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.     

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。     

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.     

AMPHOTERIC COLLOIDS : IV. THE INFLUENCE OF THE VALENCY OF CATIONS UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. A method is given by which the amount of equivalents of metal in combination with 1 gm. of a 1 per cent gelatin solution previously treated with an alkali can be ascertained when the excess of alkali is washed away and the pH is determined. The curves of metal equivalent in combination with 1 gm. of gelatin previously treated with different concentrations of LiOH, NaOH, KOH, NH₄OH, Ca(OH)₂, and Ba(OH)₂ were ascertained and plotted as ordinates, with the pH of the solution as abscissæ, and were found to be identical. This proves that twice as many univalent as bivalent cations combine with the same mass of gelatin, as was to be expected. 2. The osmotic pressure of 1 per cent solutions of metal gelatinates with univalent and bivalent cation was measured. The curves for the osmotic pressure of 1 per cent solution of gelatin salts of Li, Na, K, and NH₄ were found to be identical when plotted for pH as abscissæ, tending towards the same maximum of a pressure of about 325 mm. of the gelatin solution (for pH about 7.9). The corresponding curves for Ca and Ba gelatinate were also found to be identical but different from the preceding ones, tending towards a maximum pressure of about 125 mm. for pH about 7.0 or above. The ratio of maxi mal osmotic pressure for the two groups of gelatin salts is therefore about as 1:3 after the necessary corrections have been made. 3. When the conductivities of these solutions are plotted as ordinates against the pH as abscissæ, the curves for the conductivities of Li, Na, Ca, and Ba gelatinate are almost identical (for the same pH), while the curves for the conductivities of K and NH₄ gelatinate are only little higher. 4. The curves for the viscosity and swelling of Ba (or Ca) and Na gelatinate are approximately parallel to those for osmotic pressure. 5. The practical identity or close proximity of the conductivities of metal gelatinates with univalent and bivalent metal excludes the possibility that the differences observed in the osmotic pressure, viscosity, and swelling between metal gelatinates with univalent and bivalent metal are determined by differences in the degree of ionization (and a possible hydratation of the protein ions). 6. Another, as yet tentative, explanation is suggested.     

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 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 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 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.     

ULTRAFILTRATION : II. "BOUND" WATER (HYDRATION) OF BIOLOGICAL COLLOIDS

Assuming that "bound" water loses its solvent properties, it is shown by an ultrafiltration method that in solutions of gelatin, casein, starch, and glycogen, and in blood serum, only a very small fraction of the water can be associated with the colloids in this form.     

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.     

A TECHNIQUE FOR THE ANALYSIS OF EPISTATIC GENE ACTION II. THE APPLICATION OF THE METHOD

With a method based on probability of phenotypic ratios being produced by crosses involving two pairs of interacting genes, data from genetic studies of Impatiens Sultana Hook. f. (Balsaminaceae) were analysed. Nine genes postulated to control floral color are: P, purple; I and W, white; R, red; O, orange; F, fuchsia; D, red; E, red; S, salmon. Epistatic action was evaluated, but progeny counts were too small for linkage to be determined.     

STUDIES ON BIOLUMINESCENCE : II. THE PARTIAL PURIFICATION OF CYPRIDINA LUCIFERIN

Some solubility, oxidation, reduction, and compound-forming characteristics of extracts of Cypridina luciferin have been presented. A method of purification has been described which increased the amount of luciferin per unit of dry weight, as measured by the total light emitted, to about two thousand times that in the dry starting material. The best yields were from 50 to 65 per cent.     

AMPHOTERIC BEHAVIOR OF COMPLEX SYSTEMS : II. TITRATION OF SULFANILIC ACID-GLYCINE MIXTURES.

Electrometric titrations of glycine, sulfanilic acid, and various mixtures of the two have been made. These mixtures are shown to give a curve which, between their respective isoelectric points, is different from that of either substance. These mixtures have a maximum buffering power at a pH which can be theoretically calculated, and which has the characteristics of an "isoelectric point of the system." Other pairs of ampholytes are shown to act in an analogous manner.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

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.