Rapid report Critical pressures in multicomponent lipid monolayers

Epifluorescence microscopy has been used previously to study coexisting liquid phases in lipid monolayers of dihydrocholesterol and dimyristoylphosphatidylcholine at the air/water interface. This binary mixture has a critical point at room temperature (22°C), a monolayer pressure of approx. 10 mN/m, and a composition in the vicinity of 20-30 mol% dihydrocholesterol. It is reported here that this critical pressure can be lowered, raised, or maintained constant by systematically replacing molecules of this phosphatidylcholine with molecules of a phosphatidylethanolamine, or an unsaturated phosphatidylcholine, or mixtures of the two, while maintaining the dihydrocholesterol concentration at 20 mol%. Thus, even complex mixtures of lipids may be characterized by a single, well-defined second-order phase transition. In principle, such transitions might be found in biological membranes. Lipids fonn monolayers when spread at the air/water interface. Under certain conditions of composition, temper­ ature, and pressure these monolayers can exhibit two coex­ isting liquid phases. Fluorescence microscopy has been used to observe the shapes, sizes, and movements of domains of one such liquid phase in the background of the other liquid phase [1-4]' Monolayers composed of choles­ terol (or cholesterol analogs such as dihydrocholesteroO and phospholipids are of special interest as eukaryotic cell membranes are composed of such mixtures. There have been many previous studies of these lipid mixtures in bilayers for the purpose of describing coexisting liquid phases [5-8]. Although the existence of liquid phases in these binary systems is now well established, there has been relatively little published work seeking to discover whether or not these phases might be present in more complex lipid systems [9]. Such studies are of relevance to long-standing questions concerning the possible occur­ The present study is most conveniently discussed in tenns of the approximate phase diagram for the binary mixture of dihydrocholesterol and DMPC shown in Fig. 1. (Dihydrocholesterol, abbreviated DChol, is used because it appears to oxidize less rapidly than cholesterol. The oxida­ tion products are line active and so can strongly affect domain morphology [18].) At low pressures this binary mixture fonns two coexisting liquid phases. As the pres­ sure is raised, the system crosses a phase boundary and becomes homogeneous. These phase boundaries were lo­ cated using epifluorescence microscopy [1-3,18]' In the present study the same method is used to locate the phase boundaries in monolayers of multicomponent lipid mix­ tures containing 20 mol% dihydrocholesterol. Although the complete phase diagram for such multicomponent systems would require a multidimensional representation, we find that the systems studied behave in some respects as quasi­ binary mixtures. That is, the critical composition with respect to dihydrocholesterol remains between 20 and 30 mol% in these mixtures, as judged by the appearance of a stripe phase near the critical pressure [3]. Materials and methods. The phospholipids dimyristoyl­ L-a-phosphatidylcholine (DMPC), dimyristoyl-L-a-phos­ phatidylethanolamine (DMPE), dimyristoleoyl-L-a-phos­ phatidylcholine (DMoPC), and the fluorescent lipid probe N -(7-nitrobenz-2-oxa-l ,3-diazol-4-yI)dipalmitoyl-L-a­ phosphatidylethanolamine (NBD-DPPE) were purchased rence of phase transitions in biological membranes [10-17] from Avanti Polar Lipids. The fluorescent lipid probe N-(Texas Red sulfonyI)-1,2-dihexadecanoyl-sn-glycero-3­ phosphoethanolamine, triethylammonium salt (TR-DPPE) was purchased from Molecular Probes. The synthetic cholesterol analog dihydrocholesterol (DChoI) was pur­ chased from Sigma. All chemicals were used without further purification. All experiments were done at 20-22°C. The sub-phase was distilled, deionized water. Compression and expansion of the monolayer were carried out with a movable barrier and surface pressure was measured with a Wilhelmy plate. The monolayer was viewed with a Zeiss epifluorescence microscope fitted with a Cohu low-level video camera. The monolayer images were recorded on a lVC BR601MU video recorder. The lipids were spread from I mM chloroform solution containing 0.3 mol% of the probe TR-DPPE or I mol% of the probe NBD-DPPE. The experiments with DMoPC were done using TR-DPPE. Experiments with DMPE were done first with TR-DPPE and then repeated with NBD­ DPPE. After spreading, the monolayers were compressed while under observation. The pressure at which the two phases became homogeneous was noted. Results and discussion. Note once again the phase diagram for the binary mixture of DChol and DMPC shown in Fig. J. This diagram is adapted from earlier work; the solid curve is taken from Lee and McConnell [19]. The shaded region is adapted from Seul [20], and is only schematic. This shows a region of composition where the two phases form stripe domains, rather than the more commonly seen circular domains. This 'superstructure' stripe phase is only observed in the vicinity of a critical point [3]. Note that the phase boundary between the stripe phase and the homogeneous region is relatively flat. Con­ sequently, the mixing-demixing pressure of the stripe phase provides a measurement of the critical pressure of the system. Further, measurements of critical pressure at the