A tryptophan residue is essential to the sugar-binding site of winged bean basic lectin

Abstract

Winged bean basic lectin contains four tryptophan residues per molecule (Molecular weight 53 000). Treatment of the lectin with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide cause a concomitant loss of the hemagglutinating activity with modification of tryptophan residues. Modification of two tryptophan residues out of original four caused a complete loss of the activity. The remaining two tryptophan residues remained unoxidized under non-denaturing conditions. Although N-bromosuccinimide also oxidized tyrosine residues, the nonparticipation of this amino acid in the activity was confirmed by acetylation of the lectin with N-acetylimidazole. The change correlated to oxidation of tryptophan residues by N-bromosuccinimide was prevented by d-galactose, the sugar specific for the hemagglutination of the lectin. These results clearly suggested that the loss of the hemugglutinating activity was due specifically to oxidation of tryptophan residues. The protected tryptophan residue was calculated to be one per molecule, indicating that a single tryptophan residue was essential to the sugar-binding site of the lectin. Moreover, modification of tryptophan residue was shown by an immunological study to produce no gross change in the conformation of the protein.