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.     

On the subtransitions of deuterated derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

By means of Fourier transform infrared spectroscopy the subtransition temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the derivatives in which either or both of the acyl chains were deuterated were determined. It was found that, while the temperatures of the gel to liquid-crystal and the pre-transitions decrease with increasing deuteration, the temperature of the subtransition is independent of the degree of deuteration.     

The phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as seen by Fourier transform infrared difference spectroscopy

The potential of Fourier transform infrared difference spectroscopy for biochemical applications is demonstrated by the gel to liquid crystal phase transition of the title compound. While the changes occurring in the vibrational pattern of the hydrophobic palmitoyl chains are easily monitored, this technique also discriminates between no change in the choline moiety and a small yet significant change in the carbonyl moiety, both located in the hydrophylic head group.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

The phase transition behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membrane influenced by 2,4-dichlorophenol--an FT-Raman spectroscopy study

The effect of 2,4-dichlorophenol (DCP) on the structures and phase transitions of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes was studied using FT-Raman spectroscopy. Whereas the Raman frequency shifts of the most frequently investigated bands of C-C and C-H stretching regions only indicate the main phase transition (P(beta')-L(alpha)) of the pure DPPC/water system, the Raman shift of C-H scissoring vibration at 1440 cm(-1) was found to be able to reveal the pretransition (L(beta')-P(beta')) as well. Analyzing the spectral parameters of the trans band at 1128 cm(-1), which does not overlap with DCP vibrational modes, a continuous decrease of trans conformations was found with increasing DCP concentration at 26 degrees C accompanying the phase transitions L(beta')-P(beta') and P(beta')-L(alpha). The intensity ratio of the symmetrical and asymmetrical methylene stretching bands (at 2850 cm(-1) and 2880 cm(-1)), defined as the disorder parameter by Levin [Levin, I.W., 1985. Two types of hydrocarbon chain interdigitation in sphingomielin bilayers. Biochemistry 24, 6282-6286], indicated that in the interdigitated phase (L(I)) the order is markedly high and comparable with that of L(beta). Both the phase transition P(beta')-L(alpha) in the DCP/DPPC molar ratio range of 10/100-50/100 and the phase transition L(I)-L(alpha) led to a significant increase of disordered chains and the presence of DCP molecules induced a more disordered chain region than that observed in the L(alpha) phase of DPPC. Nevertheless, it was found that the L(alpha) phase with DCP contains approximately the same amount of trans conformers than that without DCP.     

Generation of fatty acids from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cardiolipin liposomes that stabilize recombinant human serum albumin

Abstract

Context: At elevated temperatures, studies have shown that serum albumin undergoes irreversible changes to its secondary structure. Anionic fatty acids and/or anionic surfactants have been shown to stabilize human serum albumin (HSA) against thermal denaturation through bridging hydrophobic domains and cationic amino acids residues of the protein.

Objective: As albumin can readily interact with a variety of liposomes, this study proposes that cardiolipin delivered via 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes can improve the thermal stability of recombinant HSA produced in Saccharomyces cerevisiae (ScrHSA) in a similar manner to anionic fatty acids.

Materials and methods: Thermal stability and structure of ScrHSA in the absence and presence of DPPC/cardiolipin liposomes was assessed with U/V circular dichroism spectropolarimetry and protein thermal stability was confirmed with differential scanning calorimetry.

Results: Although freshly prepared DPPC/cardiolipin liposomes did not improve the stability of ScrHSA, DPPC/cardiolipin liposomes incubated at room temperature for 7?d (7dRT) dramatically improved the thermal stability of the protein. Mass spectrometry analysis identified the presence of fatty acids in the 7dRT liposomes, not identified in freshly prepared liposomes, to which the improved stability was attributed.

Discussion and conclusion: The generation of fatty acids is attributed to either the chemical hydrolysis or oxidative cleavage of the unsaturated acyl chains of cardiolipin. By modulating the lipid composition through the introduction of lipids with higher acyl chain unsaturation, it may be possible to generate the stabilizing fatty acids in a more rapid manner.     

A Fourier transform infrared spectroscopic study of the molecular interaction of cholesterol with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

The temperature dependencies of the infrared spectra of pure and cholesterol-containing multibilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine were studied using Fourier transform infrared techniques. A comparison of the spectroscopic data showed the retention of a melting phenomenon at 60 mol% cholesterol content, and the retention of some all-trans conformations in the liquid-crystalline phase. It is also demonstrated that at temperatures less than 30 degrees C, the cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine multibilayers still contain a small amount of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, packed in an orthorhombic subcell lattice. Spectral changes were found in the absorptions characteristic of the phospholipid head groups. The addition of cholesterol results in changes in the ester bands, and demonstrates the induction by cholesterol of non-equivalent ester conformations.     

Phase transitions in 1,2- and 1,3-diacyl-sn-glycero-3-phosphocholine monolayers at the oil/water interface.

Moving the phosphatidylcholine group from the 3- to the 2-position in monolayers of distearoyl-sn-glycero-3-phosphocholine at the oil/water interface expands the surface pressure-area isotherm and markedly increases the surface pressure at which phase separation occurs with only a slight change in the monolayer surface density at the onset of the transition. This is interpreted in terms of a change in an ordering parameter in the solid-condensed state.     

Phase transitions in 1,2-and 1,3-diacyl-sn-glycerol-3-phosphocholine monolayers at the oil/water interface

Moving the phosphatidylcholine group from the 3-to the 2-position in monolayers of $\text{distearoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ at the oil/water interface expands the surface pressure-area isotherm and markedly increases the surface pressure at which phase separation occurs with only a slight change in the monolayer surface density at the onset of the transition. This is interpreted in terms of a change in an ordering parameter in the solid-condensed state.     

Biosynthesis of 1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine in Caenorhabditis elegans.

Previously, we showed that lowering the growth temperature increased the level of eicosapentaenoic acid (EPA) in the phosphatidylcholine (PtdCho) of Caenorhabditis elegans. In this study, we investigated the molecular species composition of PtdCho of C. elegans, with an emphasis on EPA-containing species. C. elegans contained a substantial amount of 1,2-dipolyunsaturated fatty acid-containing PtdCho (1,2-diPUFA-PtdCho) species, such as arachidonic acid/EPA and EPA/EPA, which are unusual phospholipids in higher animals. The EPA/EPA-PtdCho content was significantly increased in C. elegans grown at a low temperature. To examine the possibility that the acyltransferase activity involved in the remodeling of phospholipids accounts for the production of 1,2-diPUFA-PtdCho, we investigated the substrate specificity of this enzyme in C. elegans and found that it did not exhibit a preference for saturated fatty acid for acylation to the sn-1 position of PtdCho. The efficacy of the esterification of EPA to the sn-1 position was almost equal to that of stearic acid. The lack of preference for a saturated fatty acid for acylation to the sn-1 position of PtdCho is thought to result in the existence of the unusual 1,2-diEPA-PtdCho in C. elegans.     

Water binding and mobility in the phosphatidylchlone/cholesterol/water lamellar phase.

Measurements of hydration and water self diffusion in lamellar phases of the ternary system: phosphatidylcholine/cholesterol/water have been made using pulse NMR relaxation methods. Systems containing phosphatidylcholine and cholesterol in a 1:1 mol ratio with varying water contents are studied at 20.5 degrees C. The results indicate that 12 water molecules corresponds to complete hydration of the phosphatidylcholine/cholesterol unit, and in the region of this hydration a 4-fold decrease in water diffusion occurs. The nature of the bound water and its relationship to phase stability and overall water mobility in the system are discussed. It is concluded that at the stoichiometric composition the diffusion decreases due to the relative immobility of the bound water. The implications in terms of permeability regulation in the aqueous channels by water content and hydration are cited.     

Cation diffusion selectivity in a pore model. The phosphatidylcholine/water lamellar phase

The diffusion coefficients $\text{D}$ (cm²/s), of four monovalent cations K⁺, Na⁺, Rb⁺ and Cs⁺ and of Ca²⁺ have been measured in phosphatidylcholine/water lamellar phase as a function of phase hydration and temperature and in the presence of divalent cations. Diffusion rates vary strongly with phase hydration, between 10^?7 and 10^?6 cm²/s for monovalent and 10^?8 and 10^?7 for Ca²⁺. The activation energies obtained are relatively small (5–10 kcal/mol). As the phase water content increases, a series of diffusion sequences is obtained, corresponding to the sequences predicted by Eisenman's theory of alkali ion equilibrium selectivity.This diffusionnal selectivity, which depends exclusively upon non-equilibrium parameters (mobility) within the hydrophilic path is discussed in respect to current theories of pore selectivity.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems.

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions. The lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (-120 degrees C to +120 degrees C). Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids. Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H2O and 2H2O media yielded a value of 1.013 +/- 0.026 ml/g for the partial specific volume of this lipid. We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude. Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Water binding and mobility in the phosphatidylcholine/cholesterol/water lamellar phase

Measurements of hydration and water self diffusion in lamellar phases of the ternary system: phosphatidylcholine/cholesterol/water have been made using pulse NMR relaxation methods. Systems containing phosphatidylcholine and cholesterol in a 1 : 1 mol ratio with varying water contents are studied at 20.5°C. The results indicate that 12 water molecules corresponds to complete hydration of the phosphatidylcholine/cholesterol unit, and in the region of this hydration a 4-fold decrease in water diffusion occurs. The nature of the bound water and its relationship to phase stability and overall water mobility in the system are discussed. It is concluded that at the stoichiometric composition the diffusion decreases due to the relative immobility of the bound water. The implications in terms of permeability regulation in the aqueous channels by water content and hydration are cited.     

Effect of free fatty acids on the permeability of 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer at the main phase transition

We measured the influence of saturated and unsaturated free fatty acids on the permeability and partition of ions into 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers. The bilayer permeability was measured using the depletion of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine (N-NBD-PE) fluorescence as a result of its reduction by dithionite. We observed a distinct increase of dithionite permeability at the main gel-fluid phase transition of DMPC. When vesicles were formed from a mixture of DMPC and oleic acid, the membrane permeability at the phase transition was reduced drastically. Stearic acid and methyl ester of oleic acid have little effect. Similar results in the quenching of pyrene-PC in DMPC vesicles by iodide were obtained. Again, the increase of iodide partition into the lipid phase at the main phase transition of DMPC was abolished by the addition of unsaturated free fatty acids. Free fatty acids, in concentrations up to 5 mol%, do not abolish DMPC phase transition when measured by differential scanning calorimetry. It seems that unsaturated, but not saturated, free fatty acids reduce the lipid bilayer permeability to dithionite and iodide ions at the main phase transition of DMPC, without altering the thermodynamic properties of the bilayer.     

Differential thermal analysis and x-ray study of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine phases with less than 5% water

The results of a study on extremely pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) by thermal analysis and X-ray are reported. The solvent removal out of the single crystals, the effect of purity on the differential thermal analysis (DTA) scans and a new structural feature observed in the high temperature lyotropic region have been investigated.     

The temperature-composition phase diagram and mesophase structure characterization of monopentadecenoin in water.

The temperature-composition phase diagram of monopentadecenoin, a monoacylglycerol with a cis monounsaturated fatty acid 15 carbon atoms long (C15:1c10) in water was constructed using x-ray diffraction. Low- and wide-angle diffraction patterns were collected from samples of fixed hydration as a function of temperature in the heating direction on x-ray-sensitive film. The temperature and hydration ranges investigated were 0-104 degrees C and 0-60% (w/w) water, respectively. The phases identified in the system include the lamellar crystalline phase, the lamellar liquid crystalline phase, the fluid isotropic phase, and two inverted cubic phases belonging to space groups la3d (Q230) and Pn3m (Q244). Particular attention has been devoted to the issues of phase equilibrium, phase boundary verification, and structure characterization. The phase diagrams of monopentadecenoin, monomyristolein (C14:1c9), and monoolein (C18:1c9) are compared, and the impact of molecular structure on mesophase stability and structure is discussed.     

Sucrose metabolism in green algae. I. The presence of sucrose synthetase and sucrose phosphate synthetase.

The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae: Chlorella vulgaris and Scenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.     

Sucrose metabolism in green algae I. The presence of sucrose synthetase and sucrose phosphate synthetase

Summary The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae:Chlorella vulgaris andScenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.Dedicated toLuis F. Leloir on his seventieth birthday.