Binding of 4-methylumbelliferyl α-D-mannopyranoside to dimeric concanavalin A: fluorescence temperature-jump relaxation study

Abstract

The kinetics of saccharide binding to the dimer form of concanavalin A (con A) was studied at pH 5.5 with the fluorescence temperature-jump method. 4-Methylumbelliferyl .alpha.-D-mannopyranoside, a fluorescent carbohydrate derivative which is quenched upon binding to con A, was used as the ligand. Three relaxation effects were seen. The major relaxation (.tau. = 20-400 ms) was investigated at 4 different temperatures. The behavior of this relaxation as a function of reactant concentrations is consistent with a simple 1-step bimolecular association reaction. These conclusions result from the analysis of both the relaxation times and amplitudes, and from the comparison of the kinetically determined equilibrium parameters (Kass [association rate constant] = 3.5 .times. 104 M-1 at 18.5.degree. C, .DELTA.H.degree. [enthalpy] = -(6-7) kcal/mol) to those obtained from a parallel series of equilibrium experiments (Loontiens, F.G., Clegg, R.M. and Jovin, T.M. (1977)). The Kass and Kd are in the range of (6-15) .times. 10-4 M-1 s-1 and (1.5-5.6) s-1, respectively, within a temperature range of 13.5-28.1.degree. C. Activation energies for the forward and reverse reactions are .apprx. 10 and .apprx. 15 kcal/mol, respectively. The 2 additional relaxations also present in the absence of saccharides result from changes in protein fluorescence and are attributed to protein conformational changes which are not affected by binding of saccharides. These effects were further studied using succinylated, acetylated and demetallized con A. The faster relaxation (13 ms at 18.5.degree. C) was independent of the protein concentration and was not present in derivatized con A samples. The 2 derivatized forms of con A show almost identical carbohydrate binding parameters as the underivatized protein. A limited series of stopped-flow experiments yielded results fully compatible with those from the relaxation measurements.