Masters Thesis

Effects of Oxidized Lipids on the Structure of Phospholipid Bilayers

This work employs the fluorescence probe Laurdan to measure the polarity of mixed bilayer phospholipid (PL) and oxidized phospholipid (OxPL) bilayers. The polarity variation with mixture composition is examined to determine whether the bilayer PL and OxPL mix randomly or separated into domains. Three bilayer phospholipids were used, 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The mixing behavior of these phospholipids and the OxPL 1-palmitoyl-2-glutaryl phosphatidylcholine (PGPC) was investigated using Laurdan fluorescence emission experiments. Mixtures of the bilayer PL with LOPC and LPPC were also investigated to understand the effects of OxPL on the lipid bilayer. From the emission spectra of Laurdan incorporated into the mixtures, a quantity called generalized polarization (GP) was extracted. The emission peak of Laurdan is at 440nm (blue) in tightly packed, gel like domains, and at 490nm (red) in loosely packed lipid regions, where there is more water present. The difference in the fractions of the intensity between the blue and red is referred to as GP. GP varies with composition and temperature. Higher GP indicates lower polarity and vice versa. The GP results together with previously conducted Dynamic Light Scattering experiments show that POPC and PGPC form mixed vesicles and mix randomly up to 20 mol %. PGPC. DOPC and PGPC do not mix, and separate into coexisting vesicles. DPPCand PGPC mix in the gel phase but separate in the liquid phase. The main contributions of this work are: (1) novel use of Laurdan fluorescence and complementary application of Dynamic Light Scattering to distinguish random mixing versus separation into domains. (2) a new hypothesis that the mixing behavior is due to the intrinsic curvature difference between the mixing lipids. This difference in behavior is hypothesized to be due to the difference in the shape of the mixing molecules. POPC is more similar in shape to PGPC, than DPPC and DOPC. The long tail of POPC is identical to the long tail of PGPC. PGPC is inverse cone, DPPC is cylindrical, and DOPC is cone shaped. The difference in shape increases curvature stresses and the molecules separate. The degree of separation depends on the degree of the difference in shape.

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