Phase Imaging of Phosphatidylcholine Bilayer Membranes by Prodan Fluorescence.

Prodan (6-propiponyl-2-(N,N-dimethylamino)naphthalene) is well known as a polarity-sensitive fluorescent probe and has a high capability of detecting structural changes occurring within phospholipid bilayer membranes. In this study, we carried out the fluorescence spectroscopic observation of bilayer phase behavior for a series of symmetric saturated diacylphosphatidylcholines (CnPCs) with different acyl-chain length n (n = 12-15 and 19-22) using Prodan as a membrane probe to confirm the availability of Prodan along with the previous results for the CnPC bilayer membranes (n = 16-18). The results were discussed by constructing spectral three-dimensional (3D) imaging plots for visualizing the change in bilayer phase states with temperature or pressure to verify the functionality of this 3D imaging plot. It was found that the Prodan fluorescence technique is applicable to the detection of the changes in the bilayer phase states of all CnPCs with a few exceptions. One of the most crucial exceptions was that Prodan cannot be used for the detection of the bilayer-gel state of the C21PC bilayer membrane. It was also found that it is only to the CnPC bilayer membranes with n = 15-18 that the 3D imaging plot is adequately and accurately applicable as a useful graphical tool for visually detecting the bilayer phase states. This is a disadvantageous feature of this technique brought about by the high sensitivity of Prodan as a membrane probe. Further detailed studies on the molecular behavior of Prodan will enable us to find a more useful way of utilizing this membrane probe.

Subgel-phase formation of membranes of ether-linked phosphatidylcholines.

The formation of subgel (so-called hydrated crystal) phase of membranes of ether-linked phospholipids, dialkylphosphatidylcholines containing linear saturated alkyl chain (Cn = 14, 16 and 18), was examined under atmospheric and high pressure. The results of differential scanning calorimetry in 50 wt% aqueous ethylene glycol solution and water showed that these PC membranes undergo the subtransition from the subgel phase to the gel phase at a low temperature with or without the thermal pretreatment of lipid samples called annealing. The subtransition in water was clearly observed by light-transmittance measurements under high pressure and the transition temperature increased by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities of the subtransition were obtained from the phase-transition data and compared with those of membranes of ester-linked phospholipids, diacylphosphatidylcholines. The phase diagrams indicated that all gel phases of the ether-linked PC membranes exist as stable phases while parts of the gel phases of the ester-linked PC membranes are metastable. The subtransition temperatures of the ether-linked PC membranes were lower than those of the ester-linked PC membranes by more than 10 °C and the corresponding thermodynamic quantities were extremely small. Further, it was revealed by high-pressure fluorometry that the difference in subgel phase between ether- and ester-linked PC membranes results from their phase structures: the nonbilayer interdigitated structure is maintained after the conversion from the gel phase to the subgel phase in the ether-linked PC membranes whereas the ester-linked PC membranes form the bilayer subgel phase with staggered structure.

Membrane fusion of phospholipid bilayers under high pressure: Spherical and irreversible growth of giant vesicles.

Membrane fusion of giant vesicles (GVs) for binary bilayers of unsaturated phospholipids, dioleoylphosphatidyl-ethanolamine (DOPE) having an ability to promote membrane fusion, and its homolog dioleoylphosphatidylcholine (DOPC) having an ability to form GV, was investigated under atmospheric and high pressure. While DOPC formed GVs in the presence of inorganic salts with a multivalent metal ion under atmospheric pressure, an equimolar mixture of DOPE and DOPC formed GVs both in the absence and the presence of LaCl3. We examined the change in size and shape of the GVs of this binary mixture in the absence and presence of LaCl3 as a function of time under atmospheric and high pressure. The size and shape of the GVs in the absence of LaCl3 under atmospheric and high pressure and those in the presence of LaCl3 under atmospheric pressure hardly changed with time. By contrast, the GV in the presence of LaCl3 under high pressure gradually changed in the size and shape with time on a time scale of several hours. Namely, the GV became larger than the original GV due to accelerated membrane fusion and its shape became more spherical. This pressure-induced membrane fusion was completely irreversible, and the growth rate was correlated with the applied pressure. The reason for the GV growth by applying pressure was considered on the basis of thermodynamic phase diagrams. We concluded that the growth is attributable to a closer packing of lipid molecules in the bilayer resulting from their preference of smaller volumes under high pressure. Furthermore, the molecular mechanism of the pressure-induced membrane fusion was explored by observing the fusion of two GVs with almost the same size. From their morphological changes, we revealed that the fusion is caused by the actions of Laplace and osmotic pressure.

Formation of intermediate gel-liquid crystalline phase on medium-chain phosphatidylcholine bilayers: Phase transitions depending on the bilayer packing.

The bilayer phase transitions of medium-chain phosphatidylcholines with linear saturated acyl chains (Cn = 12, 13 and 14) were measured by high-pressure light-transmittance measurements and differential scanning calorimetry to investigate the formation of intermediate gel-liquid crystalline phase called Lx phase. The constructed phase diagrams showed that there existed a distinct region of the Lx phase between ripple gel (Pß') and liquid crystalline (Lα) phase for multilamellar vesicle bilayers of C12PC and C13PC. The Lx phase of the C12PC bilayer was metastable at all pressures and disappeared at a higher pressure. In the C13PC bilayer, the Lx phase was stable and also disappeared at a higher pressure but its region markedly shrunk. By contrast, the Lx phase was not detected for the C14PC bilayer. Effects of other factors such as vesicle size and solvent substitution on the Lx phase of the C13PC bilayer were also examined. A decrease in vesicle size and solvent substitution from water to 50 wt% ethylene glycol solution promoted the Lx-phase formation as opposed to the effects of acyl-chain elongation and pressurization. The fluorescence data of the C13PC bilayer with different vesicle sizes showed that the Lx phase is caused by the difference of local packing in the bilayer. Considering these facts, we concluded that the Lx phase is an intermediate gel-Lα phase that has gel-phase monolayers with negative curvature and Lα-phase monolayers with positive curvature. The formation mechanism of the Lx-phase in stacked bilayers and dispersed vesicles is also explainable by this difference in packing state.

Membrane States of Saturated Glycerophospholipids: A Thermodynamic Study of Bilayer Phase Transitions.

Bilayer membranes formed by phospholipids vary in their membrane states by undergoing phase transitions in response to various external environmental factors. Pressure is one of these important environmental factors, but there are very few studies on the effects of pressure on phospholipid bilayer membranes. It is possible to deepen our understanding of the membrane states of phospholipid bilayer membranes by combining information regarding temperature- and/or ligand-responsivity with that regarding pressure-responsivity. In this review, we thermodynamically characterize the bilayer phase transitions of three kinds of saturated glycerophospholipids, each with a different polar head group (phosphatidyl-ethanolamine (PE), -choline (PC) or -glycerol (PG)), and explain their various membrane states depending on temperature and pressure. Both temperature- and pressure-responsivity reveal inherent features of these bilayer membranes: the metastability of the gel phase for PE bilayer membranes, the polymorphism of the gel phases for PC bilayer membranes and morphological changes in bilayer aggregates for PG bilayer membranes.

Ligand partitioning into lipid bilayer membranes under high pressure: Implication of variation in phase-transition temperatures.

The variation in phase-transition temperatures of dipalmitoylphosphatidylcholine (DPPC) bilayer membrane by adding two membrane-active ligands, a long-chain fatty acid (palmitic acid (PA)) and an inhalation anesthetic (halothane (HAL)), was investigated by light-transmittance measurements and fluorometry. By assuming the thermodynamic colligative property for the bilayer membrane at low ligand concentrations, the partitioning behavior of these ligands into the DPPC bilayer membrane was considered. It was proved from the differential partition coefficients between two phases that PA has strong affinity with the gel (lamellar gel) phase in a micro-molal concentration range and makes the bilayer membrane more ordered, while HAL has strong affinity with the liquid crystalline phase in a milli-molal concentration range and does the bilayer membrane more disordered. The transfer volumes of both ligands from the aqueous solution to each phase of the DPPC bilayer membrane showed that the preferential partitioning of the PA molecule into the gel (lamellar gel) produces about 20% decrease in transfer volume as compared with the liquid crystalline phase, whereas that of the HAL molecule into the liquid crystalline phase does about twice increase in transfer volume as compared with the gel (ripple gel) phase. Furthermore, changes in thermotropic and barotropic phase behavior of the DPPC bilayer membrane by adding the ligand was discussed from the viewpoint of the ligand partitioning. Reflecting the contrastive partitioning of PA and HAL into the pressure-induced interdigitated gel phase among the gel phases, it was revealed that PA suppresses the formation of the interdigitated gel phase under high pressure while HAL promotes it. These results clearly indicate that each phase of the DPPC bilayer membrane has a potential to recognize various ligand molecules.

Thermotropic and barotropic phase transitions on diacylphosphatidylethanolamine bilayer membranes.

The bilayer phase transitions of four diacylphosphatidylethanolamines (PEs) with matched saturated acyl chains (Cn=12, 14, 16 and 18) and two PEs with matched unsaturated acyl chains containing a different kind of double bonds were observed by differential scanning calorimetry under atmospheric pressure and light-transmittance measurements under high pressure. The temperature-pressure phase diagrams for these PE bilayer membranes were constructed from the obtained phase-transition data. The saturated PE bilayer membranes underwent two different phase transitions related to the liquid crystalline (Lα) phase, the transition from the hydrated crystalline (Lc) phase and the chain melting (gel (Lß) to Lα) transition, depending on the thermal history. Pressure altered the gel-phase stability of the bilayer membranes of PEs with longer chains at a low pressure. Comparing the thermodynamic quantities of the saturated PE bilayer membranes with those of diacylphosphatidylcholine (PC) bilayer membranes, the PE bilayer membranes showed higher phase-transition temperatures and formed more stable Lc phase, which originates from the strong interaction between polar head groups of PE molecules. On the other hand, the unsaturated PE bilayer membranes underwent the transition from the Lα phase to the inverted hexagonal (HII) phase at a high temperature and this transition showed a small transition enthalpy but high pressure-responsivity. It turned out that the kind of double bonds markedly affects both bilayer-bilayer and bilayer-nonbilayer transitions and the Lα/HII transition is a volume driven transition for the reconstruction of molecular packing. Further, the phase-transition behavior was explained by chemical potential curves of bilayer phases.

Effect of pressure on bilayer phase behavior of N-methylated di-O-hexadecylphosphatidylethanolamines: relevance of head-group modification on the bilayer interdigitation.

The phase transitions of N-methylated di-O-hexadecylphosphatidylethanolamines (DHPE, DH-N-methyl-PE (DHMePE) and DH-N,N-dimethyl-PE (DHMe2PE)) were observed by differential scanning calorimetry (DSC) and fluorometry under atmospheric pressure and by light-transmittance measurements under high pressure. The DSC thermograms showed that the N-methylated DHPE bilayers underwent the phase transition from the gel phase to the liquid crystalline (Lα) phase under atmospheric pressure. The gel phase was identified by fluorometry as the lamellar gel (Lß) phase, and not interdigitated gel (LßI) phase. The gel/Lα transition temperature increased with pressure while decreased stepwise with increasing polar head-group size. This stepwise depression of the transition temperature may be caused by the inverse-proportional hydrogen-bonding capabilities of the head-group to the head-group size. The thermodynamic quantities of the gel/Lα transition were comparable for the N-methylated DHPE bilayers. The pressure-induced LßI phase was not found in these bilayers although the bilayer of di-O-hexadecylphosphatidylcholine (DHPC), which is a kind of N-methylated DHPEs, forms the LßI phase only by hydration under atmospheric pressure. Taking into account that the bilayers of diacyl-homologs of N-methylated DHPEs, N-methylated dipalmitoyl-PEs except for dipalmitoylphosphatidylcholine (DPPC), do not form the LßI phase in the whole pressure range investigated but the DPPC bilayer forms the LßI phase under high pressure, we can say that the interdigitation requires weaker interaction between large-sized head groups like the bulky choline group.

How Do Membranes Respond to Pressure?

Bilayers formed by phospholipids are fundamental structures of biological membranes. The mechanical perturbation brought about by pressure significantly affects the membrane states of phospholipid bilayers. In this chapter, we focus our attention on the pressure responsivity for bilayers of some major phospholipids contained in biological membranes. At first, the membrane states and phase transitions of phospholipid bilayers depending on water content, temperature and pressure are explained by using the bilayer phase diagrams of dipalmitoylphosphatidylcholine (DPPC), which is the most familiar phospholipid in model membrane studies. Subsequently, the thermotropic and barotropic bilayer phase behavior of various kinds of phospholipids with different molecular structures is discussed from the comparison of their temperature--pressure phase diagrams to that of the DPPC bilayer. It turns out that a slight change in the molecular structure of the phospholipids produces a significant difference in the bilayer phase behavior. The systematic pressure studies on the phase behavior of the phospholipid bilayers reveal not only the pressure responsivity for the bilayers but also the role and meaning of several important phospholipids existing in real biological membranes.

Comprehensive characterization of temperature- and pressure-induced bilayer phase transitions for saturated phosphatidylcholines containing longer chain homologs.

Complete elucidation of the phase behavior of phospholipid bilayers requires information on the subtransition from the lamellar crystal (Lc) phase to the gel phase. However, for bilayers of saturated diacylphosphatidylcholines (CnPCs), especially longer chain homologs, equilibration in the Lc phase is known to be very slow. In this study, bilayer phase transitions of three CnPCs with longer acyl chains, C19PC, C20PC and C21PC, were observed by differential scanning calorimetry under atmospheric pressure and by light-transmittance measurements under high pressure. Using lipid samples treated by thermal annealing enabled the observation of the sub-, pre- and main transitions of the C19PC and C20PC bilayers under atmospheric pressure. Only the pre- and main transitions could be observed for the C21PC bilayer due to very slow kinetics of the Lc phase formation for lipids with long acyl chains. The temperature and pressure phase diagrams constructed and phase-transitions quantities (enthalpy, entropy and volume changes) evaluated for these bilayers were compared with one another and with those of bilayers of the CnPC homologs examined in previous studies. These results allowed us (1) to clarify the temperature- and pressure-dependent phase sequence and phase stability of the CnPC (n=12-22) bilayers as a function of the hydrophobicity of the molecules, (2) to prove the presence of a shorter and a longer limit (n=13 and 21) in the acyl chain length for the pressure-induced bilayer interdigitation and (3) to reveal the chain-length dependence of the thermodynamic quantities of the subtransitions including the volume change.

Thermotropic phase behavior of hydrogenated soybean phosphatidylcholine-cholesterol binary liposome membrane.

By combination of differential scanning calorimetry (DSC) and fluorescence spectroscopy of 6-propionyl-2-(dimethylamino)naphthalene (Prodan), we elucidated the thermotropic phase behavior of hydrogenated soybean phosphatidylcholine (HSPC)-cholesterol binary liposome membrane which has similar lipid composition to Doxil®, the widely used liposome product in treatment of various tumors. We found that the characteristic points at cholesterol mole fraction (Xch)=0.023 and 0.077 correspond to the hexagonal lattice, in which cholesterol molecules are considered to be regularly distributed in all regions of HSPC lipid bilayer with 1 : 42 and 1 : 12 units, respectively, as static averaged structures. Apparent endothermic peak disappeared at Xch=0.40 in the DSC thermograms, indicating the existence of single liquid ordered phase at Xch>0.40. In addition, fluorescence measurements of Prodan and its lauroyl derivative in poly(ethylene glycol) (PEG)-modified liposomes indicated that PEG modification has a negligible effect on the phase behavior of HSPC-cholesterol binary liposome membrane. These results may provide useful information in developing novel liposome products whose stability and encapsulated drug release are controlled.

Possible role of inter-domain salt bridges in oligopeptidase B from Trypanosoma brucei: critical role of Glu172 of non-catalytic ß-propeller domain in catalytic activity and Glu490 of catalytic domain in stability of OPB.

Oligopeptidase B (OPB) is a member of the prolyl oligopeptidase (POP) family of serine proteases. OPB in trypanosomes is an important virulence factor and potential pharmaceutical target. Characteristic structural features of POP family members include lack of a propeptide and presence of a ß-propeller domain (PD), although the role of the ß-PD has yet to be fully understood. In this work, residues Glu(172), Glu(490), Glu(524) and Arg(689) in Trypanosoma brucei OPB (Tb OPB), which are predicted to form inter-domain salt bridges, were substituted for Gln and Ala, respectively. These mutants were evaluated in terms of catalytic properties and stability. A negative effect on kcat/Km was obtained following mutation of Glu(172) or Arg(689). In contrast, the E490Q mutant exhibited markedly decreased thermal stability, although this mutation had less effect on catalytic properties compared to the E172Q and R689A mutants. Trypsin digestion showed that the boundary regions between the ß-PD and catalytic domains (CDs) of the E490Q mutant are unfolded with heat treatment. These results indicated that Glu(490) in the CD plays a role in stabilization of Tb OPB, whereas Glu(172) in the ß-PD is critical for the catalytic activity of Tb OPB.

How does acyl chain length affect thermotropic phase behavior of saturated diacylphosphatidylcholine-cholesterol binary bilayers?

Thermotropic phase behavior of diacylphosphatidylcholine (CnPC)-cholesterol binary bilayers (n=14-16) was examined by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry. The former technique can detect structural changes of the bilayer in response to the changes in polarity around Prodan molecules partitioned in a relatively hydrophilic region of the bilayer, while the latter is sensitive to the conformational changes of the acyl chains. On the basis of the data from both techniques, we propose possible temperature T-cholesterol composition Xch phase diagrams for these binary bilayers. A notable feature of our phase diagrams, including our previous results for diheptadecanoylphosphatidylcholine (C17PC) and distearoylphosphatidylcholine (C18PC), is that there is a peritectic-like point around Xch=0.15, which can be interpreted as indicating the formation of a 1:6-complex of cholesterol and CnPCs within the binary bilayer irrespective of the acyl chain length. We could give a reasonable explanation for such complex formation using the modified superlattice view. Our results also showed that the Xch value of the abolition of the main transition is almost constant for n=14-17 (ca. 0.33), while it increases to ca. 0.50 for n=18. By contrast, a biphasic n-dependence of Xch was observed for the abolition of the pretransition, suggesting that there are at least two antagonistic n-dependent factors. We speculate that this could be explained by the enhancement of the van der Waals interaction with increases in n and the weakening of the repulsion between the neighboring headgroups with decreases in n.

Thermotropic and barotropic phase behavior of phosphatidylcholine bilayers.

Bilayers formed by phospholipids are frequently used as model biological membranes in various life science studies. A characteristic feature of phospholipid bilayers is to undergo a structural change called a phase transition in response to environmental changes of their surroundings. In this review, we focus our attention on phase transitions of some major phospholipids contained in biological membranes, phosphatidylcholines (PCs), depending on temperature and pressure. Bilayers of dipalmitoylphosphatidylcholine (DPPC), which is the most representative lipid in model membrane studies, will first be explained. Then, the bilayer phase behavior of various kinds of PCs with different molecular structures is revealed from the temperature-pressure phase diagrams, and the difference in phase stability among these PC bilayers is discussed in connection with the molecular structure of the PC molecules. Furthermore, the solvent effect on the phase behavior is also described briefly.

Study on the subgel-phase formation using an asymmetric phospholipid bilayer membrane by high-pressure fluorometry.

The myristoylpalmitoylphosphatidylcholine (MPPC) bilayer membrane shows a complicated temperature-pressure phase diagram. The large portion of the lamellar gel (L(ß)'), ripple gel (P(ß)'), and pressure-induced gel (L(ß)I) phases exist as metastable phases due to the extremely stable subgel (L(c)) phase. The stable L(c) phase enables us to examine the properties of the L(c) phase. The phases of the MPPC bilayers under atmospheric and high pressures were studied by small-angle neutron scattering (SANS) and fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan. The SANS measurements clearly demonstrated the existence of the metastable L(ß)I phase with the smallest lamellar repeat distance. From a second-derivative analysis of the fluorescence data, the line shape for the L(c) phase under high pressure was characterized by a broad peak with a minimum of ca. 460 nm. The line shapes and the minimum intensity wavelength (λ″(min)) values changed with pressure, indicating that the L(c) phase has highly pressure-sensible structure. The λ″(min) values of the L(c) phase spectra were split into ca. 430 and 500 nm in the L(ß)I phase region, which corresponds to the formation of a interdigitated subgel L(c) (L(c)I) phase. Moreover, the phase transitions related to the L(c) phase were reversible transitions under high pressure. Taking into account the fluorescence behavior of Prodan for the L(c) phase, we concluded that the structure of the L(c) phase is highly probably a staggered structure, which can transform into the L(c)I phase easily.

Volumetric characterization of ester- and ether-linked lipid bilayers by pressure perturbation calorimetry and densitometry.

We investigated the thermotropic volume behavior of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and dihexadecylphosphatidylcholine (DHPC) membranes using pressure perturbation calorimetry (PPC) and densitometry. The ln φ(2) vs temperature curves (φ(2): apparent molar volume of phospholipid) obtained from the PPC data using an analysis method that we developed agreed with the results from the density measurements for these lipids within the relative difference of about 0.62%. From those curves, the volume changes with the main transition were estimated at 18.0±0.49, 23.5±2.33 and 23.0±0.33 cm(3) mol(-1) for DMPC, DPPC and DHPC, respectively. For DPPC and DMPC, the average volume per methylene group of the hydrocarbon chains v(CH2) calculated by referring to the procedure by Nagle and Wilkinson was consistent with the previous result, which indicates that the DPPC bilayer in the gel state has denser hydrophobic bilayer core than the DMPC bilayer. For DHPC, the volume of the headgroup region v(H) was calculated to be 244 Å(3) by assuming that v(CH2) of DHPC equals that of DPPC above 45°C. This value was comparable to that of DPPC when the volume of the carbonyl groups was considered, which may signify that there is no significant conformational difference in the polar headgroups of both phospholipids. However, it was suggested from the consideration on v(H) of DHPC at 20°C that expansion of the headgroup region should occur as the interdigitated structure is formed, which means some conformational change of the headgroup region is induced by the interdigitation.

Thermotropic and barotropic phase transitions of dialkyldimethylammonium bromide bilayer membranes: effect of chain length.

The bilayer phase transitions of dialkyldimethylammonium bromides (2C(n)Br; n = 12, 14, 16) were observed by differential scanning calorimetry and high-pressure light-transmittance measurements. Under atmospheric pressure, the 2C(12)Br bilayer membrane underwent the stable transition from the lamellar crystal (L(c)) phase to the liquid crystalline (L(α)) phase. The 2C(14)Br bilayer underwent the main transition from the metastable lamellar gel (L(ß)) phase to the metastable L(α) phase in addition to the stable L(c)/L(α) transition. For the 2C(16)Br bilayer, moreover, three kinds of phase transitions were observed: the metastable main transition, the metastable transition from the metastable lamellar crystal (L(c(2))) phase to the metastable L(α) phase, and the stable lamellar crystal (L(c(1)))/L(α) transition. The temperatures of all the phase transitions elevated almost linearly with increasing pressure. The temperature (T)-pressure (p) phase diagrams of the 2C(12)Br and 2C(14)Br bilayers were simple, but that of the 2C(16)Br bilayer was complex; that is, the T-p curves for the metastable main transition and the L(c(2))/L(α) transition intersect at ca. 25 MPa, which means the inversion of the relative phase stability between the metastable phases of L(ß) and L(c(2)) above and below the pressure. Moreover, the T-p curve of the L(c(2))/L(α) transition was separated into two curves under high pressure, and as a result, the pressure-induced L(c(2P)) phase appeared in between. Thermodynamic quantities for phase transitions of the 2C(n)Br bilayers increased with an increase in alkyl-chain length. The chain-length dependence of the phase-transition temperature for all kinds of transitions observed suggests that the stable L(c(1))/L(α) transition incorporates the metastable L(c(2))/L(α) transition in the bilayers of 2C(n)Br with shorter alkyl chains, and the main-transition of the 2C(12)Br bilayer would occur at a temperature below 0 °C.

Hydrostatic pressure reveals bilayer phase behavior of dioctadecyldimethylammonium bromide and chloride.

Bilayer phase transitions of dioctadecyldimethylammonium bromide (2C(18)Br) and chloride (2C(18)Cl) were observed by differential scanning calorimetry and high-pressure light-transmittance measurements. The 2C(18)Br bilayer membrane showed different kinds of transitions depending on preparation methods of samples under atmospheric pressure. Under certain conditions, the 2C(18)Br bilayer underwent three kinds of transitions, the metastable transition from the metastable lamellar crystal (L(c(2))) phase to the metastable lamellar gel (L(ß)) phase at 35.4 °C, the metastable main transition from the metastable L(ß) phase to the metastable liquid crystalline (L(α)) phase at 44.5 °C, and the stable transition from the stable lamellar crystal (L(c(1))) phase to the stable L(α) phase at 52.8 °C. On the contrary, the 2C(18)Cl bilayer underwent two kinds of transitions, the stable transition from the stable L(c) phase to the stable L(ß) phase at 19.7 °C and the stable main transition from the stable L(ß) phase to the stable L(α) phase at 39.9 °C. The temperatures of the phase transitions of the 2C(18)Br and 2C(18)Cl bilayers were almost linearly elevated by applying pressure. It was found from the temperature (T)-pressure (p) phase diagram of the 2C(18)Br bilayer that the T-p curves for the main transition and the L(c(1))/L(α) transition intersect at ca. 130 MPa because of the larger slope of the former transition curve. On the other hand, the T-p phase diagram of the 2C(18)Cl bilayer took a simple shape. The thermodynamic properties for the main transition of the 2C(18)Br and 2C(18)Cl bilayers were comparable to each other, whereas those for the L(c(1))/L(α) transition of the 2C(18)Br bilayer showed considerably high values, signifying that the L(c(1)) phase of the 2C(18)Br bilayer is extremely stable. These differences observed in both bilayers are attributable to the difference in interaction between a surfactant and its counterion.

Chain elongation of diacylphosphatidylcholine induces fully bilayer interdigitation under atmospheric pressure.

The phase transitions of dibehenoylphosphatidylcholine (C22PC) bilayer membrane were observed by differential scanning calorimetry under atmospheric pressure and light-transmittance measurements under high pressure. The constructed temperature-pressure phase diagram suggests that the gel phase at low temperatures is the interdigitated gel phase. To confirm the phase state, we performed small-angle neutron scattering and fluorescence measurements using a polarity-sensitive probe Prodan for the C22PC bilayer membrane under atmospheric pressure. The peaks obtained in both measurements clearly showed the characteristic patterns of the fully interdigitated gel phase. Taking into account of previous studies on the gel phase for long-chain PC bilayers under atmospheric pressure and our studies on the pressure-induced bilayer interdigitaion of diacyl-PCs, it turned out that the interdigitation of diacyl-PC bilayer membranes occurs when the carbon number of acyl chain reaches at least 22. The present study revealed that the interdigitation of PC bilayer membranes occurs not only by weakening the attractive force of polar head groups but also by strengthening the cohesive force of acyl chains. When dominating the force of acyl chains, the interdigitation can be induced even in a diacyl-PC bilayer membrane by only hydration under atmospheric pressure.