Phase Diagram of Ternary Cholesterol/Palmitoylsphingomyelin/Palmitoyloleoyl-Phosphatidylcholine Mixtures: Spin-Label EPR Study of Lipid-Raft Formation
2012
Ionova, Irina V. | Livshits, Vsevolod A. | Marsh, Derek
For canonical lipid raft mixtures of cholesterol (chol), N-palmitoylsphingomyelin (PSM), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), electron paramagnetic resonance (EPR) of spin-labeled phospholipids—which is insensitive to domain size—is used to determine the ternary phase diagram at 23°C. No phase boundaries are found for binary POPC/chol mixtures, nor for ternary mixtures with PSM content <24 mol %. EPR lineshapes indicate that conversion from the liquid-disordered (Lα) to liquid-ordered (Lₒ) phase occurs continuously in this region. Two-component EPR spectra and several tie lines attributable to coexistence of gel (Lᵦ) and fluid phases are found for ternary mixtures with low cholesterol or low POPC content. For PSM/POPC alone, coexistence of Lα and Lᵦ phases occurs over the range 50−95.5 mol % PSM. A further tie line is found at 3 mol % chol with endpoints at 50 and ≥77 mol % PSM. For PSM/chol, Lᵦ−Lₒ coexistence occurs over the range 10−38 mol % chol and further tie lines are found at 4.5 and 7 mol % POPC. Two-component EPR spectra indicative of fluid-fluid (Lα−Lₒ) phase separation are found for lipid compositions: 25%<PSM<65%, 5%<chol<30–35%, 65%>POPC>10%, and confirmed by nonlinear EPR. Tie lines are identified in the Lα−Lₒ coexistence region, indicating that the fluid domains are of sufficient size to obey the phase rule. The three-phase triangle is bounded approximately by the compositions 40 and 75 mol % PSM with 10 mol % chol, and 60 mol % PSM with 25 mol % chol. These studies define the compositions of raft-like Lₒ phases for a minimal realistic biological lipid mixture.
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