Purification of mitogenic proteins derived from Phaseolus vulgaris: isolation of potent and weak phytohemagglutinins possessing mitogenic activity

Abstract

Commercially obtained phytohemagglutinin (PHAP) derived from Phaseolus vulgaris contains 17 different protein bands when analyzed by acrylamide gel electrophoresis. When it is subjected to CM-Sephadex chromatography followed by molecular sieving on Sephadex G150, several species of potent mitogenic proteins, which differ greatly in their hemagglutinating capacity, are obtained. A low hemagglutinating mitogen (L-PHAP), homogeneous by several different criteria, is the most potent mitogen isolated, and also possesses potent leukoagglutinating activity. It is a glycoprotein with a molecular weight of 115,000, containing glucosamine, mannose, xylose, and fucose or arabinose. Also isolated is a mixture of at least two closely related proteins possessing high hemagglutinating capacity, with hemagglutination titers 250 times more potent than L-PHAP. This material is a slightly less potent mitogen than L-PHAP and also possesses leukoagglutinating capacity, although of a lower order of magnitude. Its amino acid and carbohydrate composition are similar to L-PHAP, but it contains approximately twice as much carbohydrate and is slightly larger as determined by molecular sieving.