrogen ion concentration as determined by the conductivity ratio. 4. The addition of neutral salts increases the velocity of hydrolysis and the hydrogen ion concentration (as determined by the hydrogen electrode) to approximately the same extent. 5. The velocity in strongly alkaline solutions (pH greater than 10) is directly proportional to the hydroxyl ion concentration. 6. Between pH 2.0 and pH 10.0 the rate of hydrolysis is approximately constant and very much greater than would be calculated from the hydrogen and hydroxyl ion concentration. This may be roughly accounted for by the assumption that the uncombined gelatin hydrolyzes much more rapidly than the gelatin salt.     

THE SIGNIFICANCE OF THE HYDROGEN ION CONCENTRATION FOR THE DIGESTION OF PROTEINS BY PEPSIN

The experiments described above show that the rate of digestion and the conductivity of protein solutions are very closely parallel. If the isoelectric point of a protein is at a lower hydrogen ion concentration than that of another, the conductivity and also the rate of digestion of the first protein extends further to the alkaline side. The optimum hydrogen ion concentration for the rate of digestion and the degree of ionization (conductivity) of gelatin solutions is the same, and the curves for the ionization and rate of digestion as plotted against the pH are nearly parallel throughout. The addition of a salt with the same anion as the acid to a solution of protein already containing the optimum amount of the acid has the same depressing effect on the digestion as has the addition of the equivalent amount of acid. These facts are in quantitative agreement with the hypothesis that the determining factor in the digestion of proteins by pepsin is the amount of ionized protein present in the solution. It was shown in a previous paper that this would also account for the peculiar relation between the rate of digestion and the concentration of protein. The amount of ionized protein in the solution depends on the amount of salt formed between the protein (a weak base) and the acid. This quantity, in turn, according to the hydrolysis theory of the salts of weak bases and strong acids, is a function of the hydrogen ion concentration, up to the point at which all the protein is combined with the acid as a salt. This point is the optimum hydrogen ion concentration for digestion, since the solution now contains the maximum concentration of protein ions. The hydrogen ion concentration in this range therefore is merely a convenient indicator of the amount of ionized protein present in the solution and takes no active part in the hydrolysis. After sufficient acid has been added to combine with all the protein, i.e. at pH of about 2.0, the further addition of acid serves to depress the ionization of the protein salt by increasing the concentration of the common anion. The hydrogen ion concentration is, therefore, no longer an indicator of the amount of ionized protein present, since this quantity is now determined by the anion concentration. Hence on the acid side of the optimum the addition of the same concentration of anion should have the same influence on the rate of digestion irrespective of whether it is combined with hydrogen or some other ion (provided, of course, that there is no other secondary effect of the other ion). The proposed mechanism is very similar to that suggested by Stieglitz and his coworkers for the hydrolysis of the imido esters. Pekelharing and Ringer have shown that pure pepsin in acid solution is always negatively charged; i.e., it is an anion. The experiments described above show further that it behaves just as would be expected of any anion in the presence of a salt containing the protein ion as the cation and as has been shown by Loeb to be the case with inorganic anions. Nothing has been said in regard to the quantitative agreement between the increasing amounts of ionized protein found in the solution (as shown by the conductivity values) and the amount predicted by the hydrolysis theory of the formation of salts of weak bases and strong acids. There is little doubt that the values are in qualitative agreement with such a theory. In order to make a quantitative comparison, however, it would be necessary to know the ionization constant of the protein and of the protein salt and also the number of hydroxyl (or amino) groups in the protein molecule as well as the molecular weight of the protein. Since these values are not known with any degree of certainty there appears to be no value at present in attempting to apply the hydrolysis equations to the data obtained. It it clear that the hypothesis as outlined above for the hydrolysis of proteins by pepsin cannot be extended directly to enzymes in general, since in many cases the substrate is not known to exist in an ionized condition at all. It is possible, however, that ionization is really present or that the equilibrium instead of being ionic is between two tautomeric forms of the substrate, only one of which is attacked by the enzyme. Furthermore, it is clear that even in the case of proteins there are difficulties in the way since the pepsin obtained from young animals, or a similar enzyme preparation from yeast or other microorganisms, is said to have a different optimum hydrogen ion concentration than that found for the pepsin used in these experiments. The activity of these enzyme preparations therefore would not be found to depend on the ionization of the protein. It is possible of course that the enzyme preparations mentioned may contain several proteolytic enzymes and that the action observed is a combination of the action of several enzymes. Dernby has shown that this is a very probable explanation of the action of the autolytic enzymes. The optimum hydrogen ion concentration for the activity of the pepsin used in these experiments agrees very closely with that found by Ringer for pepsin prepared by him directly from gastric juice and very carefully purified. Ringer''s pepsin probably represents as pure an enzyme preparation as it is possible to prepare. There is every reason to suppose therefore that the enzyme used in this work was not a mixture of several enzymes.     

ON THE INFLUENCE OF AGGREGATES ON THE MEMBRANE POTENTIALS AND THE OSMOTIC PRESSURE OF PROTEIN SOLUTIONS

1. It is shown that when part of the gelatin in a solution of gelatin chloride is replaced by particles of powdered gelatin (without change of pH) the membrane potential of the solution is influenced comparatively little. 2. A measurement of the hydrogen ion concentration of the gelatin chloride solution and the outside aqueous solution with which the gelatin solution is in osmotic equilibrium, shows that the membrane potential can be calculated from this difference of hydrogen ion concentration with an accuracy of half a millivolt. This proves that the membrane potential is due to the establishment of a membrane equilibrium and that the powdered particles participate in this membrane equilibrium. 3. It is shown that a Donnan equilibrium is established between powdered particles of gelatin chloride and not too strong a solution of gelatin chloride. This is due to the fact that the powdered gelatin particles may be considered as a solid solution of gelatin with a higher concentration than that of the weak gelatin solution in which they are suspended. It follows from the theory of membrane equilibria that this difference in concentration of protein ions must give rise to potential differences between the solid particles and the weaker gelatin solution. 4. The writer had shown previously that when the gelatin in a solution of gelatin chloride is replaced by powdered gelatin (without a change in pH), the osmotic pressure of the solution is lowered the more the more dissolved gelatin is replaced by powdered gelatin. It is therefore obvious that the powdered particles of gelatin do not participate in the osmotic pressure of the solution in spite of the fact that they participate in the establishment of the Donnan equilibrium and in the membrane potentials. 5. This paradoxical phenomenon finds its explanation in the fact that as a consequence of the participation of each particle in the Donnan equilibrium, a special osmotic pressure is set up in each individual particle of powdered gelatin which leads to a swelling of that particle, and this osmotic pressure is measured by the increase in the cohesion pressure of the powdered particles required to balance the osmotic pressure inside each particle. 6. In a mixture of protein in solution and powdered protein (or protein micellæ) we have therefore two kinds of osmotic pressure, the hydrostatic pressure of the protein which is in true solution, and the cohesion pressure of the aggregates. Since only the former is noticeable in the hydrostatic pressure which serves as a measure of the osmotic pressure of a solution, it is clear why the osmotic pressure of a protein solution must be diminished when part of the protein in true solution is replaced by aggregates.     

THE COLLOIDAL BEHAVIOR OF PROTEINS

1. It is well known that neutral salts depress the osmotic pressure, swelling, and viscosity of protein-acid salts. Measurements of the P.D. between gelatin chloride solutions contained in a collodion bag and an outside aqueous solution show that the salt depresses the P.D. in the same proportion as it depresses the osmotic pressure of the gelatin chloride solution. 2. Measurements of the hydrogen ion concentration inside the gelatin chloride solution and in the outside aqueous solution show that the difference in pH of the two solutions allows us to calculate the P.D. quantitatively on the basis of the Nernst formula See PDF for Equation if we assume that the P.D. is due to a difference in the hydrogen ion concentration on the two sides of the membrane. 3. This difference in pH inside minus pH outside solution seems to be the consequence of the Donnan membrane equilibrium, which only supposes that one of the ions in solution cannot diffuse through the membrane. It is immaterial for this equilibrium whether the non-diffusible ion is a crystalloid or a colloid. 4. When acid is added to isoelectric gelatin the osmotic pressure rises at first with increasing hydrogen ion concentration, reaches a maximum at pH 3.5, and then falls again with further fall of the pH. It is shown that the P.D. of the gelatin chloride solution shows the same variation with the pH (except that it reaches its maximum at pH of about 3.9) and that the P.D. can be calculated from the difference of pH inside minus pH outside on the basis of Nernst''s formula. 5. It was found in preceding papers that the osmotic pressure of gelatin sulfate solutions is only about one-half of that of gelatin chloride or gelatin phosphate solutions of the same pH and the same concentration of originally isoelectric gelatin; and that the osmotic pressure of gelatin oxalate solutions is almost but not quite the same as that of the gelatin chloride solutions of the same pH and concentration of originally isoelectric gelatin. It was found that the curves for the values for P.D. of these four gelatin salts are parallel to the curves of their osmotic pressure and that the values for pH inside minus pH outside multiplied by 58 give approximately the millivolts of these P.D. In this preliminary note only the influence of the concentration of the hydrogen ions on the P.D. has been taken into consideration. In the fuller paper, which is to follow, the possible influence of the concentration of the anions on this quantity will have to be discussed.     

THE INFLUENCE OF ELECTROLYTES ON THE SOLUTION AND PRECIPITATION OF CASEIN AND GELATIN

1. Colloids have been divided into two groups according to the ease with which their solutions or suspensions are precipitated by electrolytes. One group (hydrophilic colloids), e.g., solutions of gelatin or crystalline egg albumin in water, requires high concentrations of electrolytes for this purpose, while the other group (hydrophobic colloids) requires low concentrations. In the latter group the precipitating ion of the salt has the opposite sign of charge as the colloidal particle (Hardy''s rule), while no such relation exists in the precipitation of colloids of the first group. 2. The influence of electrolytes on the solubility of solid Na caseinate, which belongs to the first group (hydrophilic colloids), and of solid casein chloride which belongs to the second group (hydrophobic colloids), was investigated and it was found that the forces determining the solution are entirely different in the two cases. The forces which cause the hydrophobic casein chloride to go into solution are forces regulated by the Donnan equilibrium; namely, the swelling of particles. As soon as the swelling of a solid particle of casein chloride exceeds a certain limit it is dissolved. The forces which cause the hydrophilic Na caseinate to go into solution are of a different character and may be those of residual valency. Swelling plays no rôle in this case, and the solubility of Na caseinate is not regulated by the Donnan equilibrium. 3. The stability of solutions of casein chloride (requiring low concentrations of electrolytes for precipitation) is due, first, to the osmotic pressure generated through the Donnan equilibrium between the casein ions tending to form an aggregate, whereby the protein ions of the nascent micellum are forced apart again; and second, to the potential difference between the surface of a micellum and the surrounding solution (also regulated by the Donnan equilibrium) which prevents the further coalescence of micella already formed. This latter consequence of the Donnan effect had already been suggested by J. A. Wilson. 4. The precipitation of this group of hydrophobic colloids by salts is due to the diminution or annihilation of the osmotic pressure and the P.D. just discussed. Since low concentrations of electrolytes suffice for the depression of the swelling and P.D. of the micella, it is clear why low concentrations of electrolytes suffice for the precipitation of hydrophobic colloids, such as casein chloride. 5. This also explains why only that ion of the precipitating salt is active in the precipitation of hydrophobic colloids which has the opposite sign of charge as the colloidal ion, since this is always the case in the Donnan effect. Hardy''s rule is, therefore, at least in the precipitation of casein chloride, only a consequence of the Donnan effect. 6. For the salting out of hydrophilic colloids, like gelatin, from watery solution, sulfates are more efficient than chlorides regardless of the pH of the gelatin solution. Solution experiments lead to the result that while CaCl₂ or NaCl increase the solubility of isoelectric gelatin in water, and the more, the higher the concentration of the salt, Na₂SO₄ increases the solubility of isoelectric gelatin in low concentrations, but when the concentration of Na₂SO₄ exceeds M/32 it diminishes the solubility of isoelectric gelatin the more, the higher the concentration. The reason for this difference in the action of the two salts is not yet clear. 7. There is neither any necessity nor any room for the assumption that the precipitation of proteins is due to the adsorption of the ions of the precipitating salt by the colloid.     

An ion switch regulates fusion of charged membranes

Here we identify the recruitment of solvent ions to lipid membranes as the dominant regulator of lipid phase behavior. Our data demonstrate that binding of counterions to charged lipids promotes the formation of lamellar membranes, whereas their absence can induce fusion. The mechanism applies to anionic and cationic liposomes, as well as the recently introduced amphoteric liposomes. In the latter, an additional pH-dependent lipid salt formation between anionic and cationic lipids must occur, as indicated by the depletion of membrane-bound ions in a zone around pH 5. Amphoteric liposomes fuse under these conditions but form lamellar structures at both lower and higher pH values. The integration of these observations into the classic lipid shape theory yielded a quantitative link between lipid and solvent composition and the physical state of the lipid assembly. The key parameter of the new model, κ(pH), describes the membrane phase behavior of charged membranes in response to their ion loading in a quantitative way.     

THE POTASSIUM EQUILIBRIUM IN MUSCLE

1. Analyses were made of the K and HCO₃ content, the irritability, and weight change of isolated frog sartorius muscles after immersion for 5 hours in Ringer''s solutions modified as to pH and potassium content. 2. At each pH a concentration of potassium in the solution was found which was in diffusion equilibrium with the potassium in the muscle. In greater concentrations potassium moved into the muscle against the concentration gradient and vice versa. 3. The greater the alkalinity of the solution the smaller the concentration of the potassium at equilibrium so that the product of the concentrations of OH and K in the solution at equilibrium tends to remain approximately constant. 4. The pH inside the muscle is approximately equal to that outside when first dissected but it tends to change during immersion so as to follow the changes in the pH of the solution. This finding is in direct conflict with the theory according to which the high potassium concentration inside should be accompanied by an equally high hydrogen ion concentration in relation to that outside. 5. The diffusion of potassium into the muscle makes its contents more alkaline but the increase in alkalinity is not always, nor usually, equivalent to the amount of potassium which has diffused and conversely, the pH inside can change in either direction according to the pH outside without there being any diffusion of potassium. Hence potassium is not the only penetrating ion. 6. The irritability of the muscles is at a maximum in concentrations of potassium which are greater than that in normal Ringer''s solution, or about 20 mg. per cent potassium. This optimum does not seem to be a function of pH and is therefore not dependent upon the direction of movement of the potassium but probably on the ratio of potassium outside to that inside. 7. Swelling of the muscles occurs in solutions which injure the muscle so as to permit both cations and anions to enter without permitting the organic protein anions to escape. Anion impermeability is necessary to prevent this same osmotic swelling under normal conditions. 8. An increase in the CO₂ tension in muscle and solution causes a greater increase in acidity in the solution than in the muscle and leads to a loss of potassium. One expects therefore a potassium shift from tissues to blood comparable to the chlorine shift from plasma to corpuscles.     

Donnan membrane equilibrium,sedimentation equilibrium,and coil expansion of DNA in salt solutions

This is a review of applications of the McMillan-Mayer-Hill virial theory and the ionic double-layer theory to dilute colloidal solutions, in particular, solutions of DNA. Interactions of highly charged colloidal rods are developed in terms of the second virial coefficients between two rods, and between one rod and one small co-ion. The relevant cluster integrals are evaluated with interaction potentials based on the Poisson-Boltzmann equation. The treatment is extended to the intrachain repulsion responsible for the statistical swelling of coiled DNA (excluded volume effect). The theory is compared with three sets of experimental data: The salt distribution in Donnan membrane equilibria of DNA-salt solutions, sedimentation equilibria of short DNA fragments at different ionic strengths, and the intrinsic viscosity of T7 DNA in NaCl solutions. In all cases the theory agrees well with the experiments. The agreement is not convincing for the sedimentation equilibrium at low ionic strength, because here the experimental DNA concentration is too high for the truncated dilute solution expansion of the DNA-salt repulsion.