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. 1978 Mar;75(3):1324–1328. doi: 10.1073/pnas.75.3.1324

Ligands containing heavy atoms: Perturbation of phosphorescence of a tryptophan residue in the binding site of wheat germ agglutinin

Michel Monsigny 1,2, Francis Delmotte 1,2, Claude Hélène 1,2
PMCID: PMC411463  PMID: 274721

Abstract

Information on the structure of binding sites of wheat germ agglutinin was obtained on the basis of fluorescence and phosphorescence changes of tryptophan residues induced by the binding of several thiomercuribenzoate derivatives of glycosides. The thiomercuribenzoate derivatives bind selectively to wheat germ agglutinin in the same way as the corresponding sugars. Using the thiomercuribenzoate of di-N-acetyl-β-chitobiose, it was found that: (i) the fluorescence of tryptophan residues was drastically quenched at both 298 and 77 K; (ii) the phosphorescence intensity was strongly enhanced at 77 K; (iii) the phosphorescence lifetime was markedly decreased. A similar effect was observed with the thiomercuribenzoate of N-acetyl-β-D-glucosamine. These changes were completely reversed upon addition of 1-O-methyl-di-N-acetyl-β-chitobioside. The thiomercuribenzoate of β-D-glucose had no effect at all, and the thiomercuribenzoate of tri-N-acetyl-β-chitotriose had a limited effect. These results are interpreted as a specific heavy atom effect due to a close contact between one tryptophan residue of the protein and the heavy atom of the bound ligand. They are consistent with the view that: (i) binding sites of wheat germ agglutinin may be divided in three subsites, A, B, and C; (ii) a tryptophan residue is in the binding site at subsite C; and (iii) this residue and the ligand are in close contact. This new method, using the enhancement of spin-orbit coupling due to the selective perturbation induced in a tryptophan residue by a ligand containing a heavy atom, has proved to be suitable for locating the tryptophan residue in the binding site of wheat germ agglutinin and can probably be extended to other sugar-binding proteins.

Keywords: heavy atom effect, lectin, protein-sugar interaction

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Selected References

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