Molecular Pharmacology, Vol 19, 205-216, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Anomalous Equilibrium Binding Properties of High-Affinity Racemic Radioligands

¹ Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

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 K_Dav (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 K_D(-) at low receptor concentrations to 0.5 K_D(+) at high receptor concentrations. Thus the generally measured "K_Dav" of these racemic radioligands is really a hybrid of K_D(-) and K_D(+). 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 K_D(-) is greatest at low receptor concentrations, whereas that about K_D(+) 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 K_D values. Estimated K_D values of the stereoisomers of [¹²⁵I]HYP and [³H]carazolol were virtually identical, being K_D(-) = 10-50 pM and K_D(+) 400-2000 pM at 25°. Use of the K_Dav for a racemic radioligand resulted in up to 5-fold systematic underestimation of the affinity of nonracemic competitors. The K_D 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.

Note:
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.

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