Scatchard plot and heterogeneity in binding affinity of labeled and unlabeled ligand.

In saturation analysis the Scatchard plot is a generally accepted method for calculation of the affinity constant, K, and the molar concentration, q, of the binder. However, in a system where the K's for the labeled and unlabeled ligand are unequal, a nonlinear plot can be obtained from which incorrect values for K and q may be calculated. This paper mathematically explains how the plot may deviate and under which conditions there will be a maximum in the curve. When the binding sites are homogeneous, the coordinates of this maximum can be used to calculate K and q. A general mathematical expression is derived on the basis of which a linear curve can be constructed for calculation of q and K, which is valid even when affinity for the labeled and unlabeled ligand is not identical.