A simplified analysis of scatchard plots for systems with two interacting binding sites

Abstract
A simple graphical method for calculating stoichiometric and site binding constants for systems with two initially equivalent interacting sites is derived from a modified Scatchard equation. The binding constants can be calculated from Scatchard plots (r/[A] as a function of r) using the values of r/[A] (r is the molar ratio of bound ligands to total protein and [A] is the equilibrium concentration of free ligand) when r = 0 and r = 1 (half‐saturation). The applicability of the method to the adsorption of bilirubin by peptide pendants immobilized on a polyacrylamide support is demonstrated.