Determining PPARγ-ligand binding affinity using fluores-cent assay with cis-parinaric acid as a probe

Upon the study of small-molecules binding to proteins, the traditional methods for calculating dissociation constants (K-d and K-i) have shortcomings in dealing with the single binding site models. In this paper, two equations have been derived to solve this problem. These two equations are independent of the total concentration or initial degree of saturation of receptor and the activity of the competitive molecule. Through nonlinear fitting against these two equations, K-d value of a probe can be obtained by binding assay, and K-i value of a ligand can be obtained by competitive assay. Moreover, only the total concentrations of receptor([R](t)), ligand([L](t)) and probe([P](t)) are required for the data fitting. In this work, K-i values of some typical ligands of PPAR gamma were successfully determined by use of our equations, among which the K-i value of PPAR gamma-LY171883 was reported for the first time.