Fluorescence Polarization Study on the Dynamics and Location of Peroxidized Fluorescent Phospholipids in Liposomes

Abstract

Motional properties of fluorescent substances produced by lipid peroxidation by a time-resolved fluorescence polarization technique were studied. When liposomes containing phosphatidylethanolamine (PE) and linoleic hydrocarbon chain were incubated at 37°C, fluorophores absorbing maximally at 360 nm and emitting near 430 nm were produced. Their fluorescence anisotropy decay measured at 23°C was fitted well with a sum of a fast relaxation and a time-independent residual term. With the increase of oxidation degree, the time constant of the relaxation term increased. This may be explained by alteration in the membrane structure or by modification of the fluorescent products themselves. Information on the location of the fluorescent products was obtained when their motional property was compared with those of various extrinsic probes that were incorporated at different positions of the lipid bilayer. It was found that the motional property of the fluorescent oxidation products is similar to that of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene, a rod-shaped hydrophobic probe with a charged terminal. Other probes sensing the polar region or the hydrophobic region of the membrane were characterized by a lower order parameter. It is suggested that the fluorescent oxidation products have a polar moiety located at the membrane surface and attached to the amino group of PE while the tail part being buried in the hydrophobic region of the membrane. This picture is supported by fluorescence quenching experiments with the aqueous quencher Co2+. On the other hand, fluorophores produced by the reaction of malondialdehyde and PE suggested to have a chemical structure in which the angle between the absorption and emission dipole moments is very large. On the basis of these observations, the production pathway of fluorophores in oxidized membranes is discussed.