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.

Role of hydrophobic effect and surface charge in surfactant-DNA association.

Ethidium bromide is one of the best known DNA intercalator. Upon intercalation inside DNA, the fluorescence due to ethidium bromide gets enhanced by many orders of magnitude. In this paper, we employed ethidium bromide as a probe for studying surfactant-DNA complexation using fluorescence spectroscopy and agarose gel electrophoresis. Surfactants of different charge types and chain lengths were used and the results were compared with that of the related small organic cations or salts under comparable conditions. The cationic surfactants induced destabilization of the ethidium bromide-DNA complex at concentrations in orders of magnitude lower than that of the small organic cations or salts. In contrast however, the anionic surfactants failed to promote any such destabilization of probe-DNA complex. DNA loses its ethidium bromide stainability in the presence of high concentration of cationic surfactant aggregates as revealed from agarose gel electrophoresis experiments. Inclusion of surfactants and other additives into the DNA generally enhanced the DNA double-strand to single strand transition melting temperatures by a few degrees, in a concentration-dependent manner and at high surfactant concentration melting profiles got broadened.