Competitive binding kinetics in ligand-receptor-competitor systems. Rate parameters for unlabeled ligands for the...

We describe the real-time kinetics of the competition of different ligands for the same receptor and use the computer routine SAAM to simulate this competition. Based on the simulation, we have developed two experimental approaches whereby the parameters of the interaction of nonlabeled ligands with their receptor can be detected; briefly, the analysis of the nonlabeled ligands depends on the perturbation of the kinetics of interaction of labeled ligands with the receptor with which they are in competition. The approach relies primarily upon an analysis of the kinetics of the competition between fluorescent and nonfluorescent ligands using a real-time, homogeneous binding assay in the fluorescence flow cytometer. A secondary approach depends upon an examination of the kinetic impact of antagonists on the responses of cells stimulated by agonists at the same receptor. Experimental verification of these approaches has been obtained using the N-formyl peptides (and their antagonists) which bind to receptors on human neutrophils and produce rapid cell stimulation. We find that agonistic N-formyl peptides have residence times of minutes while nonstimulatory antagonists have residence periods of, at most, a few seconds at these receptors. The limitations and general range of applicability of these procedures are discussed. The main advantage of these approaches is that they permit the evaluation of kinetic parameters of unlabeled ligands, even those which bind weakly or which have brief residence times--properties which make analyses by conventional methods difficult.