BINDING SITES ON CONCANAVALIN A 399 Concanavalin

Equilibrium dialysis of concanavalin A against methyl c¢-D-mannopyranoside and methyl C¢-D-glucopyranoside conducted at 2 ° in the presence of I M NaC1 showed that concanavalin A is bivalent. A SCATCHARD plot of the data obtained gave straight lines for both sugars with observed association constants (K') of 1. 4. lO 4 1/mole for methyl ,¢-D-mannopyranoside and o.3" IO 4 1/mole for methyl ~¢-D-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 68 ooo for concanavalin A. The binding of methyl ~-D-mannopyranoside to concanavaiin A was maximal at pH 6.2 (K' ----2.06. lO 4 1/mole). The standard free energy change (AF °) of this reactionwas 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 a-Dmannopyranoside and methyl ~-D-glucopyranoside parallels the relative activity of these sugars in hapten inhibition experiments reported in previous studies.