Thermotropic Phase Behavior of Binary Bilayer Membrane of Dipalmitoylphosphatidylcholine and Ergosterol

Abstract

We investigated the phase behavior of a binary bilayer membrane of dipalmitoylphosphatidylcholine (DPPC) and ergosterol, using Prodan fluorescence spectroscopy and differential scanning calorimetry to construct a temperaturecomposition phase diagram. We found that several important features of the phase diagram can be explained by the superlattice view, on the basis of the hexagonal lattice, which suggests the presence of a molecular cluster composed of an ergosterol molecule and its six surrounding DPPC molecules above an ergosterol composition of ca. 0.15.Cholesterol is a major component of cell membranes of animals, and the effect of cholesterol on the thermotropic bilayer phase behavior of phospholipids has long been studied. Although the phase behavior of binary bilayers containing cholesterol has still not been completely established, many important features of the cholesterol effect have been revealed. For example, cholesterol induces phase separation within a phospholipid bilayer and also an intermediate state, the so-called liquid ordered (L o ) state, between the ordered (S o ) and disordered (L d ) states above a certain cholesterol content.1 These findings led to the proposal of the raft hypothesis, 2 which provides the model of the cell membrane that is most widely accepted today. On the other hand, it is well known that other kinds of sterols exist in cell membranes of plants and fungi. Ergosterol is a sterol found in yeast and fungal cell membranes, and its chemical structure is identical to that of cholesterol, except that ergosterol has an additional ethyl group at C-24 and two double bonds, between C-7 and C-8 and between C-22 and C-23. Ergosterol is generally thought to have the same functions as cholesterol in animal cell membranes. However, there are far fewer studies of ergosterol than of cholesterol and little is known about the effect of ergosterol on the phase behavior of phospholipid bilayers.3 In this study, we examined the phase behavior of a binary bilayer of dipalmitoylphosphatidylcholine (DPPC) and ergosterol by differential scanning calorimetry (DSC) and fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) as a polarity-sensitive membrane probe to construct a temperature composition phase diagram.A synthetic phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and (22E)-ergosta-5,7,22-trien-3¢-ol were purchased from Avanti Polar Lipids (Alabaster, AL) and SigmaAldrich Co. (St. Louis, MO), respectively, and used without further purification. A fluorescence probe Prodan was purchased from Molecular Probes Inc. (Eugene, OR). Water was distilled twice from dilute alkaline permanganate for use in the preparation of all the sample dispersions. Aqueous dispersions of DPPC vesicle particles and DPPCergosterol vesicle particles with different ergosterol compositions were prepared as follows. First, stock solutions of ergosterol/chloroform and Prodan/ ethanol were added to a weighed amount of DPPC powder and the mixed solution obtained was dried in a vacuu...