Phase diagram of soybean phosphatidylcholine-diacylglycerol-water studied by x-ray diffraction and 31P- and pulsed field gradient 1H-NMR: evidence for reversed micelles in the cubic phase.

The phase equilibria of the system soybean phosphatidylcholine, diacylglycerol, and water has been determined using a combination of classical methods together with x-ray diffraction and NMR techniques. In particular, the extent of the phase regions of the lamellar, the reversed hexagonal, and the cubic phases have been determined. By pulsed field gradient 1H-NMR, the diffusion coefficients of all three components in a cubic phase composed of soybean phosphatidylcholine, diacylglycerol, and heavy water have been determined at 25 and 59 degrees C and also for the corresponding cubic phase composed of the chemically more well defined synthetic components 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoylglycerol (DOG), and heavy water. The extension of the phase region of the cubic phase did not seem to change appreciably for the two ternary systems studied. The translational diffusion coefficient of DOPC in this cubic phase is more than an order of magnitude smaller (3 x 10(-13) m2 s-1, 59 degrees C) than the lateral diffusion coefficient of DOPC in an oriented lipid bilayer (5 x 10(-12) m2 s-1, 35 degrees C), whereas the diffusion coefficients of water and DOG were found to be about two orders of magnitude larger than DOPC at 59 degrees C. It is concluded that the cubic phase is built built up of closed reversed micelles in accordance with the suggestion from previous x-ray diffraction studies.

Phase equilibria and formation of vesicles of dioleoylphosphatidylcholine in glycerol/water mixtures.

The lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) forms a lamellar liquid crystalline phase (L alpha) in arbitrary mixtures of glycerol and water. The phase has been characterized by means of X-ray diffraction, 31P-NMR spectroscopy and differential scanning calorimetry (DSC). In the L alpha state, and for DOPC concentrations greater than 50% (w/w), the thickness of the lipid bilayer decreases, while the area of the polar head group increases with increasing glycerol concentration. The phase transition from gel to L alpha state occurs in the range of 240 to 260 K. Contrary to a previous (McDaniel, R.V., McIntosh, T.J. and Simon, S.A. (1983) Biochim. Biophys. Acta 731, 97) study of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) we find that in the gel state, the thickness of the DOPC lipid bilayer is greater than that in the L alpha state. This suggests that in the gel state, the lipid acyl chains of DOPC are in extended configuration. The lamellar phase reaches its maximum swelling at about 50% (w/w) of DOPC. At lower DOPC concentrations a two-phase system is formed where the lamellar phase exists in equilibrium with excess of solvent. Unilamellar vesicles can be prepared from a diluted suspension of the lamellar phase either by using the sonicator or extruder technique. We show this by means of 31P-NMR, EPR and fluorescence spectroscopy. The mean radius of the vesicles, prepared by a sonicator, has been determined at different glycerol/water mixtures. It is found to decrease continuously from 100 A at 100% water to a minimum of 75 A at about 50% water in the solvent mixture. By further decreasing the water content in the solution, the radius rapidly increases, and a mean radius of 450 A is estimated at a water content of 10%. The rotational relaxation times of a fluorescent probe and two EPR spin probes, solubilized in DOPC vesicles, have been measured at different glycerol/water mixtures. It is found that the rotational rates are always much slower in the systems containing glycerol.

Phase equilibria in four lysophosphatidylcholine/water systems. Exceptional behaviour of 1-palmitoyl-glycerophosphocholine.

The phase equilibria in four lysophosphatidylcholine/water systems were investigated at different temperatures. Each of the 1-palmitoyl-, 1-stearoyl-, 1-oleoyl- and 1-linoleoyl-sn-glycero-3-phosphocholines was dispersed in heavy water at different concentrations. The phase structures were determined by 2H-, 14N- and 31P-NMR, polarization microscopy and low-angle X-ray diffraction. The phase diagrams of the oleoyl and linoleoyl systems were quite similar. At room temperature and with decreasing water content the isotropic micellar solution was followed by a hexagonal phase and then a cubic phase. Finally the lamellar phase appeared before the region of hydrated crystals. The same sequence of phases was observed in the stearoyl system at elevated temperatures. The palmitoyl system differed from the others: here a cubic phase followed after the micellar solution, then came a hexagonal phase and after this a lamellar phase. In general the lysophosphatidylcholines seem to behave similarly to the many soaps and detergents as they show the same sequence of isotropic micellar solution, hexagonal phase, lamellar phase with interspersed cubic phases. The presently established phase diagrams demonstrate that the major lysophosphatidylcholines which may be generated by phospholipase A2 in mammalian cell membranes, viz. 1-palmitoyl- and 1-stearoyl-glycerophosphocholines differ greatly in their packing properties. The extraordinary ability of 1-palmitoyl-glycerophosphocholine to form a cubic phase in equilibrium with a micellar solution is of particular interest with regard to the possible occurrence of cubic structures in biomembranes during the process of fusion.

The interactions between monovalent ions and phosphatidyl cholines in aqueous bilayers.

The interactions of lithium and sodium ions and water with phosphatidylcholine bilayers have been studied by means of 7Li, 23Na and 2H NMR quadrupole splittings. The experimental results are interpreted in terms of a simple three-site model with two anisotropic sites ('binding' sites) and one isotropic site ('free' ions and water molecules). The findings obtained for the zwitterionic model membrane are compared with previous investigations of lamellar phases composed of ionic and nonionic amphiphiles. It is shown that the data obtained are compatible with our previous suggestion [Lindblom, G., Persson, N.-O., and Arvidson G. (1976) Adv. Chem. Ser. 152, 121] that an increase in the salt content in the water layer induces a conformational change in the polar head group of phosphatidylcholine. Thus at high salt concentration the phosphocholine head group tends to be oriented perpendicular to the lipid bilayer surface. The study also shows that increasing the amount of salt leads to a squeezing out of water between the bilayers. This is interpreted in terms of a reduction of the repulsion forces between the bilayers.

A cubic protein-monoolein-water phase.

A cubic monoacylglycerol-protein-water phase has been identified by low-angle X-ray diffraction, and the main features of the ternary phase diagram monoolein/lysozyme/water are presented. The thermal stability of the protein in the lipid-protein cubic phase has been examined by differential scanning calorimetry. According to the physical properties of the phase it is proposed that the protein molecules are located in the water medium, i.e. in the water channel systems of the cubic structure earlier suggested. The ability of various proteins to form this cubic phase has been studied, and it was found that the formation of this phase is favoured by an isoelectric point (pI) far from pH 7 in a salt-free solution, thus by high electrostatic repulsive forces.

Molecular organization in the liquid--crystalline phases of lecithin--sodium cholate-water systems studied by nuclear magnetic resonance.

The molecular organization in the hexagonal and lamellar phases of the ternary systems lecithin--sodium cholate--water has been investigated by using a variety of nuclear magnetic resonance techniques. The main findings and conclusions are the following: (i) When calculated on a mole fraction basis, the phase equilibria are insensitive to changes in the alkyl chains of the lecithin. (ii) When incorporated into a lecithin bilayer, cholate exerts a strong perturbation on the lecithin alkyl chain order, giving a large decrease of the order parameters. (iii) This decrease of the order occurs since the average cross-sectional area per alkyl chain increases probably as a result of cholate placing itself flat on the bilayer surface. (iv) The diffusion of lecithin molecules is approximately equally rapid in the lamellar and hexagonal phases. (v) The hexagonal phase is formed by rodlike aggregates with the polar groups at the surface of the rods and with a continuous hydrocarbon core. The rods are not formed by stacking disklike mixed micelles. (vi) With the interpretations of the molecular packing and the phase structures, the observed phase equilibria are in good agreement with current theories of the factors that govern phase behavior in amphiphile--water systems.

Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and x-ray diffraction.

Water binding capability and phase structures for different lipid species extracted from Acholeplasma laidlawii A membranes have been studied using deuteron nuclear magnetic resonance and low-angle X-ray diffraction. The dominating membrane lipids are monoglucosyldiglyceride and diglucosyldiglyceride and each of them takes up limited amounts of water (bound plus trapped), i.e., up to 13% (w/w), whereas the phospholipids and phosphoglycolipids have larger hydration capacities. Addition of magnesium and calcium ions, but not sodium ions, to the diglucosyldiglyceride increases the hydration capability. This increase is accompanied by the formation of a metastable liquid crystalline phase and a hysteresis effect for the transition temperature. Large differences in water deuteron quadrupole splitting were observed between mono- and diglucosyldiglyceride. Both 2H nuclear magnetic resonance and low-angle X-ray diffraction studies on lipids containing biosynthetically incorporated omega-d3-palmitic acid clearly indicate the existence of a reverse hexagonal phase structure for the monoglucosyldiglyceride and lamellar structures for the diglucosyldiglyceride and the other membrane lipids. The low hydration capability of the large diglucosyldiglyceride polar head is discussed in terms of polar head configuration. Both mono- and diglucosyldiglyceride have several physical properties similar to those of phosphatidylethanolamine.