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 the results obtained. This problem is approached by employing the simple observation that as the concentration of receptor in the assay is reduced, ligand depletion is also reduced. Measuring apparent K(D)'s of a ligand at multiple concentrations of receptor with extrapolation to infinitely low receptor concentration takes ligand depletion into account and, depending on the binding 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 the EGF receptor expressed in intact 32D cells, using a homogeneous fluorescein-labeled preparation of EGF and measuring binding by flow cytometry. Binding isotherms were carried out at varying cell densities with each isotherm fit to the generally applied model with two independent binding sites. Examination of the variation in the K(D)'s versus cell density yields a high-affinity site that accounts for 18% of the sites and a lower affinity site that accounts for the remainder. However, further examination of these data suggests that while consistent with each individual isotherm, the simple model of two independent binding sites that is generally applied to EGF binding to the EGF receptor is inconsistent with the changes in the apparent K(D)'s seen across varying cell densities.