Determination of Surface Coverage and Orientation of Reduced Cytochrome <i>c</i> on a Silica Surface with Polarized ATR Spectroscopy

Abstract

Linearly polarized attenuated total internal reflection (ATR) spectroscopy is used to study the adsorption properties of reduced cytochrome c to a silica surface. The adsorption equilibrium constant, surface coverage, protein orientation, effect of NaCl, and pH dependence are determined for the adsorption of reduced cytochrome c onto a silica surface. Surface coverage results (at pH 7.2) show that reduced cytochrome c packs onto the silica surface at <80% of a closely packed monolayer. The protein orientation distribution as measured by an order parameter is shown to be dependent on the surface coverage and solution pH. All of the results for the surface adsorption of reduced cytochrome c on silica are indicative of an electrostatically driven interaction. The results for reduced cytochrome c are compared to surface adsorption results for oxidized cytochrome c on silica. These results indicate that there is no significant difference in the adsorption behavior correlating to the oxidation state of this heme protein.