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Davis* Department of Physics, University of Guelph, Guelph, Ontario, Canada NlG 2 W1 Received May 11, 1989; Revised Manuscript Received August 24, 1989 ABSTRACT: Deuterium nuclear magnetic resonance spectroscopy and differential scanning calorimetry are used to map the phase boundaries of mixtures of cholesterol and chain-perdeuteriated 1,2-dipalmitoyl-sn- glycero-3-phosphocholine at concentrations from 0 to 25 mol % cholesterol. Three distinct phases can be identified: the La or liquid-crystalline phase, the gel phase, and a high cholesterol concentration phase, which we call the 0 phase. The liquid-crystalline phase is characterized by highly flexible phospholipid chains with rapid axially symmetric reorientation; the gel phase has much more rigid lipid chains, and the motions are no longer axially symmetric on the 2H NMR time scale; the 0 phase is characterized by highly ordered (rigid) chains and rapid axially symmetric reorientation. In addition, we identify three regions of two-phase coexistence. The first of these is a narrow La/gel-phase coexistence region lying between 0 and about 6 mol % cholesterol at temperatures just below the chain-melting transition of the pure phospho- lipid/water dispersions, at 37.75 OC. The dramatic changes in the *H NMR line shape which occur on passing through the phase transition are used to map out the boundaries of this narrow two-phase region. The boundaries of the second two-phase region are determined by 2H NMR difference spectroscopy, one boundary lying near 7.5 mol 5% cholesterol and running from 37 down to at least 30 OC; the other boundary lies near 22 mol 5% cholesterol and covers the same temperature range. Within this region, the gel and /3 phases coexist. As the temperature is lowered below about 30 "C, the phospholipid motions reach the intermediate time scale regime of 2H NMR so that spectral subtractions become difficult and unreliable. The third two-phase region lies above 37 OC, beginning at a eutectic point somewhere between 7.5 and 10 mol % cholesterol and ending at about 20 mol %. In this region, the La and /3 phases are in equilibrium. The boundaries for this region are inferred from differential scanning calorimetry traces, for the boundary between the La- and the two-phase region, and from a dramatic sharpening of the NMR peaks on crossing the boundary between the two-phase region and the &phase region. In this region, the technique of difference spectroscopy fails, presumably because the diffusion rate in both the La- and P-phase domains is so rapid that phospholipid molecules exchange rapidly between domains on the experimental time scale. Cholesterol is a major constituent of the plasma membrane of many of the cells of higher organisms, making up as much 'Supported by grants from the Natural Sciences and Engineering Research Council of Canada and by an NSERC postgraduate scholar- ship to M.R.V. as 50 wt % of the lipid fraction in the case of the human erythrocyte membrane. Even so, its functional role within the membrane is not understood. The large changes which it induces in the physical properties of membranes suggest that part of its function may be to improve these characteristics over those of, for example, a simple phospholipid bilayer. Additionally, it may permit wider variations in composition of the other membrane constituents while maintaining the * Author to whom correspondence should be addressed. 'Present address: Theratronics International Ltd., 41 3 March Rd., Kanata, Ontario, Canada K2K 2B7. 0006-2960/90/0429-45 1 $02.50/0 0 1990 American Chemical Society452 integrity of the membrane under varying physiological con- ditions. In order to investigate these possibilities, it is first necessary to quantitate the changes in these physical properties. The first step is to study the phase equilibria of a simple two-component phospholipid/cholesterol system. The effect of cholesterol on the physical properties of phospholipid bilayers has been studied extensively. Mixtures of cholesterol with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)’ have been studied by electron paramagnetic reso- nance (EPR) (Oldfield & Chapman, 197 1 ; Schreier-Mucillo et al., 1973; Shimshick & McConnell, 1973; Delmelle et al., 1980; Presti & Chan, 1982; Kar et al., 1985), nuclear magnetic resonance (NMR) (Gally et al., 1976; Haberkorn et al., 1977; Brown & Seelig, 1978; Kuo & Wade, 1979; Lindblom et al., 1981; Jarrel et al., 1981; Wittebort et