Lateral phase separation in cholesterol/diheptadecanoylphosphatidylcholine binary bilayer membrane

We investigated the phase behavior of cholesterol/diheptadecanoylphosphatidylcholine (C17:0-PC) binary bilayer membrane as a function of the cholesterol composition (Xch) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra showed that the wavelength at the maximum intensity (λmax) changed depending on the bilayer state: ca. 440 nm for the lamellar gel (L′β or Lβ) and the liquid ordered (Lo) phases and ca. 490 nm for the liquid-crystalline (Lα) phase. The transition temperatures were determined from the temperature dependence of λmax and endothermic peaks of the DSC thermograms. Both measurements showed that the pre- and main transition disappear around Xch = 0.05 and 0.30, respectively. The constructed temperature–Xch phase diagram resembled a typical phase diagram for a eutectic binary mixture containing a peritectic point. The presence of a peritectic point at Xch = 0.15 suggested that a complex of cholesterol and C17:0-PC is stoichiometrically formed in the gel phase. Consideration based on the hexagonal lattice model revealed that the compositions of 0.05 and 0.15 correspond to the bilayer states where cholesterol molecules are regularly distributed in different ways. The former is nearly equal to the composition for the membrane occupied entirely with Units (1:18), composed of a cholesterol and 18 surrounding C17:0-PC molecules within the next–next nearest neighbor sites. The latter is represented by a Unit (1:6), including a cholesterol and 6 surrounding C17:0-PC molecules. Further, the disappearance of the main transition at Xch = 0.30 indicates that the pure Lo phase can exist in Xch > 0.30. The eutectic behavior observed in the phase diagram was explainable in terms of phase separation between two different types of regions with different types of regular distributions of cholesterol.