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. Therefore, further detailed studies on the molecular behavior of Prodan will enable us to find a more useful way of utilizing this membrane probe.


Temperature- and Pressure-Induced Bilayer Phase Transitions of an Amide-Linked Phosphatidylcholine: A Contrasting Effect of Chain Linkage Type

An unnatural phospholipid with two amide linkages, dipalmitoylamidodeoxyphosphatidylcholine (DPADPC), was synthesized and its bilayer phase transitions were observed by differential scanning calorimetry under atmospheric pressure and light-transmittance measurements under high pressure. The DPADPC bilayer membrane showed different phase transitions depending on the thermal history of the lipid sample under atmospheric pressure. The thermal-history dependent transitions were also observed under high pressure. The temperature pressure phase diagram and thermodynamic quantities of the phase transitions of the DPADPC bilayer membrane were compared with those of bilayer membranes of hydrophobic chain-linkage isomers, ester-liked dipalmitoylphosphatidylcholine (DPPC) and ether-linked dihexadecylphosphatidylcholine (DHPC). The thermodynamic quantities of the main transition for these PC bilayer membranes were similar to each other, whereas the suppression of the interdigitation and great stabilization of the hydrated crystal phase were observed for the DPADPC bilayer membrane. Furthermore, the results of nuclear magnetic resonance measurements indicated that the motility of PC molecules in both gel and liquid crystal phases of the DPADPC bilayer membranes were much lower than those of the DPPC and DHPC bilayer membranes. From the above results, we concluded that the molecular interaction in the PC bilayer membranes increases in the order of ether-, ester- and amide-linkage isomers.

Bulletin of the Chemical Society of Japan


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, dioleoylphosphatidylethanolamine (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.


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 could be 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 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 bilayer subgel phase with staggered structure.


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 (Pβ') and liquid crystalline (Lα) phases 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.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Salt effect on bilayer phase transitions of dipalmitoylphosphatidylglycerol in saline water under high pressure

The phase transitions of dipalmitoylphosphatidylglycerol (DPPG) bilayer membrane at high NaCl concentrations under high pressure were investigated to construct the temperature pressure phase diagram and determine phase-transition properties. The constructed phase diagrams exhibited the qualitative resemblance to that of the dipalmitoylphosphatidylcholine (DPPC) bilayer membrane: they underwent the gel-phase polymorphism including the pressure-induced bilayer interdigitation. The phase-transition properties of the DPPG bilayer membrane changed in a salt concentration-dependent manner, whereas qualitatively the phase behavior is not affected appreciably by the salt concentration measured. We discussed the salt effect on the DPPG bilayer membrane from the variation in interactions between the polar head group of the PG molecule.


Phase Behavior of Binary Bilayer Membrane of Dipalmitoylphosphatidylcholine and Stigmasterol

We investigated the phase behavior of a binary bilayer membrane of dipalmitoylphosphatidylcholine (DPPC) and stigmasterol (Stig) as a function of the Stig concentration (XSt) by means of differential scanning calorimetry (DSC) and fluorescence spectroscopy using 6-propionyl-2-(N,N-dimethylamino)naphthalene (Prodan) as a membrane probe. The DSC results revealed that the pretransition and the main transition are abolished at XSt = ca. 0.08 and ca. 0.50, respectively. The main transition enthalpy DeltaHm of the DPPC Stig binary bilayer decreased with XSt in a non-linear manner, which is contrast to the almost linear decrease in DeltaHm for the DPPC cholesterol (Chol) binary bilayer. This suggests that Stig has a stronger effect of disturbing the chain packing in a gel state and a weaker effect of suppressing the chain melting as compared to Chol. The XSt dependence of the peak width at half height (DeltaT1/2) suggests that a drastic change in the phase behavior occurs at XSt = ca. 0.14, which is similar to the DPPC Chol binary bilayer. The Prodan fluorescence spectra changed depending on the phase state, which was characterized by the wavelength at the emission maximum and allowed us to determine the transition temperatures of the binary bilayers. The compositional phase diagram of the DPPC Stig binary bilayer was found to be similar to that of the DPPC Chol binary bilayer. The phase behavior of the DPPC Stig binary bilayer could be well explained by applying the so-called superlattice view to interpret the phase diagram.

リン脂質ジパルミトイルホスファチジルコリン-スティグマステロール混合二分子膜の熱的相挙動を示差走査熱量測定と蛍光プローブ測定から調査した．本系に対して得られた温度-濃度相図はジパルミトイルホスファチジルコリン-コレステロール混合二分子膜のものと類似していたが，スティグマステロールはコレステロールに比べて，ゲル相では脂質アシル鎖に大きな撹乱効果を示す一方で，鎖融解の抑制効果は弱いことが明らかとなった．

Journal of Thermal Analysis and Calorimetry


Phase Behavior of Cholesterol-Containing Binary Membrane of an Ether-Linked Phospholipid, Dihexadecylphosphatidylcholine

The thermotropic phase behavior of the cholesterol (Chol)-containing binary membrane of dihexadecylphosphatidylcholine (DHPC) was investigated as a function of the mole fraction of Chol Xch by DSC and fluoresce spectroscopy using 6-propionyl-2-(N,N-dimethylamino)naphthalene (Prodan). The main-transition enthalpy Hm was almost constant at Xch < ca. 0.05, but it decreased almost linearly with increasing Xch above 0.05 until it reached 0 at Xch = ca. 0.50, suggesting that the interaction between DHPC and Chol is relatively weaker than that between dipalmitoylphosphatidylcholine (DPPC) and Chol. The Prodan fluorescence spectra changed depending on the phase state, which was characterized by the wavelength at the emission maximum. Particularly, the two-phase state of the interdigitated (LβI) and non-interdigitated gel (Lβ) was sensitively detected as the superposition of the two elementary spectra corresponding to each phase state, allowing us to explain the Xch dependent non-linear behavior of the Chol-induced Lβ phase by introducing a proportionality factor ξ(Xch) and by assuming a simple probabilistic process. By comparing the phase diagram constructed for the DHPC Chol binary membrane with that for the DPPC Chol binary bilayer, a difference was observed in the minimum Xch value for the entire formation of the liquid ordered (Lo) phase. This difference could be explained in terms of the hydrophobic mismatch at the bilayer midplane by taking into account the difference in the capability of hydrogen bonding with the 3β-OH group of Chol due to the presence or absence of the carbonyl oxygens at the glycerol backbone.

エーテル結合型リン脂質ジヘキサデシルホスファチジルコリン-コレステロール混合二分子膜の熱的相挙動を示差走査熱量測定と蛍光プローブ測定から調査した．コレステロール添加は指組み構造ゲル相の形成を抑制した．エステル結合型脂質ジパルミトイルホスファチジルコリン-コレステロール混合二分子膜系の相図との比較から，脂質頭部のカルボニル炭素の有無が二分子膜中における両分子の混和性に大きな影響を与えることが明確となった．


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.

ジパルミトイルホスファチジルコリン(DPPC)二分子膜の相転移温度への2種類の膜活性リガンド(麻酔薬ハロセン，脂肪酸パルミチン酸)の効果を調べた．DPPC二分子膜の前転移および主転移は，ハロセン添加により降下，パルミチン酸添加により逆に上昇し，加圧は相転移を温度を共に上昇させた．熱力学的解析からハロセンは液晶相，パルミチン酸はラメラゲル相に親和性を有し，ハロセン添加により液晶相が膨張し，パルミチン酸添加によりゲル相が収縮することがわかった．また，ハロセンは高圧力下における指組みゲル相形成を促進し，パルミチン酸は逆に抑制した．指組みゲル相を含む相転移挙動へのリガンド効果についての議論が熱力学観点からなされた．


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 light-transmittance measurements under high pressure. The DSC thermograms showed that the N-methylated DHPE bilayers underwent the phase transitions from the gel phase to the liquid crystalline (Lα) phase under atmospheric pressure. The gel phase was identified by fluorometry as the lamellar gel (Lβ) phase, and not interdigitated gel (LβI) phase. The gel/Lα transition temperature increased with pressure while decreased stepwise with increasing polar head-group size. This stepwise depression in 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 LβI 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 LβI 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 LβI phase in the whole pressure range investigated but the DPPC bilayer forms the LβI phase under high pressure, we can say that the interdigitation requires a weaker interaction between large-sized head groups like the bulky choline group.

極性基サイズの異なる3種類のエーテル型リン脂質N-メチル化ジヘキサデシルホスファチジルエタノールアミンが形成する二分子膜の高圧相挙動を調査した．極性基サイズの増加と共に相転移温度は段階的に降下したが，相転移熱力学量は極性基サイズにはほとんど依存しなかった．指組み構造ゲル相は，これら脂質二分子膜では常圧，高圧下共に観測されなかった．エステル型リン脂質N-メチル化ジパルミトイルホスファチジルエタノールアミン二分子膜系との結果の比較から，指組み構造形成にはコリン基のような嵩高い極性基が必須であることが明らかとなった．


Thermotropic and barotropic phase transitions on diacylphosphatidylethanolamine bilayer membranes

The bilayer phase transitions of four diacylphosphatidylethanolamines (PEs) with identical two saturated acyl chains (Cn = 12, 14, 16 and 18) and two PEs with identical two 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 (main) transition, depending on the thermal history. Pressure induced the change in 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 diacylphosphatidylcholines (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 small thermodynamic quantities 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 profiles of bilayer phases.

4種類の飽和ホスファチジルエタノールアミン(PE)および2種類の不飽和PEが形成する二分子膜の温度および圧力誘起相転移を調査した．飽和PE二分子膜は熱履歴に依存して水和結晶相/液晶相転移およびゲル相/液晶相(主)転移を示し，長鎖飽和PE二分子膜においては高圧力下でゲル相の安定性が変化した．熱力学量から，飽和PE二分子膜は同鎖長のホスファチジルコリン(PC)二分子膜よりも顕著に安定な水和結晶相を形成することがわかった．他方，不飽和PE二分子膜は，液晶相から逆ヘキサゴナル相への二分子膜/非二分子膜転移を誘起した．熱力学的な考察から，この転移は体積駆動の転移であることを示した．


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.

疎水鎖長が異なる長鎖飽和ジアシルホスファチジルコリン(CnPC: n = 19 - 21)の温度および圧力誘起二分子膜相転移を，特に水和結晶(Lc)相に着目して調査した．C20PCまではLc相を観測することができたが，C21PCでは観測できなかった．得られた結果を過去になされたCnPC同族体のものと比較して，CnPC二分子膜の相挙動を包括的に考察した．結果，CnPC(n = 12 - 22)二分子膜の温度-圧力相図，各種の相転移(副転移，前転移，主転移)に伴う熱力学量のアシル鎖長依存性および圧力誘起指組み構造形成の短鎖および長鎖限界など，CnPC二分子膜相挙動に関する有用な情報を得ることができた．


Pressure-induced interdigitation of lipid bilayer membranes: Dependence on Acyl-chain length and limitation of the formation

脂質膜の圧力誘起指組構造形成:疎水鎖長依存性と形成限界

We investigated the bilayer interdigitation of diacylphosphatidylcholines (CnPCs) with long acyl chains (n = 19 - 22) under high pressure. The temperature-pressure phase diagrams of the CnPC bilayers were constructed by the methods of differential scanning calorimetry and high-pressure light transmittance. We confirmed the bilayer interdigitation for CnPCs (n = 19 - 21) under high pressure. On the other hand, the C22PC bilayer showed peculiar behavior: two transitions were observed under atmospheric pressure while one transition under high pressure. To identify the low-temperature phase of the C22PC bilayer, we performed small angle neutron scattering measurements under atmospheric pressure and fluorescence measurements under high pressure. From these results, the phase was identified as the interdigitated gel (LβI) phase. Considering our previous results, it turned out that the acyl-chain length of CnPC that exhibits the pressure-induced interdigitation is limited within n = 14 - 21. The present results definitely indicate that the bilayer interdigitation is governed by the hydrophobic interaction as well as the hydrophilic interaction of the PC molecule in the bilayer.

Journal of the Society of Materials Science, Japan


How Does Acyl-chain Length Affect Thermotropic Phase Behavior of Saturated Diacylphosphatidylcholine-chloresterol 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 C17PC and 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.

Biochimica et Biophysica Acta (BBA) - Biomembranes


An Attempt to Reveal Viscoelastic Behavior of Lipid Bilayer Membrane by Pressure Perturbation Calorimetry


Thermotropic Phase Behavior of Binary Bilayer Membrane of Dipalmitoylphosphatidylcholine and Ergosterol


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(β)'), ripple gel (P(β)'), and pressure-induced gel (L(β)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(β)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(β)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.


Phase Behavior of Dipalmitoylphosphatidylglycerol Bilayer Membrane in Saline Water under High Pressure

International Journal of Modern Physics: Conference Series


Volumetric Study on Dioleoylphosphatidylcholine Bilayer by Pressure Perturbation Calorimetry


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.


Morphological Change of Vesicle Particle can Produce a Peculir Stepwise Transition in Dipalmitoylphosphatidylglycerol Bilayer at High NaCl Concentration


Imaging of Phosphatidylcholine Bilayers by a High-Pressure Fluorescence Technique: Detection of the Packing Difference

The packing states in phosphatidylcholine (PC) bilayers under high pressure were studied from fluorescence measurements using a polarity-sensitive probe Prodan. By using the Prodan fluorescence data, three-dimensional image plots, which can visually evaluate the bilayer packing, were constructed for the C16PC bilayer. It turned out from the shape of contours in the image plot constructed by the second-derivative spectra that the Prodan molecules can be distributed into multiple sites in the bilayer and move around the head-group region depending on the phase state. Furthermore, comparing the plot to those for the C17PC and C18PC bilayers, the slight difference in hydrophobicity among the PC molecules produced a marked difference in quantity of the Prodan molecules distributed among the bilayer gel, nonbilayer gel, and liquid crystalline phases of the PC membranes. The present study shows that the imaging using Prodan is useful for the evaluation of lipid bilayer packing.

Bulletin of the Chemical Society of Japan


Mucosal immune features to phosphorylcholine by nasal Flt3 ligand cDNA-based vaccination

Phosphorylcholine (PC) is an immunodominant epitope in some pathogens including Streptococcus pneumoniae and it is well-known that PC-specific antibodies (Abs) play a key role in the induction of protective immunity against pneumococcal infection. In this study, we examined whether nasal administration of DNA plasmid encoding Flt3 ligand gene (pFL) as a mucosal adjuvant plus PC-conjugated keyhole limpet hemocyanin (PC-KLH), would elicit PC-specific immune responses, and characterized mucosal immune responses to PC induced by this nasal vaccination. Nasal immunization with pFL plus PC-KLH enhanced induction of PC-specific IgA and IgM Abs in airway secretions when compared with mice given PC-KLH with or without empty plasmid gene (pORF) as controls; in addition to the mucosal immune responses, PC-specific immune responses in serum were also induced. Furthermore, the mucosal and serum IgA and IgM Abs in mice given pFL plus PC-KLH nasally, exhibited high-specificity for the PC molecule. Of interest, the PC-specific Abs bound dose-dependently to anti-T15 idiotype (AB1-2). Thus, the inhibition of S. pneumoniae colonization on the nasal cavity and lungs after nasal challenge with the live organism was significantly elicited in mice immunized with pFL plus PC-KLH compared to that of mice immunized with antigen with pORF. Taken together, these findings show that nasal administration of pFL with PC-KLH elicited T15-like anti-PC IgA and IgM Abs in the respiratory tracts, and further attenuated S. pneumoniae colonization on the respiratory tracts. Nasal administration of Flt3 ligand cDNA with PC may contribute to the development of nasal vaccination for prevention of S. pneumoniae infection.


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(β)) 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(β) 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.


Prodan fluorescence detects the bilayer packing of asymmetric phospholipids

The bilayer phase behavior of asymmetric phospholipids, palmitoylstearoylphosphatidylcholine (PSPC) and stearoylpalmitoylphosphatidylcholine (SPPC), with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under high pressure. The results were compared with those of a symmetric phospholipid, diheptadecanoyl PC (C17PC). The difference in phase transitions of the PSPC and SPPC bilayers and in thermodynamic quantities of the transitions was hardly observed between LMV and GMV as the case of the C17PC bilayer. On the other hand, the Prodan fluorescence showed clear differences between LMV and GMV of the asymmetric PC bilayers. From the second derivative of Prodan fluorescence spectra, the three dimensional image plots in which we can clearly see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under high pressure. We revealed from the plots that the bilayer packing is significantly dependent on not only the vesicle size but also the acyl-chain asymmetry of PC molecule in addition to the phase states. It was found that the packing of the gel phases of the asymmetric PC bilayers is weaker than that of the symmetric PC bilayer, and the size of vesicle affects the packing of the interdigitated gel phase the most markedly among three gel phases. This study suggests that the Prodan molecules can detect the effect of vesicle size on the phase states for the asymmetric PC bilayers, and they become a useful indicator for various membrane properties, especially bilayer interdigitation.


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.


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(β)) phase at 35.4 °C, the metastable main transition from the metastable L(β) 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(β) phase at 19.7 °C and the stable main transition from the stable L(β) 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.


Application of pressure perturbation calorimetry to the aqueous system of phospholipid vesicle dispersion


Packing state in bilayer membranes of diacylphosphatidylcholines with varying acyl chain lengths under high pressure


Effect of Vesicle Size on the Prodan Fluorescence in Diheptadecanoylphosphatidylcholine Bilayer Membrane under Atmospheric and High Pressures

The bilayer phase behavior of diheptadecanoylphosphatidylcholine (C17PC) with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under atmospheric and high pressures. The difference in phase transitions and thermodynamic quantities of the transition was hardly observed between LMV and GMV used here. On the contrary, the Prodan fluorescence in the bilayer membranes changed depending on the size of vesicles as well as on the phase states. From the second derivative of fluorescence spectra, the three-dimensional image plots in which we can see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under atmospheric pressure. The following characteristic behavior was found: (1) the Prodan molecules in GMV can be distributed to not only adjacent glycerol backbone region, but also near bulk-water region in the lamellar gel or ripple gel phase; (2) the blue valleys of GMV became deeper than those of LMV because of the greater surface density of the Prodan molecules per unit area of GMV than LMV; (3) the liquid crystalline phase of the bilayer excludes the Prodan molecules to a more hydrophilic region at the membrane surface with an increase in vesicle size; (4) the accurate information as to the phase transitions is gradually lost with increasing vesicle size. Under the high-pressure condition, the difference in Prodan fluorescence between LMV and GMV was essentially the same as the difference under atmospheric pressure except for the existence of the pressure-induced interdigitated gel phase. Further, we found that Prodan fluorescence spectra in the interdigitated gel phase were especially affected by the size of vesicles. This study revealed that the Prodan molecules can move around the headgroup region by responding not only to the phase state but also to the vesicle size, and they become a useful membrane probe, detecting important membrane properties such as the packing stress.


Recovery of Tobacco BY-2 Cells After Treatment of High Hydrostatic Pressure

The recovery of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells in Linsmaire and Skoog medium after treatment at high hydrostatic pressure was investigated using an Evans Blue staining method to discriminate live from dead cells. The survival of BY-2 cells just after the high-pressure treatment at 5 degrees C and 25 degrees C decreased abruptly at pressures higher than 50 MPa and 100 MPa, respectively. Furthermore, almost all of the BY-2 cells treated at 5 degrees C and 25 degrees C recovered pressures below 25 MPa and 75 MPa, respectively. However, no BY-2 cells recovered at pressures above 100 MPa at either temperature.

Annals of the New York Academy of Sciences


Pressure Study on Symmetric and Asymmetric Phospholipid Bilayers: Effect of Vesicle Size on Prodan Fluorescence

The bilayer phase behavior of symmetric and asymmetric phosphatidylcholines (PCs), 1,2-diheptadecanoyl-PC (C17PC), 1-palmitoyl-2-stearoyl-PC (PSPC), and 1-stearoyl-2-palmitoyl-PC (SPPC), with different vesicle sizes were investigated by a high-pressure fluorescence method using the polarity-sensitive fluorescent probe Prodan. The second derivative of fluorescence spectra for all the PCs of small-sized vesicle showed four minima characteristic of four membrane states on the spectra irrespective of the acyl-chain symmetry, whereas those of large-sized vesicle had one more minimum originating from the most hydrophilic site at the membrane surface. These findings indicate that Prodan molecules can distribute into multiple sites in the bilayer and move around the head-group region depending on the vesicle size. The behavior of the spectra in the SPPC bilayer suggested that the interdigitated gel phase had a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains. It turned out that the curvature of vesicles affects the distribution of the Prodan molecules in all phases, more particularly in the interdigitated gel phase.


Pressure Effect on the Bilayer Phase Transition of Asymmetric Lipids with an Unsaturated Acyl Chain

The bilayer phase transitions of mixed-chain lipids with monounsaturated acyl chain in the sn-2 position, 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and with a polyunsaturated acyl chain in the sn-2 position, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC), and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC), were observed by differential scanning calorimetry (DSC) under ambient pressure and by light-transmittance measurements under high pressure. The DSC thermogram for each lipid bilayer showed only one transition between the lamellar gel and liquid crystalline phases. The introduction of one or two cis double bonds into the sn-2 acyl chain caused the significant depression of the main-transition temperature and an obvious decrease of enthalpy and volume changes associated with the transition. These features are attributable to loose packing of saturated and unsaturated acyl chains in the bilayer gel phase. The existence of four or six double bonds in the sn-2 chain produced no further decrease in the transition temperature, and in fact six double bonds caused a slight increase in the transition temperature. Thermodynamic properties associated with the bilayer phase transition were discussed.


A critical role for highly conserved Glu610 residue of oligopeptidase B Tryoanosoma brucei in thermal stability

Oligopeptidase B from Trypanosoma brucei (Tb OPB) is a virulence factor and therapeutic target in African sleeping sickness. Three glutamic acid residues at positions 607, 609 and 610 of the catalytic domain are highly conserved in the OPB subfamily. In this study, the roles of Glu(607), Glu(609) and Glu(610) in Tb OPB were investigated by site-directed mutagenesis. A striking effect on k(cat)/K(m) was obtained following mutation of Glu(607) to glutamine. In contrast, the heat stability of Tb OPB decreased markedly following the single mutation of Glu(610) to glutamine, although this mutation had significantly less effect on catalytic properties compared with the Glu(607) mutation. Although no differences were found in the tertiary and secondary structures between wild-type (WT) OPB and the E610Q mutant prior to heat treatment, the E610Q mutant is inactivated more rapidly than WT OPB following heat treatment in a manner correlating with its attendant structural changes. Trypsin digestion showed that the boundary regions between the beta-propeller and catalytic domain of the E610Q mutant are unfolded with heat treatment. It is concluded that Glu(607) is essential for the catalytic activity of Tb OPB and that Glu(610) plays a critical role in stabilization rather than catalytic activity despite their close proximity.


A Peculiar Phase Transition of Plasmalogen Bilayer Membrane under High Pressure

The bilayer phase transition of plasmalogen, monounsaturated plasmenylcholine 1-O-1'-(Z)-octadecenyl-2-oleoyl-sn-glycero-3-phosphocholine (Plg-SOPC), was examined by differential scanning calorimetry, high-pressure transmittance, and fluorescence techniques. The bilayer properties of Plg-SOPC such as the temperature-pressure phase diagram, the thermodynamic quantities of the transition, and the location of a fluorescent membrane probe in the bilayer, were compared with those of a similar phospholipid 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). It turned out that a vinyl-ether bond in the sn-1 position of the glycerol backbone in the Plg-SOPC molecule produces a peculiar phase transition under high pressure and significantly affects the membrane properties.


Chain asymmetry alters thermotropic and barotropic properties of phospholipid bilayer membranes

The alignment of the sn-1 and sn-2 acyl chains at the terminal methyl ends generally produces significant influence on the thermodynamic properties of the bilayer phase transitions. We investigated the bilayer phase behavior of asymmetric phospholipids, myristoylpalmitoylphosphatidylcholine and palmitoylmyristoylphosphatidylcholine, by high-pressure light-transmittance and Prodan-fluorescence techniques and differential scanning calorimetry. Constructed temperature-pressure phase diagrams revealed that no stable Lbeta' phase can exist in the whole pressure range because of the formation of the most stable Lc phase. Nevertheless, the pretransition, the detection of which is severely hampered by the exceptionally prompt formation of the Lc phase, was successfully observed. Moreover, the effect of the total and difference of the sn-1 and sn-2 acyl chain lengths on minimal interdigitation pressure (MIP) was summarized in a MIP vs. chain-length inequivalence parameter plot, where the effect was proved to be classified in three zones depending on the alignment of both terminal methyl ends.


Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L(beta)) and liquid crystalline (L(alpha)) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L(c)) to the L(beta) (or L(alpha)) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L(c)/L(alpha) transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L(c) phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Interaction modes of long-chain fatty acids in dipalmitoylphosphatidylcholine bilayer membrane: contrast to mode of inhalation anesthetics

The effects of long-chain fatty acids (four saturated and two unsaturated fatty acids, one derivative) on phase transitions of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were examined in the low concentration region, and the results were compared with those for an inhalation anesthetic. The effects of all fatty acids on the pre- and main-transition temperatures of the DPPC bilayer membrane appeared in the concentration range of microM order while that of the anesthetic appeared in the mM order. The appearance modes of these ligand actions were significantly different from one another. The three differential partition coefficients of the ligands between two phases of the DPPC bilayer membrane were evaluated by applying the thermodynamic equation to the variation of the phase-transition temperatures. The DPPC bilayer membranes showed the different receptivity for the ligands; the saturated fatty acids had an affinity for gel phase whereas unsaturated fatty acids and an anesthetic had an affinity for liquid-crystalline phase to the contrary. In particular, the receptivity for the ligands in the gel phase markedly changed depending on kinds of ligands. The interaction modes between the DPPC and fatty acid molecules in the gel phase were considered from the hexagonal lattice model. The disappearance compositions of the pretransition by the fatty acids coincided with the compositions at which the membrane is all covered by the units in each of which two fatty acids molecules are regularly distributed in the hexagonal lattice in a different way, and the distribution depended on the chain length and existence of a double bond for the fatty acids. The interpretation did not hold for the case of the anesthetic at all, which proved that a number of anesthetic molecules act the surface region of the bilayer membrane nonspecifically. The present study clearly implies that DPPC bilayer membranes have high ability to recognize kinds of ligand molecules and can discriminate among them with specific interaction by the membrane states.


High-pressure study on bilayer phase behavior of oleoylmyristoyl- and myristoyloleoyl-phosphatidylcholines

We investigated the thermotropic and barotropic bilayer phase behavior of 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC) and 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (OMPC) by means of the differential scanning calorimetry (DSC) and high-pressure light-transmittance technique. Water could be used as a solvent for measurements at high pressures because of the elevation of the transition temperatures above 0 degrees C by pressurization, whereas aqueous 50 wt.% ethylene glycol solution was used mainly for those at low pressures. Only one phase transition was observed in the DSC thermogram of the MOPC bilayer membrane as an endothermic peak, and also observed at high pressures as an abrupt change of the light-transmittance. The transition was assigned as a main transition between the lamellar gel (L(beta)) and liquid-crystalline (L(alpha)) phases on the basis of the values of enthalpy change (DeltaH) and slope of the transition temperature with respect to pressure (dT/dP). The DSC thermogram of the OMPC bilayer membrane similarly showed a single endothermic peak but two kinds of phase transitions were observed at different temperatures in the light-transmittance profile at high pressures. The extrapolation of the lower-temperature transition in the high-pressure range to an ambient pressure coincided with the transition observed in the DSC thermogram. This transition was identified as a transition between the lamellar crystal (L(c)) and L(alpha) (or L(beta)) phases from the DeltaH and dT/dP values. The higher-temperature transition, appearing only at high pressures, was identified as the L(beta)/L(alpha) transition considering the topological resemblance of its temperature-pressure phase diagram as that of the dioleoylphosphatidylcholine bilayer membrane. The phase diagram of the OMPC bilayer membrane demonstrated that the L(beta) phase cannot exist at pressures below ca. 190 MPa while it can exist stably in a finite temperature range at pressures above the pressure.


Lateral phase separation in cholesterol/diheptadecanoylphosphatidylcholine binary bilayer membrane

We investigated the phase behavior of cholesterol/diheptadecanoylphosphatidylcholine (C17:0-PC) binary bilayer membrane as a function of the cholesterol composition (X(ch)) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra showed that the wavelength at the maximum intensity (lambda(max)) changed depending on the bilayer state: ca. 440 nm for the lamellar gel (L'(beta) or L(beta)) and the liquid ordered (L(o)) phases and ca. 490 nm for the liquid-crystalline (L(alpha)) phase. The transition temperatures were determined from the temperature dependence of lambda(max) and endothermic peaks of the DSC thermograms. Both measurements showed that the pre- and main transition disappear around X(ch)=0.05 and 0.30, respectively. The constructed temperature-X(ch) phase diagram resembled a typical phase diagram for a eutectic binary mixture containing a peritectic point. The presence of a peritectic point at X(ch)=0.15 suggested that a complex of cholesterol and C17:0-PC is stoichiometrically formed in the gel phase. Consideration based on the hexagonal lattice model revealed that the compositions of 0.05 and 0.15 correspond to the bilayer states where cholesterol molecules are regularly distributed in different ways. The former is nearly equal to the composition for the membrane occupied entirely with Units (1:18), composed of a cholesterol and 18 surrounding C17:0-PC molecules within the next-next nearest neighbor sites. The latter is represented by a Unit (1:6), including a cholesterol and 6 surrounding C17:0-PC molecules. Further, the disappearance of the main transition at X(ch)=0.30 indicates that the pure L(o) phase can exist in X(ch)>0.30. The eutectic behavior observed in the phase diagram was explainable in terms of phase separation between two different types of regions with different types of regular distributions of cholesterol.


エーテル結合型リン脂質における指組み構造形成

エーテル結合型リン脂質における指組み構造形成

High Pressure Bioscience and Biotechnology

高圧バイオサイエンスとバイオテクノロジー


高圧力下におけるジノナデカノイルホスファチジルコリン二分子膜の相挙動

高圧力下におけるジノナデカノイルホスファチジルコリン二分子膜の相挙動

高圧バイオサイエンスとバイオテクノロジー


Thermodynamic Consideration for Nonbilayer Formation of Phospholipid Membranes

リン脂質膜の非二分子膜形成に対する熱力学的考察

高圧バイオサイエンスとバイオテクノロジー


A new interpretation of eutectic behavior for distearoylphosphatidylcholine-cholesterol binary bilayer membrane

We investigated the thermotropic phase behavior of the distearoylphosphatidylcholine (DSPC)-cholesterol binary bilayer membrane as a function of the cholesterol composition (X(ch)) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra, each of which has a single maximum, showed that the wavelength at the maximum intensity (lambda(max)) changed depending on the bilayer state: ca. 440 nm for the lamellar gel (L(beta)' or L(beta)) and the liquid ordered (L(o)) phases, ca. 470 nm for the ripple gel (P(beta)') phase and ca. 490 nm for the liquid crystalline (L(alpha)) phase, respectively. The transition temperatures were determined from the temperature dependences of the lambda(max) and endothermic peaks of the DSC thermograms. Both measurements showed that the pretransition disappears around X(ch)=0.035. The constructed temperature-X(ch) phase diagram indicated that the phase behavior of the binary bilayer membrane at X(ch)< or =0.15 is similar to that of general liquid-solid equilibrium for a binary system where both components are completely miscible in the liquid phase and completely immiscible in the solid phase. It was also revealed that the diagram has two characteristic points: a congruent melting point at X(ch)=0.08 and a peritectic-like point at X(ch)=0.15. The hexagonal lattice model was used for the interpretation of the phase behavior of the binary bilayer membrane. These characteristic compositions well correspond to the bilayer states in each of which cholesterol molecules are regularly distributed in the hexagonal lattice in a different way. That is, each composition of 0.035, 0.08 and 0.15 is nearly equal to that for the binary bilayer membrane which is entirely occupied with units, each composed of a cholesterol and 30 surrounding DSPC molecules within the next-next-next nearest neighbor sites (Unit (1:30): L(beta)(1:30)), with units, each of a cholesterol and 12 surrounding DSPC molecules within the next nearest sites (Unit (1:12): L(beta)(1:12)) or with units, each of a cholesterol and 6 surrounding DSPC molecules at the nearest neighbor sites (Unit (1:6): L(beta)(1:6)), respectively. Therefore, the eutectic behavior observed in the phase diagram was fully explainable in terms of a kind of phase separation between two different types of regions with different types of regular distributions of cholesterol. Further, the L(o) phase was found in the higher X(ch)-region (X(ch)>0.15). No endothermic peak over the temperature range from 10 to 80 degrees C at X(ch)=0.50 suggested that the single L(o) phase can exist at X(ch)>0.50.


Thermotropic and barotropic phase transitions of dilauroylphosphatidylcholine bilayer

The bilayer phase transitions of dilauroylphosphatidylcholine (DLPC), containing two linear acyl chains with 12 carbon atoms, were observed by means of differential scanning calorimetry (DSC) under ambient pressure and light transmittance under high pressure. When the heating scan for the DLPC bilayer in 50 wt.% aqueous ethylene glycol (EG) solution began at -30 degrees C after cold storage, the DSC thermogram showed two endothermic peaks at 1.7 and 4.5 degrees C, which correspond to the transition from the lamellar crystalline (Lc) phase to the intermediate liquid crystalline (Lx) phase and the transition from the Lx phase to the liquid crystalline (L) phase, respectively. Extremely large enthalpy change (32.9 kJ mol(-1)) is characteristic of the Lc/Lx phase transition. The DSC thermogram for the heating scan beginning from -10 degrees C showed a single endothermic peak with 9.2 kJ mol(-1) at -0.4 degrees C, which was assigned as the so-called main transition between the metastable ripple gel (P'(beta)) and metastable Lalpha phases. The DLPC bilayer under high pressure underwent three kinds of transitions in EG solution, whereas only one transition was observed in water under high pressure. The middle-temperature transition in EG solution could be assigned to the main transition because of its consistency with the main transition in water. The lower-temperature transition is probably assigned as transition from the Lc phase to the P'(beta) phase. Since the slope (dT/dp) of the Lc/P'(beta) phase boundary is smaller than that for the main transition, the Lc/P'(beta) phase boundary and the main transition curves crossed each other at 40 MPa on the temperature-pressure phase diagram. The higher-temperature transition in EG solution refers to the transition from the Lx phase to the Lalpha phase. The Lx phase disappeared at about 180 MPa, and the direct transition from the P'(beta) phase to the Lalpha phase was observed at high pressures above 180 MPa.


Cholesterol Suppresses Pressure-induced Interdigitation of Dipalmitoylphosphatidylcholine Bilayer Membrane


Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (Lc), lamellar gel (L beta'), ripple gel (P beta') and liquid crystal (L alpha), in turn. The Lc phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L beta I) phase appeared above the critical interdigitation pressure (CIP) between the L beta' and P beta' phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F''497/F''430 value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L beta'/L beta I phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L beta I phase in the SPPC bilayer has a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.


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.


Barotropic phase transition between the lamellar liquid crystal phase and the inverted hexagonal phase of dioleoylphosphatidylethanolamine

The phase transition between the lamellar liquid crystal (Lalpha) phase and the inverted hexagonal (H(II)) phase of dioleoylphosphatidylethanolamine (DOPE) in aqueous NaCl solutions was observed by means of differential scanning calorimetry (DSC) under ambient pressure and light-transmittance technique under high pressure. The pressure dependence of the transition temperature (dT/dp) and the thermodynamic quantities for the Lalpha/H(II) transition were compared with those of another phase transition found in the DOPE bilayer membrane, which is the transition from the lamellar crystal (Lc) phase to the Lalpha phase. The dT/dp value of the Lalpha/H(II) transition was about 3.5 times as large as that of the Lc/Lalpha transition while the thermodynamic quantities were significantly smaller than those of the latter to the contrary. Comparing the enthalpy and volume behavior of the Lalpha/H(II) transition with that of the Lc/Lalpha transition, we concluded that the Lalpha/H(II) transition can be regarded as the volume-controlled transition for the reconstruction of molecular packing.


Barotropic Phase Transitions of 1-Palmitoyl-2-stearoylphosphatidylcholine Bilayer Membrane

非対称型リン脂質1-パルミトイル-2-ステアロイルホスファチジルコリン二分子膜の圧力誘起相転移を高圧光透過率測定および示差走査熱量測定により観測した．決定した相転移点に基づいて温度—圧力相図を構築し，3種類の対称型ホスファチジルコリン二分子膜の相図と比較した．1-パルミトイル-2-ステアロイルホスファチジルコリン二分子膜の相挙動は対称型ホスファチジルコリン二分子膜とは大きく異なり，指組み構造形成の臨界圧力は全ての脂質のうちで最も小さくなった．


Visiting Research Fellow at ETH, Paul Scherrer Institute

スイス連邦工科大学ポール・シェラー研究所留学記

The Review of High Pressure Science and Technology


人工生体膜のソフトナノテクノロジー

Soft nanotechnology is technology that treats soft and wet materials such as self-assemblysystems in a living body under a relatively moderate condition. We focus our attention on the sizecontrol of liposomes, namely lipid bilayer membranes and performed the soft nanotechnology insizing of liposomes under high pressure. Two high-pressure sizing techniques, a method ofcontinuous pressure relaxation using a phase transition between gel and liquid crystalline phases ofthe bilayer membrane and that of a pressure-induced gel phase using a phase transition betweenbilayer and nonbilayer membranes, were applied to liposomes of distearoylphosphatidylcholine, andthe effects of the both methods were considered. Further, the application of giant liposomescontrolled by high-pressure sizing to microsensor was briefly described.

Bulletin of Institute of Technology and Science, The University of Tokushima

徳島大学大学院ソシオテクノサイエンス研究部研究報告