Chemical Modification of Tryptophan Residues in Abrin-a.

Chemical modification of tryptophan residues in abrin-a with N-bromosuccinimide (NBS) was studied with regard to saccharide-binding.The number of tryptophan residues available for NBS oxidation increased with lowering pH, and ll out of the 13 tryptophan residues in abrin-a were eventually modified with NBSat pH 4.0, while 6 tryptophan residues were modified at pH 6.0 in the absence of specific saccharides.Modification of tryptophan residues at pH 6.0 greatly decreased the saccharide-binding ability of abrin-a, and only 2% of the hemagglutinating activity was retained after modification of 3 residues/mol.Whenthe modification was done in the presence of lactose or galactose, 1 out of 3 residues/mol remained unmodified with a retention of a fairly high hemagglutinating activity.However, GalNAc did not show such a protective effect.NBS-oxidation led to a great loss of the fluorescence of abrin-a, and after modification of 3 tryptophan residues/mol, the fluorescence intensity at 345 nmwas only 38%of that of the unmodified abrin-a.The binding of lactose to abrin-a altered the environment of the tryptophan residue at the saccharide-binding site of abrin-a, leading to a blue shift of the fluorescence spectrum.The ability to generate such fluorescence spectroscopic changes induced by lactose-binding was retained in the derivative in which 2 tryptophan residues/mol were oxidized in the presence of lactose, but not in the derivative in which 3 tryptophan residues/mol were oxidized in the absence of lactose.Importance of the tryptophan residue(s) in the saccharide-binding of abrin-a is suggested.* To whomcorrespondence should be addressed.Abbreviations: NBS, iV-bromosuccinimide; GalNAc, A^-acetyl-D-galactosamine; HA-site, a high affinity binding site; LA-site, a low affinity binding site; UV-, ultraviolet; CD, circular dichroism; 1 or 2-m-abrin-a (+lac), abrin-a which was modified in the presence of lactose and contained 1 or 2 oxidized tryptophan residues/mol; 3-m-abrin-a (-lac), abrin-a which was modified in the absence of lactose and contained 3 oxidized tryptophan residues/mol.