Electrophoresis of human serum albumin at pH 4.0. I. A systematic study on the effect of organic acids and alcohols upon the electrophoretic behavior of albumin.

Recently we have reported on a hitherto unknown modification of human serum albumin of which the outstanding properties are electrophoretic homogeneity at pH 4.0 and its transformation to an electrophoretically heterogeneous form (2). In continuation of this study, the question was asked whether the ordinary human serum albumin (Fraction V), known to bc electrophoretically heterogeneous at pH 4.0 (3), could be t.ransformed to an electrophoretically homogeneous form. Serum albumin, when studied at pH values acid to its isoelectxic point, eshibit.s several physicochemical abnormalities, such as the appearance of a multicomponent system on electrophoresis (3-8) ; changes of sedimentation and diffusion const.ants (9-12) and of viscosity and optical rotation (9, 12, 13); and the expansion of the molecule (11, 14). In the present study a particular aspect of one of these physicochemical changes was investigated estensively, namely the behavior of this prot.ein in pH 4.0, r/2 0.1 buffers as observed on free moving boundary clectrophoresis. The reasons for selecting these conditions are the pronounced heterogeneity at pH 4.0 and the easier interpretation of the electrophoretic analyses performed at ionic strength of 0.1. The electrophoretic behavior of serum albumin at pH 4.0 has been investigated at low ionic strength and at low protein concentration by Aoki and Foster (7, 8). The obtained data were interpreted as a configurational isomerism, e.g. the “normal” or “native” form binds H-ions that results in a faster moving, presumably higher charged form of albumin. These two modifications arc in equilibrium with each other within a pH range from 4.5 to 3.5. Phclps and Cann (4-6) studied albumin solutions at higher ionic strengths and protein concentrations and considered the different electrophoresis patterns as a result. of a specific interaction between albumin and the undissociated organic acids (5). The apparent paradox that a two-component, equilibrium system can manifest itself in two moving boundaries on free electrophoresis, provided t.hat the time of electrophoresis is less than the half-time of the reactions, has recently been resolved (16, 17).