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The liquid-ordered phase in sphingomyelincholesterol 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 (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.

Abbreviations

BSM:

bovine brain sphingomyelin

DMPC:

dimyristoylphosphatidylcholine

DPPC:

dipalmitoylphosphatidylcholine

EPR:

electron paramagnetic resonance

ESM:

egg sphingomyelin

FOT:

fast oxygen transport

l d :

liquid-disordered

l o :

liquid-ordered

PC:

phosphatidylcholine

PSM:

palmitoylsphingomyelin

5-SASL:

5-doxylstearic acid spin label

SLOT:

slow oxygen transport

s o :

solid-ordered

T 1 :

spin-lattice relaxation time

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Wisniewska, A., Subczynski, W.K. The liquid-ordered phase in sphingomyelincholesterol membranes as detected by the discrimination by oxygen transport (DOT) method. Cell Mol Biol Lett 13, 430–451 (2008). https://doi.org/10.2478/s11658-008-0012-y

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Key words

  • Liquid-ordered phase
  • Raft domain
  • Sphingomyelin
  • Cholesterol
  • Lipid bilayer
  • Spin labeling
  • EPR