Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin

Abstract

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 x 106 M-1 at 20 °C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: ΔG = -34.7 kJ·mol-1, ΔH = -37.9 kJ·mol-1 and ΔS = -10.9 J·mol-1·K-1. The specific binding of this sugar to SBA shows that the lectin can accomodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 x 105 M-1·s-1 and k-1 = 0.2 s-1 at 20 ° C. The activation parameters indicate an enthalpicly controlled association process.