STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI : II. THE CONDUCTIVITIES OF ALKALI CASEINATE SOLUTIONS.

1. The results of conductivity experiments with alkali caseinate solutions are given and a graphical method of extrapolation, which gives a straight line, is described. The results of the conductivity experiments are shown to be in accord with the results of the previous transference experiments. 2. The change of conductivity of the alkali caseinate solutions with temperature is shown to follow a straight line relationship. 3. The high value of the mobility which was obtained for the casein ion and the high temperature gradient are discussed in relation to McBain''s theory of colloidal electrolytes.     

STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI : IV. THE TRANSPORT NUMBERS OF THE COMPOUNDS OF CASEIN WITH THE ALKALI EARTH ELEMENTS.

1. Data from the results of transference experiments on solutions of the alkaline earth caseinates are given. 2. The data support the idea that part of the alkaline earth element is held by the casein in the form of complex ions. 3. Grounds are given for believing that the complex anions have a definite composition.     

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.     

THE KINETICS OF TRYPSIN DIGESTION : V. SCHUTZ'S RULE.

The rate of digestion of concentrated casein solutions by low concentrations of trypsin at 0° has been followed. Under these conditions the enzyme is inhibited by the product of the reaction and under certain conditions this effect should lead to Schütz''s rule, i.e. the amount of hydrolysis should be proportional to the square root of the product of the time into the enzyme concentration. This is the result obtained. Both Schütz''s rule and Arrhenius'' equation fail to hold accurately owing to the incorrect relation assumed to hold between the rate of hydrolysis and the substrate concentration.     

STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI : III. THE ELECTROCHEMICAL BEHAVIOR OF RACEMIC CASEIN.

1. The phenomenon of protein racemization is discussed and certain deductions are made in connection with the hypothesis of Dakin to account for this phenomenon and Robertson''s theory of the ionization of proteins. 2. Experimental data are given to show that the electrochemical behavior of racemic casein is not in accord with the deductions which have been drawn from the theory advanced by Robertson. 3. An analysis of the nitrogen groups of racemic casein is given and compared with a similar analysis of normal casein. From these analyses and from the electrochemical equivalent of racemic casein, it is concluded that except for the hydrolysis of amide groups, racemic casein is probably not a degradation product of casein. 4. Considerable evidence is presented against the view that the -COHN- groups take part in the reactions of the protein molecule with acids and with bases.     

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 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.     

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 KINETICS OF PENETRATION : XVI. THE ACCUMULATION OF AMMONIA IN LIGHT AND DARKNESS

The accumulation of ammonia takes place more rapidly in light than in darkness. The accumulation appears to go on until a steady state is attained. The steady state concentration of ammonia in the sap is about twice as great in light as in darkness. Both effects are possibly due to the fact that the external pH (and hence the concentration of undissociated ammonia) outside is raised by photosynthesis. Certain "permeability constants" have been calculated. These indicate that the rate is proportional to the concentration gradient across the protoplasm of NH₄ X which is formed by the interaction of NH₃ or NH₄OH and HX, an acid elaborated in the protoplasm. The results are interpreted to mean that HX is produced only at the sap-protoplasm interface and that on the average its concentration there is about 7 times as great as at the sea water-protoplasm interface. This ratio of HX at the two surfaces also explains why the concentration of undissociated ammonia in the steady state is about 7 times as great in the sea water as in the sap. The permeability constant P'''''' appears to be greater in the dark. This is possibly associated with an increase in the concentration of HX at both interfaces, the ratio at the two surfaces, however, remaining about the same. The pH of sap has been determined by a new method which avoids the loss of gas (CO₂), an important source of error. The results indicate that the pH rises during accumulation but the extent of this rise is smaller than has hitherto been supposed. As in previous experiments, the entering ammonia displaced a practically equivalent amount of potassium from the sap and the sodium concentration remained fairly constant. It seems probable that the pH increase is due to the entrance of small amounts of NH₃ or NH₄OH in excess of the potassium lost as a base.     

THE KINETICS OF THE DECOMPOSITION OF PEROXIDE BY CATALASE

It has been shown that the experimental results obtained by Morgulis in a study of the decomposition of hydrogen peroxide by liver catalase at 20°C. and in the presence of an excess of a relatively high concentration of peroxide are quantitatively accounted for by the following mechanisms. 1. The rate of formation of oxygen is independent of the peroxide concentration provided this is greater than about 0.10 M. 2. The rate of decomposition of the peroxide is proportional at any time to the concentration of catalase present. 3. The catalase undergoes spontaneous monomolecular decomposition during the reaction. This inactivation is independent of the concentration of catalase and inversely proportional to the original concentration of peroxide up to 0.4 M. In very high concentrations of peroxide the inactivation rate increases. 4. The following equation can be derived from the above assumptions and has been found to fit the experiments accurately. See PDF for Equation in which x is the amount of oxygen liberated at the time t, A is the total amount of oxygen liberated (not the total amount available), and K is the inactivation constant of the enzyme.     

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.     

MOLECULAR ASSOCIATION OF HEMOCYANIN PRODUCED BY X-RAYS AS OBSERVED IN THE ULTRACENTRIFUGE

1. When normal, monodisperse hemocyanin (60.5S) from Limulus Rolyphemus was irradiated in neutral buffer with x-rays, several new, more rapidly sedimenting ultracentrifugal components (86S, 107S, 122S) were produced, with a corresponding loss in the amount of the unaffected protein. The amount of the effect was roughly proportional to the amount of irradiation. 2. The new resolvable components apparently represented an association of the primary particles into aggregates of 2, 3, and 4 primary particles respectively. 3. The proportional amount of hemocyanin affected decreased almost to the vanishing point as the concentration of the protein was raised to high levels. 4. The absolute effect, i.e. the total number of particles affected in a given volume, increased with the concentration of hemocyanin, at least for concentrations below 15 per cent. 5. The presence of 33 per cent horse serum during irradiation inhibited the effect on the hemocyanin almost completely, with hemocyanin concentrations of both 0.8 and 14 per cent. 6. The presence of 2.8 per cent egg albumin during irradiation lowered the effect by about 70 per cent in the case of dilute preparations (0.8 per cent hemocyanin), but by only about 25 per cent in the case of 14 per cent solutions. 7. A lowering of the solution''s oxygen tension during irradiation enhanced the effect, almost doubling it in some cases. 8. The probable theoretical significance of these and other observations are discussed in the text.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

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.     

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED,ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES : II. KINETICS OF PROTEIN DIGESTION BY ASCLEPAIN

1. The kinetics of milk clotting by asclepain, the protease of Asclepias speciosa, were investigated. At higher concentrations of enzyme, the clotting time was inversely proportional to the enzyme concentration. 2. The digestion of casein and hemoglobin in 6.6 M urea by asclepain follows the second order reaction rate. The rate was roughly second order for casein in water. 3. Evaluation of the nature of the enzyme-substrate intermediate indicates that one molecule of asclepain combines with one molecule of casein or hemoglobin in urea solution. 4. Inhibition by the reaction products was deduced from the fact that the digestion velocity of hemoglobin in urea solution varied with the asclepain concentration in agreement with the Schütz-Borissov rule.     

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.     

AN ULTRACENTRIFUGAL ANALYSIS OF CONCENTRATED STAPHYLOCOCCUS BACTERIOPHAGE PREPARATIONS

Analytical observations have been made with the air ultracentrifuge on concentrated staphylococcus bacteriophage solutions and on these solutions inactivated by alkali, chymo-trypsin, and heat. All active solutions contain a homogeneous heavy component that sediments with a constant of s _20° = ca. 650 x 10^–13 cm. sec.^–1 dynes^–1, has an apparent density of ca. 1.20, and a molecular weight probably not less than 200 millions. There is also present some very light ultraviolet-absorbing material which is not a carrier of bacteriophage activity. The amount of the heavy component is not strictly proportional to the bacteriophage activity so that if the activity resides in it, as appears to be the case, inactivation may occur without measurable change in molecular size and shape. When the bacteriophage solutions are inactivated by chymo-trypsin, the heavy component is not disrupted but the sedimenting boundaries have always been fairly diffuse. As the activity gradually disappears from alkaline solutions, the heavy component is replaced by unsedimentable material. When a solution is inactivated by heating, a dilute gel is produced which sediments with an exceptionally sharp boundary in a relatively intense centrifugal field,     

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

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

The effect of dietary protein on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua

We compared the ability of larval H. zea (Boddie) and S. exigua (Hubner) to digest and utilize dietary protein by: (a) determining the ability of different concentrations of dietary protein to support larval growth, and (b) determining the effect of different concentrations of dietary protein on the digestive physiology of the organisms, as measured by in vivo digestion of protein and proteolytic activity. Using an artificial diet containing casein as the primary source of protein, we found that H. zea was able to grow at very low levels of casein (≤0.6%), while optimal growth occurred at 1.2% casein. For S. exigua, dietary casein levels of >0.6% were required for growth, and optimal growth occurred at ≥1.2% casein. However, optimal growth in both species was not correlated with the degree of in vivo digestion of protein. The level of in vivo digestion of protein and tryptic activity in S. exigua was proportional to the concentration of dietary protein (under both acute and chronic exposure), and not the amount of food in the gut, suggesting that enzyme synthesis and/or secretion is controlled by a secretagogue mechanism. H. zea only demonstrated a secretagogue mechanism of control of tryptic activity while under acute exposure to different concentrations of casein; under chronic exposure, tryptic activity was uniform regardless of the concentration of dietary casein. When comparing the two species of noctuid, H. zea, which is the larger of the two species, produced less tryptic activity on a unit weight basis, and also digested less of the available dietary protein than S. exigua. Hence, these closely related organisms are processing dietary protein at different efficiencies.