STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI : I. THE TRANSPORT NUMBERS OF ALKALI CASEINATE SOLUTIONS.

1. The deposition of casein on a platinum anode which takes place on the passage of a direct current through solutions of alkali caseinates was quantitatively studied, and it was found that: (a) the amount of casein which is deposited is directly proportional to the current, i.e. it obeys Faraday''s law; (b) the amount of casein deposited is inversely proportional (within the limits studied) to the amount of alkali which is combined with the casein. 2. A method of determining the transport numbers of proteins insoluble at their isoelectric point has been developed. 3. A titration method for determining the amount of alkali in a casein solution is given. 4. Data from the results of transference experiments on sodium caseinate, potassium caseinate, cesium caseinate, and rubidium caseinate solutions are given. It is shown that the data are best explained on the assumption that in these solutions the carriers of the current are alkali metal cations and casein anions. 5. On the basis of our transference results an explanation is given of the results which were obtained by Robertson and by Haas in their migration experiments.     

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.     

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 EFFECT OF TEMPERATURE UPON FACET NUMBER IN THE BAR-EYED MUTANT OF DROSOPHILA : PART III.

Three strains of the bar-eyed mutant of Drosophila melanogaster Meig have been reared at constant temperatures over a range of 15–31°C. The mean facet number in the bar-eyed mutant varies inversely with the temperature at which the larvæ develop. The temperature coefficient (Q₁₀) is of the same order as that for chemical reactions. The facet-temperature relations may be plotted as an exponential curve for temperatures from 15–31°. The rate of development of the immature stages gives a straight line temperature curve between 15 and 29°. Beyond 29° the rate decreases again with a further rise in temperature. The facet curve may be readily superimposed on the development curve between 15 and 27°. The straight line feature of the development curve is probably due to the flattening out of an exponential curve by secondary factors. Since both the straight line and the exponential curve appear simultaneously in the same living material, it is impractical to locate the secondary factors in enzyme destruction, differences in viscosity, or in the physical state of colloids. Differential temperature coefficients for the various separate processes involved in development furnish the best basis for an explanation of the straight line feature of the curve representing the effect of temperature on the rate of physiological processes. Facet number in the full-eyed wild stock is not affected by temperature to a marked degree. The mean facet number for fifteen full-eyed females raised at 27° is 859.06. The mean facet number for the Low Selected Bar females at 27° is 55.13; for the Ultra-bar females at 27° it is 21.27. A consistent sexual difference appears in all the bar stocks, the females having fewer facets. This relation may be expressed by the sex coefficient, the average value of which is 0.791. The average observed difference in mean facet number for a difference of 1°C. in the environment in which the flies developed is 3.09 for the Ultra-bar stock and 14.01 for the Low Selected stock. The average proportional differences in the mean for a difference of 1°C. are 9.22 per cent for Ultra-bar, and 14.51 for Low Selected. The differences in the number of facets per °C. are greatest at the low and least at the high temperatures. The difference in the number of facets per °C. varies with the mean. The proportional differences in the mean per °C. are greatest at the lower (15–17.5°) and higher (29–31°) temperatures and least at the intermediate temperatures. Temperature is a factor in determining facet number only during a relatively short period in larval development. This effective period, at 27°, comes between the end of the 3rd and the end of the 4th day. At 15°, this period is initiated at the end of 8 days following a 1st day at 27°. At 27° this period is approximately 18 hours long. At 15° it is approximately 72 hours long. The number of facets and the length of the immature stage (egg-larval-pupal) appear related when the whole of development is passed at one temperature. That the number of facets is not dependent upon the length of the immature stage is shown by experiments in which only a part of development was passed at one temperature and the remainder at another. Temperature affects the reaction determining the number of facets in approximately the same way that it affects the other developmental reactions, hence the apparent correlation between facet number and the length of the immature stage. Variability as expressed by the coefficient of variability has a tendency to increase with temperature. Standard deviation, on the other hand, appears to decrease with rise in temperature. Neither inheritance nor induction effects are exhibited by this material. This study shows that environment may markedly affect the somatic expression of one Mendelian factor (bar eye), while it has no visible influence on another (white eye).     

FACTORS INFLUENCING THE ABILITY OF ISOLATED CELL NUCLEI TO FORM GELS IN DILUTE ALKALI

1. Known methods for isolating cell nuclei are divided into two classes, depending on whether or not the nuclei are capable of forming gels in dilute alkali or strong saline solutions. Methods which produce nuclei that can form gels apparently prevent the action of an intramitochondrial enzyme capable of destroying the gel-forming capacity of the nuclei. Methods in the other class are believed to permit this enzyme to act on the nuclei during the isolation procedure, causing detachment of DNA from some nuclear constituent (probably protein). 2. It is shown that heating in alkaline solution and x-irradiation can destroy nuclear gels. Heating in acid or neutral solutions can destroy the capacity of isolated nuclei to form gels. 3. Chemical and biological evidence is summarized in favor of the hypothesis that DNA is normally bound firmly to some nuclear component by non-ionic linkages.     

KINETICS OF THE BIOLUMINESCENT REACTION IN CYPRIDINA. II

1. The decay curve of the light produced in the course of the luminescent reaction in Cypridina is, after the first second, in complete agreement with the theoretical expectation for a monomolecular reaction, if light intensity at any instant is assumed to be proportional to reaction velocity at that instant. It is shown that for such a reaction log I = - kt + log Ak and that the experimental values satisfy this equation. 2. The first second or two of the reaction is characterized by a brilliant initial flash, whose value is much too high to accord with the succeeding intensities and with the above formula. It is suggested that this initial high reaction velocity is an indication of a heterogeneous system. 3. Identical solutions run simultaneously give decay curves which check within the limits of the photographic error. 4. Stirring does not affect the reaction velocity or the form of the decay curve. 5. Reaction velocity is proportional to enzyme concentration, over the range of concentrations used in the study. 6. Changes in the concentration of the substrate do not affect the value of k, when all other factors are held constant. A diminution of luciferin concentration results only in a decrease in the value of the y-intercept, Log Ak, the two straight line plottings for two different concentrations being parallel. 7. The temperature coefficient is high, being about 4.5 for the 15–25° interval, and 3.0 for the 25–35° interval.     

STUDIES ON PERMEABILITY OF MEMBRANES : VII. CONDUCTIVITY OF ELECTROLYTES WITHIN THE MEMBRANE.

Two methods of measuring the electrical conductivity of the dried collodion membrane in contact with an electrolyte solution are described and the results of such measurements with different electrolytes in different ranges of concentration recorded. Some of the difficulties encountered in making these measurements are outlined. Of special interest was the fact that each membrane with each electrolyte showed a maximum level of resistance at a certain point in the dilution scale, a level which was not surpassed by further dilution. It is believed that this level was fixed by the collodion itself rather than by the contiguous electrolyte solution. Its existence limited the results available for reasonable interpretation. In relatively concentrated solutions the conductivity was shown to be approximately proportional to the concentration. With different electrolytes in the same concentration it was shown that the conductivities varied much more than in simple solutions without a membrane and that they fell in the order HCl > KCl > NaCl > LiCl. A method was described whereby the electrolyte content of a membrane in contact with different chloride solutions could be determined. It was shown that a membrane saturated with either 0.5 N HCl or 0.5 N KCl had practically the same total electrolyte content whereas the same membrane in contact with 0.5 N LiCl contained only half the quantity. These results were used in interpreting the conductivity data, the evidence presented strongly suggesting that two factors are operative in causing the widely divergent conductivities recorded with different electrolytes. The first factor depended on the quantity of electrolyte which can enter the membrane pores, a quantity dependent on the size of the pores and the volume of the larger of the two hydrated ions of the electrolyte. This factor was the chief one in determining the difference in conductivity between KCl and LiCl. The second factor was concerned with differences in the mobility of the various cations within the membrane brought about by friction between the moving ions and the pore walls. With KCl and HCl the quantity of electrolytes entering the membrane was in each case the same, being determined by the size of the larger Cl^- ion. The widely different conductivity values were explained as due to the changes in the mobility of the two cations within the membrane pores.     

THE RELATION BETWEEN CHLOROPHYLL CONTENT AND RATE OF PHOTOSYNTHESIS

1. Data are presented which support the conclusion of Emerson (1929) that the rate of photosynthesis is proportional to the chlorophyll content when the latter is varied by varying the iron supply. These data give a straight line passing through the origin, which is not true of Emerson''s results. 2. Similar data are presented which show that a similar relation exists when nitrogen controls the chlorophyll content. 3. Evidence is given which indicates that magnesium plays a part in the process of photosynthesis in addition to its effect upon the chlorophyll content.     

Alkali-labile sites and post-irradiation effects in single-stranded DNA induced by H radicals.

Single-stranded phiX174 DNA in aqueous solutions has been irradiated in the absence of oxygen, under conditions in which only H radicals react with the DNA. It was shown that H radical reactions result in breaks, which contribute approximately 10 per cent inactivation. Further, two types of alkali-labile sites are formed. One is lethal and gives rise to single-strand breaks by alkali and is most probably identical with post-irradiation heat damage and contributes about 33 per cent to the inactivation mentioned above. The other consists of non-lethal damage, partly dihydropyrimidine derivatives, and is converted to lethal damage by alkali. This follows from experiments in which the DNA was treated with osmium-tetroxide, which oxidizes thymine to 5,6-dihydroxy-dihydrothymine. Treatment with alkali of this DNA gives the same temperature dependence as found for the non-lethal alkali-labile sites in irradiated DNA. A similar temperature dependence is found for dihydrothymine and irradiated pyrimidines with alkali.     

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.     

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

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

STUDIES ON PERMEABILITY OF MEMBRANES : VIII. THE BEHAVIOR OF THE DRIED COLLODION MEMBRANE TOWARD BIVALENT CATIONS.

A study of the behavior of the dried collodion membrane toward the bivalent calcium ion showed that: 1. There is almost no potential difference established across a membrane separating two calcium chloride solutions of 0.1 and 0.01 N concentrations. 2. The transfer numbers of chlorine and calcium, as measured in electrical transfer experiments, are both close to 0.5. 3. A sample of membrane in equilibrium with a solution of calcium chloride has an extremely high electrical resistance, greater than is observed with solutions of the chlorides of any of the monovalent cations. 4. The total electrolyte content of a membrane in equilibrium with a solution of calcium chloride was only 20 per cent of that observed when the solution was lithium chloride and 10 per cent of that found when the solution was potassium chloride. In explaining these various results it is supposed that (1), (2) and (3) are all the result of (4), that is, of the inability of the calcium ion to penetrate any but the largest of the membrane pores. As the total quantity of electrolyte able to penetrate the membrane is very small the electrical conductivity must also be very small. Moreover, the few larger pores that are large enough to transport the hydrated calcium ion are too large to exert any appreciable effect in decreasing the mobility of the anion. Thus the membrane has no effect in modifying the potentials established across concentration chains with CaCl₂ and the transfer numbers determined experimentally are what one would expect if no membrane were present.     

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 DETERMINATION OF THE EQUIVALENT WEIGHT OF PROTEINS

The magnitude of the correction in the fifth column of Table III may be open to some doubt, as are all corrections of such a character, and the significance of the above experiment in the author''s mind lies not so much in the actual magnitude of the values given in the last column of this table as in their comparative magnitudes. For this reason the entire experiment reported was performed in a single session using the same gelatin solution, so that, whatever the magnitude of the correction, it would be the same in all cases. Actually the results in the case of the acid titrations are in fair agreement with those of Hitchcock (8). In the present experiment it is seen that, within the limits of experimental error, one gets the same value for the number of cc. of tenth normal acid bound by 1 gm. of gelatin whether one titrates with the acid or with the gelatin. In the case of the base there is a small difference, due probably to carbon dioxide, but this effect is in a direction opposite to that which one would expect on the assumption that it is due to appreciable adsorption. From this it is concluded that the binding due to adsorption in the case of gelatin is not significant compared to that due to chemical neutralization. The author realizes that gelatin is a poor choice for a basis of generalizations, and similar work is at present in progress on various other proteins. He does feel, however, that the conclusions of Hoffman and Gortner from their work on the prolamines may also be too widely generalized, and that, on the whole, the acid or alkali bound by adsorption in the case of proteins will not constitute the large majority of the total amounts bound, though certainly one will expect a certain amount of such binding in all cases. It also seems that before placing undue emphasis on the conclusions of these workers the possibilities of equivocal results due to specific technique should be considered. This technique consisted in introducing weighed amounts of dry protein into a definite volume of standard acid or base at the equilibrium temperature, in general, and, "after about 15 minutes, during which time the flask was shaken several times," determining the pH of the equilibrium solution. Is it possible that the actual speed of solution of the protean is such that, even though reproducible results are obtained using identical technique, actual equilibrium conditions are approached only when comparatively high concentrations of acid or alkali are employed, in which cases the solution velocity of the protein may he expected to be greater, other factors remaining constant?     

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.     

A STUDY OF THE COMPONENTS OF THE CORNIFIED EPITHELIUM OF HUMAN SKIN

Pulverized cornified epithelium of human skin was divided into a "soluble fraction" and a "residue." About half of the "soluble fraction" proved to be soluble epidermal keratin (keratin A); the remainder, dialyzable substances of low molecular weight. The "residue" contained epidermal keratin and resistant cell membranes of cornified cells. Epidermal keratin was found to form an oriented and dense submicroscopic structure in the cornified cells. It showed high resistance toward strong acid and moderately strong alkali solutions as well as concentrated urea. In strong alkali, reducing substances, alkaline urea, and mixtures of reducing substance with alkali, epidermal keratin dissociated and yielded a non-dialyzable derivative of high molecular weight (keratin B) which resembled true proteins. The cell membranes of cornified cells showed higher resistance toward strong alkali and reducing substance than did epidermal keratin.     

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 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.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. I

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