Phase diagram of ternary cholesterol/palmitoylsphingomyelin/ palmitoyloleoyl-phosphatidylcholine mixtures: Spin-label EPR study of lipid-raft formation

摘要

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 (L α) to liquid-ordered (L o) phase occurs continuously in this region. Two-component EPR spectra and several tie lines attributable to coexistence of gel (L β) and fluid phases are found for ternary mixtures with low cholesterol or low POPC content. For PSM/POPC alone, coexistence of L α and L β 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, L β-L o 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 (L α-L o) phase separation are found for lipid compositions: 25%PSM65%, 5%chol30-35%, 65%POPC10%, and confirmed by nonlinear EPR. Tie lines are identified in the L α-L o 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 L o phases for a minimal realistic biological lipid mixture.