Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds

Abstract

The potential of immunoaffinity chromatography as a means of purifying legumin from a wide range of Pisum (pea) types was assessed. The method required small amounts of highly purified legumin from a single Pisum type, and this was obtained by salting out with (NH4)2SO4 followed by zonal isoelectric precipitation, ion-exchange chromatography on DEAE-cellulose and sucrose-density-gradient centrifugation. Some physiocochemical properties of purified legumin were determined, a number of which (Strokes radius, subunit molecular weights, subunit N-terminal residues and subunit molar ratios) have not previously been reported for Pisum legumin. Examination of Pisum legumin by two-dimensional gel isoelectric focusing/electrophoresis indicated the existence of extensive subunit heterogeneity, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate showed apparent variation in the nature of this heterogeneity from one Pisum variety to another. Despite this variation, immunoaffinity chromatography on immobilized anti-legumin (which was prepared by affinity chromatography on the immubolized purified legumin from the single Pisum type) was shown to be a generally applicable method for the purification of undegraded legumin from a range of pisum types, including two primate lines.