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 l igand can introduce artifacts to the analytical Scatchard plot. The ca uses are either an incorrect assessment of the specific radioactivity of the ligand or an incorrect assessment of the equilibrium concentrat ion of free ligand, or both. When non-binding impurities are present, the apparent specific activity of the ligand, S.A.(app), and the appar ent concentration of free ligand, [S](app), are related to the true va lues by: S.A.(app) = mS.A./n and [GRAPHICS] where n is the fractional radiochemical [counts per minute (cpm)] purity of the tracer; m, the f ractional chemical (weight) purity of the ligand preparation; [P](t), the total concentration of active receptor sites; K-s = the dissociati on constant of the receptor-ligand complex; and VR, the ratio of compa rtment volumes for binding measurements performed by equilibrium dialy sis, which equals the volume of the compartment without the receptor/v olume of compartment containing the receptor. (VR = 0 for measurements performed by filtration methods.) The apparent concentration of the P S complex, [PS](app), calculated indirectly from the difference betwee n the radioactivity in the ''plus receptor'' and the ''minus receptor' ' compartments or directly from the radioactivity trapped on the filte r is related to the true concentration, [PS] by: [PS](app) = n[PS]/m. In most cases, the Scatchard plot of [PS](app)/[S](app) vs. [PS](app) will be curved (concave down). For any degree of radiochemical impurit y, the curvature increases with increasing total receptor concentratio n. At receptor levels >> K-s, the Scatchard plot and the Hill plot res emble those observed for positive co-operativity. Although linear plot s are obtained if VR is very large, or if m < 1 but n = 1, the experim ental K-s or [P](t) (or both) will be in error. Under certain conditio ns, 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 direct ion (depending on the relative K values of the competing ligands).