POLARITY OF LIPID BILAYERS - A FLUORESCENCE INVESTIGATION

Through steady-state and time-resolved fluorescence experiments, the polarity of the bilayers of egg phosphatidylcholine vesicles was studied by means of the solvatochromic 2-anthroyl fluorophore which we have recently introduced for investigating the environmental micropolarity of membranes and which was incorporated synthetically in phosphatidylcholine molecules (anthroyl-PC) in the form of 8-(2-anthroyl)octanoic acid. Fluorescence quenching experiments carried out with NN-dimethylaniline and 12-doxylstearic acid as quenchers showed that the 2-anthroyl chromophore was located in depth in the hydrophobic region of the lipid bilayer corresponding to the C-9-C-16 segment of the acyl chains. Steady-state fluorescence spectroscopy revealed a nonstructured and red-shifted (lambda(em)max = 464 nm) spectrum for the probe in egg-PC bilayers, which greatly differed from the structured and blue (lambda(em)max = 404 nm) spectrum the fluorophore was shown to display in n-hexane. While the fluorescence decays of the fluorophore in organic solvents were monoexponential, three exponentials were required to account for the fluorescence decays of anthroyl-PC in egg-PC vesicles, with average characteristic times of 1.5 ns, 5.5 ns, and 20 ns. These lifetime values were independent of the emission wavelength used. Addition of cholesterol to the lipid did not alter these tau-values. One just observed an increase in the fractional population of the 1.5-ns short-living species detrimental to the population of the 20-ns long-living ones. These observations enabled time-resolved fluorescence spectroscopy measurements to be achieved in the case of the 1/1 (mol/mol) egg-PC/cholesterol mixture. Three distinct decay associated spectra (DAS) were recorded, with maximum emission wavelengths, respectively, of 410 nm, 440 nm, and 477 nm for the 1.5-ns, 6-ns, and 20-ns lifetimes found in this system. On account of the properties and the polarity scale previously established for the 2-anthroyl chromophore in organic solvents, these data strongly suggest the occurrence of three distinct excited states for anthroyl-PC in egg-PC bilayers, corresponding to three environments for the 2-anthroyl chromophore, differing in polarity. The lifetime of 1.5 ns and the corresponding structured and blue (lambda(em)max = 410 nm) DAS account for a hydrophobic environment, with an apparent dielectric constant of 2, which is that expected for the hydrophobic core of the lipid bilayer. The lifetime of 5.5 ns indicates a more polar surrounding corresponding to an epsilon-value around 2-20, the origin of which has still to be elucidated. In organic solvents, a lifetime of 20 ns was detected in the presence of water only. In egg-PC bilayers, this long lifetime, associated with a nonstructured and red-shifted (lambda(em)max = 477 nm) DAS, is suggested to account for interaction of the fluorophore with water molecules which diffuse across the bilayer. In connection with the dynamics of lipids in bilayers, these observations are discussed with regard to the permeability of lipid bilayers to water and its modulation by cholesterol and to the potential of the probe for investigating the polarity of membranes and their permeability to water.