Models of mobility-shift assay of complexes between dimerizing protein and DNA

The theory of mass transport coupled to macromolecular interactions under chemical kinetic control forms the basis of four different models of the electrophoretic mobility-shift assay of complexes formed between dimerizing proteins and DNA. The theory of mass action was applied to the set of simultaneous dimerization (either simple or ligand-induced) and DNA-binding reactions in order to fix the initial equilibrium composition of mixtures to be assayed. Theoretical mobility-shift patterns were obtained for a range of protein concentrations at constant DNA concentration by numerical solution of the set of simultaneous transport-reaction equations appropriate for each model. In those cases in which dimerization in solution is modeled (including heterodimerization), analysis of the peaks in the patterns provides apparent binding constants, which, when extrapolated to infinite dilution of protein, yield acceptable estimates of equilibrium constants. Those for binding of dimer are products of two or three equilibrium constants, from which the equilibrium binding constant can be extracted, provided that dimerization and, where required, ligand-binding constants are determined by independent physicochemical methods. Dimerization of protein when bound to DNA is distinctive in that extrapolation to infinite dilution of protein is not required.