The kinetics of competitive radioligand binding predicted by the law of mass action.

Abstract

Although equilibrium competitive radioligand binding studies are often used to characterize hormone and neurotransmitter receptors, the kinetics of such experiments have not been extensively explored. The interactions of the radioligand and competitor with the receptors can be described by two differential equations which can be solved to yield a single equation describing the binding of the radioligand as a function of time. This equation has several applications: First, it can be used to simulate competitive binding reactions under defined conditions. Second, fitting experimental data to this equation allows one to determine the association and dissociation rate constants of the competing ligand, parameters that cannot be derived from equilibrium experiments. Furthermore, this method can be used to determine the KI of the competing drug from data acquired before equilibrium is reached. Third, mathematical analysis of the binding equation allowed us to answer two specific questions regarding the kinetics of competitive radioligand binding: how long such an incubation takes to equilibrate, and how the IC50 varies over time. The answers to these questions depended, to a large extent, on the relative values of the dissociation rate constants of the radioligand and competitor, which can be determined as noted above. When the competitor dissociates from the receptors more rapidly than the radioligand, the IC50 first decreases and then increases, but never has a value less than the KI. At low radioligand concentrations, equilibrium is reached in the same amount of time required of the radioligand to dissociate completely from the receptors as determined in an "off-rate experiment." At higher concentrations of radioligand this time is halved. When the competitor dissociates from the receptor more slowly than does the radioligand, then the time required to equilibrate depends only on the dissociation rate constant of the competitor, and the IC50 decreases over time.