Abstract Upon the study of small-molecules binding to proteins, the traditional methods for
calculating dissociation constants (Kd and Ki) 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, Kd
value of a probe can be obtained by binding assay, and Ki 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, Ki values of some typical ligands of
PPAR were successfully determined by use of our equations, among which the Ki value of
PPAR-LY171883 was reported for the first time.