Determination of protein orientation on surfaces with X-ray photoelectron spectroscopy

The use of X-ray photoelectron spectroscopy (XPS) for determination of the preferred orientation of proteins on metal surfaces is demonstrated. A myoglobin derivative in which a pentaamineruthenium (III) group is attached to a specific histidine (residue 81) is used for this purpose, with the Ru and Fe acting as a double marker. The relative intensities of the XPS photoelectron spectra for these two markers are shown to be a sensitive measure of orientation of the protein film. On both aluminum and indium-tin-oxide surfaces, XPS indicates that the Ru-Fe axis of the myoglobin derivative is preferentially oriented with the Ru furthest from the substrate.