Isoelectric Zone Research Articles

Effect of Proteins on the Diffusion of Amino Acids Through Membranes.

The concentration of amino acids in nucleated erythrocytes is known to be several times that of the surrounding plasma. The concentration in mammalian liver and muscle, as indicated by determinations on tungstic acid extracts, is 6 to 8 times that of the blood plasma. As part of an inquiry into the factors that cause this inequality in distribution we have studied the diffusion of amino acids through tubular membranes of cellophane. The progress of dialysis was measured by amino nitrogen determinations on the inner and outer liquids by the manometric method of Van Slyke. The inner liquid consisted of a 1.5% solution of gold label gelatin. The amino acid was contained in the outer liquid in an initial concentration of 0.001 N. Glycine was generally employed. After 15 to 30 hours at a temperature of 20° the experiments were terminated. The outer fluid was analyzed directly, while the inner fluid was first rendered free of protein by treatment with phosphotungstic acid. Over a wide range of H ion concentration, (pH 2.5-9.5) the equilibrium concentration of amino acid in the outer fluid was found to be over twice as great as that in the inner protein-containing solution. This inequality persisted, undiminished in magnitude, even in the isoelectric zone of the protein. The equilibrium, moreover, could be approached from the other end, that is by dissolving the amino acid in the inner fluid containing the dispersed protein. In neutral solutions aspartic acid and glutamic acid behaved like glycine in the establishment of a high concentration ratio when dialyzed against 1.5% gelatin. Dialyzed solutions of crystalline egg albumin (1.5%) and agar-agar (0.38%) affected the diffusion of glycine at pH 5 in similar fashion.

Specific Agglutination and Precipitation

Summary and Discussion Agglutinating and precipitating antibodies are a specifically altered fraction of the serum globulin. The antigen-antibody complex, whether sensitized red cells, agglutinated bacteria, or the precipitate formed by a soluble protein and the corresponding antiserum, contains this antibody globulin, demonstrable chemically, immunologically, and by a change in the cataphoretic, flocculating, interfacial, and complement-fixing properties of the antigen towards those of the protein with which it has combined. In the case of the cellular antigen, this antibody is present as an invisible film of specifically adsorbed protein, while in the precipitation reaction, it may constitute the bulk of the material formed. In both cases, the originally hydrophilic globulin has become water-insoluble (denatured), upon combination with antigen. This change in properties is not a phenonomen peculiar to the immune reactions, but is a commonly observed and as yet unexplained property of adsorbed proteins, responsible for their sensitizing effect upon otherwise stable colloidal suspensions. It is suggested that in the case of the immune reactions, this denaturation of the antibody globulin is due to the fact that its specificity is determined by hydrophilic groups. When these combine with antigen, hydrophobic groups necessarily face the water phase, determining the surface properties of the antigen-antibody complex. But when normal serum protein is adsorbed, since there are no groups with a specific affinity to antigen, the molecules naturally orient themselves at the interface so that the hydrophilic groups face the water, and the adsorbed protein acts as a protective film away from its isoelectric point. There are therefore three factors which determine specific flocculation: (1) The hydrophilic antigen is covered, with (2) a film of immune globulin, denatured by its combination with antigen. In the absence of electrolytes the charge due to the ionization of this protein suffices to prevent aggregation. Minute concentrations of (3) electrolyte, however, depress this surface charge below the critical value necessary for stability. The resultant aggregation is therefore primarily of the immune globulin surfaces, and only incidentally of the associated antigen. With insufficient immune-serum, only a very small portion of the cell surface is covered with antibody globulin; most of the impacts are between hydrophilic antigen surfaces, ineffective in producting cohesion. The more immune serum, the greater is the proportion of antigen surface covered with the sensitizing denatured protein, and the correspondingly greater the proportion of effective impacts. The optimum hydrogen ion concentration for flocculation is intermediate between that of the original cell and that of the antibody globulin, shifting towards the latter as the degree of sensitization is increased (i.e., more extensive antibody film). At the optimum reaction, ionization, and therefore the surface charge, are minimal: no added electrolytes are necessary to produce aggregation. In more acid or more basic reaction the surface charge due to the ionization of the adsorbed protein causes a mutual repulsion of the particles; but traces of electrolytes depress this charge and allow the cohesion of the denatured antibody films. The flocculating ion is always the one opposite in charge to the ionized protein, and its flocculating efficiency increases enormously with increasing valence. The further from the isoelectric zone, the greater is the degree of ionization, and the more electrolytes are necessary to depress the surface charge below the critical value. As will be shown in a forthcoming paper, the kinetics of specific flocculation, the so-called “Danysz” and “zone” phenomena, and the varying proportions of antigen and antibody in the aggregates formed, are all in keeping with the theory of specific aggregation as just presented.

A CONVENIENT METHOD FOR THE FORMOL TITRATION

The formol titration, as described by Srrensen, 1 has been most useful in the determination of amino-acids and especially in following the course of hydrolysis of proteins. The method as originally described could not be used for accurate determinations of small quantifies of amino-acids owing to the difficulty of determining the exact end-point. The value obtained also depends on the point at which the titration is started. In the light of our present knowledge the success of the method depends upon the fact that in the presence of formalin the titration curve is displaced to the acid side to such an extent that the end-point of the titration is reached at a pH of about 9.0, where a sharp end-point is easily obtained, instead of at about 12.0, where the end-point is very indefinite owing to the buffer effect of the alkali itself. With a solution of a pure amino-acid or peptide, therefore, the titration gives directly the alkali equivalent of the compound. In the case of an unknown solution, however, it is necessary to select some arbitrary pH as the starting point. I t so happens that practically all the amino-acids and peptides whose titration curves have been studied, have an isoelectric zone around pH 6 to 7, and that the proteins also have a flat place in the titration curve in this region although it is not the isoelectric point. In practically all solutions of proteins and their split products, therefore, it is possible to obtain a sharp end-point in this range of pH. I t is, consequently, a convenient point from which to start the titration. The difficulty with the alkaline end-point is largely due to the fact that the formalin affects the color of the indicators so that it is difficult to match the standard exactly. This may be overcome by taking advantage of the property of a one color indicator which makes it possible to vary the end-point