On the specificity of carbohydrate-lectin recognition: the interaction of a lectin from Ricinus communis beans with simple saccharides and concanavalin A

Abstract

A galactose-specific protein (RC₁) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8-9% of the total protein and was found to be rich in mannose. The interaction of RC₁ with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (K_a) at 5°C was calculated as 3.8 mM⁻¹ and 1.2 mM⁻¹ for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC₁ with molecular weight of 120000 was found to be 2. From the temperature-dependent K_a values for the binding of lactose, the values of -5.7 kcal/mol and -4.3 cal×mol⁻¹×K⁻¹ were calculated for ΔH and ΔS, respectively. The addition of concanavalin A to RC₁ or vice versa led to the formation of the insoluble complex RC₁·ConA₄ containing one molecule of RC₁ and one molecule of tetrameric concanavalin A (ConA₄) which could be dissociated upon addition of concanavalin A-specific sugars. The complex formation results in a time-dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time-dependent appearance and disappearance of the turbidity the formation (k_f) and dissociation (k_d) rate constants were calculated as 3 mM⁻¹×s⁻¹ and 0.07 ks⁻¹ respectively. The ratio k_f/k_d (43μM ⁻¹), that corresponds to the association constant of complex RC₁·ConA₄, is higher than that of mannoside·ConA₄ and thereby suggests that protein-protein interaction contributes significantly in stabilising glycoprotein·lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell-lectin interaction is discussed.