Estimation of binding parameters by kinetic data analysis: differentiation between one and two binding sites.

Abstract

A method that enables the discrimination between binding models and the estimation of binding parameters, based solely on kinetic data, is described. Experimental data from association and dissociation experiments were fitted simultaneously to models with mono- or biphasic kinetics with the aid of a non-linear maximum likelihood computer program. Discrimination between two models can be performed statistically. The protocol was used to study the binding of the antitussive [3H]noscapine to guinea pig brain homogenate. Two binding processes could be discriminated by their kinetics, despite the fact that [3H]noscapine apparently binds to one homogeneous population of binding sites in equilibrium binding experiments. This method might find general application when two populations of binding sites are suspected from kinetic data, but when selective ligands are lacking. Since parameter estimates are obtained independent of equilibrium binding data, our approach could also serve as an independent control of such experiments, with respect to both Kd and Bmax.