THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID

The amount of HCl combined with a given weight of gelatin has been determined by hydrogen electrode measurements in 1 per cent, 2.5 per cent, and 5 per cent solutions of gelatin in HCl of various concentrations, by correcting for the amount of HCl necessary to give the same pH to an equal volume of water without protein. The curve so obtained indicates that the amount of HCl combined with 1 gm. of gelatin is constant between pH 1 and 2, being about 0.00092 moles.     

THE COMBINATION OF DEAMINIZED GELATIN WITH HYDROCHLORIC ACID

1. The analysis of isoelectric gelatin by the Van Slyke method indicates 0.00040 equivalents of amino N per gm. gelatin. 2. If deaminized gelatin is prepared without heating, the product contains less nitrogen than the original gelatin by an amount equal to 0.00040 equivalents N per gm. protein. 3. Deaminized gelatin, prepared either with or without heating, contains no amino nitrogen detectable with certainty by either the Van Slyke or the formol titration method. 4. The isoelectric point of deaminized gelatin prepared without heating is at pH 4.0. 5. The maximum combining capacity of this protein for HCl is 0.00044 equivalents per gm. 6. The maximum combining capacity of gelatin for HCl should be corrected to 0.00089 equivalents per gm. 7. The difference between these maximum combining capacities, 0.00045, is nearly equivalent to the loss in amino or total nitrogen occurring in the deaminizing reaction. 8. This equivalence constitutes a new indication that the combination of protein with acid is chemical combination.     

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 CERTAIN PROTEINS WITH HYDROCHLORIC ACID

Electromotive force measurements of cells without liquid junction, of the type Ag, AgCl, HCl + protein, H₂, have been made at 30°C. with the proteins gelatin, edestin, and casein in 0.1 M hydrochloric acid. The data are consistent with the assumptions of a constant combining capacity of each protein for hydrogen ion, no combination with chloride ion, and Failey''s principle of a linear variation of the logarithm of the mean activity coefficient of the acid with increasing protein concentration. The combining capacities for hydrogen ion so obtained are 13.4 x 10^–4 for edestin, 9.6 x 10^–4 for gelatin, and 8.0 x 10^–4 for casein, in equivalents of combined H⁺ per gm. of protein.     

THE FLOCCULATION OF GELATIN AT THE ISOELECTRIC POINT

The results of this investigation show that a gelatin solution consists of a considerable number of constituents. At a particular temperature, certain gelatin constituents tend to aggregate and to flocculate from solution. When these particular gelatin constituents have completely flocculated, no further change occurs in the system and an apparent equilibrium exists. This is not a dynamic equilibrium between the gelatin flocculate as a whole and the gelatin remaining in the solution but a steady state determined for that system by the temperature. It is also shown that gelatin can be separated into fractions in which the gelatin constituents are more nearly uniform and tend to flocculate over a much narrower temperature range. It should be possible to obtain a number of fractions in which all of the gelatin would flocculate at a definite temperature. The aggregation of the various gelatin constituents is presumably due to loss of thermal energy, and the temperature at which this occurs must be some function of the mass of the constituent. It is natural to assume, then, that the constituents which flocculate at a given temperature are larger than those which remain in solution at that temperature. Recently, Krishnamurti and Svedberg (1930) have obtained evidence with the ultra-centrifuge that the constituents of a gelatin solution are heterogeneous as to mass, even at a pH value at which there is no tendency toward aggregation. There is much reason to suppose that the gelatin constituents do not differ very greatly chemically since different fractions have the same refractive index and the same isoelectric point. The data as a whole are best explained by considering the gelatin constituents to be different degrees of association of the same or very similar molecular structural units. This is in agreement with Sheppard and Houck (1930), who consider that "the molecules of gelatin are fundamentally identical with those of collagen, the difference being only in the degree of association and orientation". Meyer and Mark (1928) have interpreted the x-ray data obtained from collagen as indicating that the micelles of the collagen fiber are built up of main valency chains of anhydro-amino acids. It may be supposed that during peptization of these fibers, the amino acid chains become separated, disorientated, and partially broken up, so producing the heterogeneous system which we know as gelatin. It is evident that the manner in which this breaking-up proceeds depends upon the chemical treatment previous to the peptization process and the gelatin produced from lime-treated collagen would be expected to differ from that from acid-treated collagen. From the results herein reported it seems evident that the technique of isoelectric flocculation of electrolyte-free gelatin offers a profitable method for the study of gelatin and an extended investigation along these lines should yield much valuable information concerning the nature of gelatin. It is possible that this method may also be extended to other hydrophilic colloids.     

THE ISOELECTRIC POINT OF A STANDARD GELATIN PREPARATION1

Two samples of a standard gelatin were studied, both prepared according to published specifications and washed free from diffusible electrolytes. The isoelectric point of this material was determined in four ways. 1. The pH values of solutions of gelatin in water approached the limit 4.86 ± 0.01 as the concentration of gelatin was increased. 2. The pH values of acetate buffers were unchanged by the addition of gelatin only at pH 4.85 ± 0.01. This gives the isoionic point of Sørensen, which is the isoelectric point with respect only to hydrogen and hydroxyl ions. 3. Gels of this gelatin made up in dilute HCl or NaOH, or in dilute acetate buffers, exhibited maximum turbidity at pH 4.85 ± 0.03. 4. Very dilute suspensions of collodion particles in 0.1 per cent gelatin solutions made up in acetate buffers showed zero velocity in cataphoresis experiments only at pH 4.80 ± 0.01. No evidence was found for the assumption that gelatin has two isoelectric points at widely separated pH values. It is concluded that the isoelectric point of this standard gelatin is not far from pH 4.85.     

THE MOLECULAR WEIGHT AND ISOELECTRIC POINT OF THYROGLOBULIN

1. The sedimentation constant of hog thyroglobulin is 19.2ċ10^–13. That of human thyroglobulin is essentially the same. 2. The specific volume of hog thyroglobulin is 0.72. 3. The isoelectric point of native hog thyroglobulin is at pH 4.58, that of denatured thyroglobulin at pH 5.0. 4. The molecular weight of hog thyroglobulin is, in round numbers, 700,000, as calculated from the sedimentation and diffusion constants, or 650,000, as calculated from the sedimentation equilibrium data. 5. The thyroglobulin molecule deviates markedly from the spherical.     

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 CHLOROPHYLL-PROTEIN COMPLEX : I. ELECTROPHORETIC PROPERTIES AND ISOELECTRIC POINT

1. Reported effects of different conditions on the stability of the purified chlorophyll-protein complex have been confirmed. 2. The electrophoretic behavior of the chlorophyll-protein complex prepared from two unrelated species of plants (Aspidistra elatior and Phaseolus vulgaris) has been investigated and shown to be dissimilar. In M/50 acetate buffer at 25°C, the isoelectric point of the complex from Phaseolus is at pH 4.70, whereas that from Aspidistra is at pH 3.9 (extrapolated). These values fall within the usual range of protein isoelectric points. 3. Treatment with weak acids causes an irreversible denaturation of the protein complex from both species, with a resultant shift in the mobility-pH curves to more basic values. 4. Differences in electrophoretic behavior between the chlorophyll-protein complex and the cytoplasmic proteins of Phaseolus have been demonstrated. The isoelectric point of the latter is at pH 4.22.     

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.     

IONIZING INFLUENCE OF SALTS WITH TRIVALENT AND TETRAVALENT IONS ON CRYSTALLINE EGG ALBUMIN AT THE ISOELECTRIC POINT

1. While crystalline egg albumin is highly soluble in water at low temperature at the pH of its isoelectric point, it is coagulated by heating. It has long been known that this coagulation can be prevented by adding either acid or alkali, whereby the protein is ionized. 2. It is shown in this paper that salts with trivalent or tetravalent ions, e.g. LaCl₃ or Na₄Fe(CN)₆, are also able to prevent the heat coagulation of albumin at the isoelectric point (i.e. pH 4.8), while salts with a divalent ion, e.g. CaCl₂, BaCl₄, Na₂SO₄, or salts like NaCl, have no such effect. 3. This is in harmony with the fact shown in a preceding paper that salts with trivalent or tetravalent ions can cause the ionization of proteins at its isoelectric point and thus give rise to a membrane potential between micellæ of isoelectric protein and surrounding aqueous solution, while the above mentioned salts with divalent and monovalent ions have apparently no such effect.     

THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na₂HPO₄. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied.     

THE ISOELECTRIC POINT OF RED BLOOD CELLS AND ITS RELATION TO AGGLUTINATION

1. The movement of normal and sensitized red blood cells in the electric field is a function of the hydrogen ion concentration. The isoelectric point, at which no movement occurs, corresponds with pH 4.6. 2. On the alkaline side of the isoelectric point the charge carried is negative and increases with the alkalinity. On the acid side the charge is positive and increases with the acidity. 3. On the alkaline side at least the charge carried by sensitized cells is smaller and increases less rapidly with the alkalinity than the charge of normal cells. 4. Both normal and sensitized cells combine chemically with inorganic ions, and the isoelectric point is a turning point for this chemical behavior. On the acid side the cells combine with the hydrogen and chlorine ions, and in much larger amount than on the alkaline side; on the alkaline side the cells combine with a cation (Ba), and in larger amount than on the acid side. This behavior corresponds with that found by Loeb for gelatin. 5. The optimum for agglutination of normal cells is at pH 4.75, so that at this point the cells exist most nearly pure, or least combined with anion and cation. 6. The optimum for agglutination of sensitized cells is at pH 5.3. This point is probably connected with the optimum for flocculation of the immune serum body.     

AMPHOTERIC COLLOIDS : II. VOLUMETRIC ANALYSIS OF ION-PROTEIN COMPOUNDS; THE SIGNIFICANCE OF THE ISOELECTRIC POINT FOR THE PURIFICATION OF AMPHOTERIC COLLOIDS.

1. It is shown by volumetric analysis that on the alkaline side from its isoelectric point gelatin combines with cations only, but not with anions; that on the more acid side from its isoelectric point it combines only with anions but not with cations; and that at the isoelectric point, pH = 4.7, it combines with neither anion nor cation. This confirms our statement made in a previous paper that gelatin can exist only as an anion on the alkaline side from its isoelectric point and only as a cation on the more acid side of its isoelectric point, and practically as neither anion nor cation at the isoelectric point. 2. Since at the isoelectric point gelatin (and probably amphoteric colloids generally) must give off any ion with which it was combined, the simplest method of obtaining amphoteric colloids approximately free from ionogenic impurities would seem to consist in bringing them to the hydrogen ion concentration characteristic of their isoelectric point (i.e., at which they migrate neither to the cathode nor anode of an electric field). 3. It is shown by volumetric analysis that when gelatin is in combination with a monovalent ion (Ag, Br, CNS), the curve representing the amount of ion-gelatin formed is approximately parallel to the curve for swelling, osmotic pressure, and viscosity. This fact proves that the influence of ions upon these properties is determined by the chemical or stoichiometrical and not by the "colloidal" condition of gelatin. 4. The sharp drop of these curves at the isoelectric point finds its explanation in an equal drop of the water solubility of pure gelatin, which is proved by the formation of a precipitate. It is not yet possible to state whether this drop of the solubility is merely due to lack of ionization of the gelatin or also to the formation of an insoluble tautomeric or polymeric compound of gelatin at the isoelectric point. 5. On account of this sudden drop slight changes in the hydrogen ion concentration have a considerably greater chemical and physical effect in the region of the isoelectric point than at some distance from this point. This fact may be of biological significance since a number of amphoteric colloids in the body seem to have their isoelectric point inside the range of the normal variation of the hydrogen ion concentration of blood, lymph, or cell sap. 6. Our experiments show that while a slight change in the hydrogen ion concentration increases the water solubility of gelatin near the isoelectric point, no increase in the solubility can be produced by treating gelatin at the isoelectric point with any other kind of monovalent or polyvalent ion; a fact apparently not in harmony with the adsorption theory of colloids, but in harmony with a chemical conception of proteins.     

草菇蛋白酶的分离、纯化及等电点的测定

草菇是我国食用菌主要栽培品种之一,属典型高温真菌。低温将诱导草菇细胞内的蛋白质降解,导致草菇菌丝自溶、死亡。蛋白酶在草菇低温自溶过程中起了重要作用。在分离、纯化草菇蛋白酶的基础上,采用等电点聚焦电泳测定了蛋白酶的等电点,为草菇低温自溶与蛋白酶之间的关系的研究奠定了基础。     

运动后尿液蛋白质分子量与等电点的变化特征

通过对９名男性受试者在分别完成１００－２００ｍ,４００－８００ｍ和１ ５００－３ ０００ｍ跑步间歇训练后尿蛋白分子量和等电点的测定发现：①运动时尿液高、低分子量蛋白质排泄率均较运动前明显增加,但以高分子量蛋白质排泄为主;②运动时尿液高、低分子量蛋白质排泄率均以４００－８００ｍ间歇训练时最高,１００－２００ｍ间歇训练时次之,１ ５００－３ ０００ｍ间歇训练时最低;③运动时尿液排出的蛋白质以负离子为主     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : III. THE RELATION BETWEEN THE AMINO ACID COMPOSITION OF CASEIN AND ITS CAPACITY TO COMBINE WITH BASE.

1. The methods of measuring the base-combining capacities of proteins have been considered, and the constants and corrections that are employed in their calculation have been critically examined. 2. The base-combining capacities of ten casein preparations have been determined. These differed from each other to a far greater extent than can be attributed to the experimental errors involved in their measurement and calculation. The variations were, moreover, systematic in manner, and can be explained as dependent upon the method employed in the preparation of the casein. 3. Casein that had never been exposed to greater alkalinities than those in which it exists in nature combined with approximately 0.0014 mols of sodium hydroxide per gm., while casein prepared nach Hammarsten, and casein that was saturated with base during its preparation, combined with approximately 0.0018 mols of sodium hydroxide per gm. 4. 1 mol of sodium hydroxide, therefore, combined with 735 gm. of casein that had not previously been exposed to alkaline reactions, or with 535 gm. of casein that had previously been saturated with base. 5. If the minimal molecular weight of casein, based upon its tryptophane content, is placed at 12,800, the native protein must, therefore, contain approximately eighteen acid groups, and in addition six acid groups that are released in alkaline solutions, and presumably represent internally bound groups. The total base-combining capacity therefore represents that of a substance with a molecular weight of 12,800 and containing twenty-four acid valences. 6. This base-combining capacity is no greater than can be accounted for on the basis of our knowledge of the structure and composition of casein. On the basis of a molecular weight of 12,800 casein contains at least 19 molecules of glutamic acid, 4 of aspartic, and 8 of hydroxyglutamic acid. If the amino acids in the protein molecule are bound to each other in polypeptide linkage, each of these thirty-one dicarboxylic acids should yield terminal groups. The ammonia in casein suggests that twelve of these groups are bound as amides. As many as nineteen carboxyl groups may, therefore, be free in the protein molecule. 7. Casein contains phosphorus. If this phosphorus represents phosphoric acid, and if we consider that all of the valences of this acid are either themselves free, or that they have liberated carboxyl groups by entering into the structure of the protein molecule, casein should contain nine additional acid groups. 8. Recent analytical results, therefore, indicate that casein contains at least nineteen, and possibly twenty-eight, free acid groups. The physicochemical measurements presented suggest that casein combines with base as though it contained twenty-four acid groups, of which six, or one-fourth, appear to be bound in the native protein. These experimental results are therefore in close agreement with the expectation on the basis of the classical theory of protein structure.     

METABOLIC PROPERTIES OF A PURIFIED PREPARATION OF LARGE FRAGMENTS OF THE CEREBELLAR GLOMERULI: GLUCOSE METABOLISM AND AMINO ACID UPTAKE

Glomerulus particle preparations contain large fragments of the cerebellar glomeruli and are composed almost exclusively of well-defined neuronal processes (Balázs et al., 1975). The metabolic competence of the glomerulus particles was demonstrated by their ability to convert [¹⁴C]glucose to ¹⁴CO₂ and lactate at a linear rate for over 1 h. The preparations also transported deoxyglucose via an high affinity uptake system (K_T= 0.2-0.5 mM). The kinetics of uptake of various labelled amino acids were also studied. Apparently high affinity uptake systems (K_T values about 10^-5 M) were found for thc putative transmitters GABA, glycine, glutamate, and aspartate, but not for leucine, serine, and tyrosine. The maximal velocity of high affinity uptake was the greatest for GABA (about 15 nmol/mg protein per 10 min), while glycine was taken up at about 50%, and aspartate and glutamate at only 13% of the rate obtained with GABA. High affinity uptake of glycine required Na⁺ (half maximal uptake at 70 mM-NaCl). Inhibition of glucose transport and glycolysis, electron transport, or oxidative phosphorylation also depressed high affinity uptake of glycine. 2,4-Diaminobutyric acid was a potent competitive inhibitor of GABA uptake (K₁ approx 22 μM), while β-alanine and glycine had a relatively minor inhibitory effect on the uptake of GABA.     

THE INFLUENCE OF ELECTROLYTES ON THE CATAPHORETIC CHARGE OF COLLOIDAL PARTICLES AND THE STABILITY OF THEIR SUSPENSIONS : II. EXPERIMENTS WITH PARTICLES OF GELATIN,CASEIN, AND DENATURED EGG ALBUMIN.

1. This paper gives measurements of the influence of various electrolytes on the cataphoretic P.D. of particles of collodion coated with gelatin, of particles of casein, and of particles of boiled egg albumin in water at different pH. The influence of the same electrolyte was about the same in all three proteins. 2. It was found that the salts can be divided into two groups according to their effect on the P.D. at the isoelectric point. The salts of the first group including salts of the type of NaCl, CaCl₂, and Na₂SO₄ affect the P.D. of proteins at the isoelectric point but little; the second group includes salts with a trivalent or tetravalent ion such as LaCl₃ or Na₄Fe(CN)₆. These latter salts produce a high P.D. on the isoelectric particles, LaCl₃ making them positively and Na₄Fe(CN)₆ making them negatively charged. This difference in the action of the two groups of salts agrees with the observations on the effect of the same salts on the anomalous osmosis through collodion membranes coated with gelatin. 3. At pH 4.0 the three proteins have a positive cataphoretic charge which is increased by LaCl₃ but not by NaCl or CaCl₂, and which is reversed by Na₄Fe(CN)₆, the latter salt making the cataphoretic charge of the particles strongly negative. 4. At pH 5.8 the protein particles have a negative cataphoretic charge which is strongly increased by Na₄Fe(CN)₆ but practically not at all by Na₂SO₄ or NaCl, and which is reversed by LaCl₃. the latter salt making the cataphoretic charge of the particles strongly positive. 5. The fact that electrolytes affect the cataphoretic P.D. of protein particles in the same way, no matter whether the protein is denatured egg albumin or a genuine protein like gelatin, furnishes proof that the solutions of genuine proteins such as crystalline egg albumin or gelatin are not diaphasic systems, since we shall show in a subsequent paper that proteins insoluble in water, e.g. denatured egg albumin, are precipitated when the cataphoretic P.D. falls below a certain critical value, while water-soluble proteins, e.g. genuine crystalline egg albumin or gelatin, stay in solution even if the P.D. of the particles falls below the critical P.D.