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Pressure and Temperature Effects on Conformational and Hydrational Properties of Lamellar and Bicontinuous Cubic Phases of the Fully Hydrated Monoacylglyceride MonoelaidinsA Fourier Transform-Infrared Spectroscopy Study Using the Diamond Anvil Technique O. Reis and R. Winter* University of Dortmund, Department of Chemistry, Physical Chemistry I, D-44221 Dortmund, Germany Received November 5, 1997. In Final Form: February 2, 1998 We report on the effects of pressure and temperature on the phase behavior and structural properties of aqueous dispersions of monoelaidin (ME) using Fourier transform-infrared spectroscopy (FT-IR) in combination with the diamond anvil technique. The IR spectral parameters, such as frequencies, intensities, band shapes, and band splittings, were used to detect structural and dynamic changes upon change of pressure and temperature. Analysis of these spectral parameters yields information on conformer population, reorientational fluctuations, interchain interaction, hydrogen bonding, and phase transformations. The monoacyglyceride ME was chosen for investigation because it exhibits various mesomorphic liquidcrystalline phases of different dimensionality, including lamellar, a cubic-primitive (QIID), and a bodycentered cubic phase (QIIP). The latter two are inverse bicontinuous cubic phases. We have established the excess water p,T-phase diagram of ME over the 1-23-kbar range at ∼15-95 °C. In the high-pressure region, enhanced interchain interaction leads to a correlation field splitting of the CH2 bending and rocking modes, which is expected when entering the lamellar crystalline Lc phase that has the smallest partial molar volume. As revealed by the phase diagram, the energetic degeneracy of the cubic phases is broken. With increasing temperature or decreasing pressure, the cubic phases QIIP and QIID are formed. Interpretation of the CH2 stretching and wagging modes for evaluation of conformational states in the fluidlike disordered (LR, QIIP, and QIID) phases reveals different populations of gauche conformers and kinks in these fluidlike phases. From the analysis of the carbonyl stretching mode vibrations we have been also able to detect small but marked differences in the level of hydration of different bicontinuous cubic phases. Compared with the QIID phase of ME, the lipid chains of the body-centered cubic phase QIIP seem to contain a slightly higher population of gauche sequences and a slightly lower level of hydration of the carbonyl group. The results are compared with recent energetic models for intercubic phase transitions.
Introduction Although most biological amphiphiles in excess water exist in lamellar bilayer phases, certain lipids, including the monoacylglycerides, can also form nonlamellar liquidcrystalline phases, such as the inverse hexagonal (HII) and cubic phases.1-8 Most of the cubic liquid-crystalline phases consist of bicontinuous regions of water and hydrocarbon, which can be described by infinite periodic minimal surfaces (IPMSs). An IPMS is an intersectionfree surface periodic in three dimensions with a mean curvature that is everywhere zero. The surface, that sits at the lipid bilayer midplane, separates two interpenetrating but not connected water networks. Nonlamellar phases, which occur for a number of membrane lipids, probably play an important functional role as local and transient intermediates in some cell processes, such as (1) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1. (2) Seddon, J. M. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 380. (3) Seddon, J. M.; Templer, R. H. Philos. Trans. R. Soc. London A 1993, 344, 377. (4) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221. (5) Cevc, G.; Marsh, D. Phospholipid Bilayers; John Wiley & Sons: New York., 1987. (6) Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993. (7) Tate, M. W.; Eikenberry, E. F.; Turner, D. C.; Shyamsunder, E.; Gruner, S. M. Chem. Phys. Lipids 1991, 57, 147. (8) Winter, R.; Landwehr, A.; Brauns, Th.; Erbes, J.; Czeslik, C.; Reis, O. In High Pressure Effects in Molecular Biophysics and Enzymology; Markley, J. L., Northrop, D. B., Royer, C. A., Eds.; Oxford University: New York, 1996; p 274.
cell fusion and division and fat digestion.9-11 Interestingly, a recent reanalysis of a large number of published electron micrographs of cell membranes has shown that inverse bicontinuous cubic structures might also occur in biological cells.12 Given these observations, it is clear that understanding lyotropic lamellar and nonlamellar phase behavior of model systems is crucial in understanding the properties of the more complex biological membranes. So far, no generally recognized full theoretical description of lyotropic phase behavior exists, though some progress has been made in recent years.3,13-18 It is a frequent assumption that the phase behavior is dominated by the interfacial curvature energetics, and a stretching term must be added to quantify the packing frustration. To probe the concept of any energetic description and the resultant set of parameters necessary to provide a general (9) Mariani, P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165. (10) Gruner, S. M. In Liposomes, From Biophysics to Therapeutics; Ostro, M. J., Ed.; Marcel Dekker: New York, 1987; p 1. (11) Bouligand, Y. Colloq. Phys. 1990, C7, 35. (12) Landh, T. FEBS Lett. 1995, 369, 13. (13) Templer, R. H.; Seddon., J. M.; Warrender, N. A. Biophys. Chem. 1994, 49, 1. (14) Templer, R. H.; Turner, D. C.; Harper, P.; Seddon., J. M. J. Phys. II France 1995, 5, 1053. (15) Templer, R. H. Langmuir 1995, 11, 334. (16) Templer, R. H.; Seddon, J. M.; Duesing, P. M.; Winter, R.; Erbes, J., submitted for publication in J. Phys. Chem. (17) Chung, H.; Caffrey, M. Nature 1994, 368, 224. (18) Mariani, P.; Paci, B.; Bo¨secke, P.; Ferrero, C.; Lorenzen, M.; Caciuffo, R. Phys. Rev. E 1996, 54, 5840.
S0743-7463(97)01213-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/11/1998
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explanation of universal lyotropic phase behavior, one needs to scan the appropriate parameter space experimentally. Most of the experimental work so far has relied on temperature and sample composition as the tools to attack this problem. A further important thermodynamic variable is pressure. Remarkably, little pressure work on curved lipid phases has been done to date, in particular at higher pressures above the 1-2-kbar range.8,19-23 In addition to the general physicochemical interest in using high pressure as a tool for understanding phase behavior, structure and energetics of amphiphilic molecules, high pressure is also of considerable physiological and biotechnological (e.g., high-pressure food processing) interest.24-29 The monoacylglyceride monoelaidin (ME; C18:1t9) is a neutral ester-linked 18-carbon-atom-long fatty acid with a trans double bond at the C9 position of the acyl chain. ME was chosen for investigation in this study, because it exhibits a variety of mesomorphic phases with different lattice dimensionality at rather moderate conditions of temperature and pressure.22,30,31 Several of the mesophases ME exhibits when dispersed in water are metastable, and the thermal history of the sample has been found to have a profound influence on the phase behavior of the system.22,30 Such features are rather often observed in lyotropic systems and are, in fact, well-known in surfactants. In biological systems, these features might even enable certain organisms to survive fluctuations in environmental conditions. The temperature-water concentration phase diagram of ME has largely been established.30,31 The polymorphic and metastable phase behavior of dry ME and ME in excess water was extensively studied recently by using high-sensitivity differential scanning calorimetry (DSC) and time-resolved X-ray diffraction in the temperature range 4-60 °C.31 The first pressure-dependent studies in the lower pressure region up to 1.5 kbar were obtained by X-ray and neutron diffraction.22 It is clear from these studies that the polymorphic and mesomorphic behavior of ME dispersed in excess water is quite complex. On the other hand, this complex phase behavior permits the study of a variety of structures for one and the same system by change of temperature and pressure only without having to change the chemical composition and level of hydration of the sample. By these means, one is able to detect slight structural changes between topologically similar lyotropic mesophases, such as the liquid-crystalline lamellar and different bicontinuous cubic phases. At low temperatures, the lamellar crystalline Lc phase of ME is probably the thermodynamically stable phase. (19) So, P. T. C.; Gruner, S. M.; Shyamsunder, E. S. Phys. Rev. Lett. 1993, 70, 3455. (20) Chang, E. L.; Yager, P. Mol. Cryst. Liq. Cryst. 1983, 98, 125. (21) Landwehr, A.; Winter, R. Ber. Bunsen-Ges. Phys. Chem. 1994, 98, 214. (22) Czeslik, C.; Winter, R.; Rapp, G., Bartels, K. Biophys. J. 1995, 68, 1423. (23) Duesing, P. M.; Seddon, J. M.; Templer, R. H.; Mannok, D. A. Langmuir 1997, 13, 2655. (24) High Pressure and Biotechnology, Balny, C., Hayashi, R., Heremans, K., Masson, P., Eds.; Colloque Inseram, Vol. 224; John Libbey Eurotext: Montrouge, France, 1992. (25) High-Pressure Chemistry, Biochemistry and Materials Science, NATO ASI C 401; Winter, R., Jonas, J., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1993. (26) High-Pressure Nervous Syndrome - 20 Years Later; Rostain, J. C., Martinez, E., Lemaire, C., Eds.; ARAS-SNHP Publications: Marseille, France, 1989. (27) Weber, G.; Drickamer, H. G. Quart. Rev. Biophys. 1983, 16, 89. (28) Yayanos, A. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 9542. (29) Jaenicke, R. Annu. Rev. Biophys. Bioeng. 1981, 10, 1. (30) Lutton, E. S. J. Am. Oil Chem. Soc. 1965, 42, 1068. (31) Chung, H.; Caffrey, M. Biophys. J. 1995, 69, 1951.
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In the Lc phase, most molecular motions are frozen out. When heated, the Lc phase transforms to the lamellar liquid-crystalline LR phase at ∼32 °C. DSC measurements reveal an enthalpy change ∆H of 52 kJ/mol for the transition. With continued heating, the LR phase converts to the bicontinuous cubic phase QIIP (space group Im3m) at ∼39 °C. At ∼55 °C, a second bicontinuous cubic phase, the QIID (space group Pn3m) phase, is formed. Metastable phases can easily be induced in ME when cooling the system down from temperatures >60 °C. The QIID phase can be undercooled to 27 °C, and the QIIP phase appears between 29 and 21 °C. Further decrease of temperature leads to the formation of the Lβ phase and no LR phase is observed.22 The Lβ phase seems to be a metastable phase at room temperature. Above 11 °C, the Lβ phase is stable for days, at 9 oC, it exists for about 30 min, and at 4 °C, it converts to the Lc phase within 10 min. This transition is accompanied by an enthalpy change ∆H of -21 kJ/mol. Qualitatively similar results have been obtained by Chung et al.31 Differences might be due to using D2O instead of H2O as solvent in this study. The reasoning for the formation of metastable cubic phases in the cooling direction is that bulk water must access all the three-dimensional (3D) narrow labyrinths of the cubic mesophases to allow the unit cell to swell to its equilibrium level of hydration. Indeed, the fact that the lattice paramater remains constant during a cooling scan performed at a rate of -5 °C/min suggests that water uptake does not occur on this time scale.22,31 Similar to the observation that metastable cubic and lamellar phases can easily be obtained by cooling, metastable phases can also been obtained by an increase of pressure at constant temperature. To decipher the conformational differences of the various (in particular liquid-crystalline) mesophases of ME in excess water, we have applied Fourier transform-infrared (FT-IR) spectroscopy. The IR spectroscopy is a nonperturbing technique that monitors molecular vibrations and thus operates on a very short time scale. Vibrational spectra of lipid systems consist of bands arising from the transitions between vibrational energy levels of various types of intra- and intermolecular vibrations in the ground electronic state. It has been extensively shown in the literature, that many IR spectral parameters, particularly the frequencies, widths, intensities, shapes, and splittings of the IR bands, are very sensitive to the structural and dynamical properties of membrane lipid molecules.32-42 In particular cases, such as by analyzing the CH2 wagging vibrations, even quantitative conformational information can be obtained. We present FT-IR data of ME in excess D2O in the pressure range from 0.001 to 23 kbar at temperatures from 15 to 95 °C. (32) Wong, P. T. T.; Siminovitch, D. J.; Mantsch, H. H. Biochim. Biophys. Acta 1988, 947, 139. (33) Auger, M.; Jarrell, H. C.; Smith, I. C. P.; Siminovitch, D. J.; Mantsch, H. H.; Wong, P. T. T. Biochemistry 1988, 27, 6086. (34) Nilsson, A.; Holmgren, A.; Lindblom, G. Chem. Phys. Lip. 1994, 69, 219. (35) Tuchtenhagen, J.; Ziegler, W.; Blume, A. Eur. Biophys. J. 1994, 23, 323. (36) Mendelsohn, R.; Davies, M. A.; Brauner, J. W.; Schuster, H. F.; Dluhy, R. A. Biochemisty 1989, 29, 8934. (37) Davies, M. A.; Schuster, H. F.; Brauner, J. W.; Mendelsohn, R. Biochemistry 1990, 29, 4368. (38) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213. (39) Casal, H. L.; McElhaney, R. N. Biochemistry 1990, 29, 5423. (40) Blume, A.; Hu¨bner, W.; Messner, G. Biochemistry 1988, 27, 8239. (41) Lewis, R. N. A. H.; McElhaney, R. N.; Pohle, W.; Mantsch, H. H. Biophys. J. 1994, 67, 2367. (42) Reis, O.; Winter, R.; Zerda, T. W. Biochim. Biophys. Acta 1996, 1279, 5.
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Materials and Methods The monoacylglyceride monoelaidin (ME) was purchased from Sigma Chemical Company (Deisenhofen, Germany) and was used without further treatment. Fully hydrated ME/D2O dispersions (10-30 wt % lipid) were prepared for the IR experiments by weighing the appropriate amounts of lipid and D2O. The samples were then subjected to five freeze/thaw cycles in a closed vessel to yield homogeneous lipid dispersions. For the temperature-dependent studies, lipid dispersions were filled into an IR cell with CaF2 windows and an optical path length of 25 µm. For the pressure-dependent work, small amounts of the sample were placed at room temperature in a 0.5-mm diameter hole of a 0.5-mm thick stainless steel gasket of a diamond anvil cell,42 along with powdered R-quartz, which was used as internal pressure calibrant.25,43 The diamond anvil cell (Diamond Optics, Tucson, AZ) was connected to an external resistance heating element that allowed measurements up to temperatures of 95 °C. The IR spectra were collected on a Nicolet Magna 550 FT-IR spectrometer equipped with a mercury cadmium telluride detector operated at -196 °C. The IR beam was condensed by a spectra-bench onto the pinhole of the diamond anvil cell. For each spectrum, typically 512 scans were coadded, at a spectral resolution of 2 cm-1, and apodized with a Happ-Genzel function, leading to a total measuring time per spectrum of ∼8 min. The sample chamber was purged with dry and carbon dioxide-free air to avoid contamination of atmospheric gases. Determination of peak positions and further data treatment was done with the OMNIC sofware developed by the Nicolet Instrument Corporation.
Figure 1. The IR spectra of ME in excess water (D2O) in the region of the CH2 stretching vibrations in the temperature range from 15 to 68 °C (p ) 1 bar).
Results and Discussion The peak positions, intensities, half-widths, and splitting of the various IR absorption bands were analyzed to monitor phase transitions and the conformation and hydration changes accompanied by the various phase transformations of ME. Prior to the measurements, the lipid was hydrated at 70 °C and then cooled to 15 °C. Symmetric CH2 Stretching Mode. The vibrational modes due to hydrocarbon chains are easily identified on the basis of the well-studied polymethylene and polymethylene chain compounds.44-46 The carbon hydrogen stretching vibrations give rise to bands in the spectral region between 2800 and 3100 cm-1. In general, the CH2 antisymmetric stretching mode at ∼2920 cm-1 and the CH2 symmetric stretching mode at ∼2850 cm-1 are the strongest bands that can be observed in lipid IR spectra. The positions of these bands are conformation sensitive and thus give qualitative information about temperatureand pressure-induced changes of the trans/gauche ratio in the lipid acyl chains. This is also the case for the symmetric and antisymmetric stretching vibrations originating from the terminal CH3 group at ∼2870 cm-1 and ∼2956 cm-1, however, the frequency dependence of these modes is less sensitive. The temperature-pressure phase diagram of ME in excess water in the lower pressure range up to ∼1.5 kbar has recently been determined by Czeslik et al.22 with smallangle X-ray diffraction. The stable and metastable phases found include the lamellar crystalline (Lc) phase, the gel (Lβ) phase, the liquid-crystalline (LR) phase, and two cubic phases (QIIP and QIID) belonging to the crystallographic space groups Im3m and Pn3m, respectively. Transitions between the different lipid phases are accompanied by structural rearrangements and changes (43) Wong, P. T. T.; Moffatt, D. J.; Baudais, F. L. Appl. Spectroscopy 1985, 39, 733. (44) Snyder, R. G. J. Mol. Spectrosc. 1961, 7, 116. (45) Snyder, R. G. J. Chem. Phys. 1967, 47, 1316. (46) Snyder, R. G.; Maroncelli, M.; Strauss, H. L. J. Am. Chem. Soc. 1983, 105, 133.
Figure 2. Temperature dependence of the wavenumber of the CH2 symmetric stretching band in the IR spectra of aqueous dispersions of ME at ambient pressure.
in the hydration of the bilayer/water interface, which lead to a significant change in the IR spectra. Figure 1 exhibits part of the IR spectrum of ME in excess D2O in the region of the CH2 stretching region as a function of temperature. At 15 °C, the symmetric stretching mode occurs at 2850.8 cm-1. The position shifts to larger wavenumbers with increasing temperature. At ∼23 °C a signifiant shift to larger frequences is observed. A further change in the IR spectrum occurs at 44 °C. Figure 2 shows the wavenumber of the symmetric CH2 stretching vibration as a function of temperature. At ∼23.5 °C a sharp increase in the band position is observed. The transition can be ascribed to a conversion from a lamellar gel (Lβ) to the lamellar liquidcrystalline (LR) phase.22 Phases have been identified by small- and wide-angle X-ray diffraction. A further increase, however to a lesser extent, can be observed at ∼44 °C, where the body-centered cubic phase QIIP is formed. At a temperature around 55 °C, the position of the C-H stretching vibration is slightly moving to lower wavenumbers again, which can be correlated to the intercubic QIIP-to-QIID transition. The position of the symmetric C-H stretching band is a qualitative measure of the number of gauche conformers in the acyl chains. It is well-known for diacylphospholipids that when all methylene groups are in the trans conformation, the band is observed at low wavenumbers of ∼2849 cm-1.47 Introducing gauche conformers into the acyl chains leads to a shift to higher wavenumbers. At the Lβ-LR phase transition, which is accompanied by a “melting” of the acyl chains, the band shifts by ∼2 cm-1, (47) Umemura, J.; Cameron, D. G.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 602, 32.
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Figure 3. Pressure dependence of the CH2 symmetric stretching mode wavenumber of ME in excess D2O at T ) 50 °C.
indicating the introduction of a significant amount of gauche conformers. Because conformational changes are much smaller at the fluid LR-to-fluid QIIP transition, the shift of wavenumber is less pronounced (∼0.8 cm-1). Obviously, the cubic QIIP phase is conformationally more disordered than the lamellar LR phase due to an enhanced gauche/trans ratio in the acyl chain region. Above 55 °C, the QIID cubic phase emerges, which leads to a slight decrease of the frequency of the symmetric CH2 stretching band. Therefore, it seems to be clear from our experiments that there are small but marked differences in the gauche population between the two bicontinuous cubic phases. The pressure dependence of the symmetric CH2 stretching band at 50 °C is shown in Figure 3. Over most of the pressure range studied, the frequency varies linearly with increasing pressure within experimental error, a behavior which is observed for the other mode frequencies as well. Such a pressure-induced linear blue shift is generally observed when elastic repulsive forces dominate the system.42 Phase transitions can easily be detected by welldefined changes from this linear pressure dependence, manifested either by discontinuities or by changes of slope of ν(p). At ∼1.6 kbar, an abrupt drop of the wavenumber of the symmetric stretching mode can be detected, being indicative of the pressure-induced LR-Lβ transition, which is accompanied by a drastic decrease of the population of gauche conformers. A second discontinuity appears at a pressure of ∼3.1 kbar, which is due to the Lβ-Lc transition. Here again, an increase of conformational order is caused by the pressure-induced reduction of gauche conformers. These phase transitions are also observed at higher temperatures of 65 and 90 °C. The corresponding transition pressures for the LR-Lβ transition are ∼2.5 and ∼3.2 kbar, and for the Lβ-Lc transition are ∼4.6 and ∼5.1 kbar, respectively. Because conformational changes due to the intercubic QIID-QIIP and the cubic-to-lamellar QIIP-LR transition are very small, these transformations are not detectable with sufficient accuracy in the high-pressure sample environment of the diamond anvil cell. The combined results of our X-ray diffraction and FT-IR spectroscopy measurements for the transition pressures of ME/water are depicted in the temperature-pressure phase diagram shown in Figure 4. The Wagging Progression Region. The conformation-dependent wagging modes of interest in this work appear at wavenumbers of ∼1368, 1356, and 1341 cm-1.42 They arise from gauche-trans-gauche, double gauche, and end gauche sequences within the acyl chain. The most intense band in the wagging region between 1300 and 1400 cm-1 is the methyl “umbrella” deformation mode at 1378 cm-1. This mode is insensitive to the lipid conformational state and is used as an internal intensity
Reis and Winter
Figure 4. Temperature-pressure phase diagram of ME in excess D2O. Key: (b) X-ray data; (0) FT-IR data of this work. The lamellar Lβ phase is probably metastable (dashed phase lines).
Figure 5. The IR spectrum of ME/D2O in the CH2 wagging region at T ) 62 °C. Key: (full curve) experimental IR spectrum; (dashed curve) simulated spectrum based on the different component bands; (dotted lines) different component bands.
Figure 6. Temperature dependence of the relative amount (I1368/I1378) of g-t-g′ conformers (kinks) of ME in D2O as obtained from the analysis of the CH2 wagging region.
standard to which the methylene wagging bands are normalized. The spectral region was simulated with mixed Gaussian-Lorentzian functions over the 13301395-cm-1 spectral range. The curve fit was optimized using a reduced chi-square algorithm (Figure 5). Figure 6 exhibits the relative integral intensity of the 1368 cm-1 band, which is due to CH2 deformation modes in kink (g-t-g′) and g-t-g sequences, as a function of temperature. Because transition dipole moments differ for the g-t-g′ and g-t-g modes, the 1368 cm-1 feature is not suitable for quantitative evaluation of disordering due to kinks alone.48 Therefore, the I (1368)/I (1378) intensity ratio is only used as a qualitative marker for kink formation. Starting at 25 °C in the liquid-crystalline
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Figure 8. Pressure dependence of the τCH2 rocking mode wavenumber of ME in excess D2O at selected temperatures. Figure 7. Pressure contours of the τCH2 rocking band of ME in excess D2O at (a) T ) 50 °C and (b) T ) 65 °C.
LR phase, the relative contribution of kinks does not change much with temperature. When the body-centered cubic QIIP phase is formed at ∼42 °C, the intensity increases rapidly by ∼60%. Further increase of temperature leads to a drop of integral intensity of ∼70%, where the cubic primitive QIID phase emerges at >50 °C. Obviously, the mean population of gauche conformers in kink sequences is significantly greater in the QIIP phase compared with the lamellar liquid-crystalline LR phase, whereas it is slightly smaller in the QIID phase. These results support in a more quantitative way the findings that were already discussed when interpreting the band shifts of the CH2 stretching vibrations. Correlation Field Splitting. Pressure-induced correlation field splitting of the CH2 bending (δCH2) and rocking (τCH2) modes of the methylene chains can be used to monitor the structural and dynamic properties of lipid bilayers. The δCH2 bending vibration is located in the wavenumber region between 1460 and 1480 cm-1, whereas the τCH2 rocking band is located at lower wavenumbers around 720-730 cm-1. At ambient pressure, both vibrational modes consist of a single band that splits into two at sufficiently high pressures. This so-called correlation field splitting originates from the vibrational coupling interaction between the fully extended methylene chains with different site symmetry along each bilayer leaflet.49 The observation of only a single CH2 rocking band at atmospheric pressure reflects the fact that under these conditions of temperature and pressure, the orientation of the methylene chains is highly disordered due to significant reorientational fluctuations of the acyl chains. At high enough pressures, these fluctuations can be dampened, which leads to an increase in interchain interaction and thus the correlation field splitting appears in the spectra, provided that the equilibrium orientations of neighboring chains are nonequivalent. Figure 7 shows a sequence of IR spectra of the terminal CH2 rocking modes of the ME/D2O dispersion as a function of pressure at 50 and 65 °C. The band at 695 cm-1 is the phonon band of R-quartz, which was used for the pressure calibration (see Materials and Methods). It is clearly seen that the τCH2 mode splits into two branches at pressures >2.7 kbar for T ) 50 °C and ∼3.8 kbar at T ) 65 °C, and the intensity of the correlation field components increases with increasing pressure. One can assume that at these conditions the crystalline Lc phase is reached. The wavenumbers of the τCH2 bands for the various temper(48) Senak, L.; Davies, M. A.; Mendelsohn, R. J. Phys. Chem. 1991, 95, 2565.
atures studied are plotted as a function of pressure in Figure 8. It is clearly seen that the pressure at which the onset of the correlation field splitting, pcf, occurs increases with increasing temperature. The splitting starts at 2.7 kbar at 50 °C, and pcf increases to 3.8 kbar at 65 °C and 4.6 kbar at 90 °C. Because the magnitude of the required pressure is a measure for the order/disorder dynamics of the methylene chains it is evident that at higher temperatures, orientation of these is more disordered due to reorientational fluctuations and torsion/twisting motions. After all gauche conformers are gradually removed from the chains, the high-frequency branch of the split τCH2 band increases linearly with pressure, whereas the wavenumber of the low-frequency component is pressure independent to a first approximation. The absolute position of the wavenumbers of both components is the same for all temperatures at high pressures. When all gauche conformers are removed by pressure, the magnitude of the correlation field splitting is a measure of the degree of interchain interaction in the lipid bilayer. Thus, in the case of ME/ water, these interactions are obviously temperature independent when all fluctuations are dampened at high enough pressures. Analysis of the correlation field splitting of the δCH2 bending mode (data not shown) led to the same values for the correlation field pressure pcf and the overall magnitude of splitting. Carbonyl Stretching Mode. The CdO stretching vibration of ME is located at a wavenumber of ∼1737 cm-1. The contour and position of the carbonyl band is sensitive and responsive to conformational changes in the interfacial region and to hydration or nonhydration of the headgroup. Thus, interpretation of spectral changes of this band is not straightforward, as already discussed elsewhere.40,41 However, the conformational effect, as shown by reverse isotopic labeling, is negligible with only 1-2 cm-1, and therefore changes in position and intensity of the CdO stretching band are mainly caused by carbonyl groups taking part in hydrogen bonding to water.40,41 Figure 9 depicts the contour of the carbonyl stretching vibration as a function of temperature. It is clearly seen, that the band is not symmetric but shows a shoulder at low wavenumbers that increases drastically at a temperature of 23.5 °C, where the Lβ-LR phase transition takes place. At ∼44 °C, where the QIIP phase is formed, the maxima of the two components shift to higher frequencies with increasing temperature. In the region where the QIID phase appears, the opposite effect is observed. Analysis of the band by Fourier self-deconvolution gives rise to two overlapping bands centered at ∼1737 and ∼1718 cm-1, which can be assigned to stretching vibrations of free carbonyl groups that are not
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Figure 9. The CdO band contour of ME in excess D2O at temperatures from 15 to 68 °C (p ) 1 bar).
Figure 10. Carbonyl spectral region of the IR spectrum of ME/excess D2O at 20 °C. Key: (full line) experimental IR spectrum; (dashed line) calculated band shape with two Gaussian-Lorentzian functions; (dotted lines) GaussianLorentzian functions.
involved in hydrogen bonding and to hydrogen-bonded CdO groups whose vibrations are red-shifted to lower wavenumbers, respectively. Assuming that water is the primary source of hydrogen donors to which carbonyl groups can bond, which is certainly reasonable in this case, it is possible to yield quantitative information about the water penetration within the different lamellar and nonlamellar phases. The carbonyl stretching band was fitted with two Gaussian-Lorentzian functions using the positions determined by the Fourier self-deconvolution technique as starting parameters. Figure 10 shows the 1650-1800 cm-1 spectral region of the IR spectrum of ME/D2O at 20 °C, where the dashed line is the calculated band shape and the dotted lines are the two Gaussian-Lorentzian functions. The integral intensity ratio of these two bands is a measure for the degree of hydration of the bilayer apolar interface. Figure 11 depicts the intensity ratio of the two carbonyl stretching band components as a function of temperature. At the Lβ-LR phase transformation at 23.5 °C, the intensity of the high-frequency component decreases drastically, indicating an increasing contribution of CdO groups that take part in hydrogen bonds with water due to the lateral expansion of the lipid lattice. Formation of the cubic QIIP phase at ∼44 °C leads to a slight decrease of the population of hydrated carbonyl groups. A slight increase is observed when the QIID phase emerges at ∼58 °C. Obviously, compared with the bodycentered cubic QIIP phase, the CdO groups in the cubic primitive QIID phase are slightly more accessible to water. The various liquid-crystalline phases of ME in water thus not only differ in mean acyl chain conformation, but also slightly in carbonyl ester hydration.
Reis and Winter
Figure 11. Intensity ratio of the high (nonhydrated CdO groups) to low (hydrated CdO groups) frequency component of the carbonyl stretching contour band of ME/excess D2O as a function of temperature (p ) 1 bar).
Figure 12. The pressure dependence of the intensity ratio between the high- and low-frequency component of the carbonyl stretching vibration of ME/excess D2O at T ) 50 °C.
The pressure dependence of the intensity ratio between the high- and low-frequency component of the carbonyl stretching vibration is shown in Figure 12 at a temperature of 50 °C. At ∼1.6 kbar the LR-Lβ phase transition leads to a decrease of hydrated CdO group, which is expressed by an increase of the intensity ratio. The level of hydration decreases again at ∼3.2 kbar, where the crystalline lamellar Lc phase is formed. Further increase of pressure leads to a slight increase of carbonyl groups involved in hydrogen bondings. The same analysis, done for temperatures of 65 and 90 °C (data not shown), gave analogous results for the phase transitions LR-Lβ and Lβ-Lc. Differences in the hydration level between fluidlike phases cannot be detected with sufficient accuracy in these pressure studies in the diamond anvil cell. Conclusions Fourier transform-infrared spectroscopy has been used to characterize differences in conformation and hydration between the different lamellar and nonlamellar phases of ME in excess water and, in combination with synchrotron X-ray diffraction results,28 to establish the T,p-phase diagram of the system over an extended temperature and pressure range. At ambient pressure, the Lβ-LR transition occurs at ∼23.5 °C and the LR-QIIP transition occurs at ∼44 °C. At temperatures above ∼55 °C, the cubic primitive phase QIID emerges. The two bicontinuous cubic phases exhibit small but significant differences in absolute band positions of the CH2 stretching modes that can be correlated to changes in the average population of gauche conformers. The lipid chains of the cubic phase QIIP seem to contain a slightly higher population of gauche conformers. Analysis of the CH2 wagging progression region was used to
Pressure and Temperature Effects on Monoelaidin
yield more quantitative information about the distribution of gauche-trans-gauche sequences in the acyl chain. The integral intensity of the 1368 cm-1 band was normalized to the intensity of the CH3 deformation mode at 1378 cm-1 and was used as a measure for the amount of kink sequences. Compared with the lamellar LR phase, the relative amount of kink sequences is increased in the QIIP phase by ∼60% and decreased by ∼70% in the cubic phase QIID. The latter phase thus seems to have the smallest conformational acyl chain disorder of the three liquidcrystalline phases of ME in excess water. Information about hydrational effects in the lipid headgroup region is reflected by the carbonyl stretching vibration at 1737 cm-1. This broad absorption band consists of two overlapping bands, that can be assigned to vibrations of free and hydrogen-bonded CdO groups, assuming that conformational changes are negligible. Contribution of hydrogen-bonded carbonyl groups increases drastically at the phase transition from the Lβ gel to the LR liquid-crystalline phase, which can be explained in terms of the large lateral expansion of the 2D lipid lattice. Analysis of the CdO stretching mode within the cubic QIIP phase shows a very small decrease of the average level of hydration compared with that of the LR phase. At high temperatures >55 °C, the QIID phase is formed. This form exhibits slightly more CdO groups that take part in hydrogen bonds with water. Obviously, part of the bound water is withdrawn when entering the QIIP phase, whereas the average hydration level of the LR and QIID phase is, to a first approximation, similar. The barotropic phase behavior was studied at three different temperatures (50, 65, and 90 °C). The LR-Lβ and Lβ-Lc phase transitions could easily be monitored by marked deviations from the linear pressure dependence of the CH2 stretching vibration mode. The intercubic QIIDQIIP and QIIP-LR transformation were not detectable within the accuracy of the experiment in the high-pressure sample environment. Pressure-induced correlation field splitting of the δCH2 bending and τCH2 rocking mode was analyzed to detect structural and dynamic changes of the system as a function of pressure. The splitting of the bands results from enhanced interchain interactions between oriented chains, which is expected when entering the crystalline lamellar Lc phase. Increasing temperature leads to an increase of the onset of the correlation field splitting, which is indicative of increasing reorientational fluctuations and torsion/twisting motions of the hydrocarbon chain. The overall magnitude of splittings at high pressures is the same for all temperatures, showing that the interchain interactions are temperature independent when all fluctuations have been dampened by pressure. In the lower temperature region of the T,p-phase diagram of ME/water, the phase sequence as a function of pressure is, as expected, LR-Lβ-Lc, due to the decreasing partial molar lipid volumes in this series. The Lβ-Lc phase
Langmuir, Vol. 14, No. 10, 1998 2909
boundary has a slope of ∼20 °C/kbar. For the LR-Lβ transition line, a slope of ∼20 °C/kbar is also found, which is similar to that of the corresponding phospholipid system dielaidoylphosphatidylcholine and to saturated phospholipids of chain length C14-C18.50 Increasing the temperature will introduce greater disorder in the hydrocarbon chain, thus leading to a larger chain splay and greater desire for the lipid interface to curve toward the aqueous region. As a consequence, bicontinuous cubic phases are formed. The dominant effect of pressure will then be a straightening of the chains as the molecular volume of the system is reduced by pressurization. The lattice constant of the lamellar Lβ phase changes at a rate of 0.24 Å/°C.22 This increase of lattice constant with increasing temperature indicates an increase of the interlamellar water layer and an increase in headgroup hydration, which is also observed in the IR data. The lattice constant of the LR phase changes at a rate of -0.12 Å/°C.22 The decrease in lattice constant in the LR phase is due to a marked increase of conformational disorder. It is clear that an understanding of the form of the T,pphase diagram of ME/water would require a detailed consideration of all the complex interactions involved, such as interfacial, hydration, and van der Waals forces, steric repulsion, hydrogen bonding, as well as the geometry of the lipid molecule as a function of the thermodynamic parameters temperature and pressure. At present, modeling of these contributions is still in its infancy9 and one is not yet able to predict lyotropic phase stability and phase sequence as a function of temperature and pressure. Current energetic models predict that the different inverse bicontinuous cubic phases QIIG, QIID, and QIIP, which are based on the G, D, and P minimal surfaces, are energetically degenerate. In fact, this energetic degeneracy is not observed in the experimental results for establishing the T,p-phase diagram of this and other lipid systems.8,22,23. Recently, it has been shown by Templer et al.16 that the energetic degeneracy of the bicontinuous cubic phases can indeed be broken by changes in the geometry of the interface. Analysis of 2:1 fatty acid/phospholipid mixtures suggests that the destabilization of QIIP with respect to QIID as one increases the temperature may be understood in terms of the requirements for the interface to alter geometry as it goes from one of almost constant mean curvature to one that approaches a constant thickness monolayer. The experimental data presented in this study on ME/water seem to support these ideas. Acknowledgment. We are grateful to the DFG and the Fonds der Chemischen Industrie for financial support. LA971213D (49) Wong, P. T. T. Biophys. J. 1994, 66, 1505. (50) Winter, R.; Pilgrim, W.-C. Ber. Bunsen-Ges. Phys. Chem. 1989, 93, 708.
