Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the 'multivalency effect'

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A and Dioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal α-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3,6-di-O-(α-D-mannopyranosyl)-α-D- mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence, n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandiflora lectin are observed. The enthalpy of binding, ΔH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, H of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases in K(a) values of the multivalent carbohydrates relative to monovalent analogs, known as the 'multivalency effect,' are shown to be due to more positive entropy (TAS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsites versus binding to separate receptor molecules is given.