Equilibrium ligand binding assays using labeled substrates: Nature of the errors introduced by radiochemical impurities

The effects of radiochemical impurities in a labeled substrate on the characteristics of the experimental equilibrium binding plots were examined. The protein (receptor) was assumed to be a monomer or an oligomer composed of identical, noninteracting subunits. The substrate was assumed to be chemically and radiochemically impure (Case I) or just radiochemically impure (Case II). In both cases, the apparent free substrate concentration required for half-saturation of the protein, [S] 0.5,app, increases linearly with increasing total protein concentration. Reciprocal plots and Scatchard plots are nonlinear. The curvature of both plots is opposite to that observed for the heterogeneity of binding sites. Hill plots are curved with average slopes >1 in the region of half-saturation. If the radiochemical impurity goes undetected, the experimental data might lead an investigator to suggest a number of unnecessarily complicated binding models. The plots obtained in the presence of a radiochemical impurity are very similar to those seen when the receptor protein is a dissociable dimer and K s (monomer) < K s (dimer). However, in the dimer model the variation of [S] 0.5 with total protein concentration is nonlinear. The most direct way of assessing the radiochemical purity of a labeled substrate is to vary the binding protein concentration at a fixed concentration of S ∗. If all of the radioactivity resides in S ∗, the concentration of the PS ∗ complex will approach [S ∗] t as [P] t increases, while the free unbound radioactivity will be driven toward zero. If the labeled substrate is radiochemically impure, “saturating” protein will not bind all of the label. This procedure will detect some types of major impurities missed by paper chromatography (e.g., 3H 2O and nonreactive isomers of S ∗).