Equilibrium dialysis and carbohydrate-binding studies on the 2-acetamido-2-deoxy--glucopyranosyl-binding lectin from Bandeiraea simplicifolia seeds

Abstract

The carbohydrate-binding specificity of Bandeiraea simplicifolia lectin II (BS II lectin) has been studied by quantitative precipitin and hapten-inhibition analysis. The BS II lectin precipitated biopolymers having nonreducing 2-acetamido-2-deoxy--glucopyranosyl residues, such as antigen A. Dextran B-1355-S and rabbit-liver glycogen also afforded precipitin curves with high concentrations of the BS II lectin. Phenyl 2-acetamido-2-deoxy-[alpha]--glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-[alpha]--glucopyranoside, the best inhibitors of the BS II lectin-p-azophenyl 2-acetamido-2-deoxy-[beta]--glucopyranoside--bovine serum albumin conjugate precipitin-system, were 4 times as active as 2-acetamido-2-deoxy--glucopyranose. Of the free monosaccharides tested, 2-acetamido-2-deoxy--glucopyranose was the most potent inhibitor, being over 100 times better than -fructose and 400 times better than -glucose. Comparison of the inhibiting capacity of methyl or p-nitrophenyl 2-acetamido-2-deoxy-[alpha]--glucopyranoside with their corresponding [beta] anomers showed that the [alpha] anomer was bound 6 to 8 times more avidly than the [beta] anomer. Replacement of the C-3, C-4, or C-6 hydroxyl group of -glucose by a methoxyl group or a fluorine atom abolished the capacity of the resulting sugar to bind the BS II lectin, but substitution of the C-2 hydroxyl group of -glucose, by either a methoxyl group or a fluorine group, had no appreciable effect on binding to the lectin. N,N'-Diacetylchitobiose was as active as N,N'N''-triacetylchitotriose, and they were both twice as potent as disaccharides having a nonreducing 2-acetamido-2-deoxy-[alpha]--glucopyranosyl residue. Disaccharides having [beta]--(1 --> 6) glycosidic bonds were very poor inhibitors. Equilibrium-dialysis experiments with p-nitrophenyl 2-acetamido-2-deoxy-[alpha]--glucopyranoside as binding ligand indicated that the BS II lectin possesses approximately one carbohydrate-binding site per subunit for the tetrameric protein (Mr 113,000), with association constants of 1.3 x 105 M-1 at 4[deg], and 0.4 X 105 M-1 at 37[deg].