Anomalous equilibrium binding properties of high-affinity racemic radioligands
Bürgisser, E.; Hancock, A.A.; Lefkowitz, R.J.; De Lean, A.
Molecular Pharmacology 19(2): 205-216
1981
ISSN/ISBN: 0026-895X PMID: 6262614 Document Number: 169346
In receptor binding studies, high-affinity racemic radioligands are often used as tracers, neglecting the difference in affinity of their stereoisomers. An experimental and theoretical study comparing the binding of (.+-.)-[3H]carazolol and (.+-.)-[125I]hydroxybenzylpindolol (HYP) to their pure respective isomers in the frog erythrocyte .beta.-adrenergic system was presented. 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 Kd) obtained by considering the racemic radioligand as a single ligand, as has generally been done in the literature, varied with the receptor concentration from .apprx. 2 Kd(-) [Kd for the levo-isomer] at low receptor concentrations to .simeq. 0.5 Kd(+) [Kd for the dextro-isomer] 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 Kd of high-affinity racemic radioligands reported in the literature. Experimental data and simulations indicate that information about the Kd(-) is greatest at low receptor concentrations, but 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(+) .simeq. 400-2000 pM at 25.degree. C. Use of the Kdav for a racemic radioligand caused up to 5-fold systematic underestimation of the affinity of nonracemic competitors. The Kd values of all high-affinity competitors were misestimated by as much as 10-fold using the commonly employed Cheng and Prusoff approximation when the affinity of the radioligand was significantly lower than that of the competitor. Under such circumstances, slope factors of .simeq. 2 were obtained for competition curves in the absence of cooperativity.