Probing Low Affinity and Multivalent Interactions with Surface Plasmon Resonance: Ligands for Concanavalin A

Abstract

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 complications that arise from the importance of multivalency in these interactions. We describe a convenient method to rapidly evaluate the inhibitory constants for a panel of different ligands, both monovalent and multivalent, for low-affinity receptors, such as the carbohydrate-binding protein Con A. A nonnatural, mannose-substituted glycolipid was synthesized, and self-assembled monolayers of varying carbohydrate density were generated. The synthetic surfaces bind Con A. Competition experiments that employed monovalent ligands in solution yielded Ki values similar to equilibrium binding constants obtained in titration microcalorimetry experiments. In addition, this assay could be used to examine various polymeric ligands of defined le...