Orientation of adsorbed cytochrome c as a function of the electrical potential of the interface studied by total internal reflection fluorescence.

A method for determination of the orientation of adsorbed structure-stable proteins using Total Internal Reflection Fluorescence is outlined. The theory has been elaborated for orientation studies on adsorbed free base cytochrome c, of which the prophyrin can be used as an intrinsic fluorescent label. The ratio of fluorescence intensities at two polarization modes of the incident light (the transverse magnetic and the transverse electric polarization mode, respectively) gives a relation between the orientation angles of the porphyrin relative to the interface. As an illustration of the theory, experimental results on the adsorption of cytochrome c at an optically transparent SnO2 film electrode are presented. It is concluded that the orientation of the molecules can only be affected by the interfacial potential during the process of adsorption, but, once adsorbed, the orientation cannot be changed anymore by variation of the potential.