Equilibrium dialysis and carborydrate-binding studies on the 2-acetamido-2-deoxy- d-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- d-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-α- d-glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-α- d-glucopyranoside, the best inhibitors of the BS II lectin- p-azophenyl 2-acetamido-2-deoxy-β- d-glucopyranoside-bovine serum albumin conjugate precipitin-system, were 4 times as active as 2-acetamido-2-deoxy- d-glucopyranose. Of the free monosaccharides tested, 2-acetamido-2-deoxy- d-glucopyranose was the most potent inhibitor, being over 100 times better than d-fructose and 400 times better than d-glucose. Comparison of the inhibiting capacity of methyl or p-nitrophenyl 2-acetamido-2-deoxy-α- d-glucopyranoside with their corresponding β anomers showed that the α anomer was bound 6 to 8 times more avidly than the β anomer. Replacement of the C-3, C-4, or C-6 hydroxyl group of d-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 d-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-β- d-glucopyranosyl residue. Disaccharides having β- d-(1 → 6) glycosidic bonds were very poor inhibitors. Equilibrium-dialysis experiments with p-nitrophenyl 2-acetamido-2-deoxy-α- d-glucopyranoside as binding ligand indicated that the BS II lectin possesses approximately one carbohydrate-binding siteper subunit for the tetrameric protein ( M r 113,000), with association constants of 1.3 x 10 5 M −1 at 4°, and 0.4 x 10 5 M −1 at 37°.