A gel retardation assay for the interaction of proteins and carbohydrates by fluorophore-assisted carbohydrate electrophoresis

Abstract

A gel retardation method for studying the interaction between proteins and carbohydrates employing fluorophore-assisted carbohydrate electrophoresis (FACE) has been developed. A carbohydrate (or carbohydrate mixture) is labeled fluorescently at its reducing end with 8-aminonaphthalene-1,3,6-trisulfonic acid, incubated with its binding protein(s), separated, visualized, and quantified by the FACE system. Protein-bound carbohydrate remains at the top of the high-percentage polyacrylamide gel, whereas free carbohydrate migrates into the gel and is thereby separated. Employing mixtures of carbohydrates, a side-by-side comparison between protein-present and protein-absent samples easily identifies the carbohydrate ligands of the protein. The method proposed provides a rapid, convenient, and visual alternative for screening and identifying protein-carbohydrate interactions. The feasibilities of the method and of the determination of the apparent association constants of protein-carbohydrate complexes were examined by the use of lectins and their known carbohydrate ligands. The potential of determining association constants for proteins and native carbohydrates through competition experiments with unlabeled carbohydrate is also discussed.