Theory of Zone Electrophoresis of Reversibly Interacting Systems. Two Zones from a Single Macromolecule.

where P represents a macromolecular ion in solution and P(HA), its complex with n moles of a small uncharged constituent, HA, of the solvent medium. It was assumed that the complex migrates with a greater electrophoretic mobility than the uncomplexed macromolecule and that equilibrium is established instantaneously. Computer solution of the conservation equations showed that under appropriate conditions the electrophoretic patterns will resolve into two moving peaks as a result of production of gradients of HA concentration in the electrophoresis column. The computations predicted the essential features of the electrophoretic behavior of proteins in acidic media containing varying concentrations of carboxylic acid buffer and provided a theoretical basis for our previous interpretation in terms of binding of undissociated buffer acid by the protein with concomitant increase in electrophoretic mobility. These computations have now been extended to include zone electrophoresis on a solid supporting medium or in a density gradient. A result with important implications for zone electrophoretic analysis is that under appropriate conditions a single macromolecule interacting with an uncharged constituent of the solvent medium can give two zones. Other conditions may yield a single but very broad, trailing zone.