An artifactual component of drug-protein binding generated in vitro

In the course of investigating the binding of imipramine to soluble cellular fractions from brain and leukocytes using equilibrium dialysis, an artifactual binding component was produced. On Scatchard and double-reciprocal plots of the data, the component appeared as a homogenous population of sites displaying a binding affinity of 170 nM. The obtained pattern of biphasic interaction bore a marked resemblance to reported Scatchard plots representing the interaction of drugs with bovine serum albumin, and depicting two components of widely differing binding affinity and capacity. The artifact occurred when solutions were transferred after dialysis and before quantitation to intermediate containers, and resulted from binding of 3H-imipramine to the walls of these containers. The latter interaction decreased the concentration of radiolabeled drug in the dialysate but not in the dialyzed solution, and thus mimicked increased imipramine binding to the biological material under study. The effect was particularly pronounced at low drug concentrations, and was particularly pronounced at low drug concentrations, and was prevented by the presence of either proteinaceous material, or of an excess of another basic compound such as methadone. The concentration dependence of the phenomenon led to its appearance as a discrete binding component. The artifact was eliminated either by applying an appropriate correction factor, or by transferring the dialyzed solutions directly into scintillation vials for counting.