PROBING LOW-AFFINITY AND MULTIVALENT INTERACTIONS WITH SURFACE-PLASMON RESONANCE - LIGANDS FOR CONCANAVALIN-A

The affinities of the carbohydrate-binding protein concanavalin A (Con A) for mono- and multivalent ligands were measured by surface plasmon resonance (SPR) detection. Assessing protein-carbohydrate affinities is typically difficult due to weak affinities observed and the complic ations that arise from the importance of multivalency in these interac tions. We describe a convenient method to rapidly evaluate the inhibit ory constants for a panel of different ligands, both monovalent and mu ltivalent, for low-affinity receptors, such as the carbohydrate-bindin g protein Con A. A nonnatural, mannose-substituted glycolipid was synt hesized, and self-assembled monolayers of varying carbohydrate density were generated. The synthetic surfaces bind Con A. Competition experi ments that employed monovalent ligands in solution yielded K-i values similar to equilibrium binding constants obtained in titration microca lorimetry experiments. In addition, this assay could be used to examin e various polymeric ligands of defined lengths, generated by ring-open ing metathesis polymerization (ROMP). This study demonstrates the util ity of this method for rapidly screening ligands that engage in low af finity interactions with their target receptors. Our results emphasize that those molecules that can simultaneously occupy two or more sacch aride binding sites within a lectin oligomer are effective inhibitors of protein-carbohydrate interactions.