Simple method of isolation of the major storage protein of bean seed (Phaseolus vulgaris) and its characterization.

Abstract

Fractional extraction of the bean seed major storage protein (MSP) by ammonium sulphate solutions at a concentration corresponding to 65-75% saturation was shown to be a simple and effective method for its isolation. The extraction with 70-75% saturated solutions permits to obtain directly a chromatographic and electrophoretic homogeneous preparation of MSP. The extract obtained with 65% saturated solution contains small quantity of contaminants. One-step DEAE-cellulose chromatography was found to be sufficient for their complete removal. On PAGE the MSP gives one wide, diffuse band with a relative mobility to bromphenol blue of Rm 0.38-0.42, and one additional band with Rm 0.18-0.20. The latter corresponds to tetrameric form of MSP. Reversible pH-dependent protomer-tetramer association was demonstrated by sedimentation velocity behaviour. At pH 7.2 the MSP gives one peak for 7S protomeric form. Below pH 7.0 a second peak for 18S tetrameric appears. SDS-PAGE analysis of subunit pattern shows the existence of 3 main components with molecular weights 53,000, 49,000 and 46,000. Some minor, lower MW polypeptides are also found. It seems that they are products of further dissociation of the main subunits.