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 immobilization substrate on the apparent affinity of immobilized receptors for their soluble ligands. In particular, we report measurements of the interaction between cytochrome c or cytochrome b 5 bound to an oriented streptavidin monolayer and its soluble reaction partner. Surface plasmon resonance measurements demonstrate that the charge on the underlying streptavidin monolayer has a pronounced impact on the measured equilibrium constants for the heterologous interaction between cyt c and cyt b 5 . The pH dependence of the equilibrium binding constant is similar qualitatively to that reported for the protein interaction in solution. However, the pH optima of the affinities measured with the immobilized proteins are shifted by up to 1.0 pH units. Whether the shift is to acidic or basic pH depends on which of the two proteins is immobilized. The observed changes are consistent with the long-range repulsion or attraction between the soluble ligand and the supporting streptavidin monolayer.