An analytical approach to the measurement of equilibrium binding constants: Application to EGF binding to EGF receptors in intact cells measured by flow cytometry

In ligand binding studies, ligand depletion often limits the accuracy of th e results obtained. This problem is approached by employing the simple obse rvation that as the concentration of receptor in the assay is reduced, liga nd depletion is also reduced. Measuring apparent K-D's of a ligand at multi ple concentrations of receptor with extrapolation to infinitely low recepto r concentration takes ligand depletion into account and, depending on the b inding model employed, yields a K-D within the defined limits of accuracy. We apply this analysis to the binding of epidermal growth factor (EGF) to t he EGF receptor expressed in intact 32D cells, using a homogeneous fluoresc ein-labeled preparation of EGF and measuring binding by flow cytometry. Bin ding isotherms were carried out at varying cell densities with each isother m fit to the generally applied model with two independent binding sites. Ex amination of the variation in the K-D's versus cell density yields a high-a ffinity site that accounts for 18% of the sites and a lower affinity site t hat accounts for the remainder. However, further examination of these data suggests that while consistent with each individual isotherm, the simple mo del of two independent binding sites that is generally applied to EGF bindi ng to the EGF receptor is inconsistent with the changes in the apparent K-D 's seen across varying cell densities.