Capillary affinophoresis of fluorescence-labeled pea lectin with laser-induced fluorescence detection.

Abstract

Affinophoresis is a type of affinity electrophoresis in which an affinophore, a conjugate of an affinity ligand and a multiply charged soluble matrix, causes a mobility change in proteins that have specific affinity for the ligand. This technique enables, not only the separation of a specific molecule, but also the analysis of specific interaction between biomolecules. The affinity constant between fluorescence-labeled pea lectin and affinophores bearing mannoside as an affinity ligand, was determined by capillary electrophoresis using laser-induced-fluorescence detection. Although the labeling of the protein caused microheterogeneity as evidenced by electrophoretic mobility, the affinity constant determined for the main peak in the electropherogram of the labeled lectin agreed with those obtained for unlabeled lectin by the same type of analysis using ultraviolet-absorption detection. As a result of the use of the fluorescence detection, the consumption of the protein sample is greatly reduced to several pg per run.