Investigation of the competition of two ligands for the same binding sites on a protein.

The authors describe an experimentally simple method for investigating the competition of two ligands for the same binding sites on a protein. The free and bound concentrations of one ligand are modified by the presence of the other; binding isotherms are therefore deformed. The authors present a theoretical study based on the analysis of these isotherms obtained with different known concentrations of the competing ligand. It is shown that deformation of the isotherms can be used to obtain the affinity constant of the competitor. The method was applied to competition between different ligands for the binding sites of testosterone-estradiol-binding globulin, a serum protein specific for the transport of steroid hormones as essential as testosterone and estradiol.