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.     

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.     

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 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.     

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.     

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.     

Raman spectroscopic study of an interdigitated lipid bilayer. Dipalmitoylphosphatidylcholine dispersed in glycerol

Dipalmitoylphosphatidylcholine (DPPC) dispersed in perdeuterated glycerol was investigated in order to determine the effects on the Raman spectra of hydrocarbon chain interdigitation in gel-phase lipid bilayers. Interdigitated DPPC bilayers formed from glycerol dispersions in the gel phase showed a decrease in the peak height intensity $\text{I}{}_{2850}\text{/I}{}_{2880}$ ratio, for the symmetric and asymmetric methylene CH stretching modes, respectively, as compared to non-interdigitated DPPC/water gel-phase dispersions. The decrease in this spectral ratio is interpreted as an increase in chain-chain lateral interactions. Spectra recorded in the 700–740 cm^?1 CN stretching mode region, the 1000–1200 cm^?1 CC stretching mode region and the 1700–1800 cm^? CO stretching mode region were identical for both the interdigitated and non-interdigitated hydrocarbon chain systems. At low temperatures the Raman peak height intensity ratios $\text{I}{}_{2935}\text{/I}{}_{2880}$ were identical for the DPPC/glycerol and DPPC/water dispersions, indicating that this specific index for monitoring bilayer behavior is insensitive to acyl chain interdigitation. The increase, however, in the change of this index at the gel-liquid crystalline phase transition temperature for the DPPC/glycerol dispersions implies a larger entropy of transition in comparison to the non-interdigitated DPPC/water bilayer system.     

Interaction of amphiphilic chlorin-based photosensitizers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers

The drawbacks of the presently used photosensitizers include their relatively low selectivity toward cancer cells, and long-lasting accumulation in healthy tissues. Our recent results indicate that conjugating a photosensitizer with folic acid both enhances the active uptake by cells, and decreases the accumulation in healthy tissue. Here, the interaction between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers used as model membranes, and three different photosensitizers were studied; the derivatives were the non-conjugated meta-tetrahydroxyphenylchlorin (m-THPC, CHL1) and tris(3-hydroxyphenyl)-4-carboxyphenylchlorin (CHL2), as well as a folic acid-conjugated m-THPC-like molecule (CHL3). The results obtained indicate that the folate moiety present in the conjugated derivative CHL3 is involved in the interaction with the phospholipid polar heads. This interaction may be responsible for a better miscibility of CHL3 with the DPPC films compared to CHL1 and CHL2, while elimination of CHL3 from the tissue may be due rather to specific, biological processes and not to its polarity.     

DFT study of structure and vibrational spectra of ceramide 3: comparison to experimental data

The Fourier transform Raman and infrared (IR) spectra of the Ceramide 3 (CER3) have been recorded in the regions 200–3500 cm^? 1 and 680–4000 cm^? 1, respectively. We have calculated the equilibrium geometry, harmonic vibrational wavenumbers, electrostatic potential surfaces, absolute Raman scattering activities and IR absorption intensities by the density functional theory with B3LYP functionals having extended basis set 6-311G. This work is undertaken to study the vibrational spectra of CER3 completely and to identify the various normal modes with better wavenumber accuracy. Good consistency is found between the calculated results and experimental data for the IR and Raman spectra.     

A vibrational study of the CD2 stretching bands of selectively deuterated palmitic and stearic acids

The C-D stretching regions of the infrared and Raman spectra of 14 different palmitic and stearic acids containing isolated CD₂ groups are reported. Anomalous behaviour is observed when substitution occurs near the terminal methyl group, which behaviour can not be explained in terms of crystal field effects.     

Conformational order of the hydrocarbon chains in lipid bilayers. A raman spectroscopic study

Raman spectra of dihexadecylphosphatidic acid (DHPA) and of dimyristoylphosphatidylcholine (DMPC) and its longer chain homologues have been obtained as a function of temperature in order to study the conformational order of the hydrocarbon chains in lipid bilayers. The frequency of the longitudinal acoustical (LA) vibration band is evaluated in terms of the length of all-trans chain segments. In the ordered phase, the chains are found to be overwhelmingly in the all-trans conformation. In the fluid phase, definite all-trans segments occur predominantly, the length of which coincides with the extension of the order parameter plateau known from deuterium magnetic resonance (DMR). The frequency of the skeletal optical (SO) trans vibration band leads to the same result, if evaluated under the assumption of vibrational decoupling by gauche bands in the fluid phase, thus lending support to this assumption. The intensity of this band determined from the band area increases linearly with chain length in the ordered phase and is independent of chain length in the fluid phase. Evaluating the intensity for the length of all-trans segments, the same result for the chain conformation is obtained as derived from the frequencies, with the additional information that the length of the all-trans segments in the fluid phase does not vary with chain length.     

Fourier transform infrared spectroscopic studies of lipid-protein interaction in native and reconstituted sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been used to monitor lipid-protein interaction and protein secondary structure in native and reconstituted sarcoplasmic reticulum vesicles. Studies of the temperature dependence of the CH₂ symmetric stretching frequency reveal no cooperative phase transitions in purified sarcoplasmic reticulum or in vesicles reconstituted with dioleoylphosphatidylcholine, although a continuous introduction of disorder into the lipid acyl chains is observed as the temperature is raised. In addition, temperature-dependent changes are observed in the Amide I and Amide II vibrations arising from protein peptide bonds. A comparison of lipid order in native sarcoplasmic reticulum and its lipid extract showed that the introduction of protein is accompanied by a slight increase in lipid order. Reconstitution of Ca²⁺-ATPase from sarcoplasmic reticulum with dipalmitoylphosphatidylcholine (lipid/protein ratio 30:1), reveals a perturbed lipid melting event broadened and reduced in midpoint temperature from multilamellar lipid vesicles. The onset of melting (27–28°C) correlates well with the onset of ATPase activity and confirms a suggestion (Hesketh, T.R., Smith G.A., Houslay M.D., McGill, K.A., Birdsall, N.J.M., Metcalfe, J.C. and Warren, G.B. (1976) Biochemistry 15, 4145–4151) that a liquid crystalline environment is a requirement for optimal protein function. Finally, Ca²⁺-ATPase has been reconstituted into binary lipid mixtures of DOPC and acyl-chain perdeuterated DPPC. The effect of protein on the structure and melting behavior of each lipid component was monitored. The protein appears to preferentially interact with the DOPC component.     

A high-pressure, Raman spectroscopic study of bis(pyrophosphato)tetrazinc(II) and -tetramanganese(II) decahydrate salts

The Raman spectra of crystalline bis(pyrophosphato)tetrazinc(II) and -manganese(II) decahydrates, M₄(P₂O₇)₂ · 10H₂O (M = Zn and Mn), have been investigated at room temperature. The Mn(II) salt readily underwent photodecomposition upon extended exposure to 785-nm laser irradiation and so could not be examined under high pressures in a diamond-anvil cell. The Zn(II) salt was much more stable and was studied successfully at pressures up to ∼35 kbar. The pressure dependences of the δ_sPO₂, ν_sPOP, ν_asPOP, ν_sPO₂, and ν_asPO₂ internal modes of the pyrophosphate group and of an external H₂O mode indicated the occurrence of a pressure-induced phase transition in the 15-20 kbar region. Further confirmation of this structural change came from the appearance of four initially forbidden modes in the 19-26 kbar range. This phase transition proved to be reversible upon release of the applied pressure. The pressure dependences before and after the phase transition were −0.22 to 0.66 and 0.06 to 0.33 cm⁻¹/kbar, respectively.     

DNA protection by aminothiols: study of the cysteamine - DNA interaction by vibrational spectroscopy

Infrared linear dichroism measurements and Roman scattering spectra show that the cysteamine molecule binds strongly to the DNA stabilizing the double helix in a B geometry conformation. The B→A conformational transition is not observed for a cysteamine/DNA ratio of one cysteamine molecule per two phosphate sites. No evidence of interaction has been found between the radioprotector and the DNA bases. A model is proposed in which the cysteamine molecule is bound by its two ends through electrostatic interaction to two consecutive phosphate groups along the same DNA strand.     

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.     

Raman and FTIR microscopic imaging of colon tissue: a comparative study

Colon tissue constitutes a valid model for the comparative analysis of soft tissue by Raman and Fourier transform infrared (FTIR) imaging because it contains four major tissue types such as muscle tissue, connective tissue, epithelium and nerve cells. Raman microscopic images were recorded in the mapping mode using 785 nm laser excitation and a step size of 10 μm from three regions within a thin section that encompassed mucus, mucosa, submucosa, and longitudinal and circular muscle layers. FTIR microscopic images that were composed of 4, 8 and 9 individual images of 4096 spectra each were recorded from the same regions using a FTIR spectrometer coupled to a microscope with a focal plane array detector. Furthermore, Raman microscopic images were recorded at a step size of 2.5 μm from three ganglia that belong to the myenteric plexus. The results are discussed with respect to lateral resolution, spectral resolution, acquisition time and sensitivity of both modalities. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Thermomyces lanuginosus lipase in the liquid-crystalline phases of aqueous phytantriol: X-ray diffraction and vibrational spectroscopic studies

The influence of Thermomyces lanuginosus lipase (TLL) on the phase behaviour of liquid-crystalline phases of aqueous phytantriol as well as conformational changes of TLL entrapped in the cubic Q230 phase have been studied by small angle X-ray diffraction (SAXD), FT-Raman, and FT-IR techniques. It was found that the lipidic Q230 phase is able to accommodate up to 10 wt.% of TLL, and the temperature of phase transition to the inverted hexagonal phase H(II) increases indicating stabilizing effect of the protein. FT-Raman analysis of Trp amino acid marker band W3 revealed that the average rotation angle around the C3-Cbeta bond of four Trp residues of TLL in the Q230 phase increases. Reasoning from available TLL crystallographic data, this result is explained by structural transition of entrapped protein to so-called "open" and more related to the enzymatically-active conformation. TLL secondary structure analysis by amide I and amide III vibrational bands showed that content of alpha-helixes does not change, while a part of beta-sheet structures transforms to less ordered elements upon incorporation of protein into the Q230 phase of aqueous phytantriol.     

Raman spectroscopic study of DNA after photosensitive damage caused by hypocrellins A and B

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The kinetics of the main phase transition of aqueous dispersions of phospholipids induced by pressure jump and monitored by raman spectroscopy

The sensitivity of the melting transition temperature of aqueous dispersions of dipalmitoyl- and distearoylphosphatidylcholine to hydrostatic pressure is used to allow measurement of the rates of isothermal freezing and melting of the lipids by rapidly changing the pressure. The degree of order of the lipids is measured by monitoring a ratio of two points in the Raman spectrum of the lipids which changes sharply at the melting temperature. Use of this Raman order ratio allows correlation between the order of the sample and the rates of transition in a manner which is impossible by monitoring only turbidity. Our longest relaxation times range upwards from a few seconds for both compounds. The freezing rates are slowest when the samples are initially fully melted, and the melting rates are slowest when the samples are initially frozen. These results imply that nucleation of the growing phase dominates the kinetics of both freezing and melting.     

Investigations of thermotropic phase behavior of newly developed synthetic PEGylated lipids using Raman spectro-microscopy

In this article, a temperature-controlled Raman spectro-microscopic technique has been utilized to detect and analyze the phase behaviors of two newly developed synthetic PEGylated lipids trademarked as QuSomestrade mark, which spontaneously form liposomes upon hydration in contrast to conventional lipids. The amphiphiles considered in this study differ in their hydrophobic hydrocarbon chain length and contain different units of polyethylene glycol (PEG) hydrophilic headgroups. Raman spectra of these new artificial lipids have been recorded in the spectral range of 500-3100 cm(-1) by using a Raman microscope system in conjunction with a temperature-controlled sample holder. The gel to liquid phase transitions of the sample lipids composed of pure 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23) have been revealed by plotting peak intensity ratios in the C--H stretching region as a function of temperature. From this study, we have found that the main phase transitions occur at a temperature of approximately 5.2 and 21.2 degrees C for pure GDM-12 and GDS-23, respectively. Furthermore, the lipid GDS-23 also shows a postphase transition temperature at 33.6 degrees C. To verify our results, differential scanning calorimetry (DSC) experiments have been conducted and the results are found to be in an excellent agreement with Raman scattering data. This important information may find application in various studies including the development of lipid-based novel substances and drug delivery systems. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1012-1020, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.