Determination of Association Rate Constants by an Optical Biosensor Using Initial Rate Analysis

Abstract

We show that initial rate analysis can be successfully applied to analyze experimental binding data generated by an optical biosensor. The initial rates of binding obtained from linear regression are concentration dependent, and plots of initial rate against ligate concentration yield a straight line that passes through the origin. The slope of this graph is the product of the association constant times the maximal binding capacity of the immobilized ligand. This latter parameter is easily obtained from a single binding curve at high ligate concentration, allowing rapid determination of the association rate constant. The association rate constant obtained in this manner is found to be in good agreement with that obtained by the more customary method of nonlinear regression analysis of the entire binding profile. Initial rate analysis is more simple than fitting the full association profile and needs less data collection time. It also requires fewer assumptions about the functional form of the association profile. This can be advantageous when fitting biosensor-derived data, which often show complex association kinetics. Furthermore, it avoids the potential complication of second-order kinetics which may be found at low ligate concentrations with high-affinity interactions.