Identification of the Tryptophan Residue Present in the Low Affinity Saccharide-binding Site of Ricin E

Abstract

The tryptophan residue located at the low affinity saccharide-binding site (LA-site) of ricin E was identified using two derivatives of ricin E, in which tryptophan residues were oxidized with N-bromosuccinimide (NBS) in the absence and presence of lactose. From the lysyl endopeptidase and tryptic digests of the B-chains of the respective derivatives, peptides containing oxidized tryptophan residue(s) were isolated by gel filtration on Bio Gel P-2 and HPLC. Analysis of the peptides containing oxidized tryptophan showed that tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative, in which the saccharide-binding ability at the LA-site was abrogated by modification of tryptophan residues with NBS in the absence of lactose. On the other hand, two tryptophan residues at positions 93 and 160 in the B-chain were found to be oxidized in the active derivative that retained the saccharide-binding ability at the LA-site after NBS-oxidation in the presence of lactose, indicating that the tryptophan residue at position 37 is protected from NBS-oxidation in the complex with lactose. The results suggest that the loss of the saccharide-binding ability of the LA-site of ricin E by NBS-oxidation is principally due to the oxidation of the tryptophan residue at position 37 on the B-chain.