TY - JOUR T1 - Anomalous Equilibrium Binding Properties of High-Affinity Racemic Radioligands JF - Molecular Pharmacology JO - Mol Pharmacol SP - 205 LP - 216 VL - 19 IS - 2 AU - ERNST BÜRGISSER AU - ARTHUR A. HANCOCK AU - ROBERT J. LEFKOWITZ AU - ANDRE DE LEAN Y1 - 1981/03/01 UR - http://molpharm.aspetjournals.org/content/19/2/205.abstract N2 - In receptor binding studies, high-affinity racemic radioligands are often used as tracers, neglecting the difference in affinity of their stereoisomers. We present an experimental and theoretical study comparing the binding of (±)-[3H]carazolol and (±)-[125I]hydroxy-benzylpindolol (HYP) to their pure respective isomers in the frog erythrocyte beta-adrenergic system. Saturation binding curves with the racemic radioligands showed deviations from a binding isotherm for a single ligand which were accentuated at higher receptor concentrations. When different affinity constants for both isomers were considered, significant improvement in the fits of the data were obtained by computer-modeling procedures. The KDav (average dissociation constant) obtained by considering the racemic radioligand as a single ligand, as has generally been done in the literature, varied with the receptor concentration from approximately 2 KD(-) at low receptor concentrations to ≅0.5 KD(+) at high receptor concentrations. Thus the generally measured "KDav" of these racemic radioligands is really a hybrid of KD(-) and KD(+). These experimental findings are in very good agreement with Monte Carlo simulations and may help to explain the discrepancies in dissociation constants of high-affinity racemic radioligands reported in the literature. Experimental data and simulations also indicate that information about the KD(-) is greatest at low receptor concentrations, whereas that about KD(+) is greatest at high receptor concentrations. Simultaneous computer fitting of saturation curves from racemic [125I]HYP and the (+)-isomer, isolated by repeated incubations with frog erythrocyte membranes under appropriate conditions, indicates approximately a 20-fold ratio for the individual isomer KD values. Estimated KD values of the stereoisomers of [125I]HYP and [3H]carazolol were virtually identical, being KD(-) = 10-50 pM and KD(+) ≅ 400-2000 pM at 25°. Use of the KDav for a racemic radioligand resulted in up to 5-fold systematic underestimation of the affinity of nonracemic competitors. The KD values of all high-affinity competitors were also found to be misestimated by as much as 10-fold using the commonly employed Cheng and Prusoff [Biochem. Pharmacol. 22: 3099-3108 (1973)] approximation when the affinity of the radioligand was significantly lower than that of the competitor. Under such circumstances, slope factors of ≅2 were obtained for competition curves in the absence of cooperativity. ACKNOWLEDGMENTS A. D. L. wishes to thank Frank Starmer and Bruce Wright of the Department of Clinical Epidemiology, Duke University, for generously providing access and guidance to the PDP 11/45 computer. The authors also wish to thank Mrs. Elsie Priest and Mrs. Donna Addison for preparing the manuscript. ER -