The Effects of Labeled and Unlabeled Impurities on the Analysis of Equilibrium Binding and Initial Velocity Data by Means of Scatchard Plots

Chemical and/or radiochemical impurities in a preparation of labeled ligand can introduce artifacts to the analytical Scatchard plot. The causes are either an incorrect assessment of the specific radioactivity of the ligand or an incorrect assessment of the equilibrium concentration of free ligand, or both. When non-binding impurities are present, the apparent specific activity of the ligand, S.A.app, and the apparent concentration of free ligand, [S]app, are related to the true values by: p = mS.A./n = (1 - n )[P]tm(1 + VR)(1 + Ks[S]) + Ksm, n is the fractional radiochemical [counts per minute (cpm)] purity of the tracer; m, the fractional chemical (weight) purity of the ligand preparation; [P]t, the total concentration of active receptor sites; Ks = the dissociation constant of the receptor-ligand complex; and VR, the ratio of compartment volumes for binding measurements performed by equilibrium dialysis, which equals the volume of the compartment without the receptor/volume of compartment containing the receptor. (VR = 0 for measurements performed by filtration methods.) parent concentration of the PS complex, [PS]app, calculated indirectly from the difference between the radioactivity in the "plus receptor" and the "minus receptor" compartments or directly from the radioactivity trapped on the filter is related to the true concentration, [PS] by: p = n[PS]/m. t cases, the Scatchard plot of [PS]app/[S]app vs. [PS]app will be curved (concave down). For any degree of radiochemical impurity, the curvature increases with increasing total receptor concentration. At receptor levels ⪢ Ks, the Scatchard plot and the Hill plot resemble those observed for positive co-operativity. Although linear plots are obtained if VR is very large, or if m < 1 but n = 1, the experimental Ks or [P]t (or both) will be in error. Under certain conditions, the binding of an unlabeled or labeled contaminant in competition with S can produce linear plots. Contaminant binding can also produce plots that are concave down or bend backwards in the horizontal direction (depending on the relative K values of the competing ligands).