Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis

Abstract

A method has been found which distinguishes between a size isomer family of proteins (e.g. bovine serum albumin polymers) and a charge isomer family of proteins (e.g. lactate dehydrogenase isoenzymes), utilizing disc gel electrophoresis. When the log of protein mobility relative to the dye front was plotted versus acrylamide gel concentration, size isomeric proteins gave a family of nonparallel lines extrapolating to a common point in the vicinity of 0% gel concentration; charge isomeric proteins gave a parallel family of lines. Proteins differing in both charge and size gave non-parallel lines intersecting at gel concentrations other than 0% gel concentration. The slope of such a plot is related to molecular weight. The molecular weight-slope relation was established utilizing 17 well-characterized proteins as standards. From this relation, it is possible to determine the molecular weight of a protein with an average precision of ±4%. This molecular weight method can be applied to a single protein in a mixture of proteins provided a specific detection test is available. This method should find uses in distinguishing between size and charge isomer families of proteins, for the rapid, easy, and accurate determination of protein molecular weights, and as a valuable aid in indicating the procedures to be used in enzyme purifications.