CRYSTALLINE TRYPSIN : II. GENERAL PROPERTIES

A method is described for isolating a crystalline protein of high tryptic activity from beef pancreas. The protein has constant proteolytic activity and optical activity under various conditions and no indication of further fractionation could be obtained. The loss in activity corresponds to the decrease in native protein when the protein is denatured by heat, digested by pepsin, or hydrolyzed in dilute alkali. The enzyme digests casein, gelatin, edestin, and denatured hemoglobin, but not native hemoglobin. It accelerates the coagulation of blood but has little effect on the clotting of milk. It digests peptone prepared by the action of pepsin on casein, edestin or gelatin. The extent of the digestion of gelatin caused by this enzyme is the same as that caused by crystalline pepsin and is approximately equivalent to tripling the number of carboxyl groups present in the solution. The activity of the preparation is not increased by enterokinase. The molecular weight by osmotic pressure measure is about 34,000. The diffusion coefficient in ½ saturated magnesium sulfate at 6°C. is 0.020 ±0.001 cm.² per day, corresponding to a molecular radius of 2.6 x 10^–7 cm. The isoelectric point is probably between pH 7.0 and pH 8.0. The optimum pH for the digestion of casein is from 8.0–9.0. The optimum stability is at pH 1.8.     

THE COMBINATION OF EDESTIN WITH HYDROCHLORIC ACID

Electromotive force measurements of cells without liquid junction, of the type Ag, AgCl, HCl + protein, H₂, lead to the conclusion that 1 gm. of edestin (or, more probably, edestan) combines with a maximum of 13.4 x 10^–4 equivalents of H⁺ and 3.9 x 10^–4 equivalents of Cl^-, when the protein is dissolved in 0.1 M HCl.     

THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID : II. NEW DETERMINATIONS OF THE ISOELECTRIC POINT AND COMBINING CAPACITY OF A PURIFIED GELATIN.

1. Cooper''s gelatin purified according to Northrop and Kunitz exhibited a minimum of osmotic pressure and a maximum of opacity at pH 5.05 ±0.05. The pH of solutions of this gelatin in water was also close to this value. It is inferred that such gelatin is isoelectric at this pH and not at pH 4.70. 2. Hydrogen electrode measurements with KCl-agar junctions were made with concentrated solutions of this gelatin in HCl up to 0.1 M. The combination curve calculated from these data is quite exactly horizontal between pH 2 and 1, indicating that 1 gm. of this gelatin can combine with a maximum of 9.35 x 10^–4 equivalents of H⁺. 3. Conductivity titrations of this gelatin with HCl gave an endpoint at 9.41 (±0.05) x 10^–4 equivalents of HCl per gram gelatin. 4. E.M.F. measurements of the cell without liquid junction, Ag, AgCl, HCl + gelatin, H₂, lead to the conclusion that this gelatin in 0.1 M HCl combines with a maximum of 9.4 x 10^–4 equivalents of H⁺ and 1.7 x 10^–4 equivalents of Cl^- per gram gelatin.     

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.     

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 EFFECT OF VARIOUS ACIDS ON THE DIGESTION OF PROTEINS BY PEPSIN

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

STIMULATION BY MINERAL AND FATTY ACIDS IN THE BARNACLE BALANUS BALANOIDES

1. Stimulation in the rock barnacle Balanus balanoides by hydrochloric, sulfuric, and nitric acids, and by the first seven members of the normal aliphatic acid series has been studied. The hydrogen ion concentrations of the solutions tested varied from 3.2 x 10^–8 to 5.889 x 10^–6. The criterion of response was percentage closure in groups of individuals, recorded at 1 minute intervals until maximum closure occurred. 2. The intensity of stimulation by these acids is proportional to the effects of two forces, one related to the change in the (H⁺), and the other to the field of force around the anion of the acid added to the environment. 3. A preliminary interpretation of the results led to the development of the following expression which fits approximately the data obtained at the end of 4 minutes: Per cent closure = 100 – 100e ^{–0.1z+(0.003125)2–0.1z+(0.003125)2n(z–0.4)} where z is the (H⁺) x 10⁷ and n is the number of carbon atoms (if present) in the anion of the acid. This equation assumes that the anions of the mineral acids enter into the reaction stoichiometrically, and emphasizes the difference in the fields of force around the anion of the fatty acids, a difference which is correlated with the length of the carbon chain. 4. A further analysis of the data revealed the presence of three or more receptor groups which appeared to be differentially affected by forces originating from the anions of the acids. 5. The order of stimulating efficiency for the mineral acids was found to be: HCl>H₂SO₄>HNO₃. 6. The order of stimulating efficiency for the fatty acids was found to be: heptylic>caproic>valeric>butyric = acetic>propionic = formic.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS : II. THE ACTION OF SUBSTITUTED BENZOIC ACIDS AND OF BENZOIC AND SALICYLIC ACIDS AS INFLUENCED BY THEIR SALTS.

1. Comparison of the rates of activation of unfertilized starfish eggs in pure solutions of a variety of parthenogenetically effective organic acids (fatty acids, carbonic acid, benzoic and salicylic acids, chloro- and nitrobenzoic acids) shows that solutions which activate the eggs at the same rate, although widely different in molecular concentration, tend to be closely similar in C_H. The dissociation constants of these acids range from 3.2 x 10^–7 to 1.32 x 10^–3. 2. In the case of each of the fourteen acids showing parthenogenetic action the rate of activation (within the favorable range of concentration) proved nearly proportional to the concentration of acid. The estimated C_H of solutions exhibiting an optimum action with exposures of 10 minutes (at 20°) lay typically between 1.1 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.96), and in most cases between 1.6 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.8). Formic acid (C_H = 4.2 x 10^–4 M) and o-chlorobenzoic acid (C_H = 3.5 x 10^–4 M) are exceptions; o-nitrobenzoic acid is ineffective, apparently because of slow penetration. 3. Activation is not dependent on the penetration of H ions into the egg from without, as is shown by the effects following the addition of its Na salt to the solution of the activating acid (acetic, benzoic, salicylic). The rate of activation is increased by such addition, to a degree indicating that the parthenogenetically effective component of the external solution is the undissociated free acid. Apparently the undissociated molecules alone penetrate the egg freely. It is assumed that, having penetrated, they dissociate in the interior of the egg, furnishing there the H ions which effect activation. 4. Attention is drawn to certain parallels between the physiological conditions controlling activation in the starfish egg and in the vertebrate respiratory center.     

THE COLLOIDAL BEHAVIOR OF SERUM GLOBULIN

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

THE IONIZATION OF PROTEIN CHLORIDES

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

AMPHOTERIC BEHAVIOR OF COMPLEX SYSTEMS : IV. NOTE ON THE ISOELECTRIC POINT AND IONIZATION CONSTANTS OF SULFANILIC ACID.

From the solubility minimum the value of the basic ionization constant of sulfanilic acid is shown to lie probably between the values 1.7 x 10^–15 and 3.2 x 10^–15. From solubility measurements the value of this same constant is shown to lie probably between 2.0 and 2.2 x 10^–15, and the isoelectric point of sulfanilic acid is thus at a cH of 0.056 or a pH of 1.25. From conductivity ratios the acid ionization constant of sulfanilic acid is shown to be 7.05 x 10^–4 at room temperature (21°C.). Calculations are made, from data published in preceding papers, of the ionization constants of glycine, K_a being 2.3 x 10^–10, and K_b being 2.2 x 10^–12.     

THE EFFECT OF DETERGENTS ON THE CHLOROPHYLL-PROTEIN COMPOUND OF SPINACH AS STUDIED IN THE ULTRACENTRIFUGE

1. The chlorophyll-protein compound of the spinach leaf has been studied in the air-driven ultracentrifuge using the Svedberg light-absorption method, and a direct-reading refractive index method. 2. When the untreated extracts are centrifuged at low speeds, the green protein sediments with a purely random spread of particle sizes confirming the fact that the protein is not in true solution. 3. In the presence of digitonin, bile salts, and sodium desoxycholate, the extracts are clarified. These detergents split the chlorophyll from the protein and the protein itself shows a sedimentation constant of 13.5 x 10^–13 equivalent to a molecular weight of at least 265,000 as calculated from Stokes'' law. This probably represents the minimum size of the protein in native form. 4. Sodium dodecyl sulfate, a detergent which also clarifies the leaf extracts, shows a different behavior. The prosthetic group remains attached to the protein but the protein is split into smaller units. In 0.25 per cent SDS, S ₂₀ is 2.6 x 10^–13 over a pH range of 5 to 9, although at the acid pH chlorophyll is converted to phaeophytin. In 2.5 per cent SDS, S ₂₀ is 1.7 x 10^–13 suggesting a further splitting of the protein. 5. No differences in behavior were found for the various chloroplast pigments.     

ELECTRIFICATION OF WATER AND OSMOTIC PRESSURE

1. Amphoteric electrolytes form salts with both acids and alkalies. It is shown for two amphoteric electrolytes, Al(OH)₃ and gelatin, that in the presence of an acid salt water diffuses through a collodion membrane into a solution of these substances as if its particles were negatively charged, while water diffuses into solutions of these electrolytes, when they exist as monovalent or bivalent metal salts, as if the particles of water were positively charged. The turning point for the sign of the electrification of water seems to be near or to coincide with the isoelectric point of these two ampholytes which is a hydrogen ion concentration of about 2 x 10^–5 N for gelatin and about 10^–7 for Al(OH)₃. 2. In conformity with the rules given in a preceding paper the apparently positively charged water diffuses with less rapidity through a collodion membrane into a solution of Ca and Ba gelatinate than into a solution of Li, Na, K, or NH₄ gelatinate of the same concentration of gelatin and of hydrogen ions. Apparently negatively charged water diffuses also with less rapidity through a collodion membrane into a solution of gelatin sulfate than into a solution of gelatin chloride or nitrate of the same concentration of gelatin and of hydrogen ions. 3. If we define osmotic pressure as that additional pressure upon the solution required to cause as many molecules of water to diffuse from solution to the pure water as diffuse simultaneously in the opposite direction through the membrane, it follows that the osmotic pressure cannot depend only on the concentration of the solute but must depend also on the electrostatic effects of the ions present and that the influence of ions on the osmotic pressure must be the same as that on the initial velocity of diffusion. This assumption was put to a test in experiments with gelatin salts for which a collodion membrane is strictly semipermeable and the tests confirmed the expectation.     

THE SOLUBILITY OF TYROSINE IN ACID AND IN ALKALI

Measurements have been made of the solubility at 25°C. of tyrosine in hydrochloric acid and in sodium hydroxide solutions varying from 0.001 to 0.05 M, and also in distilled water. The pH of the saturated solutions was measured with the hydrogen electrode. The following values for the ionization constants of tyrosine have been obtained from the measurements: k_b = 1.57 x 10^–12, k_a1 = 7.8 x 10^–10, k_a2 = 8.5 x 10^–11. The changes in solubility with pH can be satisfactorily explained by the use of these ionization constants.     

PEPSIN ACTIVITY UNITS AND METHODS FOR DETERMINING PEPTIC ACTIVITY

Experimental methods are described for determining the activity of pepsin preparations by means of changes in the viscosity of gelatin, casein, edestin, and powdered milk solutions, and by the rate of formation of non-protein nitrogen from casein and edestin solutions, or by the increase in formol titration of casein, edestin or gelatin. Activity units for pepsin are defined in terms of these measurements.     

STIMULATION BY THE MINERAL ACIDS,HYDROCHLORIC, SULFURIC,AND NITRIC,IN THE SUNFISH EUPOMOTIS

1. The stimulating efficiency of hydrochloric, sulfuric, and nitric acids has been measured in the sunfish Eupomotis gibbosus, by a method which reduces experimental errors to a minimum. 2. The results show that stimulation by these acids is primarily dependent upon the (H⁺) produced in the animal''s aquatic environment, and that the reaction time is a logarithmic function of the (H⁺) within the range tested expressed by the equation: (RT–5) = –4.3 log (H⁺ x 10⁴) + 9.118. 3. Any effect of the chloride, sulfate, and nitrate ions must itself be measured by the (H⁺). 4. Variation in the reaction time is also a logarithmic function of the (H⁺), and the percentage variation is independent of the (H⁺) over the range tested. 5. Freshly collected fish show a lower threshold for stimulation as determined by the (H⁺) than do fish adapted to laboratory conditions, but relatively the reaction times of the two groups are the same.     

THE BEHAVIOR OF THE NUCLEIC ACIDS DURING THE EARLY DEVELOPMENT OF THE SEA URCHIN EGG (ARBACIA)

The authors wish to correct an error in the paper "The behavior of the nucleic acids during the early development of the sea urchin egg (Arbacia)" (J. Gen. Physiol., 1947–48, 31, 203). Owing to an oversight, the figures for the amounts of various P fractions in a single Arbacia egg have been erroneously expressed in γ x 10^–3 units (Tables I and II, page 205; the last two lines of page 206). The figures should have been expressed in γ x 10^–5 units. Thus, the fertilized Arbacia egg contains an average of 20 γ x 10^–5 ribonucleic acid P and 0.7 to 1 γ x 10^–5 desoxyribonucleic acid P.     

THE IONIC ACTIVITY OF GELATIN

2.5 and 1.25 per cent gelatin have been titrated potentiometrically in the absence of salts and in the presence of two concentrations (0.0750 and 0.0375µ) of NaCl, MgCl₂, K₂SO₄, and MgSO₄. The data have been used to calculate values of ± S = v^z – (v – 1)^z, where v^z = v ² – (v ² – v) r_x/18. The maximum and minimum values of S with NaCl were used to calculate the mean distance (r_x) between like charges in gelatin. This is found to be 18 Å.u. or over (between acid or basic groups) which agrees with the probable value and the titration index dispersion. Thus the data with NaCl are shown to be normal and to obey the equation found to hold for simple weak electrolytes; namely, pK'' – pK = Sa See PDF for Equation where S is related to the valence and distance by the above equations. Using the NaCl data as a standard the deviations (ΔS) produced by the other salts are calculated and are found to agree quantitatively with the deviations calculated from equations derived for the simple weak electrolytes. This shows that in gelatin, as in the simple electrolytes, the deviations are related to the "apparent valences" (values which are a function of the true valence and the distance between the groups). The maximum "apparent valences" of gelatin are 2.4 for acid groups (in alkaline solution) and 1.8 for basic groups (in acid solution). These values correspond to the hypothetical condition of zero distance between the groups. They have no physical significance but have a practical utility first as mentioned above, and second in that they may be used in the unmodified Debye-Hückel equation to give the maximum effect of gelatin on the ionic strength. The true effect is probably even lower than these values would indicate. The data indicate that gelatin is a weak polyvalent ampholyte having distant groups and that the molecule has an arborescent structure with interstices permeated by molecules of the solvent and other solutes. The size and shape probably vary with the pH.     

THE SIZE OF PORES IN COLLODION MEMBRANES

By the application of Poiseuille''s law to the rate of flow of water through collodion membranes, it is calculated that the membranes used had pore radii of the order of 0.3 to 2 x 10^–6 cm. On the same basis the number of pores per sq. cm. appears to vary from 270 x 10¹⁰ to 7 x 10¹⁰, decreasing with increase in pore size. Reasons are given for preferring these figures for the radii to figures, 100 times as large, which were calculated by others. Microscopic examination of the membranes, with dark-field illumination, indicates that they are made up of solid granules or filaments of collodion much less than 1 x 10^–4 cm. in thickness.