Background and purpose: 

The dissociation rate of unlabelled ligand-receptor complexes can be estimated indirectly by radioligand binding. To this end, we developed a simple to use and interpret "two-step competition" binding experiment.

Experimental approach: 

The experiment consists of pre-incubating the receptor-preparation with a wide range of ligand concentrations, washing off free ligand molecules, adding radioligand and monitoring its receptor binding after a fixed time. The ability of this approach to yield the unlabelled ligand's dissociation rate is evaluated both by simulations and experimentally.

Key results: 

When binding of both ligands is mutually exclusive, already bound unlabelled ligand molecules need to dissociate before radioligand binding can take place. Based on this principle, simulations suggest that the unlabelled ligand's dissociation rate can be estimated from the upward shift that the competition curve experiences after washing. Experimental confirmation is provided by comparing the dissociation rates of unlabelled D₂ dopamine-receptor antagonists according to this and alternative approaches. Additionally, the "two-step competition" approach could also disclose the ability of unlabelled ligands to partition in the cell membrane.

Conclusions: 

Compared to the previously published indirect approaches, the "two-step competition" approach gets around the discomfort of measuring radioligand binding at different time intervals and it stands out by the simplified analysis of the binding data.