Ligand depletion helps identify the low affinity site when NSB is significant in competition data. A. Increasing concentrations of binding sites and [3H]EB samples a wide range of fractional contributions of the two binding sites and NSB to total binding. The combination [3H]EB = 0.013 nM and R1T = 0.13 nM mostly samples behavior of the high affinity site. The low affinity site contributes at most one-hundredth of the total binding; its contribution is always smaller than NSB. In contrast, the combination [3H]EB = 20 nM and R1T = 20 nM more effectively samples behavior of the low affinity site. The low affinity site contributes a maximum of one-tenth of the total binding and contributes more than NSB does to total binding up to about 20 nM cold EB. The y-axis values are calculated as Q/(R1L+R2L+NSB) where Q = R1L, R2L, or NSB. These results suggest this approach might adequately sample the contribution from the low affinity site to total binding during fitting of noisy data. B. The p values compare fits from one site modeltotal and two sites modeltotal to competition data generated with α = 0.1. One set (Δ) used [3H]EB = R1T = 20 nM; the second set (□), [3H]EB = 0.013, 0.3, and 20 nM and R1T = 0.13, 1, and 20 nM. Lines show average log(p). At S/N = 50 for the first set, CIs included the true values (Kd1 = 0.016 nM (CI: 0.010-0.025 nM); Kd2 = 14.9 nM (CI: 6.3-35 nM); R1T = 20.2 nM (CI: 19.7-20.6 nM); R2T = 4.7 nM (CI: 2.8-6.6 nM); α = 0.096 (CI: 0.091-0.100) (n = 5 for each CI). At S/N = 25 for the second set, CIs included the true values (Kd1 = 0.012 nM (CI: 0.009-0.014 nM); Kd2 = 17.0 nM (CI: 4.3-66 nM); fraction of R2T = 0.28 (CI: 0.11-0.45); α = 0.10 (CI: 0.093-0.108).
Person and Wells BMC Biophysics 2011 4:19 doi:10.1186/2046-1682-4-19