Effect of the microenvironment on the recognition of immobilized cytochromes by soluble redox proteins

This work examines the effect of the electrostatic properties of an immobil ization substrate on the apparent affinity of immobilized receptors for the ir soluble ligands. In particular, we report measurements of the interactio n between cytochrome c or cytochrome bs bound to an oriented streptavidin m onolayer and its soluble reaction partner. Surface plasmon resonance measur ements demonstrate that the charge on the underlying streptavidin monolayer has a pronounced impact on the measured equilibrium constants for the hete rologous interaction between cyt c and cyt bg The pH dependence of the equi librium binding constant is similar qualitatively to that reported for the protein interaction in solution. However, the DPI optima of the affinities measured with the immobilized proteins are shifted by up to 1.0 pH units. W hether the shift is to acidic or basic pH depends on which of the two prote ins is immobilized. The observed changes are consistent with the long-range repulsion or attraction between the soluble ligand and the supporting stre ptavidin monolayer.