The liquid-ordered phase in sphingomyelin-cholesterol membranes as detected by the discrimination by oxygen transport (DOT) method

Abstract

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 (lo), liquid-disordered (ld), and solid-ordered (so) 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 lo phase, which at 50 mol% cholesterol becomes ∼5 times smaller than in the pure ESM membrane in the ld phase, and ∼2 times smaller than in the pure ESM membrane in the so 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 ld and lo phases or so and lo phases and the region with a single lo phase. The obtained results allowed for the construction of a phase diagram for the ESM-cholesterol membrane. © 2008 by the University of Wrocław.