Competitive binding studies with multiple sites. Effects arising from depletion of the free radioligand

Apparently anomalous behaviour arises if a radiolabelled drug and a non-radioactive drug compete for binding to a membrane-bound receptor when (a) there is severe depletion of the radiolabelled drug and (b) the non-radioactive drug binds to a heterogeneous population of binding sites. In extreme cases, binding of the non-radioactive drug to the sites of high affinity can be obscured completely. This phenomenon is illustrated by the binding of carbachol to muscarinic receptors, estimated by inhibition of a radiolabelled antagonist.