STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : I. THE SOLUBILITY OF CERTAIN PROTEINS AT THEIR ISOELECTRIC POINTS.

1. Two proteins of the globulin type, serum globulin and tuberin, and the protein of milk, casein, have been purified (a) of the other proteins and (b) of the inorganic electrolytes with which they exist in nature. The methods that were employed are described. 2. All three proteins were found to be only very slightly soluble in water in the pure uncombined state. The solubility of each was accurately measured at 25.0° ± 0.1°C. The most probable solubility of the pseudoglobulin of serum was found to be 0.07 gm. in 1 liter; of tuberin 0.1 gm. and of casein 0.11 gm. The methods that were employed in their determination are described. 3. Each protein investigated dissolved in water to a constant and characteristic extent when the amount of protein precipitate with which the solution was in heterogeneous equilibrium was varied within wide limits. The solubility of a pure protein is therefore proposed as a fundamental physicochemical constant, which may be used in identifying and in classifying proteins. 4. The concentration of protein dissolved must be the sum of the concentration of the undissociated protein molecule which is in heterogeneous equilibrium with the protein precipitate, and of the concentration of the dissociated protein ions. 5. The dissociated ions of the dissolved protein give a hydrogen ion concentration to water that is also a characteristic of each protein.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : II. THE RELATION BETWEEN THE SOLUBILITY OF CASEIN AND ITS CAPACITY TO COMBINE WITH BASE. THE SOLUBILITY OF CASEIN IN SYSTEMS CONTAINING THE PROTEIN AND SODIUM HYDROXIDE.

1. The solubility in water of purified, uncombined casein has previously been reported to be 0.11 gm. in 1 liter at 25°C. This solubility represents the sum of the concentrations of the casein molecule and of the soluble ions into which it dissociates. 2. The solubility of casein has now been studied in systems containing the protein and varying amounts of sodium hydroxide. It was found that casein forms a well defined soluble disodium compound, and that solubility was completely determined by (a) the solubility of the casein molecule, and (b) the concentration of the disodium casein compound. 3. In our experiments each mol of sodium hydroxide combined with approximately 2,100 gm. of casein. 4. The equivalent combining weight of casein for this base is just half the minimal molecular weight as calculated from the sulfur and phosphorus content, and one-sixth the minimal molecular weight calculated from the tryptophane content of casein. 5. From the study of systems containing the protein and very small amounts of sodium hydroxide it was possible to determine the solubility of the casein molecule, and also the degree to which it dissociated as a divalent acid and combined with base. 6. Solubility in such systems increased in direct proportion to the amount of sodium hydroxide they contained. 7. The concentration of the soluble casein compound varied inversely as the square of the hydrogen ion concentration, directly as the solubility of the casein molecule, Su, and as the constants Ka₁ and Ka₂ defining its acid dissociation. 8. The product of the solubility of the casein molecule and its acid dissociation constants yields the solubility product constant, Su·Ka₁·Ka₂ = 2.2 x 10^–12 gm. casein per liter at 25°C. 9. The solubility of the casein molecule has been estimated from this constant, and also from the relation between the solubility of the casein and the sodium hydroxide concentration, to be approximately 0.09 gm. per liter at 25°C. 10. The product of the acid dissociation constants, Ka₁ and Ka₂, must therefore be 24 x 10^–12N. 11. It is believed that these constants completely characterize the solubility of casein in systems containing the protein and small amounts of sodium hydroxide.     

THE SOLUBILITY OF d-VALINE IN WATER

1. The solubility of d-valine in water has been determined over a range of 0–60°. 2. The solubility of this amino acid varies with the mode of crystallization, indicating a dependence of solubility on the crystal form.     

FRACTIONATION OF PEPSIN : I. ISOLATION OF CRYSTALLINE PEPSIN OF CONSTANT ACTIVITY AND SOLUBILITY FROM PEPSINOGEN OR COMMERCIAL PEPSIN PREPARATIONS. II. PREPARATION OF A LESS SOLUBLE FRACTION. III. SOLUBILITY CURVES OF MIXTURES OF THE SOLUBLE AND INSOLUBLE FRACTIONS. IV. PREPARATION OF HIGHLY ACTIVE PEPSIN FROM PEPSINOGEN

1. Solubility curves of crude pepsin preparations indicate the presence of more than one protein. 2. One of these proteins has been isolated and crystallized and found to have constant activity and constant solubility in several solvents. 3. The solubility measurements are complicated by the unstable nature of the protein and the fact that in certain solvents the solubility of the protein is markedly affected by the presence of non-protein nitrogen decomposition products while in others this is not the case. 4. A more insoluble protein has been prepared of lower solubility and lower activity, as measured by hemoglobin digestion. The activity, as measured by the digestion of other proteins, is about the same as the more soluble fraction. This insoluble fraction does not have constant solubility. 5. Mixtures of the insoluble and the soluble fractions give preparations having rounded solubility curves typical of solid solutions and resembling very closely those of the original preparation. 6. A small amount of pepsinogen and pepsin from pepsinogen has been separated which has nearly twice the enzymatic activity on hemoglobin as does the most active pepsin previously isolated.     

A PHASE RULE STUDY OF THE PROTEINS OF BLOOD SERUM: THE EFFECT OF CHANGES IN CERTAIN VARIABLES

Changes were studied in the standard solubility curve of fresh serum proteins by alterations in pH, temperature, concentration of protein, and nature of the salt used for precipitation. The principal factor affecting the precipitation of protein fractions was a change in temperature. In order to investigate the proteins in their original states low temperatures are necessary. Protein fraction A is altered by a change in pH and with the use of (NH₄)₂SO₄ as a precipitant, fraction B by a change in pH and temperature, and use of (NH₄)₂SO₄, C by a change in temperature and concentration of the protein, and D by a change in temperature and pH. The solubility of D is independent of the amount of protein in solution in high concentrations of salt.     

A PHASE RULE STUDY OF THE PROTEINS OF THE BLOOD SERUM: A COMPARISON OF THE PROTEINS OF HUMAN,RAT, AND HORSE SERUM

There are four different kinds of protein in blood serum as shown by the solubility curves. They must be either single proteins, several continuous series of compounds, or solid solutions. The solid protein phases are hydrated. There are definite sex and species differences.     

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.     

A PHASE RULE STUDY OF THE PROTEINS OF BLOOD SERUM : III. GLOBULIN

A method has been developed for applying the phase rule to systems of several protein components in serum. The globulin fractions which have been investigated appear to be homogeneous substances.     

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 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.     

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.     

THE INACTIVATION OF TRYPSIN : II. THE EQUILIBRIUM BETWEEN TRYPSIN AND THE INHIBITING SUBSTANCE FORMED BY ITS ACTION ON PROTEINS.

1. A study has been made of the equilibrium existing between trypsin and the substances formed in the digestion of proteins which inhibit its action. 2. This substance could not be obtained by the hydrolysis of the proteins by acid or alkali. It is dialyzable. 3. The equilibrium between this substance (inhibitor) and trypsin is found to agree with the equation, trypsin + inhibitor ⇌ trypsin-inhibitor The equilibrium is reached instantaneously and is independent of the substrate concentration. If it be further assumed that the rate of hydrolysis is proportional to the concentration of the free trypsin and that the equilibrium conforms to the law of mass action, it is possible to calculate the experimental results by the application of the law of mass action. 4. The equilibrium has been studied by varying (a) the concentration of the inhibiting substance, (b) the concentration of trypsin, (c) the concentration of gelatin, and (d) the concentration of trypsin and inhibitor (the relative concentration of the two remaining the same). In all cases the results agree quantitatively with those predicted by the law of mass action. 5. It was found that the percentage retarding effect of the inhibiting substance on the rate of hydrolysis is independent of the hydrogen ion concentration between pH 6.3 and 10.0. 6. The fact that the experimental results agree with the mechanism outlined under 3, is contrary to the assumption that any appreciable amount of trypsin is combined with the gelatin at any one time; i.e., the velocity of the hydrolysis must depend on the time required for such a compound to form rather than for it to decompose. 7. The experiments may be considered as experimental proof of the validity of Arrhenius'' explanation of Schütz''s rule as applied to trypsin digestion. 8. Inactivated trypsin does not enter into the equilibrium.     

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.     

TRANSPORT AND TURNOVER OF NEUROHYPOPHYSIAL PROTEINS OF THE RAT

Axonal transport and turnover rate of proteins in the supraoptico-neurohypo-physial tract were studied after injection of ³⁵S cysteine into the region of the supraoptic nucleus. The proximo-distal migration of labelled proteins from the nerve cell bodies to the axon terminals in the neurohypophysis was followed by measuring the radioactivity of neurohypophysial proteins at various time intervals (4 h to 30 days) after isotope injection. A rapidly transported phase of proteins with a minimal transport rate of approximately 60 mm/day was demonstrated. An accumulation of protein-bound radioactivity was also observed in the neural lobe at 9 days after isotope injection, representing slowly transported proteins (0-5 mm/day). In addition, an intermediate phase of axonal transport (1-5 mm/day) was found. Fractionation of neurohypophysial proteins by polyacrylamide gel disc electrophoresis revealed that a predominating portion of the radioactivity was recovered in a single protein component (fraction A) at 4 h as well as at 30 days after isotope injection. This protein component was shown to be a constituent both of the rapid and the slow phase of axonal transport. With time an increasing amount of radioactivity was found in another protein component (fraction B), which reached a maximum at 14 days after injection and then remained fairly constant up to 30 days. When the turnover rates of neurohypophysial proteins were estimated, a half-life of 1-2 days and 8 days was calculated for the rapidly and slowly transported proteins, respectively.     

THE EFFECT OF RENNIN UPON CASEIN : I. THE SOLUBILITY OF PARACASEIN IN SODIUM HYDROXIDE.

1. The preparation and purification of paracasein was described and certain criteria for the absence of free enzyme provided for. 2. The solubility of purified paracasein in water at low temperature was studied, and found practically identical with the solubility of casein. 3. The capacity of paracasein to bind base was investigated by means of its solubility in NaOH at 5° and at 23° ± 2°C., and found to be distinctly different from that of casein. 4. At these two temperature levels paracasein had a 1.5 greater capacity to bind base than casein. The equivalent combining weights of paracasein and casein were found to stand each to the other, apapproximately, as 2 to 3. 5. This relationship suggested that the temperature coefficients of the solubility of paracasein and casein in NaOH are identical. 6. This evidence indicates that paracasein is a modification of casein, distinguishable by physicochemical means.     

THE EFFECT OF TEMPERATURE UPON SOME OF THE PROPERTIES OF CASEIN

1. The investigations dealing with the properties of casein as an acid were reviewed. 2. The solubility of uncombined casein in water was measured at 5°C. and found to be 0.70±0.1 mg. of N per 100 gm. of water. 3. Robertson''s solubility measurements of casein in bases at various temperatures were recalculated and found to agree well with more recent measurements. 4. By combining the observations of several investigators, as well as the author''s measurements of the solubility of casein, in base, at various temperatures, the following conclusions were reached: (a) The solubility of casein in base is affected by the temperature in a discontinuous manner. (b) There exist two ranges of temperature, one, extending from about 21° to 37°C. and the other from about 60° to 85°C. where the solubility of casein in base is practically independent of temperature. (c) From 37° to 60° the equivalent combining weight of casein rises from the value 2100 to about 3700 gm. 5. By comparing the values of base bound by 1 gm. of casein at the two temperature ranges with a constant, the value of base necessary to saturate the same amount of casein, it was found that the latter value is a common multiple of the former values, indicating the stoichiometric nature of the effect of temperature.     

Protein solubility modeling.

A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : VI. THE ACTIVITY COEFFICIENTS OF THE IONS IN CERTAIN OXYHEMOGLOBIN SOLUTIONS.

1. The solvent action of a neutral salt upon a protein, oxyhemoglobin, has been found identical to the solvent action of a neutral salt upon a bi-bivalent or uni-quadrivalent compound. 2. The solubility of oxyhemoglobin in phosphate solutions of varying ionic strength has been defined by the equation: log See PDF for Equation in which µ is the ionic strength, and S ₀ is the solubility in the absence of salt. 3. The values of S ₀ have been calculated to be 12.2, 11.2, and 13.1 gm. per liter respectively at pH 6.4, 6.6, and 6.8. 4. The relatively great solubility of oxyhemoglobin in water has been ascribed to the strong affinity constants for acid and base of certain groups in oxyhemoglobin. 5. The small change in the solubility of oxyhemoglobin effected by neutral salts suggests that but few such groups are dissociated in oxyhemoglobin in the state in which it crystallizes near its isoelectric point. 6. Certain of the other properties of oxyhemoglobin, such as its low viscosity, are considered in the light of its molecular weight and its valence type.     

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.     

SOLUBILITY STUDIES ON PURIFIED TOBACCO MOSAIC VIRUS

Different samples of purified tobacco mosaic virus show a relatively wide variation in solubility in ammonium sulfate solution. This variation and the type of solubility curve obtained in the presence of varying amounts of solid phase show that the purified virus whether isolated by mild treatment with ammonium sulfate or by ultracentrifugation is not a homogeneous chemical substance but contains more soluble and less soluble virus fractions of comparable specific activities. Long contact with strong ammonium sulfate solutions or 0.1 M phosphate buffer results in a decrease in solubility. The variation in the solubility of samples isolated from different plants by the same method seems to depend in part on the length of time the plants are inoculated before they are cut, and probably also on the conditions under which they are grown. Virus preparations isolated from plants of different genera grown under the same conditions and inoculated at the same time, however, behaved like identical substances in solubility experiments.