The interpretation of ligand displacement experiments: calculations for multisite acceptors

A method is described for calculating the degree of competition for binding between two ligands which are bound at any number of site classes on a binding protein from a generalization of the equilibrium competitive binding equations, the protein's binding parameters for each of the ligands, and the total protein and ligand concentrations. Theoretical displacement curves thus obtained for each of the possible competitive binding models with a multisite protein can then be compared with experimentally determined ligand displacements in order to find which model is most realistic or if measured displacements are due rather to negative cooperativity effects. The binding parameters used for the calculations have a statistical error attached to them, since they have been obtained experimentally, so here we also propose a method for calculating the standard deviations of the theoretical displacement curves deriving from these errors. This permits the use of statistical hypothesis testing in the comparison of theoretical and experimental results. An example is shown in which this method permits the verification that two drugs (phenylbutazone and azapropazone) are both bound by the same high- and low-affinity sites of a protein (alpha-fetoprotein).