Surface potential on purple membranes and its sidedness studied by a resonance Raman dye probe

A new technique for the measurement of membrane surface potential is proposed and demonstrated. The method is based on the fact that a positively charged styryl dye molecule aggregates when present at high concentration in the Debye layer near a membrane bearing a negative surface potential. The dye in its aggregated form exhibits marked differences in its resonance Raman spectrum relative to the free dye molecules. This method was used to study the potential on the surfaces of the purple membrane that contains the pigment bacteriorhodopsin. A value of -29.5 mV was found for membranes with bacteriorhodopsin in its relaxed, light-adapted state, and the potential decreased to -34.5 mV when most of the bacteriorhodopsin was converted to the M412 intermediate. Because the dye probe does not diffuse through the lipid bilayer, it can be used to probe the potential on the external or internal surface of a vesicle. Thus, we found that the potential on the purple membrane was asymmetric and was localized mainly on the surface that faces the cytoplasm in the cell.