Biochemistry 1992, 31, 6739-6747 6739 Lateral Diffusion in the Liquid Phases of Dimyristoylphosphatidylcholine/ Cholesterol Lipid Bilayers: A Free Volume Analysis? Paulo F. F. Almeida,* Winchil L. C. Vaz,LII and T. E. Thompson'J Department of Biochemistry, University of Virginia, Charlottemille. Virginia 22908, and Max- Planck- Institut fir biophysikalische Chemie, Postfach 2841, D W-3400 Gbttingen, FRG Received March 23, 1992 ABSTRACT: The technique of fluorescence recovery after photobleaching is used to perform an extensive study of the lateral diffusion of a phospholipid probe in the binary mixture dimyristoylphosphatidylcholine/ cholesterol, above the melting temperature of the phospholipid. In the regions of the phase diagram where a single liquid phase exists, diffusion can be quantitatively described by free volume theory, using a modified Macedo-Litovitz hybrid equation. In the liquid-liquid immiscibility region, the temperature dependence of the diffusion coefficient is in excellent agreement with current theories of generalized diffusivities in composite two-phase media. A consistent interpretation of the diffusion data can be provided based essentially on the idea that the primary effect of cholesterol addition to the bilayer is to occupy free volume. On this basis, a general interpretation of the phase behavior of this mixture is also proposed. The properties of lipid bilayers composed of phospholipids and cholesterol have been studied over the past 2 decades. Two considerations have contributed to this continued interest. First, the lipid component of mammalian cell plasma mem- branes consists primarily of these two types of molecules (Rouser et al., 1968; Houslay & Stanley, 1982; Cullis & Hope, 1985). Second, these mixed bilayers are liquid crystals accessible to direct experimentation with techniques of physical chemistry. In order for quantitative studies to be possible, a knowledge of the phase diagram of these systems is essential. Binary mixtures of dimyristoylphosphatidylcholine (DMPC)' or dipalmitoylphosphatidylcholine (DPPC) and cholesterol have received much attention in this respect. Early studies using differential scanning calorimetry (DSC) (Mabrey et al., 1978;Estepet al., 1978) andelectronspinresonance(ESR) (Shimshick & McConnell, 1973; Recktenwald & McConnell, 198 1) indicated the existence of both solid-liquid and liquid- liquid immiscibility regions. Theoretical work (Ipsen et al., 1987,1989) is in agreement with the general features of these experimental studies and suggests moreover the existence of a critical point in the liquid stateof these mixtures. However, only recently have rather complete experimental phase diagrams become available for DMPC/cholesterol (Figure 1) and DPPC/cholesterol (Vist & Davis, 1990; Sankaram & Thompson, 1990a,b, 199 l), obtained using nuclear magnetic resonance (NMR), DSC, and ESR techniques. Above the melting temperature (Tm) of DMPC (23.9 OC; Mabrey & Sturtevant, 1976), this system can exist in one of two possible liquid phases: a liquid-disordered (Ld) phase, at low cholesterol Supported in part by Grants GM-14625 and GM-23573 from the National Institutes of Health and in part by the Max-Planck-Institut ftir biophysikalische Chemie. University of Virginia. Max-Planck-Institut fiir biophysikalische Chemie. 11 Present address: Unidade de Cidncias Exactas e Humanas, Uni- versidade do Algarve, Campus de Gambelas, P-8000 Faro, Portugal. Abbreviations: DMPC, dimyristoylphosphatidylcholine; DPPC, di- palmitoylphosphatidyIcholine; NBD-C( 10)PE. N-(7-nitro-2,1,3-benzox- adiazol-4-yl)didecanoylphosphatidylethanolamine; PC, phosphatidyl- choline; FW, fluoresccncerecovery after photobleaching; ESR, electron spin resonance; NMR, nuclear magnetic resonance; DSC, differential scanning calorimetry; Ld, liquid-disordered (phase); Lo, liquid-ordered (phase); D, diffusion coefficient; E., activation energy; Tm, melting tem- perature. 0006-2960/92/043 1-6739$03.00/0 lot f I 01 " 'I 'I ' 'I 0 10 20 30 40 50 Mol% Cholesterol FIGURE 1: DMPC/cholesterol phase diagram. Filled points cor- respond to discontinuities in ESR experiments using a 5-PC spin- label (.) and a 16-PC biradical label (V) (Sankaram & Thompson, 1991). (0) are breaks in the behavior of the diffusion coefficient (Figure 4). The lines drawn through the points represent our best judgement. concentrations, and a liquid-ordered (Lo) phase, at high cholesterol concentrations, or these two phases can coexist at the same temperature and pressure (Figure 1). Recently, new models have been proposed for the molecular arrange- ments in each phase: in the Ld phase, the cholesterol molecules are believed to span the hydrocarbon core of both leaflets of the bilayer; in the Lo phase, cholesterol is thought to pack like the phospholipid molecules in each leaflet (Sankaram & Thompson 1990a, 1991). Prior to this knowledge of the phase diagram, there were several studies which examined lateral diffusion in phosphati- dylcholine (PC)/cholesterol binary mixtures (Rubenstein et al., 1979; Alecio et al., 1982; Galla et al., 1979; Lindblom et al., 1981; Shin & Freed, 1989). Recently, we have used fluorescence recovery after photobleaching (FRAP) to study the connectivity of the liquid and solid phases in several lipid binary mixtures in the solid-liquid coexistence region (Vaz et al., l989,1990;Bultmannetal., 1991;Almeidaet al., 1992). In the present study, we have used this technique to study 0 1992 American Chemical Society6740 Biochemistry, Vol. 31, No. 29, 1992 phospholipid lateral diffusion of N-(7-nitro-2,1,3-benzoxa- diazol-4-y1)didecanoylphosphatidylethanolamhe [ NDB-C( 1 0)- PE] in the liquid phases of DMPC/cholesterol in a systematic way. We are interested in understanding diffusion in the one-phase regions on the basis of free volume theory (Cohen & Turnbull, 1959; Macedo & Litovitz, 1964). It is generally believed that cholesterol causes an increase in the density of lipid bilayers or, in other words, a decrease in free volume. We can show that a modified Macedo-Litovitz diffusion equation provides a convenient means of understanding quantitatively the relation between the diffusion coefficient of a phospho- lipid and the concentration of cholesterol, through the mo- lecular area occupied by this molecule in the plane of the bilayer. An interpretation is proposed of the DMPC/ cholesterol