Fluorescence Studies of Saccharide Binding to Wheat‐Germ Agglutinin (Lectin)

Wheat germ agglutinin was isolated in pure form by a method adapted from D. LeVine, M. J. Kaplan and P. J. Greenaway, Biochem. J. 129 , 847–856 (1972). Amino‐acid composition calculated for a molecular weight of 23 500 showed that the protein contained four tryptophan residues and was rich in half‐cystine (38 residues) and glycine (51 residues). Analysis of the fluorescence spectrum showed a characteristic tryptophan emission with a maximum centered at 348 nm. The spectrum is almost the same between pH 3.5 and 9.0. By adding N ‐acetylglucosaminides (β‐ d ‐GlcNAc 1 → 4) n ‐ d ‐GlcNAc, an enhancement of the fluorescence intensity was detected. Moreover, binding of the dimer and higher GlcNAc oligomers induced a 10‐nm shift of the emission maximum towards shorter wavelengths indicating that the environment of one or several tryptophan residues was altered by addition of ligands. The pH dependence of the fluorescence quantum yields shows that the tryptophan fluorescence is not altered by any ionisable group which possesses a p K in the range 4.0–9.0 in both the free and the complexed protein. Association constants were determined by measuring the change in fluorescence intensity produced by addition of different glucosaminides. These constants increased with chain length up to the trimer. No cooperative effect could be found with any ligand. It was shown that the carbohydrate binding is abolished below pH 2.8 and that a protein carboxyl group of p K a = 3.9 is involved. Our results are discussed and compared with the model proposed by A. K. Allen, A. Neuberger and N. Sharon, Biochem. J. 131 , 155–162 (1973).