For the most frequently used two-site model, an exact binding equation is presented in terms of the total ligand concentration. This equation has been extended to analyze the spectroscopic titration experiment where the dilution of protein solution cannot be neglected, the displacement study, and the effect of non-specific binding. Thus, with a non-linear regression program, all unknown binding parameters can be determined correctly by fitting these equations to the experimental data without any data transformation. As an example of the use of the new equations, the experimental data for receptor-insulin binding were taken from literature and reanalyzed by using a non-linear regression data analysis program.
