Determination of the affinity constants of concanavalin A for monosaccharides by fluorescence affinity probe capillary electrophoresis.

Dissociation constants between a legume lectin, concanavalin A (Con A), and unlabeled neutral monosaccharides were determined by affinity probe capillary electrophoresis (APCE). An affinity probe was synthesized by coupling Lissamine rhodamine B and p-amino-phenyl-alpha-D-mannopyranoside, with glutathione as a negatively charged linker. Electrophoresis of the affinity probe was carried out at neutral pH in a fused silica capillary coated with succinylpolylysine on its inner surface, using laser-induced fluorescence detection. In the presence of Con A, the mobility of the affinity probe decreased and this decrement was analyzed as a function of the concentration of Con A. In the presence of a mixture of Con A and an unlabeled monosaccharide, APCE can thus measure the concentration of Con A unoccupied by the unlabeled monosaccharide, and this allows the determination of the dissociation constant of the Con A-monosaccharide complex. The method should be useful for the analysis of a wide range of molecular interactions on a microscale.