Experimental artifacts and the analysis of ligand binding data: results of a computer simulation

Use of computerized analysis techniques for ligand binding data have recently become generally available, and are now used quite routinely. When used appropriately, these tools can improve the precision of the estimated parameters for binding affinity, K, and capacity, R. Furthermore, such programs can also calculate the uncertainty of the estimates e.g., as a percent coefficient of variation (%CV). However, because of unmeasured variability in specific activity, tracer purity, counting efficiency, counter background, efficiency of separation, etc., the actual uncertainty in the parameters K and R is usually much larger than stated. In an attempt to examine the effects of such artifacts, we have developed a computer program which simulates data arising from a number of commonly used experimental designs, and then intentionally distorted with each of these artifacts. Finally, the data are converted to B/F and B and plotted in the conventional Scatchard plot. Distortions revealed in this graph are indicative of the effect each artifact has on the parameter estimates. The computer program is generally written to stimulate the binding of 2 or more ligands to one, two or many classes of independent or cooperative specific sites as well as to nonspecific sites. Thus, the program is applicable in a wide variety of situations. Results show that low tracer purity ("bindability") or low filtration efficiency will significantly alter the measured R value. Poorly determined specific radioactivity may significantly alter the measured K value as well. Imprecise measurement of machine background may result in the spacious appearance of positive cooperativity, or of additional high or low affinity classes of binding sites. Finally, under some circumstances, it is possible to detect and correct for the presence of these artifacts.