Immunochemical and spectral studies on Vicia faba agglutinin.

Affinity chromatographic purification of Vicia faba agglutinin (VFA) was performed with a Sephadex G-150 column according to the method of Allen and Johnson [Biochim. Biophys. Acta (1976)]. VFA has 10 tryptophanyl residues per molecule on the assumption that its molecular weight is 50,000 daltons. Equilibrium dialysis with methyl a-D-[glucose-14C(U)]glucopyranoside showed that VFA has two sugar binding sites per molecule with a binding constant of 220 M-1. Upon interaction with specific sugars, VFA induced UV-difference spectra which are typical of the perturbation of tryptophanyl residues. Therefore, the binding constants of VFA with specific sugars could be calculated from the intensity changes in the difference spectra induced by various concentrations of the sugars. The results obtained were in good agreement with the results of hemagglutination inhibition assays. 3-O-Methyl-D-glucose had the highest binding constant (1.9 x 10(3) M-1) among the sugars examined. The binding constants of VFA with glucose, mannose, methyl a-D-glucopyranoside, methyl a-D-mannopyranoside, and maltose were 290, 900, 220, 500, and 220 M-1, respectively, which are lower than those of concanavalin A. VFA did not bind with mucopolysaccharides containing 2-acetamide-2-deoxy-a-, or -beta-D-glucopyranosyl residues, such as heparin, heparan sulfate, and hyaluronic acid. The far UV-CD spectrum of VFA was similar to that of concanavalin A.