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Membranes made from binary mixtures of egg sphingomyelin (ESM) and cholesterol were investigated using conventional and saturation-recovery EPR observations of the 5-doxylstearic acid spin label (5-SASL). The effects of cholesterol on membrane order and the oxygen transport parameter (bimolecular collision rate of molecular oxygen with the nitroxide spin label) were monitored at the depth of the fifth carbon in fluid- and gel-phase ESM membranes. The saturation-recovery EPR discrimination by oxygen transport (DOT) method allowed the discrimination of the liquid-ordered (l o), liquid-disordered (l d), and solid-ordered (s o) phases because the bimolecular collision rates of the molecular oxygen with the nitroxide spin label differ in these phases. Additionally, oxygen collision rates (the oxygen transport parameter) were obtained in coexisting phases without the need for their separation, which provides information about the internal dynamics of each phase. The addition of cholesterol causes a dramatic decrease in the oxygen transport parameter around the nitroxide moiety of 5-SASL in the l o phase, which at 50 mol% cholesterol becomes ∼5 times smaller than in the pure ESM membrane in the l d phase, and ∼2 times smaller than in the pure ESM membrane in the s o phase. The overall change in the oxygen transport parameter is as large as ∼20-fold. Conventional EPR spectra show that 5-SASL is maximally immobilized at the phase boundary between regions with coexisting l d and l o phases or s o and l o phases and the region with a single l o phase. The obtained results all owed for the construction of a phase diagram for the ESM-cholesterol membrane.
The effects of ozone at different concentrations (10, 30, 45 g/m3) on fluidity and thermotropic properties of erythrocyte membranes were investigated by EPR using two spin probes: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA). The effect of ozone on the erythrocyte membrane fluidity was a dose-dependent process. The ozone at concentration of 10 g/m3 caused rigidization of the membrane while at concentration of 45 g/m3 increased fluidity both on the surface and in the deeper hydrocarbon region of the membrane. Temperature transitions close to the polar heads region (monitored by 5-DSA) were not sensitive to an increase in ozone concentration. In the case of 16-DSA, low temperature thermotropic transition (around 20°C) gradually decreased with the increase of ozone concentration. High temperature transition (around 40°C) significantly differed at the ozone concentration of 10 g/m3 and 45 g/m3, being higher and lower, respectively, as compared to untreated cells. For the ozone concentration of 45 g/m3 the disappearance of the low temperature break and the appearance of two breaks at 37°C and 16°C were observed.
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