Binding of methylumbelliferyl [formula omitted] and 4-methylumbelliferyl N, N′-diacetyl- β-chitobioside to rice lectin: studies by equilibrium dialysis and fluorescence quenching titrations

Abstract

The binding of two fuorescent sugars 4-methylumbelliferyl N- actyl-β- d- glucopyranoside (MeUmbGlcNac) and 4-methylumbelliferyl N, N′-diacetyl- β-chitobioside (MeUmb(GlcNAc) 2) to rice lectine was studied by equilibrium dialysis and extrinsic fluorescence titrations. Rice lectin contains four identical and independent carbohydrate binding sites per 36 000 M r protein. Equilibrium dialysis experiments at 25°C gave an association constant value of (4.82 ± 0.32) · 10 4 M −1 whereas fluorescence quenching titrations at 15°C yielded a value of 8.17 +- 0.25) · 10 4 M −1 with MeUmbGlcNAc. The relative fluorescence quenching efficiency of the sugar increased with the saccharide chain lenght. The calculated quenching of fluorescence in both cases was 100%, indicating that the sugar binding site on the lectin is hydrophobic in nature. Binding constants and thermodynamic parameters of the binding of MeUmb glycosides were determined at 10, 15, 20 and 25°C. In both cases, binding constants decreased with increasing temperature, indicating that the binding of sugar to the lectin is exothermic in nature. The ΔH° and Δ° values for MeUmbGlcNAc-rice lectin interaction were −8.51 kcal·mol −1 and −7.07 e.u., respectively, and for that of MeUmb(GlcNAc) 2-rice lectin the corresponding values were −11.89 kcal·mol −1 and −16.72 e.u.