The affinity constants of recombinant human galectin-1 and galectin-3 for sugars were determined by capillary affinophoresis. The monoliganded affinophore contains p-aminophenyl-beta-lactoside as an affinity ligand in the matrix of succinylglutathione and has three negative charges. An analysis of the mobility change of the lectins caused by the affinophore and its inhibition by neutral sugars allowed, for the first time, a determination of the affinity constants between the binding sites of the lectins and sugars. The relative magnitude of the affinity constants for each of the sugars in terms of dissociation constants found to be consistent with previously reported data on the concentrations of sugars that caused a 50% inhibition (I50) in the binding assay of the lectin to oligosaccharide-immobilized agarose beads but the absolute values of the dissociation constants were considerably smaller than the I50 values. Capillary affinophoresis indicated microheterogeneity of the lectin preparations and enabled the separate analysis of the affinity of each component simultaneously showing the advantage in using a separation method for analysis of bioaffinity.
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