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. 2008 Apr 2;13(3):430–451. doi: 10.2478/s11658-008-0012-y

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

Anna Wisniewska 1,2,✉, Witold K Subczynski 1
PMCID: PMC6275794  PMID: 18385950

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 (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.

Key words: Liquid-ordered phase, Raft domain, Sphingomyelin, Cholesterol, Lipid bilayer, Spin labeling, EPR

Full Text

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Abbreviations used

BSM

bovine brain sphingomyelin

DMPC

dimyristoylphosphatidylcholine

DPPC

dipalmitoylphosphatidylcholine

EPR

electron paramagnetic resonance

ESM

egg sphingomyelin

FOT

fast oxygen transport

ld

liquid-disordered

lo

liquid-ordered

PC

phosphatidylcholine

PSM

palmitoylsphingomyelin

5-SASL

5-doxylstearic acid spin label

SLOT

slow oxygen transport

so

solid-ordered

T1

spin-lattice relaxation time

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