Hydration pressure and phase transitions of phospholipids: I. Piezotropic approach

Dehydration reduces the main phase transition pressure of phospholipids. An analysis based on the Gibbs-Duhem equation shows how the shift of the transition pressure is correlated to the hydration pressure.By using Fourier transform infrared (FT-IR) spectroscopy we determined the hydration-dependent phase transition pressure. The application of our new approach gives hydration pressure values which agree with the values obtained with the osmotic stress method.     

Hydration pressure and phase transitions of phospholipids. II. Thermotropic approach

It is widely known that dehydration increases the main phase transition temperature of phospholipids. A mathematical analysis now shows that hydration pressure can be calculated by the dehydration-induced shift of the phase transition temperature. The hydration-dependent piezotropic and thermotropic phase transitions were determined by using calorimetry and FT-IR spectroscopy, and the application of our approach gives hydration pressure parameters that agree very well with the values obtained with the osmotic stress method.     

Electron diffraction study of hydrated phospholipid single bilayers. Effects of temperature,hydration and surface pressure of the “precursor” monolayer

The molecular packing and phase transition of hydrated dipalmitoylphosphatidylcholine single bilayers are studied by electron diffraction, using an electron microscope equipped with a hydration stage. The phase transition and area per molecule are measured as functions of temperature, hydration and the surface pressure of the monolayer from which the bilayer is formed. The transition temperature of a bilayer agrees with calorimetric measurements on bulk lipid/water mixtures. The molecular packing of a bilayer corresponds to that of the precursor monolayer at a surface pressure of 47 dyne/cm.     

Hydration pressure and phase transitions of phospholipids. I. Piezotropic approach

Dehydration reduces the main phase transition pressure of phospholipids. An analysis based on the Gibbs-Duhem equation shows how the shift of the transition pressure is correlated to the hydration pressure. By using Fourier transform infrared (FT-IR) spectroscopy we determined the hydration-dependent phase transition pressure. The application of our new approach gives hydration pressure values which agree with the values obtained with the osmotic stress method.     

Modulation of the bilayer to hexagonal phase transition and solvation of phosphatidylethanolamines in aqueous salt solutions

Several salts affect the temperature of the bilayer to hexagonal phase transition of phosphatidylethanolamines. Their effects are dependent on the anion as well as the cation of the salt. Salt effects on this transition can be explained by preferential hydration and ion binding. Those salts which are excluded from the solvation sphere of the membrane promote hexagonal phase formation. For example, Na2SO4 promotes preferential hydration and is a hexagonal phase promoter while NaSCN does not do this and is a bilayer stabilizer. Unlike amphiphiles and hydrocarbons, salts can shift the bilayer to hexagonal phase transition temperature without altering the cooperativity of the transition. The effect of these salts on the gel to liquid-crystal transition is opposite to their effect on the bilayer to hexagonal phase transition. We also find that MnCl2 markedly raises the gel to liquid-crystal transition temperature. This effect is due to binding of the cation to the membrane surface. The effect is reduced with MnSO4 because of preferential hydration. Our results demonstrate that the nature of the anion as well as the cation can alter the effect of salts on lipid phase transition properties. The observed effects can be explained as resulting from preferential hydration and ion binding.     

A theoretical model of the temperature- and pressure-induced phase transition of phospholipid bilayers

A statistical thermodynamic model of phospholipid bilayers is developed. In the model, a new concept of a closely packed system is applied, i.e., a system of hard cylinders of equal radii, the radius being a function of the average number of gauche rotations in a hydrocarbon chain. Using this concept of a closely packed system, reasonable values are obtained for the change in specific volume at the order-disorder transition of lecithin bilayers. In addition to interactions between the lipid matrix and water molecules, between the head groups themselves and between hydrocarbon chains, as well as the intramolecular energy associated with chain conformation, the Hamiltonian of the membrane also includes the energy of the pressure field. Thus, the phase transition of phospholipid membranes induced not only by temperature hut also by hydrostatic pressure is described by this model simultaneously. In accordance with the experimental results, a linear relationship is obtained between the phase transition temperature and phase transition pressure. The other calculated phase transition properties of lecithin homologues. e.g., changes in enthalpy, surface area. thickness and gauche number per chain are in agreement with the available experimental data. The ratio of kink to interstitial conduction of bilayers is also estimated.     

Factors determining pressure perturbation calorimetry measurements: evidence for the formation of metastable states at lipid phase transitions

The factors that influence the application of pressure perturbation calorimetry in studying the volume change of the phase transition of lipids are discussed. These factors include a correction for the temperature-shift induced by perturbation, the kinetic irreversibility of the phase transition and the magnitude of the pressure perturbation. We take into account the fact that the dependence of the phase transition temperature on pressure will affect the temperature-shift induced by pressure. As a result, there is a discrepancy between the compression part of the cycle and the expansion. In addition, sequential cycles lead to a gradual loss in magnitude of the heat effect upon pressure perturbation. We suggest that these phenomena can be explained by the formation of a metastable glass-like state that converts to a stable phase at temperatures removed from the region of the phase transition.     

Kinetics of the barotropic ripple (P beta'')/lamellar liquid crystal (L alpha) phase transition in fully hydrated dimyristoylphosphatidylcholine (DMPC) monitored by time-resolved x-ray diffraction.

We present here the first study of the use of a pressure-jump to induce the ripple (P beta')/lamellar liquid crystal (L alpha) phase transition in fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The transition was monitored by using time-resolved x-ray diffraction (TRXRD). Applying a pressure-jump from atmospheric to 11.3 MPa (1640 psig, 111.6 atm) in 2.5 s induces the L alpha to P beta' phase transition which takes place in two stages. The lamellar repeat spacing initially increases from a value of 66.0 +/- 0.1 A (n = 4) to a maximum value of 70.3 +/- 0.8 A (n = 4) after 10 s and after a further 100-150 s decreases slightly to 68.5 +/- 0.3 A (n = 4). The reverse transition takes place following a pressure jump in 5.5 s from 11.3 MPa to atmospheric pressure. Again, the transition occurs in two stages with the repeat spacing steadily decreasing from an initial value of 68.5 +/- 0.3 A (n = 3) to a minimum value of 66.6 +/- 0.3 A (n = 3) after 50 s and then increasing by approximately 0.5 A over a period of 100 s. The transition temperature increases linearly with pressure up to 14.1 MPa in accordance with the Clapeyron relation, giving a dT/dP value of 0.285 degrees C/MPa (28.5 degrees C/kbar) and an associated volume change of 40 microliters/g. A dynamic compressibility of 0.13 +/- 0.01 A/MPa has been determined for the L alpha phase. This value is compared with the equilibrium compressibilities of bilayer and nonbilayer phases reported in the literature. The results suggest testable mechanisms for the pressure-induced transition involving changes in periodicity, phase hydration, chain order, and orientation. A more complete understanding of the transition mechanism will require improvement in detector spatial resolution and sensitivity, and data on the pressure sensitivity of phase hydration.     

Energetics of a hexagonal-lamellar-hexagonal-phase transition sequence in dioleoylphosphatidylethanolamine membranes.

The phase diagram of DOPE/water dispersions was investigated by NMR and X-ray diffraction in the water concentration range from 2 to 20 water molecules per lipid and in the temperature range from -5 to +50 degrees C. At temperatures above 22 degrees C, the dispersions form an inverse (HII) phase at all water concentrations. Below 25 degrees C, an HII phase occurs at high water concentrations, an L alpha phase is formed at intermediate water concentrations, and finally the system switches back to an HII phase at low water concentrations. The enthalpy of the L alpha-HII-phase transition is +0.3 kcal/mol as measured by differential scanning calorimetry. Using 31P and 2H NMR and X-ray diffraction, we measured the trapped water volumes in HII and L alpha phases as a function of osmotic pressure. The change of the HII-phase free energy as a function of hydration was calculated by integrating the osmotic pressure vs trapped water volume curve. The phase diagram calculated on the basis of the known enthalpy of transition and the osmotic pressure vs water volume curves is in good agreement with the measured one. The HII-L alpha-HII double-phase transition at temperatures below 22 degrees C can be shown to be a consequence of (i) the greater degree of hydration of the HII phase in excess water and (ii) the relative sensitivities with which the lamellar and hexagonal phases dehydrate with increasing osmotic pressure. These results demonstrate the usefulness of osmotic stress measurements to understand lipid-phase diagrams.     

The hydration pressure between lipid bilayers. Comparison of measurements using x-ray diffraction and calorimetry.

The hydration pressure between dipalmitoyl phosphatidyl-N,N-dimethylethanolamine (DPPE-Me2) bilayers has been analyzed by both x-ray diffraction measurements of osmotically stressed liposomes and by differential scanning calorimetry. By the x-ray method, we obtain a magnitude (Po) and decay length (lambda) for the hydration pressure which are both quite similar to those found for bilayers of other zwitterionic lipids, such as phosphatidylcholines. That is, x-ray analysis of DPPE-Me2 in the gel phase gives lambda = 1.3 A, the same as that previously measured for the analogous gel phase lipid dipalmitoylphosphatidylcholine (DPPC), and Po = 3.9 x 10(9) dyn/cm2, which is in excellent agreement with the value of 3.6 x 10(9) dyn/cm2 calculated from the measured Volta potential of DPPE-Me2 monolayers in equilibrium with liposomes. These results indicate that the removal of one methyl group to convert DPPC to DPPE-Me2 does not markedly alter the range or magnitude of the hydration pressure. Calorimetry shows that the main gel to liquid-crystalline phase transition temperature of DPPE-Me2 is approximately constant for water contents ranging from 80 to 10 water molecules per lipid molecule, but increases monotonically with decreasing water content below 10 waters per lipid. A theoretical fit to these temperature vs. water content data predicts lambda = 6.7 A. The difference in observed values of lambda for x-ray and calorimetry measurements can be explained by effects on the thermograms of additional intra- and intermolecular interactions which occur at low water contents where apposing bilayers are in contact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral pressures in biomembranes estimated from the dynamics of fluorescent probes

A theoretical model is proposed which states that the time-independent fluorescence anisotropy of the rod-shaped molecule diphenylhexatriene incorporated into lipid bilayers is a direct result of forces constraining the diphenylhexatriene molecule. These forces are postulated as equating with the lateral pressure operating within the bilayer independently of the probe molecule.Insertion into the model of experimental observations (recorded in the literature) on anisotropy of diphenylhexatriene in lipid bilayers as a function of temperature yielded values of lateral pressure, which decreased with temperature, and sharply at the temperature defining the transition from gel phase to fluid phase. The values so predicted for the mid-point of the transition and for the entirely fluid phase, respectively, compared favourably with estimates of the lateral pressures in these physical states, that have been reported elsewhere and arrived at either from theories describing lipid chain behaviour or from lipid monolayer compression experiments. Previously documented effects on anisotropy induced by incorporation of cholesterol into fluid lipid bilayers have been interpreted as reflections of rises in intramembranal lateral pressure.     

The inverse hexagonal - inverse ribbon - lamellar gel phase transition sequence in low hydration DOPC:DOPE phospholipid mixtures

The inverse hexagonal to inverse ribbon phase transition in a mixed phosphatidylcholine-phosphatidylethanolamine system at low hydration is studied using small and wide angle X-ray scattering. It is found that the structural parameters of the inverse hexagonal phase are independent of temperature. By contrast the length of each ribbon of the inverse ribbon phase increases continuously with decreasing temperature over a range of 50 °C. At low temperatures the inverse ribbon phase is observed to have a transition to a gel lamellar phase, with no intermediate fluid lamellar phase. This phase transition is confirmed by differential scanning calorimetry.     

Bending, hydration and interstitial energies quantitatively account for the hexagonal-lamellar-hexagonal reentrant phase transition in dioleoylphosphatidylethanolamine.

We have accounted for the unusual structural change wherein dioleoylphosphatidylethanolamine undergoes a hexagonal-lamellar-hexagonal transition sequence as the water content is reduced systematically. We describe the role played by the energies of bending, hydration, voids in hexagonal interstices, and van der Waals interaction in this transition sequence. We have used the X-ray diffraction and osmotic stress experiments on the two phases to derive the structural parameters and all of the force constants defining the energetics of the hexagonal and lamellar phases. We have calculated the chemical potentials of lipid and water in both phases and derived the phase diagram of the lipid with no free, adjustable parameters. The calculated temperature/osmotic stress and temperature/composition diagrams quantitatively agree with experiment. The reentrant transition appears to be driven by a delicate balance between the hydration energy in the lamellar phase and bending energy in the hexagonal phase, whereas the energy of voids in hexagonal interstices defines its energy scale and temperature range. Van der Waals attraction between the bilayers in the lamellar phase does not appear to be important in this transition.     

Nuclear magnetic resonance studies of amphiphile hydration: Effects of the gel-to-liquid crystalline phase transition on the hydration of dioleoyl lecithin

The hydration of dioleoyl lecithin (DOL) and dimyristoyl lecithin (DML) has been measured as a function of temperature between ?15 and ?30 °C, using low-temperature proton magnetic resonance. The hydration of DOL is considerably higher than that of DML. We detect 9 mol of unfrozen water/mol of phospholipid at ?25 °C (our “standard” temperature) for DOL, and only 6 mol of water/mol of phospholipid for DML. The gel-to-liquid crystalline phase transition in DOL centered at ca. ?19 °C is manifested by a 70% increase in hydration for both vesicles and dispersions. Preparations of either DML vesicles or vesicles of DOL which contain 33 mol% cholesterol would not be expected to undergo this phase transition, and the hydration increase observed for these preparations in the same range of temperature is less than 20%.     

Destabilization of a lipid non-bilayer phase by high pressure

Pressure is found to destabilize the non-bilayer phase with respect to the bilayer in a model lipid system. The lamellar to inverted hexagonal (H₁₁) phase transition of aqueous egg phosphatidylethanolamine is shifted to higher temperatures by hydrostatic pressure. The slope of the increase in transition temperature is constant to beyond 300 bar, and is greater than that seen for other lipid phase transitions. This behavior is consistent with the hypothesis that increasing chain disorder drives the conversion from the bilayer into the hexagonal phase. If this non-bilayer lipid phase is an intermediate in membrane fusion, then pressure should inhibit the process. This may explain the inhibition of chemical transmission at neural synapses by pressure.     

Redetermination of the pressure dependence of the lipid bilayer phase transition.

The effect of pressure on the phase transition temperature for the dipalmitoyllecithin bilayer was redetermined by following the volume change accompanying the transition. These measurements were carried out isothermally with the transition from the ordered to the disordered phase induced by decreasing the pressure. This contrasts with our previous measurements which were carried out at constant pressure and increasing temperature. The transition at every temperature was sharp and confirmed our previous observation that the volume change associated with the transition (0.033 mL g-1) is invariant with pressure. However, our present measurements, in contrast to our previous results, indicate that dP m/dTm at all pressures is in agreement with the 1 atm value of delta H/Tm delta V within experimental error where Tm and Pm are the temperature and pressure of the phase transition, respectively. These results, which are now in agreement with all other known pressure data, indicate that the entropy change associated with the transition is invariant with pressure.     

Structure and interactions of ether- and ester-linked phosphatidylethanolamines

The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bilayer phase transitions of N-methylated dioleoylphosphatidylethanolamines under high pressure

The bilayer phase transitions of four kinds of unsaturated phospholipids with different-sized polar head groups, dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyl-N-methylethanolamine (DOMePE), dioleoylphosphatidyl-N,N-dimethylethanolamine (DOMe2PE) and dioleoylphosphatidylcholine (DOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light-transmittance. DSC thermogram and light-transmittance curve for each phospholipid vesicle solution exhibited only one phase transition under ambient pressure, respectively. The light-transmittance of DOPC solution at pressure higher than 234 MPa abruptly increased stepwise at two temperatures, which corresponds to the appearance of stable subgel and lamellar gel phases under high pressure in addition to the liquid crystal phase. The constructed temperature (T)-pressure (p) phase diagrams were compared among these phospholipids. The phase-transition temperatures of the phospholipids decreased stepwise by N-methylation of the head group. The slops of the T-p phase boundary (dT/dp) of DOPE, DOMePE and DOMe2PE bilayers (0.127-0.145 K MPa-1) were found to be close to that of the transition from the lamellar crystal (or subgel; Lc) phase to the liquid crystal (Lalpha) phase for DOPC bilayer (0.131 K MPa-1). On the other hand, the dT/dp value of the main transition from the lamellar gel (Lbeta) phase to the Lalpha phase for DOPC bilayer (0.233 K MPa-1) was significantly different from that of the Lc/Lalpha transition, hence both curves intersected with each other at 234 MPa. The thermodynamic quantities associated with the phase transition of DOPE, DOMePE and DOMe2PE bilayers had also similar values to those for the Lc/Lalpha transition of DOPC bilayer. Taking into account of the values of transition temperature, dT/dp and thermodynamic quantities compared with the corresponding results of saturated phospholipids, we identified the phase transitions observed in the DOPE, DOMePE and DOMe2PE bilayers as the transition from the Lc phase to the Lalpha phase although they have been regarded as the main transition in the previous studies. The Lbeta phase is probably unstable for DOPE, DOMePE and DOMe2PE bilayers at all pressures, it exists as a metastable phase at pressures below 234 MPa while as a stable phase at pressures above 234 MPa in DOPC bilayer. The difference in phase stability among the phospholipid bilayers is originated from that in hydration structure of the polar head groups.     

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).     

Phase behavior and structure of aqueous dispersions of sphingomyelin

The phase behavior of bovine brain sphingomyelin in water has been determined by polarizing light microscopy, differential scanning calorimetry, and X-ray diffraction. Lamellar phases, in which water is intercalated between sheets of lipid molecules arranged in the classical bilayer fashion, are present over much of the phase diagram. An order-disorder transition separates the high temperature, liquid crystalline, lamellar phase from a more ordered lamellar phase at low temperatures. The hydration characteristics of sphingomyelin are similar to the structurally related lecithin in that only limited amounts of water are incorporated above and below the transition. Above the transition at 47 degrees C, a maximum of 35% by weight of water can be incorporated between the lipid bilayers, the total thickness at maximum hydration being 60.2 A, the lipid thickness 38 A, and the surface area per lipid molecule at the interface 60 A(2). Water in excess of 35% by weight is present as a separate phase. Below the phase transition, at 25 degrees C a maximum of 42% by weight of water may be incorporated between the lipid bilayers. On increasing the hydration, the lamellar repeat distance increases from 63.5 A to a limiting value of 76 A. Within this hydration range the calculated lipid thickness decreases from 63.5 to 42.5 A, and the surface area per lipid molecule increases from 36.1 to 53.6 A(2). Although these changes may be accounted for by a structure in which the hexagonally packed ordered hydrocarbon chains tilt progressively with respect to the normal to the bilayer plane on increasing hydration, it is possible that changes in other more complex lamellar structures may be responsible for these variations in lipid thickness and surface area.