Theory of Moving Boundary Electrophoresis of Reversibly Interacting Systems. Reaction of Proteins with Small Uncharged Molecules Such as Undissociated Buffer Acid.

The theory of moving boundary electrophoresis and ultracentrifugation of associating-dissociating and two-component isomerizing systems seems to be rather well understood (1-12). However, until now little was known about the behavior expected of systems in which reversible changes in the electrophoretic mobility of a macromolecular ion are mediated by binding of small uncharged molecules such as undissociated buffer acid. Theoretical understanding of such systems is especially important for quantitative interpretation of the nonenantiographic electrophoretic patterns shown by a variety of proteins (e.g. ovalbumin, y-pseudoglobulin, insulin, and serum albumin) in acidic media containing acetate or other carboxylic acid buffers (13-19). The nature of the patterns depends upon the concentration of buffer in the supporting medium. Increasing the concentration at constant pH and ionic strength results in progressive and characteristic changes, notably in the appearance and growth of fast moving peaks at the expense of slow ones. Fractionation and other measurements (14, 16, 18) have shown that the peaks in a given pattern constitute a reaction boundary modified in some instances by mild convective disturbance. Evidence has been advanced (13, 14, 16, 18, 19) to support interpretation of these observations in terms of reversible complexing of the protein molecules with undissociated buffer acid, with concomitant subtle structural changes which increase the net positive electrical charge on the protein’ but do not change its frictional coeffi-