Impairment by covalent modification of the ability of Phaseolus vulgaris phytohemagglutinin to stimulate cultured human lymphocytes: a comparison of different forms of covalent modification.

Phytohemagglutinin (PHA) isolated from Phaseolus vulgaris has been modified by treatment with various chemical reagents and the modified proteins have been tested for their ability to stimulate peripheral lymphocytes from two healthy human donors, in vitro. Reaction of PHA with citraconic anhydride, S-methyl isothiourea, or 2-hydroxy-5-nitrobenzyl bromide produced derivatives which retained the ability to stimulate lymphocytes, at low concentrations. Acylation of the lectin with acetic anhydride or masking of the carboxyl side chains by reaction with glycinamide-carbodiimide impaired stimulation. When PHA was treated with N-bromosuccinimide or with tetranitromethane, the derivatives were ineffective as lymphocyte stimulants. Chemical modifications affected, in some cases, the quaternary structure of the lectin. Glycinamide-, homoarginine-, and nitro-PHA were tetramers whereas acetyl-, citraconyl-, and N-bromosuccinimide-treated lectin were dimers. Antinative lectin antiserum cross-reacted with all the modified proteins, except in the case of the N-bromosuccinimide derivative. The results show that, in the human lymphocyte transformation assay, the mitogenic property of PHA may depend on intact aspartic, glutamic, and tyrosine residues whereas lysine residues do not appear to be essential.