Thermodynamic analysis of binding of 4-methylumbelliferyl-a- and b-D-galactopyranosides to momordica charantia lectin

Abstract

Binding of 4-methylumbelliferyl-α-D-galactopyranoside (MeUmbαGal) and the corresponding bβ-anomer (MeUmbβGal) to the Momordica charantia (bitter gourd) lectin (MCL) has been investigated by fluorescence spectroscopy. Binding of MeUmbαGal to MCL resulted in a decrease in the fluorescence intensity of the ligand. Saturation binding at 25° C resulted in a 17.8% decrease in the fluorescence intensity of the ligand. Quenching of the ligand fluorescence intensity was temperature-dependent and decreased with increase in temperature. Addition of lactose reversed the quenching due to binding, indicating that decrease in the fluorescence intensity of MeUmbαGal is due to the interaction of its carbohydrate moiety with the lectin. The changes in the fluorescence intensity of MeUmbaGal resulting from the binding were analysed to obtain the association constants for the binding process at different temperatures. At 25° C, the association constant, Ka, was determined to be 1.14 ´´104 M^–1, and from the temperature dependence of the K_a values the enthalpy and entropy of binding were estimated as ΔH° = –25.9 kJ mol^–1 and ΔS° = –9.1 J mol^–1 K^–1. A comparison of these values with the ΔH° and ΔS° values obtained for the binding of MeUmbβGal revealed that the higher affinity of the β-anomer is due to a larger enthalpy of binding, which overrides a larger negative entropy of binding for the latter.