Characterization of the Fluorophore 4-Heptadecyl-7-hydroxycoumarin: A Probe for the Head-Group Region of Lipid Bilayers and Biological Membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature. In the membrane of vesicular stomatitis virus, the fluorophore showed a large change in steady-state fluorescence anisotropy with temperature, but its lifetime remained unaffected. In lipid vesicles as well as in the virion membrane, the tangent defects were too large to be explained only by the anisotropy in the depolarizing rotations; hindered isotropic torsional motions of the probe also accounted for tangent defects. Depletion of cholesterol from the membrane of the virus decreased the steady-state fluorescence anisotropy of the hydroxycoumarin fluorophore embedded in the bilayer while no significant effect was noted in cholesterol-enriched virions. Limiting anisotropy (r∊) of 4-heptadecyl-7-hydroxycoumarin was measured in liposomes reconstituted with either the integral glycoprotein or the peripheral matrix protein of vesicular stomatitis virus. The glycoprotein had little effect on the order of the lipid in the liquid-crystalline state but disordered the bilayer significantly in the gel state. The matrix protein, on the other hand, ordered the bilayer in the gel state. Incorporation of either of these proteins in the bilayer had no effect on the emission lifetime of the probe.