Binding of 4-Methylumbelliferyl alpha-d-Mannopyranoside to Tetrameric Concanavalin A. Fluorescence Temperature-Jump Relaxation Study

Abstract

The kinetics of saccharide binding to the tetramer form of concanavalin A have been studied at pH 7.2 with the temperature-jump method. 4-Methylumbelliferyl α-d-mannopyranoside was used as a ligand; its fluorescence is totally quenched upon binding. A single relaxation of ligand fluorescence (τ= 20–400 ms) was observed and was investigated at three different temperatures, using kinetic titration and dilution types of experiments. The concentration dependence of the relaxation time and amplitude were consistent with a single-step bimolecular association and independent binding sites. In the temperature range 13–24 C the association and dissociation rate parameters are in the range (6–10) × 10⁴ M⁻¹ s⁻¹ and (1.4–3.2) s⁻¹ respectively, corresponding to activation energies for the forward and reverse reactions equal to approx. 13 and 8 kcal/mol (54 and 33 kJ/mol) respectively. Two additional relaxations of protein fluorescence (3 ms and larger than 1 s at 25°C) were unaffected by carbohydrate binding. Tetrameric concanavalin A shows carbohydrate binding parameters that are almost identical to those of native or derivatized dimeric concanavalin A.