Chemical modification of the tryptophan residues of wheat-germ agglutinin. Effect on fluorescence and saccharide-binding properties.

The oxidation of the tryptophan residues of wheat germ agglutinin by N-bromosuccinimide was investigated under non-denaturing and denaturing conditions. All three tryptophan residues present in wheat germ agglutinin subunit (molecular weight 18 000) could be modified in 0.1 M acetic acid/8 M urea, pH 3.9. One of the residues failed, however, to react with N-bromosuccinimide when the modification was in 0.1 M citrate buffer, pH 6.0. Tryptophan fluorescence of the protein was quenched concomitantly with the oxidation of two tryptophan residues even when the modification was carried out in acetic acid urea. After oxidation of two tryptophan residues per subunit of wheat germ agglutinin, only 15% of the original tryptophan fluorescence remained; upon excitation at 280 nm, tyrosine fluorescence centered at 305 nm could be resolved. The results suggest that there are only two emitters in the protein and that the third tryptophan residue is buried in the native protein and can be modified only in acetic acid urea. This tryptophan residue is quenched in the native protein. Saturation of wheat germ agglutinin with tri-N-acetylchitotriose did not protect the tryptophan residues from oxidation by N-bromosuccinimide. Under these conditions, however, the reactivity of the tryptophan residues towards N-bromosuccinimide was reduced and a higher concentration of the reagent was required to achieve the same extent of oxidation as in the absence of the saccharide. Oxidation of one tryptophan residue per subunit in acetic acid urea led to almost complete loss (97%) of hemagglutinating activity, a 3.5-fold decrease in the affinity constant for tri-N-acetylchitotriose and loss of ability of the subunits (SO20,w = 2.0 S) to reassociate to the native dimer (So20,w = 3.5 S) after dialysis against a non-denaturing buffer. No significant changes in the circular dichroism spectrum of wheat germ agglutinin were observed after oxidation of the three tryptophan residues, suggesting that no gross conformational changes occurred. The steric relationships between the fluorescent tryptophan residues of wheat germ agglutinin and saccharides are discussed.