Improved evaluation of binding of ligands to membranes containing several receptor-subtypes

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Abstract
In order to evaluate accurately affinity characteristics and relative size of populations of receptor-subtypes in one system we analysed three relevant problems encountered in binding assays. Binding to receptors caused a decrease in the free ligand concentration (i.e. “depletion”). The neglect of depletion may lead to significant distortions of the estimates of affinity and size of receptor-subtype population when the concentrations of both receptor and ligand are of similar magnitude. The distortion is particularly marked when the affinity of a competing ligand is higher than the affinity of the radioligand. We present a formula that describes binding inhibition in a system with receptor-subtypes under conditions of depletion. Binding data usually exhibit heteroscedasticity (i.e. heterogeneous variance), which can not be neglected especially in a system with receptor heterogeneity. Assuming a log normal distribution of experimental errors and a Poisson distribution for errors due to radioactivity counting we derived a function for the transformation of binding data. Transformed data show homoscedasticity, as illustrated with experiments on membranes of guinea-pig lung using ICI 118,551 as inhibitor of 3H-(−)-bupranolol binding to β 1- and β 2-adrenoceptors. The hypothesis that affinity characteristics of receptor subtypes are independent of the tissue class can not be tested accurately by the use of standard methods because of interferences of errors between experiments. We propose a method to account for differences between experiments. Assuming invariance of affinity characteristics one is able to perform common fits of data from different tissue classes. This procedure provides more certain estimates of affinities and subtype fractions, as documented with binding inhibition experiments on β 1- and β 2-adrenoceptors of guinea pig heart and lung.

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