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.

Potato lectin (Solanum tuberosum agglutinin, STA) was found to contain fluorescent tryptophan residues highly exposed to solvent. The binding of chitin oligosaccharides to STA induced fluorescence quenching, a shift of the fluorescence maximum to shorter wavelength, a decrease in the quenching constant of iodide ion and a decrease of the number of tryptophan residues modifiable by N-bromosuccinimide. The results suggested that one tryptophan residues is located at or near a sugar binding site of STA, and that its environment is altered from hydrophilic to relatively more hydrophobic upon interaction with specific sugars. The binding constants of STA with chitin oligosaccharides were determined by measuring the peak-trough heights in the fluorescence difference spectra induced by various concentrations of sugars. The inhibition constants of chitin oligosaccharides for the hemagglutinating activity of STA were obtained by the method of Pitts and Yang [(1981) Biochem. J. 195, 435-439] and the results were in good agreement with those obtained by the fluorescence spectral method. Standard and unitary free energy changes (delta G0 and delta Gu) and standard enthalpy changes (delta H0) were also obtained. These values decreased with sugar chain length up to at least the tetramer. Thus, it was assumed that there are at least 4 subsites, A, B, C, and D, in the sugar binding site of STA. The contributions to the binding energy (delta Gu) were -17.0, -12.6, -7.3, and -4.4 kJ/mol at subsites A, B, C, and D, respectively, and the bindings of chitin monomer (GlcNAc), dimer, trimer, and tetramer were assumed to occur at subsite A, AB, ABC, and ABCD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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The role of a tyrosyl residue in the binding of Arachis hypogaea (peanut) agglutinin, AHA, to lactose has been studied using two techniques, titration of the phenolic hydroxyl group of the tyrosine residue and chemical modification of the tyrosine with iodine. More than three tyrosyl residues per mol of AHA were masked when AHA was titrated in the presence of lactose. Lactose also protected some tyrosyl residues of AHA from the modification with iodine. Upon interaction with lactose, AHA iodinated in the presence of lactose gave a UV-difference spectrum with similar peaks to those of native AHA, while AHA iodinated in the absence of lactose gave a spectrum without such peaks. Though not only native AHA but also iodinated AHA was completely adsorbed on a column of lactamyl-Sepharose 6B, equilibrium dialysis showed that the binding constant and the number of binding sites of native AHA and iodinated AHA with lactose were 4.3 x 10(3) and 3.0 x 10(3) M-1, and 3.2 and 1.8, respectively. These results suggest that about two of four sugar binding sites have tyrosyl residues which induce the UV-difference spectra upon binding with lactose, and that the iodination of these tyrosyl residues results in a decrease of the number of binding sites on AHA.