INTERPRETATION OF DEVIATIONS FROM PSEUDO-FIRST-ORDER KINETIC-BEHAVIORIN THE CHARACTERIZATION OF LIGAND-BINDING BY BIOSENSOR TECHNOLOGY

Macromolecular interactions observed using surface plasmon resonance t echnology (BIAcore, Pharmacia) often display kinetic behavior which de viates from the pseudo-first-order time dependence that has been predi cted for 1:1 interactions of ligand and ligate. In the present study w e review the major reasons for such deviations, and present results wh ich suggest that the most common source of deviations from the pseudo- first-order kinetic approximation of BIAcore kinetic data is likely to be heterogeneity of the immobilized ligand sites. A simplified analys is of the adsorption stage of BIAcore data is presented in terms of th e net observed pseudo-first-order rate constant, k(obs), rather than i n terms of the association and dissociation rate constants, k(a) and k (d). The analysis is then extended to the determination of the dissoci ation equilibrium constant for the interaction of ligand and ligate in the solution phase from sensorgrams reflecting competition between so luble and immobilized forms of ligand for ligate. (C) 1996 Academic Pr ess, Inc.