Kinetic and equilibrium studies of Ah receptor-ligand binding: use of [125I]2-iodo-7,8-dibromodibenzo-p-dioxin

Abstract

In this report, we have used the radioligand [125I]2-iodo-7,8-dibromo-dibenzo-p-dioxin to describe the kinetics of ligand binding to the Ah receptor prepared from C57BL/6J mouse liver. The higher specific activity of this radioligand (2176 Ci/mmol), compared with the usual tritiated ligand [1,6-3H]2,3,7,8-tetrachloro-dibenzo-p-dioxin (58 Ci/mmol) permitted the study of ligand-receptor interactions at much lower component concentrations. For this radioiodinated ligand, Scatchard analysis of saturation binding curves, determined at six different protein concentrations, indicated that the apparent equilibrium dissociation constant, KD, was directly related to the dilution of the receptor preparation; for example, at 1160 micrograms of protein/ml, KD = 1.6 x 10(-10) M; at 36 micrograms of protein/ml, KD = 1.2 x 10(-11) M. Extrapolation of this function to infinite receptor dilution yielded KD = 6 x 10(-12) M. The addition of 70 micrograms/ml of bovine serum albumin to a receptor preparation of 30 micrograms of protein/ml produced a 10-fold decrease in the slope of the Scatchard plot (i.e., 10-fold increase in the apparent KD). Conversely, enrichment of the receptor by high performance liquid chromatography led to an increased slope and thus decreased estimate of KD. The association rate constant (k1), calculated from the integrated second-order rate equation, was 2.8 x 10(10) M-1 hr-1 and, from the initial velocity equation, had a value of 5.25 x 10(10) M-1 hr-1. The dissociation rate constant was biphasic, consisting of a predominant fast component with a rate constant of 0.36 hr-1 (k-1) and a slower component with a rate constant of 4.2-9.4 x 10(-3) hr-1 (k-2). Higher protein concentrations produced a decrease in estimates of k1 but not k-1 or k-2. The KD determined from the ratio of the kinetic rate constant, k-1/k1 = 6.9 x 10(-12) M, is in excellent agreement with that derived from the results of equilibrium binding experiments extrapolated to infinite dilution, KD = 6 x 10(-12) M. The decrease in KD, observed in equilibrium binding studies upon dilution of the receptor preparation, is best explained by a more accurate classification of "free" radioligand at lower protein concentrations. Finally, ligand binding to the Ah receptor is best described by a two-step process, the formation of an initial complex, characterized by rapid ligand dissociation, which undergoes transformation to a second distinct complex displaying a much slower ligand dissociation rate.