Pressure jump relaxation investigations of lipid membranes using FTIR spectroscopy

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) were investigated using time-resolved FTIR spectroscopy. The methylene stretching vibrational band and the carbonyl band were analyzed to detect changes in conformational order of the hydrocarbon chains and to follow the degree of hydration of the head group, respectively. The kinetics of the transition was found to consist of multiple processes with relaxation constants from seconds down to milliseconds. Faster processes are also present, but could not be resolved by our instrument. This is the first investigation showing directly the time resolved change in chain order in lipid bilayers induced by a p-jump. The results obtained with this IR detection method support previous results that the change in chain order after a perturbation is a multi-step process with the initial molecular events occurring with time constants shorter than milliseconds.     

Fluorescence temperature jump relaxations of dansylphosphatidylethanolamine in aqueous dispersions of dipalmitoylphosphatidylcholine during the gel to liquid-crystal transition

The change of fluorescence of the probe dansylphosphatidylethanolamine embedded in multilamellar liposomes of dipalmitoylphosphatidylcholine was used to investigate the dynamics of the thermotropic gel to liquid-crystalline phase transition by use of the temperature-jump technique. The results are discussed and compared to published observations on the same system in which the phenomenon was reported by turbidity changes.     

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

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

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C_n = 12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Temperature acclimation and phospholipid phase transition in hypothalamic membrane phospholipids of garden lizard, Calotes versicolor

Garden lizards, Calotes versicolor, were acclimated to three different temperatures, i.e., 16°C, 26°C and 36°C for a period of 30 days in ‘walk-in-environmental chambers’. The phospholipid profile and fatty acid pattern were analysed in the hypothalamus and brain of the acclimated animals. Hypothalamic and brain membrane phospholipids were prepared and their phase-transition temperatures were recorded using differential scanning calorimetry. Acclimation temperature, phospholipid composition, fatty acids of these phospholipids and the physical state and fluidity of the specific model membranes of hypothalamus (and brain) are intimately inter-related. Evidence is presented for the first time to show a possible correlation between acclimation temperature and phase-transition temperature of hypothalamic phospholipid membrane. A direct physico-chemical basis is suggested for the thermoregulatory process of hypothalamus leading to a better understanding of our knowledge on the origin of thermoregulation.     

Kinetic investigations on the phase transition of phospholipid bilayers

Pressure-jump experiments were performed on vesicles and liposomes of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine following the time course of solution turbidity. For both lipids two relaxation effects were evaluated the time constants of which exhibit clear maxima at the midpoint of the phase transition. The time constants lie for vesicles in the 100 μs and 1 ms ranges and for liposomes in the 1 ms and 10 ms ranges. The processes are slightly faster for dimyristoyl phosphatidylcholine than for dipalmitoyl phosphatidylcholine. All relaxation times are concentration-independent. The time constant and amplitude behaviours indicate that all processes are cooperative in agreement with previous interpretations. It is demonstrated that cooperative units can be evaluated from the relaxation amplitudes. These are of the same order of magnitude as those obtained from static experiments. On the grounds of the present kinetic investigation we can state that the application of the linear Ising model to two-dimensional processes as attempted for the static lipid phase transition is inadequate.     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

Dynamic fluorescence measurements on the main phase transition of dipalmytoylphosphatidylcholine vesicles

The kinetics of the main phase transition in dipalmytoylphosphatidylcholine (DPPC) vesicles have been investigated using our iodine laser-Tjump technique with fluorescence detection. A set of three fluorescent probes has been used to sense different parts of the bilayer hydrocarbon chain region. The well established membrane probes DPH and TMADPH as well as DPHPC, a labelled DPPC molecule. We report three relaxation signals in the s and ms time range, which are detected with all three probes. This result supports our model of the main phase transition in DPPC vesicles.Abbreviations DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmytoylphosphatidylcholine - DPH 1,6-Diphenylhexa-1,3,5-triene - TMADPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene - DPHPC Diphenylhexatriene-phosphatidylcholine - T_m Temperature of the main phase transition     

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 (L_c), lamellar gel (L_β′), ripple gel (P_β′) and liquid crystal (L_α), in turn. The L_c 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_βI) phase appeared above the critical interdigitation pressure (CIP) between the L_β′ and P_β′ 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_β′/L_βI 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_βI 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.     

Deuterated phospholipids as raman spectroscopic probes of membrane structure: Phase diagrams for the dipalmitoyl phosphatidylcholine(and its d62 derivative)-dipalmitoyl phosphatidylethanolamine system

Raman spectroscopic techniques have been used to construct phase diagrams for the binary phospholipid systems, DPPC-d₆₂/DPPE and DPPC/DPPE (DPPC, dipalmitoyl phosphatidylcholine; DPPE, dipalmitoyl phosphatidylethanolamine). For the former, the half-width of the C-²H stretching modes of the deuterated component near 2100 cm^?1 serves as an indicator of phospholipid fluidity. The phase behavior is described semi-quantitatively using regular solution theory with the following non-ideality parameters:

$\text{ρ}{}_0{}^{\mathrm{(1)}}\text{=0.75}\text{kcal/mol and}\text{ρ}{}_0{}^{\mathrm{(s)}}\text{=1.05}\text{kcal/mol}$

The use of deuterated phospholipids as one component of a binary mixture permits direct evaluation of the conformation of a particular component in the mixture throughout the phase separation region. The approach is demonstrated with the help of a simple model correlating the half-width of the symmetric C-²H stretching mode with the fraction of DPPC-d₆₂ hydrocarbon chains in the liquid crystalline state.The effect of chain perdeuteration on the phase behavior of DPPC with DPPE is evaluated by comparison of the phase diagram of the DPPC-d₆₂/DPPE system with that of DPPC-DPPE. The latter has been constructed previously from both probe and calorimetric techniques, and is created from the Raman spectroscopic data using the $\text{I(}\text{1130}\text{1100}\text{)}$ ratio to characterize the transgauche population ratio in non-deuterated hydrocarbon chains. A reasonable fit to the phase behavior is obtained using:

$\text{ρ}{}_0{}^{\mathrm{(1)}}\text{=0.85}\text{kcal/mol and}\text{ρ}{}_0{}^{\mathrm{(s)}}\text{=0.90}\text{kcal/mol}$

The similarities of the non-ideality parameters in the two phase diagrams indicate that the effect of perdeuteration on the phase behavior of DPPC is not extensive. The use of deuterated phospholipids as essentially unperturbed components of a model membrane system is justified.     

The influence of structure and redox state of prenylquinones on thermotropic phase behaviour of phospholipids in model membranes

Our study was aimed to investigate the significance of the isoprenoid side chain size as well as redox state of the quinone ring for interaction of two main classes of prenylquinones: plastoquinones (PQ) and ubiquinones (UQ) with lipid bilayers. By use of differential scanning calorimetry (DSC) we have followed the thermotropic behaviour of multilamellar vesicles prepared from dipalmitoylphosphatidylcholine (DPPC) upon incorporation of increasing amount (1.3-12 mol%) of quinone (quinol) molecules. Our studies reveal that as the side chain is shorter (from 9 to 2 isoprenoid units) the height of the calorimetric profiles is reduced and the temperature of the main transition of DPPC (T(m)) decreases (T(m)=39.4 degrees C for a sample with 12 mol% of PQ-2), and then increases up to 39.8 degrees C for PQ-1. For the samples containing quinols the effect is more pronounced even at lower concentration. The greater influence of the added prenylquinones on the pretransition demonstrates a stronger distortion of the DPPC packing in the gel state. It seems that this is the isoprenoid side chain length rather than the redox state of prenylquinones that determines their effectiveness in perturbation of thermotropic properties of lipid bilayer.     

Effects of pressure on the phase transition of bilayers in liposomes influence of cholesterol and α-tocopherol

The effects of presure on the gel-to-liquid crystalline phase transition temperature of dimyristoylphosphatidylcholine (DMPC) bilayers containing cholesterol, α-tocopherol and α-tocopheryl acetate were studied by fluorescence depolarizalion. The transition temperature of cholesterol mixtures ( > 7.5 mol%) was lower than that of 100% DMPC at atmospheric pressure, but it became higher than the latter on increase in pressure. The thermodynamic parameters of the transition (ΔV,ΔS,ΔH) were estimated and the functions of cholesterol and α-tocopherols in the bilayers are discussed.     

Theoretical conformational analysis of phospholipids II. Role of hydration in the gel to liquid crystal transition of phospholipids

To obtain a satisfactory agreement between computed transition temperatures and those determined experimentally, we introduce explicitly water molecules which hydrate the polar headgroup of dipalmitoylphosphatidylethanolamine molecules. The calculated free energy curves as a function of the intermolecular interchain distance and the degree of hydration of the polar groups permit the determination of the transition of the phospholipid system from the gel to the liquid crystalline phase. The detailed structure of the hydration shell is defined using the supermolecular approach.     

The effect of surface curvature on the head-group structure and phase transition properties of phospholipid bilayer vesicles

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.     

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.     

Asymmetric antagonistic effects of an inhalation anesthetic and high pressure on the phase transition temperature of dipalmitoyl phosphatidic acid bilayers

The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. II. Kinetic studies

Kinetic resonance Raman spectra of native and isotopically labelled purple membranes are compared. Using these data and the assignments of the previous paper in this sequence, we have confirmed that the Schiff base is deprotonated at times that are short in comparison to M₄₁₂ evolution. In addition, by monitoring the kinetic resonance Raman spectra in ²H₂O with 488.0 nm excitation we have been able to characterize in more detail the vibrational features associated with this unprotonated intermediate that precedes M₄₁₂. Furthermore, the kinetic spectra of fully deuterated purple membranes in H₂O have allowed us to assign the 1465 cm⁻¹ band in these spectra to the C=C stretching frequency of BR₅₇₀ and the 1512 cm⁻¹ band to the C=C stretching frequency of M₄₁₂. These spectra have also provided an indication of a Raman spectral feature associated with O₆₄₀ and, finally, our kinetic spectra have provided evidence that there is a significant alteration in the rate constants for the evolution of the various intermediates when the non-exchangeable protons on the membrane are replaced by deuterons.     

Phase transition kinetics of phosphatidic acid bilayers A stopped-flow study of the electrostatically induced transition

The kinetics of the electrostatically induced phase transition of dimyristoyl phosphatidic acid bilayers was followed using the stopped-flow technique. The phase transition was triggered by a fast change in the pH or the magnesium ion concentration and followed by recording the time dependence of the absorbance. When the phase transition was induced by a pH jump the time course of the absorbance could be described by two exponentials, their time constants displaying the for cooperative processes characteristic maximum at the transition midpoint. The time constants are in the 10 and 100 ms range for the H⁺ triggered transition from the fluid to the ordered state. A third slower process shows no appreciable temperature dependence and is probably caused by vesicle aggregation. For the OH^--induced transition fron the ordered to the fluid state the time constants are in the 100 and 1000 ms range. The fluid-ordered transition could also be triggered by addition of magnesium ions. Of the several observed processes only the fastest in the 10–100 ms time range could definitely be assigned to the fluid-ordered transition while the others are due to aggregation phenomena. The experimental data were compared with results obtained from pressure jump experiments and could be interpreted on the basis of theories for non-equilibrium relaxation.     

Effect of deuterium oxide on the thermodynamic quantities associated with phase transitions of phosphatidylcholine bilayer membranes

The bilayer phase transitions of three kinds of phospholipids, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dihexadecylphosphatidylcholine (DHPC), in deuterium oxide (D₂O) and hydrogen oxide (H₂O) were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The DSC measurements showed that the substitution of H₂O by D₂O affected the pretransition temperatures and the main-transition enthalpies of all PC bilayers. The temperature-pressure phase diagrams for these PC bilayer membranes in both solvents were constructed by use of the data of light-transmittance measurements. Regarding the main transition of all PC bilayer membranes, there was no appreciable difference between the transition temperatures in D₂O and H₂O under high pressure. On the other hand, the phase transitions among the gel phases including the pretransition were significantly affected by the solvent substitution. The thermodynamic quantities of phase transitions for the PC bilayer membranes were evaluated and the differences in thermodynamic properties by the water substitution were considered from the difference of interfacial-free energy per molecule in the bilayer in both solvents. It was proved that the substitution of H₂O by D₂O causes shrinkage of the molecular area of phospholipid at bilayer interface due to the difference in bond strength between deuterium and hydrogen bonds and produces the great influence on the bilayer phase with the smaller area. Further, the induction of bilayer interdigitation in D₂O turned out to need higher pressures than in H₂O.     

The internal aqueous volume of small unilamellar vesicles changes at the phase transition temperature of the phospholipid

Changes in the fluorescence of partially self-quenched 5(6)-carboxyfluorescein trapped within the internal aqueous compartment of small unilamellar dipalmitoylphosphatidylcholine vesicles indicate that the trapped volume of these vesicles decreases when the phospholipid undergoes the liquid crystalline to gel state transition. This volume change is completely reversible and is not caused by vesicle-vesicle fusion. Furthermore, this decrease in volume of the internal aqueous compartment may be attributed to a change in vesicle shape upon undergoing the phase transition.