Studies on phytohemagglutinins: XX. Isolation and characterization of hemagglutinins from scarlet runner seeds ( Phaseolus coccineus L.)

Abstract

Two phytohemagglutinins have been isolated from normal saline extract of scarlet runner seeds ( Phaseolus coccineus L., syn. Phaseolus multiflorus Lam.) by (NH 4) 2SO 4 precipitation of the active protein fraction and subsequent chromatography on CM-cellulose and DEAE-Sephadex. Phytohemagglutinin I was shown to be homogeneous by electrophoresis on starch and polyacrylamide gels as well as on cellulose acetate strips at different pH values and by thin-layer gel filtration on Sephadex. It gives a single symmetrical peak on ultracentrifugation with a s 20,w 0 of 7.2 S. Both the sedimentation analysis and the thin-layer gel filtration method indicate a molecular weight of 120 000. Its approximate p I is 6.9. Dodecylsulfate polyacrylamide disc electrophoresis shows a single subunit band with approximate molecular weight of 34 000. The amino acid analysis shows substantial amounts of aspartic acid, serine and threonine. The substance is a glycoprotein containing glucosamine and 7.3% neutral sugar with mannose as the principal component. It also contains 0.24% of metals (0.16% Ca, 0.056% Zn, 0.019% Mg and 0.007% Mn). Phytohemagglutinin II is homogeneous on starch and polyacrylamide gel electrophoresis at acidic pH as well as on thin-layer gel filtration on Sephadex, which gives approximate molecular weight of 120 000. Both phytohemagglutinins show non specific agglutinating activity towards all ABO types of human erythrocytes; they exhibit perceptible erythroagglutination at concentrations of 0.625 and 2.5 μg/ml, respectively. Phytohemagglutinin I does not stimulate mitosis of mouse spleen lymphocytes in the concentration range of 10–200 μg/ml whereas phytohemagglutinin II is mitogenically active at concentrations of 25 μg/ml and higher.