Quantitative studies of the binding of wheat germ agglutinin (WGA) to chitinoligosaccharides and partially N-acetylated chitosans suggest inequivalence of binding sites

We have investigated the interactions between wheat germ agglutinin (WGA) and highly de-N-acetylated chitosans of low molecular weight (prepared by nitrous acid degradation and subsequent reduction) by 1H and 19F NMR spectroscopy. Three isolectins were separated from an unfractionated WGA preparation. No significant differences among isolectins were found with respect to binding properties towards GlcNAc or towards chitosans. We observed a specific interaction between WGA and Nacetylated sugar residues in highly de-N-acetylated chitosans of low molecular weight, as observed by line-broadening of the N-acetyl 1H NMR resonance. The interaction was not inhibited by fully de-N-acetylated chitosan of low molecular weight, although evidence of weak interaction between WGA and de-N-acetylated residues was found. We estimated a dissociation constant, KD, of 0.15¯0.20 mM for the complex between WGA and a single N-acetylated residue in the chitosan chain (i.e. an N-acetylated residue surrounded primarily by de-N-acetylated residues). Competitive binding experiments with tri-N-fluoroacetylglucosamine (F3-GlcNAc) were performed by 19F NMR spectroscopy. Surprisingly, the N-acetylated residues of highly de-N-acetylated chitosans were much less effective competitors for binding to WGA than expected from KD-values, while GlcNAc and (GlcNAc)3 showed the expected competition with F3GlcNAc for binding to WGA. These data suggest that internal N-acetylated sugar residues in chitosans bind primarily to other sites of WGA than chitin-oligosaccharides. Support of this suggestion is found in recent reports on the structure of WGA.