Palmitin System. A 2H NMR Study

Cédric Gaillard , Bruno Novales , François Jérôme and Jean-Paul Douliez ... Jean-Paul Douliez , Joël Barrault and François Jerome , Antonio Heredia ...
0 downloads 0 Views 374KB Size
Download PDF
Langmuir 2004, 20, 1543-1550

1543

Articles Phase Behavior of the Palmitic Acid/Palmitin System. A 2H NMR Study Jean-Paul Douliez† Unite´ de Recherche sur les Prote´ ines Ve´ ge´ tales et leurs Interactions, INRA, rue de la Ge´ raudie` re, 44316 Nantes, France Received September 15, 2003. In Final Form: December 11, 2003 The phase behavior of mixtures of palmitic acid (PA) and 1-monohexadecanoyl-rac-glycerol, palmitin, was studied by phase contrast microscopy and deuterium solid-state NMR. At pH 5, mixtures remained precipitated as lumps in solution. The NMR spectrum of the perdeuterated PA (PAd31) at 300 K exhibited a shape and quadrupolar splittings, ∆ν, characteristic of lipids embedded in a gel phase. The alkyl chains remained in a trans conformation with their long molecular axis oriented at about 15° with respect to the bilayer normal. However, gauche defects were shown to occur at the end of the alkyl chain. At 330 K, the system underwent a phase transition to a hexagonal phase followed by an isotropic phase at 340 K. Upon cooling to 330 K, the spectrum in the hexagonal phase was oriented at 0° showing that the cylinders were oriented with their long axis parallel to the field. Up to 11 positions (from 15) of PAd31 could be assigned. At pH 7 and 9 at room temperature, the mixtures were fully dispersed in a viscous solution of vesicles. The system underwent a phase transition at 320 K from a gel phase to a fluid phase with the bilayer normal oriented at 90° with respect to the field. Analogous experiments performed with PA selectively labeled on carbon C2 allowed for the assignment of ∆ν for that position and suggested different conformations of the headgroup in the gel and fluid or hexagonal phases. The implications of these findings for the bio-availability of these fatty acids, in the understanding of the contribution of hydroxyl and carboxyl groups in the membrane formation, and for the production of simple self-oriented systems are discussed.

Introduction Most of the phospholipids bearing two alkyl chains are known to form bilayers that can assemble to form vesicles.1 Fatty acids also exhibit such properties depending on the pH and the concentration.2-5 Bilayer formation occurs at a pH around the pKa of the fatty acid carboxylic group, suggesting that hydrogen bonds between the carboxylic and carboxylate groups are determinant. Moreover, incorporation of an alcohol at high pH promotes the fatty acid self-assembly as vesicles and decreases the critical vesicular concentration at a neutral pH.3 Other ligands such as alkylamine6 or cholesterol7,8 have been studied in mixtures with fatty acids and also promote the formation of membranes. It was then prospective to study the systems constituted by a fatty acid and its analogue of monoglyceride. Monoglycerides that consist of a fatty acid esterified to the glycerol are of importance in both industrial and biological contexts. The hydroxyl groups of the glycerol moiety should provide the hydrogen bonds to †

Phone: 33 240 67 50 56. Fax: 33 240 67 50 25. E-mail address: [email protected]. (1) Koynova, R.; Caffrey, M. Chem. Phys. Lipids 2002, 115, 107219. (2) Fukuda, H.; Goto, A.; Yoshioka, H.; Goto, R.; Morigaki, K.; Walde, P. Langmuir 2001, 17, 4223-4231. (3) Apel, C. L.; Deamer, D. W.; Mautner, M. N. Biochim. Biophys. Acta 2002, 1559, 1-9. (4) Morigaki, K.; Walde, P. Langmuir 2002, 18, 10509-10511. (5) Morigaki, K.; Walde, P.; Misran, M.; Robinson, B. H. Colloids Surf., A 2003, 213, 37-44. (6) Karlsson, S.; Friman, R.; Lindstrom, B.; Backlund, S. J. Colloid Interface Sci. 2001, 243, 241-247. (7) Pare, C.; Lafleur, M. Langmuir 2001, 17, 5587-5594. (8) Ouimet, J.; Croft, S.; Pare, C.; Katsaras, J.; Lafleur, M. Langmuir 2003, 19, 1089-1097.

stabilize the fatty acid membrane formation. Monoglycerides exhibit a broad polymorphism including coagel, gel, fluid, cubic, and hexagonal phases.9-15 No homogeneous dispersion of long-chain 1-monoglyceride can be obtained because it rather forms lumps of precipitated material.16 Comparatively, the monoglyceride isomer at position 2, 2-monohexadecanoyl-rac-glycerol, exhibits a lamellar crystalline phase in excess water.17 Beyond the fundamental aspect, studying mixtures of fatty acids and monoglycerides is of biological importance because these lipids are precursors of plant hydrophobic polymers, cutin, and suberin. These polymers are constituted by interesterified mono-, di-, and trihydroxyl; mono- and dicarboxyl palmitic and stearic; and glycerol, which also behaves as a hydroxyl group donor for the esterification of monomers.18-20 The routing of these fatty acids from their synthesis in the cell to the extracellular matrix is still unknown, although lipid binding proteins or lipid cor(9) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223-234. (10) Chupin, V.; Boots, J.-W. P.; Killian, J. A.; Demel, R. A.; de Kruijff, B. Chem. Phys. Lipids 2001, 109, 15-28. (11) Briggs, J.; Caffrey, M. Biophys. J. 1994, 67, 1594-1602. (12) Briggs, J.; Caffrey, M. Biophys. J. 1994, 66, 573-587. (13) Chung, H.; Caffrey, M. Biophys. J. 1995, 69, 1951-1963. (14) Clogston, J.; Rathman, J.; Tomasko, D.; Walker, H.; Caffrey, M. Chem. Phys. Lipids 2000, 107, 191-220. (15) Aota-Nakano, Y.; Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96-102. (16) Boots, J.-W. P.; Chupin, V.; Killian, J. A.; Demel, R. A.; de Kruijff, B. Biochim. Biophys. Acta 1999, 1420, 241-251. (17) Ljusberg-Wahren, H.; Herslof, M.; Larsson, K. Chem. Phys. Lipids 1983, 33, 211-214. (18) Heredia, A. Biochim. Biophys. Acta 2003, 1620, 1-7. (19) Graca, J.; Schreiber, L.; Rodrigues, J.; Pereira, H. Phytochemistry 2002, 61, 205-215. (20) Graca, J.; Pereira, H. J. Agric. Food Sci. 2000, 48, 5476-5483.

10.1021/la035719+ CCC: $27.50 © 2004 American Chemical Society Published on Web 01/27/2004

1544

Langmuir, Vol. 20, No. 5, 2004

puscles are supposed to be involved.18,21,22 Moreover, these monomers are poorly water soluble and determining the physicochemical parameters required for their dispersion in water is of practical and industrial importance, especially if one wants to increase their bio-availability and use them as a substrate for enzymes. As in the case of phospholipids,23-25 solid-state NMR has proven to be a powerful tool for investigating the phase behavior of monoglycerides10,26 or fatty acid-containing systems.7,8 Deuterium NMR is sensitive to the dynamic of alkyl chains, that is, the gradient of mobility along the alkyl chains.23,24,27,28 It can probe phase transitions and spontaneous self-orientation of molecules.10,29,30 Fourier transform infrared (FTIR) spectroscopy is also widely used to follow the thermotropism of lipid dispersions. In the case of mixtures of deuterated labeled and nonlabeled lipids, this allows for the monitoring of both the νCH and the νCD bands as a function of the temperature and, then, the melting of both lipid alkyl chains, separately.31 Interestingly, information on the thermotropism of the lipid mixture can be obtained in a very fast way. In the present paper, FTIR and deuterium NMR were successfully used to follow the phase behavior of mixtures of palmitic acid (PA) and 1-monohexadecanoylrac-glycerol, palmitin (PAgly), as a function of the pH and the molar ratio PA/PAgly, with the aim of determining the polymorphism of this system and the various parameters allowing the full dispersions of these lipids in water. Materials and Methods Lipids from Sigma-Aldrich, both 99% purity, were weighed exactly (10 or 100 mg total lipids) in a tube at molar ratios of PA/PAgly of 1, 2, or 10. The mixtures were melted at 70 °C for 10 min until all components were liquid and then vigorously vortexed. This procedure allowed for going beyond the utilization of solvents, which is of industrial importance because their use is often prohibited. Then, samples were prepared by hydration with 1 mL of either Borate buffer (10 mM, pH 9) or 2-(Nmorpholino)ethanesulfonic acid (10 mM, pH 5) and subsequent heating and vortexing at 70 °C for at least 5 min. For samples at pH 9, the pH had to be readjusted with a 1 M NaOH solution (about 100, 150, and 200 µL for molar ratios of PA/PAgly of 1, 2, and 10, respectively). All samples were then freeze-dried. Mixtures were rehydrated with 500 µL of nondeuterium-depleted water and 500 µL of the same desired buffer. All mixtures were submitted to three additional cycles of heating plus vigorous vortexing (60 °C, 10 min) and cooling (-20 °C, 30 min). The pH was checked, and samples were stored at -20 °C. Prior to being used, each sample was heated at 70 °C for 10 min. FTIR Measurements. FTIR spectra (250 scans) were recorded as a function of increasing temperature at a resolution of 1 cm-1 on a Nicolet Magna IR 550 spectrometer. After heating to 70 °C, a drop (100 µL) of sample at pH 9 or a lump of sample at pH 5 was deposited on the crystal and then covered with a homemade cup, allowing for control of the (21) Douliez, J.-P.; Michon, T.; Marion, D. Biochim. Biophys. Acta 2000, 1467, 65-72. (22) Douliez, J.-P.; Michon, T.; Elmorjani, K.; Marion, D. J. Cer. Sci. 2000, 32, 1-20. (23) Seelig, J. Q. Rev. Biophys. 1977, 10, 353-418. (24) Davis, J. Biochim. Biophys. Acta 1983, 737, 117-171. (25) Smith, R. L.; Oldfield, E. Science 1984, 225, 280-288. (26) Cassin, G.; de Costa, C.; van Duynhoven, J. P. M.; Agterof, W. G. M. Langmuir 1998, 14, 5757-5763. (27) Douliez, J.; Leonard, A.; Dufourc, E. Biophys. J. 1995, 68, 17271739. (28) Lafleur, M.; Fine, B.; Sternin, E.; Cullis, P.; Bloom, M. Biophys. J. 1989, 56, 1037-1041. (29) Carlotti, C.; Aussenac, F.; Dufourc, E. J. Biochim. Biophys. Acta 2002, 1564, 156-164. (30) Seelig, J.; Borle, F.; Cross, T. A. Biochim. Biophys. Acta 1985, 814, 195-198. (31) Velkova, V.; Lafleur, M. Chem. Phys. Lipids 2002, 117, 63-74.

Douliez hydration. Signals were obtained by attenuated total reflection using a single reflection accessory fitted with a thermostated diamond crystal. Solid-State NMR. Deuterium solid-state NMR experiments were performed at several temperatures from 300 to 350 K on a 400-MHz Bruker spectrometer operating at 61 MHz for deuterium using a static double-channel probe to which the sample coil was adapted to load a 7-mm rotor such as those used for magic-angle spinning (MAS) probes equipped with a stretched stator. Typically, lipid dispersions were previously heated at 70 °C, and 700 µL were transferred in the rotor, which was sealed and then end-capped. A Hahn quadrupolar echo sequence24 was used with an interpulse delay of 40 µs. A total of8000 points in 1000 accumulations (every 2 s) were done with a 90° pulse and spectral width of 8 µs and 250 kHz. The free induction decay signals were zero-filled to 16 000 points prior to Fourier transform after a broad line exponential multiplication of 200 Hz. For deuterium spectroscopy, the general theory for lipid systems can be found in the literature.23,24 Briefly, the deuterium NMR signal is composed of doublets separated by a quadrupolar splitting, ∆ν. In an anisotropic medium, these doublets are superimposed, forming a powder spectrum having two peaks with an increased intensity that correspond to the 90° orientation, separated by ∆ν90. The edge of the spectrum corresponds to the 0° orientation with a splitting of ∆ν0, the value of which is 2∆ν90. In the case of perdeuterated systems, the spectrum is composed of the superimposition of signals from each labeled position. In the case of oriented samples, the spectrum no longer exhibits a powder shape but is composed of doublets corresponding to the given orientation, one for each labeled position. From the powder spectrum, one may obtain the corresponding oriented spectrum by using a computer calculation, the so-called depaking procedure.32 Order parameters, SCD, are generally defined as the ratio between the quadrupolar splitting at the 90° orientation, ∆ν90, and a quadrupolar constant, AQ ) 125 kHz. Phase Contrast Microscopy. Observations were made at room temperature at 20× magnification using an optical microscope in the phase contrast mode (Nikon Eclipse E-400, Tokyo, Japan) equipped with a 3-CCD JVC camera allowing digital images (768 × 512 pixels) to be collected. A drop of the lipid dispersion (about 20 µL) was deposited on the glass slide surface (76 × 26 × 1.1 mm, RS, France) and covered with a cover slide (22 × 22 mm, Menzel-Glaser, Germany). The glass slides were previously cleaned with ethanol and acetone.

Results Visual Inspection and Phase Contrast Microscopy. Whatever the pH at a concentration of 10 or 100 mg lipids, one observed by visual inspection and phase contrast microscopy that pure PA remained precipitated as crystals in solution at room temperature (not shown). At a temperature of about 60 °C, PA melted as droplets in water, as already observed by infrared spectroscopy, 13 C MAS, or 2H NMR7,8 and as indicated by the manufacturer. In the same way, no homogeneous dispersion could be prepared in the case of pure PAgly. The material remained precipitated and formed lumps. Such an observation was already reported in the case of 1-stearin (1-monooctadecanoyl-rac-glycerol), a monoglyceride with a stearic chain length.16 Figure 1A depicts the observation made by phase contrast microscopy for mixtures of PA and PAgly at pH 5. Lumps of aggregated membranes are clearly visible as patches of various gray scale. The uniform patch corresponds to water and attests the heterogeneity of the solution. The appearance is very similar to that observed for pure PAgly (not shown). There are no crystals of PA, suggesting that it was fully embedded in the PAgly matrix. Contrary to this, at pH 7 or 9, mixtures of PA and PAgly were perfectly dispersed as turbid viscous solutions at room temperature. Black dots were observed by phase (32) Bloom, M.; Davis, J.; Mackay, A. L. Chem. Phys. Lett. 1981, 80, 198-201.

Palmitic Acid/Palmitin System

Figure 1. Micrographs obtained by phase contrast microscopy for samples at pH 5 (A) and 9 (B), at a total lipid concentration of 100 mg/mL and Ra ) 1. The scale bar corresponds to 10 µm.

Figure 2. Thermotropism as probed by the νCH (circles) and νCD (squares) frequencies for equimolar mixtures of PAd31/PAgly at pH 5 (closed symbols) and 9 (open symbol) at a total lipid concentration of 100 mg/mL. The solid lines represent a guide to the eye.

contrast microscopy characteristic of vesicles having a diameter of several micrometers (Figure 1B). At room temperature, samples were viscous even at a low lipid concentration (10 mg/mL). At a molar ratio of PA/PAgly of 10, some crystals of PA were also visible (not shown), suggesting that there exists a limit of solubility of PA in the PAgly matrix at room temperature. FTIR Measurements. The shift of the νCH and νCD bands as a function of temperature is a nice probe to follow the dynamic of the lipid alkyl chains. This brings, in a fast way, interesting information on the thermotropism of the mixtures prior to the NMR investigation. Figure 2 depicts the variation of these frequencies as a function of temperature for the equimolar mixtures constituted by perdeuterated PA (PAd31)/PAgly at pH 5 and 9. For all the investigated systems, at 300 K, the νCH and νCD bands have frequencies of about 2850 and 2089 cm-1, respec-

Langmuir, Vol. 20, No. 5, 2004 1545

tively, indicative of ordered membranes.31 Upon heating, at 320 and 330 K for mixtures at pH 9 and 5, respectively, both bands shifted in the same way toward a higher wavenumber by about 3 cm-1. These results reveal, at a given pH, the disordering of the PA and PAgly alkyl chains representative of lipids embedded in a fluid phase. Experiments performed with unlabeled PA provided similar results (not shown). No shift of the phase transition to a higher temperature was detected. Such a shift is generally expected because of the isotopic labeling between unlabeled and deuterated molecules.24 However, the present experiments were performed every 5 °C, and the shift is then probably weaker. Interestingly, the shift observed for the melting of pure PA is about 5 °C,7 whereas it is only 2 °C in the case of phospholipids.24 Altogether, the latter results revealed that this technique should be relevant for further studies of lipid mixtures involving unlabeled material such as fatty acids extracted from crude cutin or suberin. Deuterium NMR. To have insight into the phase behavior and the mobility of PA chains in these mixtures, 2 H NMR was employed. 2H NMR spectra were collected from samples prepared with PA selectively labeled at position C2 (PAd2) and fully deuterated (PAd31). Figures 3 and 4 show typical spectra as a function of the temperature obtained for a molar ratio of PA/PAgly of 1 at pH 9. Similar spectra were recorded at a molar ratio of PA/PAgly of 2 or at pH 7 (not shown). Spectra were composed of one isotropic line associated with the signal of deuterated water in natural abundance and powder components having two major peaks separated by a socalled quadrupolar splitting.23,24 The signal of water was significantly shifted on the left with respect to the middle of the powder spectra consistent with the difference in the deuterium chemical shift between water and aliphatic chains. At 300 K, the largest broad powder component for PAd31 exhibited a quadrupolar splitting of 56 kHz (plateau) and the lowest 13 kHz. These values are slightly higher than those reported for pure stearin in the gel phase.10 At the same temperature, PAd2 exhibited a broad spectrum (Figure 4) with a quadrupolar splitting of 47 kHz. Note that this value is markedly lower than the 56 kHz previously measured. Another interesting feature of these spectra is the broad nonaxially symmetric line shape of the signal. Although this is less marked than in the case of pure stearin,10 this suggests that alkyl chains exhibit motions not fast enough on the NMR time scale, yielding to a nonzero asymmetry parameter.33 Then, by analogy with pure stearin,10 PA alkyl chains are supposed to be in the all-trans conformation with their long axis, ∆, tilted with respect to the bilayer normal, n. The molecule rotates around the axis ∆, and as a consequence, the quadrupolar splittings are lower than the one expected in the static case.33 The largest powder component of the PAd31 spectrum is then attributed to the CD2 groups from carbon C3 to carbon C15, while the lowest stands for the methyl terminal group.10 With this hypothesis, one can estimate R, the tilt angle between ∆ and n. In a first assumption, one should consider that PA rotates with an axial symmetry around its long molecular axis without wobbling.27 Although this is not rigorously the case, this is not expected to markedly change the result because the asymmetry parameter is weak enough. Moreover, lipids can freely diffuse in the membrane what is equivalent to a rotation around the bilayer normal. These two motions require two transformation of (33) Davis, J. H. Biophys. J. 1979, 27, 339-358.

1546

Langmuir, Vol. 20, No. 5, 2004

Douliez

Figure 4. Selected NMR spectra of equimolar mixtures of PAd2/ PAgly at pH 9 as a function of the temperature at 300 (top) and 330 (bottom) K. For the sake of clarity, the frequency scale is reduced to -60 to +60 kHz to allow a better display of the spectrum at 330 K.

three C-D bonds make an angle of 111° with the C15-C16 bond, one can calculate the methyl order parameter, SCD 16 , and then the corresponding quadrupolar splitting.27 This may simply be written as Figure 3. Selected NMR spectra of equimolar mixtures of PAd31/PAgly at pH 9 as a function of the temperature at 300 (A), 320 (B), and 330 (C) K. For the sake of clarity, the frequency scale is reduced to -60 to +60 kHz to allow a better display of the spectrum at 330 K.

coordinates,24 and the quadrupolar splitting can then be simply written as

∆ν ) 56 kHz ) AQ[3 cos2(90°) - 1]/2 × [3 cos2(R) - 1]/2 This returns a value for R of about 15°. The hypothesis of the all-trans conformation may be tested by using the concept of carbon-carbon order parameters, SCC, previously developed.27 This implies that CC CD SCC k + Sk+1 ) -2Sk

where k is the carbon position and k ) 1 and 16 being the carbonyl and the methyl groups, respectively. SCC are the order parameters for the carbon-carbon bonds defined as in the case of a C-D bond. In the present case, with the hypothesis of the all-trans conformation, for symmetry CC reasons, this implies for instance that SCD 15 ) S16 . By assuming that the terminal methyl group rotates around the C15-C16 bond with an axial symmetry and that the

2 CD CD ∆νCD 16 ) ∆ν15 [3 cos (111°) - 1]/2 ) -0.31∆ν15

which returns an absolute value of 17 kHz, slightly higher than that obtained experimentally (13 kHz). This reveals that the hypothesis of an all-trans conformation is not strictly valid and that there exists some mobility along the alkyl chains in the gel phase yielding to a lower quadrupolar splitting for the methyl terminal. At 320 K, PAd31 exhibited a signal characteristic of the coexistence of two phases. Let us remember that this temperature corresponds to a phase transition, as observed by FTIR. The outermost doublet can be assigned to the gel phase, which has now a quadrupolar splitting of 55 kHz, which is slightly lower than that just described, meaning that the mobility increased or that the tilt angle was slightly modified. This signal is not as broad as that observed at 300 K, suggesting that the asymmetry parameter vanished and showing that motions became fast enough on the NMR time scale. The rest of the spectrum is composed of several overlapping signals with an increased 90° intensity characteristic of an oriented spectrum in which nine quadrupolar splitting may be resolved. This feature is clearly evident at higher temperatures. Increasing the temperature to 330 K yielded a single phase that can be attributed to a fluid lamellar phase in which the bilayer normal is oriented at 90° with

Palmitic Acid/Palmitin System

Langmuir, Vol. 20, No. 5, 2004 1547

Figure 5. Selected NMR spectra of equimolar mixtures of PAd31/PAgly at pH 5 as a function of increasing temperature at 320 (A), 330 (B and C), and 340 (D) K. Spectra E (330 K) and F (320 K) are obtained upon cooling from 340 K. The frequency scale for the spectra on the right (C-E) are magnified, from -12 to +12 kHz, to better display the quadrupolar splittings.

respect to the magnetic field. The outermost doublet exhibited a value of 27 kHz, usually defined as the “plateau”. This generally includes the first positions of the alkyl chains. The other positions are assigned by assuming an increase of disorder when going toward the alkyl chain.28 A similar spectrum was recorded at 340 K (not shown) with lower ∆ν than those obtained at 320 or 330 K in coherence with an expected increase of the mobility with the temperature. Except for the signal of water, no one observed any isotropic line centered in the middle of the spectra that would characterize the appearance of a cubic phase, as observed for pure monoglyceride10 or in mixtures with fatty acids.15 The signal of PAd2 at 330 K was also characteristic of an oriented spectrum with increased 90° intensity (Figure 4), and a quadrupolar splitting of 27 kHz could be measured. This value is identical to that obtained for the plateau in the case of PAd31. The comparison with the data obtained in the gel phase suggests a different orientation and mobility of the polar head at the lipid-water interface in both the gel and fluid phases. For samples at a molar ratio of PA/PAgly of 10, similar spectra were recorded (not shown), except that, at 300 K, two additional overlapping signals with about a 10% relative intensity were observed. These exhibited a large

splitting of about 125 and 36 kHz. The largest value is that obtained for carbon-deuterium bonds in the static case, suggesting that about 10% of PAd31 was in the crystalline form. Such an observation has already been made in mixtures of PA with cholesterol.7,8 The lowest splitting was assigned to the crystal PA methyl terminal group in agreement with the scaling factor of about 3 due to its rotation (see previous text). This emphasizes, as observed by phase contrast microscopy, that there exists a limit of solubility of PA in the PAgly matrix at room temperature. Upon heating, these two signals vanished, showing that PA crystals disappeared and that the fatty acid was now fully embedded in the membranes. At pH 5, the PAd31 spectrum at 320 K was similar to that obtained at higher pH. A plateau having a quadrupolar splitting of 56 kHz was observed. However, three other positions could be well-resolved with respectively 50, 46.5, and 38 kHz (Figure 5). The signal for PAd2 exhibited a single doublet of 47 kHz lower than the value obtained for the plateau (56 kHz), as observed previously at a higher pH. On the PAd31 spectrum, the methyl position has a splitting of 10 kHz, a value lower than at pH 9, suggesting an increase of the alkyl chain mobility. By assuming a gradient of mobility toward the methyl end, the plateau of 56 kHz extends from carbon C3 to carbon

1548

Langmuir, Vol. 20, No. 5, 2004

CD C13, ∆νCD 14 ) 50 kHz and ∆ν15 ) 38 kHz. The value of 46.5 kHz obtained from the PAd31 signal could then be assigned to carbon C2. From those data, with the similar hypothesis made at pH 9 and using the concept of SCC, one can calculate a value for ∆νCD 16 . Briefly, for symmetry reasons

CD SCC 14 ) S13 ) 56 kHz/AQ

Then, CD CC SCC 15 ) -2S14 - S14 ) (2 × 50 - 56)/AQ ) 44 kHz/AQ

where it must be reminded that C-D order parameters have negative values.27 In the same way, CD CC SCC 16 ) -2S15 - S15 ) (2 × 38 - 44)/AQ ) 32 kHz/AQ

It follows that ∆νCD 16 ) 0.31 × 32 ) 9.9 kHz, a value very close to the experimental splitting. This emphasizes that, at pH 5, positions C3 to carbon C13 stand in an all-trans conformation with the long axis oriented at about 15° with respect to the long molecular axis. The disorder, that is, gauche conformers, occurs from position C14 to the methyl end. Increasing the temperature to 330 K resulted in a marked decrease of the splitting (Figure 5B,C). Once again, this temperature corresponded to a phase transition as monitored by FTIR. The magnification of this spectrum depicted the overlapping of several signals where several splittings could be resolved. The order varies more uniformly than in the case of the fluid phase. The outermost doublet and the methyl group had a splitting at the 90° orientation, ∆ν90 of 7.5 kHz and 580 Hz, respectively. These values are markedly lower that those previously obtained for PA embedded in the fluid phase. This feature is characteristic of alkyl chains embedded in a hexagonal phase.34-38 For lipids in a hexagonal phase, there is an additional axis of symmetry due to the translational diffusion of molecules over the surface of the cylinders. Moreover, the quadrupolar splittings in this phase are more than a factor of 2 smaller than those in the fluid phase, as theoretically expected,24 because of the combination between the motion mentioned above and the increased orientational freedom of the alkyl chains in such a phase.34-37 At 340 K, the system underwent another transition because an isotropic line was obtained (Figure 5D). This phase was probably a cubic phase, the curvature of which is such that lipids undergo fast isotropic motions, or indicates that PA melted with solubilized PAgly. Upon cooling to 330 K, one obtained a spectrum, the aspect of which was rather different from that observed upon heating (Figure 5E). Several overlapping doublets were recorded characteristic of an oriented spectrum for which up to 11 positions could be resolved. The outermost doublet and the methyl group exhibited a splitting of 14.9 and 1.4 kHz, respectively, that is, exactly twice the values previously determined from the powder spectrum obtained upon heating from the gel phase. In other words, the present signal corresponds to a spectrum oriented at 0° (34) Thurmond, R. L.; Lindblom, G.; Brown, M. F. Biochem. Biophys. Res. Commun. 1990, 173, 1231-1238. (35) Lafleur, M.; Bloom, M.; Eikenberry, E. F.; Gruner, S. M.; Han, Y.; Cullis, P. R. Biophys. J. 1996, 70, 2747-2757. (36) Lafleur, M.; Cullis, P. R.; Fine, B.; Bloom, M. Biochemistry 1990, 29, 8325-8333. (37) Schorn, K.; Marsh, D. Biophys. J. 1996, 71, 3320-3329. (38) Armstrong, D. L.; Borchardt, D. B.; Zidovetzki, R. Biochem. Biophys. Res. Commun. 2002, 296, 806-812.

Douliez Table 1. Phase Behavior of the PA/PAgly Mixtures as a Function of the Temperature, T (K), and pH at Molar Ratios of PA/PAgly of 1 and 2 pH

T ) 300

T ) 310

T ) 320

T ) 330

T ) 340

5 7 and 9

gela gel

gela gel

gela gel/fluidb

hexagonala fluidb

isotrope fluidb

b

a The system self-orients upon cooling from the isotropic phase. The system self-orients at the given temperature.

with respect to the field. This finding emphasizes that, at this temperature, lipids are embedded in a hexagonal phase. In such a case, the long cylinder axis is oriented at 0° with respect to the field, and then, the normal of the lipid-water interface is oriented at 90° in coherence with the result obtained in the fluid phase at a higher pH. In the case of PAd2, the same behavior was observed upon heating and cooling and the splitting at 0° orientation in the hexagonal phase was 15 kHz (not shown). This value is similar to that determined for the outermost doublet in the case of the PAd31 spectrum. Upon further cooling to 320 K, one obtained a spectrum analogous to that previously observed in the gel phase but with an increased 90° orientation (Figure 5F), showing that the membrane bilayer normal was oriented at 90° with respect to the field. The splitting was in excellent agreement with those previously determined in Figure 5A. Discussion From the present data, it is clearly shown that the dispersion and the phase behavior of the PA/PAgly mixtures are clearly modulated by the pH and that the system self-orients in the magnetic field. The results are summarized in Table 1 and are discussed hereafter separately. Full Dispersion. Pure monoglycerides are not fully dispersed in water,10 and for such a task, addition of a surfactant is required.39 In the same way, PA forms crystals in aqueous solution. In the present work, at pH 7 or 9, the full dispersion of PAgly was accomplished by incorporating PA. The formation of membranes in such mixtures is facilitated by the capacity of the ionized carboxyl group of the fatty acid and the glycerol to form a hydrogen-bond network. This has already been demonstrated in the literature for mixtures of monocarboxylic acids and alcohols.3 However, at pH 5, membranes of PA and PAgly also formed although the carboxylic group of the fatty acid is protonated. Lamellar phases have also been observed at such a low pH in related mixtures.7,8 Then, hydrogen-bond formation between the carboxylic group and the alcohol could also help for the self-assembly in the bilayer. At a high pH, the electrostatic repulsion between PA polar heads allows the system to swell and incorporate water, allowing the full dispersion of the lipid mixtures. At a low pH, although self-assembly occurs, membranes or cylinders in the hexagonal phase stack together without swelling enough for the system be fully dispersed. The present findings may have some impact in biological areas because the full dispersion of such lipids is accomplished at neutral or basic pH, increasing their bioavailability. For instance, PA and PAgly are known as precursors of plant polymers, cutin, and suberin. The perception of free cutin monomers by plant cells has already been reported, and the weak solubility of the (39) Chupin, V.; Boots, J. W. P.; Killian, J. A.; Demel, R. A.; de Kruijff, B. Biophys. J. 2002, 82, 843-851.

Palmitic Acid/Palmitin System

Langmuir, Vol. 20, No. 5, 2004 1549

monomers was shown to be a limit for the experiments.40 Moreover, their use in enzymatic reactions is also hampered for the same reasons.18,41 Fatty acids and monoglycerides are also known to exhibit antibacterial activity42 so that, once again, increasing their bio-availability appears of practical interest. For instance, the effect of PA and PAgly on cell cultures, pathogens, or plant organs can now be evaluated by using such dispersions at pH 7 or 9 to induce the plant defense mechanism,40 pathogen inhibition,42 or cutin biosynthesis.18 Other enzymes such as a hydroxylase that participate in the monomer biosynthesis18 can now be used in solution to transform these cutin precursors into the hydroxyl monomers. In addition, the viscous character of these mixtures may have some additional interests in industrial fields, including cosmetics. Finally, the use of PA or PAgly for the production of emulsions is hampered because of the low solubility of these lipids in both water and the oil phase. The use of the PA/PAgly mixtures in buffer at pH 7 or 9 for such a task is promising and is under progress in the laboratory. Phase Behavior. Deuterium solid-state NMR has already been shown to be a powerful tool to follow the phase behavior in lipid systems.7,8,10,24,43 In the present work, this allowed for the identification of gel, fluid, hexagonal, and cubic phases. FTIR also provides valuable information on the alkyl chain mobility, although the type of the phase cannot be inferred from those data. However, it appears as an interesting probe of phase transitions prior to the NMR investigations. Moreover, in the case of PAd31-containing samples, this allows for the monitoring of the alkyl chain order of both PA and PAgly separately. This is not the case for NMR, which only probes the labeled chain. At a low temperature at pH 5, 7, or 9, the polymorphism of PA resembles that of pure stearin.10 However, no coagel phase was identified, which probably occurs at lower temperatures. In the gel phase, PA is more or less extended in the all-trans conformation, although some mobility at the end of the alkyl chain could be demonstrated. A similar conclusion can be made for stearin embedded in the gel phase10 from the experimental data in combination with the concept of carbon-carbon order parameters27 (not shown). The evidence of the occurrence of gauche defects in such a phase is more clearly demonstrated at pH 5. The calculated splitting of the methyl end matches rather well the experimental one, allowing that gauche conformers occurs from carbon C14 to be shown. In this phase, the long axis of the PA molecules is tilted with an angle of about 15° with respect to the bilayer normal. Such a value is similar to that obtained for other related lipid systems. It corresponds to an ideal stacking of the alkyl chains. The mobility and conformation of the polar head is such that the quadrupolar splitting of carbon C2 is different from that for the further carbon positions. It must be noticed that the NMR spectrum of the selectively labeled PAd2 is composed of a unique signal showing that both deuterons on this carbon are equivalents.24,43 This is not the case for carbon C2 on the sn-2 chains in some phospholipids embedded in membranes43,44 for which two signals are obtained. Interestingly, in the above case, the quadrupolar splitting for C2 is lower than for the plateau

because of a peculiar orientation.44 Altogether, the present findings rather suggest that the PA polar head exhibits gauche conformers around the C2-C3 bond. The unique condition is that the probability for the occurrence of a gauche + conformer is equal to that for a gauche so that both deuterons are indeed equivalents. Such a rapid motion is sufficient to induce a lower quadrupolar splitting for that particular carbon position compared to the plateau. Interestingly, the quadrupolar splitting for this carbon is identical whatever the pH, suggesting that the difference in mobility does not arise from electrostatic interactions. Such conformers should be in favor of a carboxylic group orientation more or less parallel to the interface, which is in agreement with findings from monolayer experiments of pure fatty acids.45 At pH 9 in the fluid phase, quadrupolar splittings are similar to those obtained for phospholipids.44 PA is embedded in the PAgly matrix, forming a relatively fluid membrane compared to the mixtures with cholesterol.7,8 The alkyl PA chains exhibit a typical gradient of disorder, and the gauche defects increase toward the end chain. At pH 5, the system undergoes a transition to a hexagonal phase. Note that such nonbilayer phases are often related to biological functions.33-36,38 This difference in the phase behavior as a function of the pH may be related to the difference of molecular shape. At a low pH, PA is protonated and the increase of the alkyl chain mobility with the temperature yields a conic molecular form that is in favor of a hexagonal phase.36 Contrary to this, at a higher pH, the carboxylate form of PA induces electrostatic repulsion between molecules, that is, a higher polar head area compared to that at a low pH, and the molecular shape is then a cylinder that is in favor of the formation of membranes. It is presently shown that the pH plays an important role in influencing the phase behavior. Interestingly, possible changes of pH as a function of temperature may occur. Moreover, the pK of the fatty acid is also temperature-dependent. As a consequence, changes in the phase behavior of such systems can occur upon varying the temperature because of the protonation state of PA. However, the present mixtures were made in a buffer solution so that the phase transitions observed at a given pH are more probably due to the melting of the alkyl chains rather than to a change in the pH value. The use of nondepleted deuterium water allows for the possible identification of trapped molecule of waters at the membranes/water interface. This has already been investigated in related systems.26 However, in the present case, the signal of water remains isotropic even at a high lipid concentration. Orientation in the Magnetic Field. The synthesis of magnetically self-orienting membranes is of strong importance for solid-state NMR structural studies of membrane proteins.46 Magnetic self-orientation of monoglyceride membranes has already been described.10 To date, there exist two other ways for producing oriented membranes such as the confinement of bilayers between glass plates47 and bicelles, discoidal membranes.29,48 In the present study, self-orientation of membranes is obtained with a rather simple method. Moreover, the self-orientation of membranes was observed for a wide range of

(40) Schweizer, P.; Felix, G.; Buchala, A.; Mu¨ller, C.; Me´traux, J. P. Plant J. 1996, 10, 331-341. (41) Reina, J. J.; Heredia, A. Trends Plant Sci. 2001, 6, 296. (42) Sun, C. Q.; O’Connor, C. J.; Roberton, A. M. FEMS Immunol. Med. Microbiol. 2003, 36, 9-17. (43) Seelig, A.; Seelig, J. Biochemistry 1974, 13, 4839-4845. (44) Douliez, J.-P.; Le´onard, A.; Dufourc, E. J. J. Phys. Chem. 1996, 100, 18450-18457.

(45) Peng, J. B.; Barnes, G. T.; Gentle, I. R. Adv. Colloid Interface Sci. 2001, 91, 163-219. (46) Cross, T. A.; Opella, S. J. Curr. Opin. Struct. Biol. 1994, 4, 574581. (47) Marassi, F. M.; Crowell, K. J. J. Magn. Reson. 2003, 161, 6469. (48) Arnold, A.; Labrot, T.; Oda, R.; Dufourc, E. J. Biophys. J. 2002, 83, 2667-2680.

1550

Langmuir, Vol. 20, No. 5, 2004

temperatures. Up to 9 methylene positions can be resolved on the PAd31 spectrum at pH 7 or 9 in the fluid phase. Similar resolution was obtained for phospholipids embedded in bicelles.48 Magnetic alignment occurs from the anisotropy of the diamagnetic susceptibilities of lipid molecules.30 Monoglycerides exhibit such a property,10 and it may not be surprising that mixtures of PA and PAgly also orients in the field. However, it must be noticed that mixtures of PA with other fatty acids also self-orient in the field (unpublished results). However, while pure monoglycerides form membrane sheets, the micrograph Figure 1 shows the formation of vesicles. This suggests that PA/PAgly vesicles adopt an oblate structure for which the relative proportion of the membrane oriented at 90° with respect to the field is increased. The deformation of spherical liposomes toward an ellipsoidal or cylindrical geometry has already been demonstrated in the literature.49 Interestingly, the system also self-orients at pH 5. However, this requires that the sample be heated above the isotropic transition and further cooled. This allows for the first time the self-orientation of a hexagonal phase to be evidenced. Even upon cooling to the gel phase, the system was shown to conserve the magnetic alignment. Altogether, this finding shows that simple mixtures of lipids, which can be fully dispersed in buffer, may selforient in a magnetic field, which could provide, in addition to the other methods mentioned previously, simple systems for structural studies of membrane proteins. An additional interesting feature of the present system is the (49) Speyer, J.; Sripada, P.; Das Gupta, S.; Shipley, G.; Griffin, R. Biophys. J. 1987, 51, 687-691.

Douliez

ease at which fully deuterated PA and PAgly are produced compared to phospholipids. This is required for proton NMR structural studies of membrane proteins.46 In summary, the present mixtures of lipids were shown to be fully dispersed at neutral or basic pH, whereas both pure components are not. The system self-assembles and exhibits a broad polymorphism and thermotropism, which are modulated by the pH. At a high pH, the electrostatic repulsion between the PA polar heads allows for the swelling of membranes, which can form vesicles. This helps for the incorporation of water, yielding a fully dispersed system. At a low pH, there is no more electrostatic repulsion between the PA molecules and the system still forms membranes at low temperature that stack together without incorporating so much water, yielding a nondispersed system. At a higher temperature, the conclusion is similar because in the hexagonal phase, a weak proportion of water is incorporated into the cylinders. These results should help and guide us for the preparation of full dispersions of mixtures of other fatty acids, for instance, other cutin and suberin monomers. The investigation of this aspect is currently under progress in the laboratory. Acknowledgment. I would like to thank Dr. Ce´cile Mangavel for her help with the FTIR experiments and interpretations and Mr. Nicolas Joubert (IUT Saint Nazaire) and Mrs. Laetitia Lavazay (BTS anabiotec) for their help with the preparation of samples. LA035719+