Protein-carbohydrate interaction XX. On the number of combining sites of concanavalin A, the phytohemagglutinin of the jack bean

Abstract

Equilibrium dialysis of concanavalin A against methyl [alpha]--mannopyranoside and methyl [alpha]--glucopyranoside conducted at 2[deg] in the presence of 1 M NaCl showed that concanavalin A is bivalent. plot of the data obtained gave straight lines for both sugars with observed association constants (K') of 1.4[middle dot]104 1/mole for methyl [alpha]--mannopyranoside and 0.3 [middle dot] 104 1/mole for methyl [alpha]--glucopyranoside in the pH range 4.7-5.3. Binding studies carried out at various pH values (5, 6.2, 7.3) also indicated 2 binding sites on the concanavalin A molecule. Calculations were based on a molecular weight of 6800 for concanavalin A. The binding of methyl [alpha]--mannopyranoside to concanavalin A was maximal at pH 6.2 (K' = 2.06 [middle dot] 104 1/mole). The standard free energy change ([Delta]F[deg]) of this reaction was estimated to be -5.4 kcal/mole. No appreciable binding was observed when metal-free concanavalin A was employed in the dialysis experiment. The relative affinity of concanavalin A for methyl [alpha]--mannopyranoside and methyl [alpha]--glucopyranoside parallels the relative activity of these sugars in hapten inhibition experiments reported in previous studies.