Studies on phytohemagglutinins III. Isolation and characterization of hemagglutinins from the pea ( Pisum sativum L.)

Abstract

A mixture of two closely related phytohemagglutinins have been isolated from crude water extracts of the garden pea seeds ( Pisum sativum L. var. Pyram) by (NH 4) 2SO 4 precipitation of the active protein fraction, chromatography on hydroxylapatite and DEAE-cellulose as well as by specific adsorption on Sephadex. The two phytohemagglutinins were separated by further chromatography on DEAE-cellulose. Both are homogenous proteins exhibiting a single band by electrophoresis on starch and polyacrylamide gels as well as on cellulose-acetate strips at different pH values. Their electrophoretic mobility is similar to that of human IgG globulin, their isoelectric point lies at pH 7.7–7.9. They both reveal a single symmetrical peak on ultra-centrifugation and a s 20. w of 3.5 S. The molecular weights of 54 000 and 53 000 have been found for phytohemagglutinin 1 and phytohemagglutinin II, respectively, by the sedimentation equilibrium method. Both phytohemagglutinins show the same degree of nonspecific hemagglutinating activity against all types of human red blood cells of the ABO system. They have perceptible activity up to a dilution of 2.5 μg/ml. The amino acid compositions of both pea phytohemagglutinins are very similar. They contain high amounts of aspartic acid and threonine, but no cystine and methionine. The DNP method of N-terminal amino acid analysis of phytohemagglutinin H and the mixture of both phytohemagglutinins shows the presence of equal amounts of valine and threonine.