Safranine-O as membrane potential probe: a mechanistic study using fluorescence spectroscopy

Use of safranine-o has been examined as membrane potential probe in 1-palmitoyl-2-oleoyl-3-phosphatidylcholine (POPC) vesicles both in presence and absence of cholesterol. The fluorescence signal increases in presence of vesicles and the increase in fluorescence intensity on hyperpolarization with valinomycin is diffusion potential dependent. The fluorescence spectra recorded after time driven experiments reveals the blue shift in gamma max of fluorescence with increasing diffusion potential. The fluorescence spectra of vesicles-associated dye is at variance with those of the safranine-o in organic solvents. In organic solvents with increasing hydrophobic character of the solvent the gamma max is slightly red shifted. The electronic spectra of the dye molecule and the charges on different atomic centers have been calculated by quantum chemical method GRINDOL. The predicted first excited state originating from the phenazine moiety is in very good agreement with the excitation wavelength. On the basis of charges on various atoms the binding of safranine with vesicles has been discussed. The nonlinear behaviour of fluorescence signal with delta phi, anisotropy measurements and the computational results, reveal the penetration of bound dye molecules (along with orientation) as a function of diffusion potential. Addition of microaliquots of 1.5 M K2SO4 to already hyperpolarized vesicles decreases the fluorescence signal and the fluorescence spectra recorded on stabilization of signal after each addition showed a shift in gamma max of fluorescence in opposite direction i.e. red shifted.