THE COMBINATION OF ENZYME AND SUBSTRATE : I. A METHOD FOR THE QUANTITATIVE DETERMINATION OF PEPSIN. II. THE EFFECT OF THE HYDROGEN ION CONCENTRATION.

1. A quantitative method for the determination of pepsin is described depending on the change in conductivity of a digesting egg albumin solution. 2. The combination of pepsin with an insoluble substrate has been followed by this method. 3. The amount of pepsin removed from solution by a given weight of substrate is independent of the size of the particles of the substrate. 4. There is an optimum zone of hydrogen ion concentration for the combination of enzyme and substrate corresponding to the optimum for digestion. 5. It is suggested that the pepsin combines largely or entirely with the ionized protein.     

THE INFLUENCE OF HYDROGEN ION CONCENTRATION ON THE INACTIVATION OF PEPSIN SOLUTIONS

1. Pepsin in solution at 38°C. is most stable at a hydrogen ion concentration of about 10^–5 (pH 5.0). 2. Increasing the hydrogen ion concentration above pH 5.0 causes a slow increase in the rate of destruction of pepsin. 3. Decreasing the hydrogen ion concentration below pH 5.0 causes a very rapid increase in the rate of destruction of the enzyme. 4. Neither the purity of the enzyme solution nor the anion of the acid used has any marked effect on the rate of destruction or on the zone of hydrogen ion concentration in which the enzyme is most stable. 5. The existence of an optimum range of hydrogen ion concentration for the digestion of proteins by pepsin cannot be explained by the destruction of the enzyme by either too weak or too strong acid.     

THE MECHANISM OF THE INFLUENCE OF ACIDS AND ALKALIES ON THE DIGESTION OF PROTEINS BY PEPSIN OR TRYPSIN

1. The effect of the addition of acid on the amount of ionized protein has been compared with the effect on the rate of digestion of gelatin, casein, and hemoglobin by pepsin. 2. A similar comparison has been made of the addition of alkali in the case of trypsin with gelatin, casein, hemoglobin, globin, and edestin. 3. In general, the rate of digestion may be predicted from the amount of ionized protein as determined by the titration curve or conductivity. The rate of digestion is a minimum at the isoelectric point of the protein and a maximum at that pH at which the protein is completely combined with acid or alkali to form a salt. 4. The physical properties of the protein solution have little or no effect on the rate of digestion.     

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 SUBSTRATE IN PEPTIC SYNTHESIS OF PROTEIN

1. Experiments are described in which it was observed that the yield of protein that can be synthesized by pepsin from a given peptic digest is highest when the hydrolyzing action of the pepsin is stopped as soon as all the protein has disappeared from the solution; and that the longer the digest is permitted to contain active enzyme the more the yield diminishes. 2. Exposure of the digest to a hydrogen ion concentration of pH 1.6 in the absence of active enzyme, does not cause a diminution in the amount of protein which can be synthesized from that digest. 3. Synthesis can be effected also in concentrated solutions of isolated fractions of a peptic digest, i.e. of proteose and of peptone. The yields are approximately the same as in similar concentrations of the whole digest, though the proteins so synthesized differ in some respects from those obtained from the whole digest. 4. The cessation of synthesis in any one digest is due to the attainment of equilibrium and not to the complete utilization of available synthesizeable material. The amount of the equilibrium yield, on the other hand, is dependent on the amount of synthesizeable material in the digest. 5. These observations are taken to show that the synthesizeability of a given mixture of protein cleavage products by pepsin depends upon its possession of a special complex in these products. This complex appears as a result of the primary hydrolysis of the protein molecule by pepsin and is decomposed in the slow secondary hydrolysis which ensues as digestion is prolonged.     

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 EFFECT OF THE CONCENTRATION OF ENZYME ON THE RATE OF DIGESTION OF PROTEINS BY PEPSIN

1. In certain cases the rate of digestion of proteins by pepsin is not proportional to the total concentration of pepsin. 2. It is suggested that this is due to the fact that the enzyme in solution is in equilibrium with another substance (called peptone for convenience) and that the equilibrium is quantitatively expressed by the law of mass action, according to the following equation. See PDF for Equation It is assumed that only the uncombined pepsin affects the hydrolysis of the protein. 3. The hypothesis has been put in the form of a differential equation and found to agree quantitatively with the experimental results when the concentration of pepsin, peptone, or both is varied. 4. Pepsin inactivated with alkali enters the equilibrium to the same extent as active pepsin. 5. Under certain conditions (concentration of peptone large with respect to pepsin, and concentration of substrate relatively constant) the relative change in the amount of active pepsin is inversely proportional to the concentration of peptone and the equation simplifies to Schütz''s rule. 6. An integral equation is obtained which holds for the entire course of the digestion (except for the first few minutes) with varying enzyme concentration. This equation is identical in form with the one derived by Arrhenius for the action of ammonia on ethyl acetate. 7. It is pointed out that there are many analogies between the action of pepsin on albumin solutions and the action of toxins on an organism.     

THE AUTODESTRUCTION OF PEPSIN IN RELATION TO ITS IONIZATION

1. Evidence is presented that pepsin is a univalent acid with a value for pK of 6.85 (or a base, with pK 7.39). 2. The autodestruction of the pepsin is shown to be dependent in part upon an instantaneous irreversible change occurring in the ionized form of the enzyme (if it be an acid) or in the unionized form (if it be a base). 3. A further progressive autodestruction of pepsin at any given hydrogen ion concentration and temperature is defined by the mass law equation for a monomolecular reaction 4. The velocity of autodestruction of pepsin is directly proportional to the hydroxyl ion concentration. It is much less in the range of hydroxyl ion concentration from pOH 9.89-7.7, than in the range greater than pOH 7.7. In both of these ranges variations in pK with pOH may be represented by straight lines.     

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).     

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 COLLOIDAL BEHAVIOR OF EDESTIN

1. It has been shown by titration experiments that the globulin edestin behaves like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of edestin chloride or acetate separated by a collodion membrane from an acid solution free from protein was found to be influenced by salt concentration and hydrogen ion concentration in the way predicted by Donnan''s theory of membrane equilibrium. 3. The osmotic pressure of such edestin-acid salt solutions was found to be influenced by salt concentration and by hydrogen ion concentration in the same way as is the potential difference. 4. The colloidal behavior of edestin is thus completely analogous to that observed by Loeb with gelatin, casein, and egg albumin, and may be explained by Loeb''s theory of colloidal behavior, which is based on the idea that proteins react stoichiometrically as amphoteric electrolytes and on Donnan''s theory of membrane equilibrium.     

THE INFLUENCE OF THE SUBSTRATE CONCENTRATION ON THE RATE OF HYDROLYSIS OF PROTEINS BY PEPSIN

1. It is pointed out that the apparent exceptions to the law of mass action found in enzyme reactions may be found in catalytic reactions in strictly homogeneous solutions. 2. These deviations in the rate of reaction from the law of mass action may be explained by the hypothesis that the active mass of the reacting substances is not directly proportional to the total concentration of substance taken. 3. In support of this suggestion it is shown that for any given concentration of pepsin the relative rate of digestion of concentrated and of dilute protein solutions is always the same. If the rate of digestion depended on the saturation of the surface of the enzyme by substrate the relative rate of digestion of concentrated protein solutions should increase more rapidly with the concentration of enzyme than that of dilute solutions. This was found not to be true, even when the enzyme could not be considered saturated in the dilute protein solutions. 4. The rate of digestion and the conductivity of egg albumin solutions of different concentration were found to be approximately proportional at the same pH. This agrees with the hypothesis first expressed by Pauli that the ionized protein is largely or entirely the form which is attacked by the enzyme. 5. The rate of digestion is diminished by a very large increase in the viscosity of the protein solution. This effect is probably a mechanical one due to the retardation of the diffusion of the enzyme.     

REGULATION OF THE HYDROGEN ION CONCENTRATION AND ITS RELATION TO METABOLISM AND RESPIRATION IN THE STARFISH

The normal reaction of the cœlomic fluid in Patiria miniata and Asterias ochraceus is pH 7.6, and of the cæca, 6.7, compared with sea water at 8.3, all without salt error correction. A medium at pH 6.7–7.0 is optimum for the cæca for ciliary survival and digestion of protein, and is maintained by carbon dioxide production. The optimum pH found for carbon dioxide production is a true one for the effect of hydrogen ion concentration on the tissue. It does not represent an elimination gradient for carbon dioxide. Because the normal excised cæca maintain a definite hydrogen ion concentration and change their internal environment toward that as an optimum during life, there exists a regulatory process which is an important vital function.     

STUDIES ON THE REGULATION OF OSMOTIC PRESSURE : I. THE EFFECT OF INCREASING CONCENTRATIONS OF GELATIN ON THE CONDUCTIVITY OF A SODIUM CHLORIDE SOLUTION.

1. In pure gelatin solutions the conductivity of the solution increases with increasing concentrations, regardless of the hydrogen ion concentration. The actual value of the specific conductivity is greater at that reaction where the degree of ionization is greater. 2. The addition of gelatin in increasing concentrations to a 0.6 per cent sodium chloride solution affects the conductivity of that solution in two ways: (a) At pH 3.3, (where gelatin is highly ionized) the conductivity increases with each added increment of gelatin. (b) At pH 5.1 and 7.4 (where gelatin is less highly ionized) the conductivity decreases with each added increment of gelatin. A similar study is being made of crystalline egg albumin.     

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.     

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 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.     

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 INFLUENCE OF THE BACKWARD REACTION IN THE PEPTIC HYDROLYSIS OF ALBUMIN

1. No destruction of pepsin by heat is demonstrable at pH 1.6 until a temperature of 40°C. is exceeded. 2. The influence of the backward reaction in peptic hydrolysis is shown in the diminishing rate at which increasing concentrations of protein are hydrolyzed. 3. The backward reaction causes the optimum for the hydrolysis of higher concentrations of protein to be attained at a lower temperature than with more dilute solutions. 4. The proteose and peptone associated with commercial pepsin retard hydrolysis in the same sense as the products due to the action of the enzyme.     

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.