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 × 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = −34.7 kJ × mol-1, delta H = −37.9 kJ × mol-1 and delta S = −10.9 J × mol-1 × K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate 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 × 10(5) M-1 × s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.
Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin