Determination of binding parameters in the presence of coupled reactions.

Components of a binding reaction may undergo nonbinding reactions: receptors may be degraded, internalized, or exchanged with cryptic sites; ligand may be degraded or compartmented. In such cases the parameters that characterize the system are not obtained from the usual equilibrium analyses. We have simulated the reactions of such systems and generated association curves, "Scatchard" plots, and "Scatchard-like" plots that permit the calculation of binding affinity and receptor number not normally calculable under nonequilibrium binding conditions. In particular, we show that certain coupled reactions produce local maxima and sigmoid shapes in association curves and that the maxima can be used to obtain affinities and receptor numbers.