pubs.acs.org/Langmuir © 2009 American Chemical Society
A Peculiar Phase Transition of Plasmalogen Bilayer Membrane under High Pressure Agnieszka Broniec,† Masaki Goto,‡ and Hitoshi Matsuki*,‡ †
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krak ow, Poland, and ‡Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 minamijosanjima-cho, Tokushima 770-8506, Japan Received July 10, 2009. Revised Manuscript Received August 10, 2009 The bilayer phase transition of plasmalogen, monounsaturated plasmenylcholine 1-O-10 -(Z)-octadecenyl-2-oleoylsn-glycero-3-phosphocholine (Plg-SOPC), was examined by differential scanning calorimetry, high-pressure transmittance, and fluorescence techniques. The bilayer properties of Plg-SOPC such as the temperature-pressure phase diagram, the thermodynamic quantities of the transition, and the location of a fluorescent membrane probe in the bilayer, were compared with those of a similar phospholipid 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). It turned out that a vinyl-ether bond in the sn-1 position of the glycerol backbone in the Plg-SOPC molecule produces a peculiar phase transition under high pressure and significantly affects the membrane properties.
Introduction Plasmalogens are unique phospholipids, which contain a vinylether bond in the sn-1 position of the glycerol backbone in the molecule. They are found in animal and some bacterial cells, mainly as molecular species of plasmenylethanolamine (PlgPE) and plasmenylcholine (PlgPC).1 The type of plasmalogen molecule in cell membranes, namely, the headgroup distribution, and the sn-2 position esterification profile are highly cell-type specific. Approximately 18% of total phospholipids in human cells are plasmalogens, but their percentage varies depending on the cell types. For example, PlgPE reaches 70% of total PEs in brain white matter, while only about 5% in hepatocytes. The high parcentages (35-41%) of PlgPC in total PC class are characteristic for cardiac tissues.1 There are many proposals for the main role of plasmalogens in vivo. They may act as antioxidants in photosensitized oxidative stress in cell cultures2 or in model lipid monolayer systems irradiated by UV.3 The correlation between cholesterol transport and its oxidation susceptibility in membranes containing plasmalogen was also found.4 It was further reported5-7 that they influence membrane trafficking, membrane-bound protein activity, and membrane stability. In addition, significant pathological states such as Alzheimer disease, ischemia-reperfusion injury, Down’s syndrome,1 and Gaucher disease8 are closely related to the decrease in plasmalogen content or high accumulation of its degradation products in cell membranes. These broad functions of plasmalogens make them *Corresponding author. Address: Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 minamijosanjima-cho, Tokushima 770-8506, Japan. TEL: þ81-88-656-7513. FAX: þ81-88-655-3162. E-mail address:
[email protected]. (1) Nagan, N.; Zoeller, R. A. Prog. Lipid Res. 2001, 40, 199–229. (2) Zoeller, R. A.; Morand, O. H.; Raetz, C. R. J. Biol. Chem. 1988, 263, 11590– 11596. (3) Morandat, S.; Bortolato, M.; Anker, G.; Doutheau, A.; Lagarde, M.; Chauvet, J. P.; Roux, B. Biochim. Biophys. Acta 2003, 1616, 137–146. (4) Maeba, R.; Ueta, N. J. Lipid Res. 2003, 44, 164–171. (5) Thai, T. P.; Rodemer, C.; Jauch, A.; Hunziker, A.; Moser, A.; Gorgas, K.; Just, W. Hum. Mol. Genet. 2001, 10, 127–136. (6) Chen, X.; Gross, R. W. Biochemistry 1995, 34, 7356–7364. (7) Ginsberg, L.; Xuereb, J. H.; Gershfeld, N. L. J. Neurochem. 1998, 70, 2533– 2538. (8) Moraitou, M.; Dimitriou, E.; Zafeiriou, D.; Reppa, C.; Marinakis, T.; Sarafidou, J.; Michelakakis, H. Blood Cells Mol. Dis. 2008, 41, 196–199.
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important constituents of properly functioning organisms; however, the information on physicochemical properties of plasmalogens is considerably lacking as compared with that on glycero- and sphingo-phospholipids, although they are essential phospholipids in cell membranes as mentioned above. This paper reports the phase transition of a bilayer membrane of monounsaturated plasmenylcholine, 1-O-10 -(Z)-octadecenyl2-oleoyl-sn-glycero-3-phosphocholine (Plg-SOPC). There are some studies on phase transitons of PlgPE9-12 membranes showing the formation of nonbilayer structures of membranes, especially the inverted hexagonal phase. On the other hand, there are only few reports on the properties of PlgPC bilayer membranes,13-15 so the knowledge is rather limited. We investigated the bilayer phase transition of Plg-SOPC by using differential scanning calorimetry (DSC) under atmospheric pressure and applying high-pressure techniques of light-transmittance and fluorescence detection. As a result, we obtained the temperaturepressure phase diagram, the thermodynamic quantities of the phase transition, and the location of the fluorescent probe Prodan in the bilayer membrane of Plg-SOPC. We compared the bilayer phase behavior of Plg-SOPC with that of its diacyl analog phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), which allowed us to recognize characteristic features of the phase transition of the Plg-SOPC bilayer.
Experimental Section Materials and Sample Preparation. Monounsaturated phosphatidylcholines, Plg-SOPC and SOPC, were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), and they were used without further purification. The molecular structures of two lipids are demonstrated in Figure 1. Fluorescent probe 6-propionyl-2-(dimethyloamino)naphtalene (Prodan) was obtained from Molecular Probes, Inc. (Eugene, OR). Water was distilled twice (9) Lohner, K.; Hermetter, A.; Paltauf, F. Chem. Phys. Lipids 1984, 34, 163–170. (10) Lohner, K.; Balgavy, P.; Hermetter, A.; Paltauf, F.; Laggner, P. Biochim. Biophys. Acta 1991, 1061, 132–40. (11) Glaser, P. E.; Gross, R. W. Biochemistry 1994, 33, 5805–5812. (12) Lohner, K. Chem. Phys. Lipids 1996, 81, 167–184. (13) Han, X.; Gross, R. W. Biochemistry 1990, 29, 4992–4996. (14) Han, X.; Chen, X.; Gross, R. W. J. Am. Chem. Soc. 1991, 113, 7104–7109. (15) Han, X.; Gross, R. W. Biochim. Biophys. Acta 1991, 1069, 37–45.
Published on Web 08/21/2009
DOI: 10.1021/la902503n
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Figure 1. Molecular structures of (A) Plg-SOPC and (B) SOPC.
Figure 3. A temperature-pressure phase diagram for a bilayer membrane of Plg-SOPC. Open circles and triangles refer to lowtemperature and high-temperature transitions, respectively. The inset shows a temperature-pressure phase diagram for a bilayer membrane of SOPC.
changes in isobaric thermotropic mode, using a high-pressure cell assembly attached to a U-3010 spectrophotometer (Hitachi HighTechnologies Corp., Tokyo, Japan). The detailed procedure of the measurements under high pressure was described elsewhere.16 The changes in light transmittance at 560 nm at various pressures and with increasing temperature (heating rate 0.33 K min-1) were recorded. The pressure was controlled within an accuracy of 0.2 MPa by using a Heise gauge. We also observed the phase behavior of lipid bilayer membranes under high pressure by applying the polarity-sensitive fluorescence probe Prodan, using an F-2500 spectrofluorometer (Hitachi HighTechnologies Corp., Tokyo, Japan). The experiment was done under isobaric thermotropic conditions as described previously.17 The excitation wavelength was 361 nm, and the emission spectra were recorded approximately every 1 K in the wavelength range from 400 to 600 nm. The heating rate was set to 0.5 K min-1. The second derivative of the fluorescence emission spectrum was obtained by attached software (FL-solutions) and Origin 7.0 (Lightstone Corp., Tokyo Japan). Figure 2. (A) DSC thermograms for bilayer membranes of (1) PlgSOPC and (2) SOPC. (B) Transmittance-temperature curves for bilayer membranes of Plg-SOPC: (1) 50 MPa, (2) 210 MPa. after a deionization, where the second step was done from dilute alkaline permanganate solution. Liposomes preparation for all measurements was performed by Bangham’s method. The chloroform stock solution of a lipid with or without ethanol solution of a probe Prodan was dried under vacuum at least 2 h, and then the dry film was hydrated. The suspension was sonicated by a waterbath-type sonicator (Branson 3510J-DTH with output 130 W) at the temperature above the main-transition temperature of each lipid for ca. 5 min. The lipid concentration was 5 mM (0.364 wt %) for the DSC measurements and 1 mM (0.073 wt %) for the transmittance and fluorescence measurements. The molar ratio of the lipid to Prodan was 500:1. Methods. The phase transitions of lipid bilayer membranes under atmospheric pressure were observed using a SSC 5200-DSC 120 calorimeter (SII Nanotechnology Co. Ltd., Chiba, Japan). The sample and reference solutions of 60 μL were sealed up in DSC silver cells. After the suitable thermal equilibration, the measurements were started with the heating rate of 0.3 K min-1. The results were analyzed by attached software for the apparatus. The phase transitions of lipid bilayer membranes under high pressure were observed by an optical method of light transmittance 11266 DOI: 10.1021/la902503n
Results and Discussion The DSC thermogram of the Plg-SOPC bilayer membrane is shown together with that of the SOPC bilayer membrane in Figure 2A. We detected only one broad endothermic peak at about 5 °C in the Plg-SOPC bilayer, whereas, for the SOPC bilayer, the thermal phase transition was shifted to higher temperatures (at about 7 °C).18,19 The profile of the endothermic peak was considerably sharper for the SOPC bilayer. The bilayers of PCs with unsaturated acyl chains are often known to undergo a phase transition associated with the formation of lamellar crystal (Lc) phase19-21 when annealing the samples thermally or adding an antifreeze substance such as ethylene glycol. We performed the (16) Kusube, M.; Tamai, N.; Matsuki, H.; Kaneshina, S. Biochim. Biophys. Acta 2005, 1668, 25–32. (17) Goto, M.; Kusube, M.; Tamai, N.; Matsuki, H.; Kaneshina, S. Biochim. Biophys. Acta 2008, 1778, 1067–1078. (18) Kaneshina, S.; Ichimori, H.; Hata, T.; Matsuki, H. Biochim. Biophys. Acta 1998, 1374, 1–8. (19) Tada, K.; Miyazaki, E.; Goto, M.; Nobutake, T.; Matsuki, H.; Kaneshina, S. Biochim. Biophys. Acta 2009, 1788, 1056–1063. (20) Kusube, M.; Goto, M.; Nobutake, T.; Matsuki, H.; Kaneshina, S. Chem. Phys. Lipids 2006, 142, 94–102. (21) Ichimori, H.; Hata, T.; Matsuki, H.; Kaneshina, S. Chem. Phys. Lipids 1999, 100, 151–164.
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Letter Table 1. Thermodynamic Properties of the Main Transition for Plg-SOPC and SOPC Bilayer Membranes
lipid
T (K)
T (°C)
dT/dP (K MPa-1)
ΔH (kJ mol-1)
ΔS (J K-1 mol-1)
ΔV (cm3 mol-1)
Plg-SOPC SOPC
278.0 279.9
5.1 6.7
0.193 0.201
10.9 ( 2.00 24.8 ( 3.89
39 89
7.5 19.2
DSC measurements of the Plg-SOPC liposomes after several cycles of thermal annealing between freeze storage at -30 °C (for 1 h) and cold storage at 4 °C (for 1 day) or using aqueous 50 wt % ethylene glycol solution. As seen previously, only one peak was found on the thermogram in both cases, although there was a minor variation in the temperature and area of the peak. The phase transition of the Plg-SOPC bilayer membrane under high pressure was monitored by measuring the light-transmittance changes of liposome solutions. Two examples of the transmittance curves at 50 and 210 MPa are demonstrated in Figure 2B. The interesting discovery was that, at high pressures, above 80 MPa, it was possible to record two consecutive light transmittance changes, indicating a two-phase transition of this lipid system. To summarize, using the phase-transition data obtained by DSC and light-transmittance measurements, we constructed the temperature (T)-pressure (p) phase diagram of the Plg-SOPC bilayer membrane. The resulting diagram is shown together with that of the SOPC bilayer membrane19 in Figure 3. The phasetransition temperature of the Plg-SOPC bilayer membrane increased with applying pressure, and another pressure-sensitive transition emerged at higher temperature. The behavior of the lower-temperature transition curve was almost the same as that of the SOPC bilayer membrane shown in the inset of Figure 3. There is only a minor difference in transition temperature between the Plg-SOPC and SOPC bilayers under atmospheric pressure, and the pressure dependence (dT/dp) is similar 0.193 and 0.201 K MPa-1, respectively. We have shown in our previous studies22,23 that the dT/dp slope reaches the characteristic values for different types of phase transitions. Taking into account that the kinetics of Lc phase formation is very slow19,24 and the detailed knowledge on the main transition of the SOPC bilayer confirms that this is lamellar gel (Lβ) to liquid crystalline (LR) phase reorganization,18,19 we suggest that the low-temperature phase transition of the PlgSOPC bilayer membrane is the same Lβ/LR transition. The thermodynamic quantities of the main transition, namely enthalpy (ΔH), entropy (ΔS), and volume (ΔV) changes, were obtained from the DSC data and by using Clapeyron’s equation with the dT/dp value. The results for the Plg-SOPC and SOPC bilayers are summarized in Table 1. An unexpected discovery was that the ΔH, ΔS, and ΔV values for the Plg-SOPC bilayer are approximately half of those for the SOPC bilayer, although the main-transition temperatures and the dT/dp values for both bilayers are very similar, as shown in Figure 3. Since the difference in molecular structure between Plg-SOPC and SOPC is either the existence of a vinyl-ether bond or an ester bond in the sn-1 position of the glycerol backbone, only this part of the PC molecule is responsible for its different poperties. We have shown from the comparative studies on the SOPC and distearoylphosphatidylcholine (DSPC) bilayer membranes18 that the introduction of the cis double bond into the acyl chain of lipids causes a significant reduction of thermodynamic quantities mainly by
promoting the disordered packing of unsaturated chains in the gel state of lipid bilayer. We have also revealed from studies on ether-linked PC bilayers23 that the attractive interaction in the gel phase for ether-linked PC bilayers is weaker than that for esterlinked PC bilayers. Taking into consideration that the effect of the vinyl-ether bond could be a sum of the effects imposed by the double bond and the ether bond, the existence of this linkage in PC molecule may cause a further decrease in the packing order of both chains in the gel phase of the Plg-SOPC bilayer membrane. However, the main-transition temperatures are very close between both bilayer membranes. Previous data indicate that the maintransition temperatures of bilayers of ester- and ether-linked phospholipids with the same hydrophobic chain length are almost equal to each other,23 and the introduction of a double bond into the C1-C2 position has only a minor effect on the main-transition temperature compared to the C9-C10 position.25 This is consistent with our data obtained for the Plg-SOPC bilayer, showing that the vinyl-ether bond has a relatively small effect on melting temperature but is still very important in modulating the ΔH, ΔS, and ΔV values of the phase transition process. In order to clarify the peculiar phase transition of the PlgSOPC bilayer membrane observed in the high-pressure region, we measured the fluorescence emission spectra of membrane-anchored probe Prodan under high-pressure conditions. Prodan molecule can be freely incorporated into the headgroup region of lipid molecules at bilayer interface. We have shown in the previous study17 that the second derivative of the Prodan fluorescence spectrum is useful for determination of the distribution of Prodan molecules in the bilayer membrane. Figure 4 depicts the second derivatives of the emission spectra for the Plg-SOPC and SOPC bilayer membranes under high pressure. It is possible to distinguish two minima centered at 430 and 490 nm in both types of PC bilayers. The distribution of Prodan molecules is bimodal in our system, that is, around the glycerol backbone in the Lβ phase (430 nm minimum) and around the phosphate group in the LR phase (490 nm minimum).26,27 The main difference observed between the Plg-SOPC and SOPC bilayers was the relative intensity of two minima and its temperature dependence. In the case of the Plg-SOPC bilayer, the 430 nm minimum first became very shallow, while the 490 nm minimum went deeper with increasing temperature. Finally, after several temperature scans, we achieved complete disappearance of the 430 nm minimum. In contrast to this, both minima of the second derivative fluorescence spectra of Prodan in the SOPC bilayer varied regularly with increasing temperature. It should be noted that the spectra of the Plg-SOPC bilayer showed the peculiar variation in a very narrow temperature range, as seen in the inset of Figure 4. Namely, the minima of the spectra at 430 nm, beginning from ca. 25 °C, which is the main transition temperature of this lipid at 98 MPa, became much deeper with increasing temperature. Interestingly, the deepest minimum in that particular temperature range corresponds well with the temperature of higher phase transition at 98 MPa (Figure 3).
(22) Ichimori, H.; Hata, T.; Matsuki, H.; Kaneshina, S. Biochim. Biophys. Acta 1998, 1414, 165–174. (23) Matsuki, H.; Miyazaki, E.; Sakano, F.; Tamai, N.; Kaneshina, S. Biochim. Biophys. Acta 2007, 1768, 479–489. (24) Lewis, R. N. A. H.; Mak, N.; McElhaney, R. N. Biochemistry 1987, 26, 6118–6126.
(25) Lewis, R. N. A. H.; McElhaney, R. N. In The Structure of Biological Membranes, 2nd ed.; Yeagle, P. L., Ed.; CRC Press: London, 2005; pp 66-69. (26) Kusube, M.; Tamai, N.; Matsuki, H.; Kaneshina, S. Biophys. Chem. 2005, 117, 199–206. (27) Kusube, M.; Matsuki, H.; Kaneshina, S. Colloids Surf., B: Biointerfaces 2005, 42, 79–88.
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Figure 4. Second derivatives of Prodan fluorescence spectra in the bilayers of (A) Plg-SOPC at 98 MPa and (B) SOPC at 102 MPa. Here the second derivative curves are depicted by temperature intervals of ca. 1 K. The inset in the panel A shows the magnification of second derivatives of Prodan fluorescence spectra in the gel phase around the main transition temperature (indicated by a dotted arrow): (1) 24.4 °C, (2) 25.4 °C, (3) 26.4 °C, (4) 27.4 °C. Solid black arrows indicate the direction of temperature increase.
that the molecular conformation of the glycerol backbone and the orientation of the acyl chains and headgroup are considerably different between plasmalogens and diacyl phospholipids because of the existence of the vinyl-ether bond of the former. They showed that the sn-2 acyl chain of plasmalogens is perpendicular to the membrane surface at all chain segments, while that of diacyl phospholipids bends at the carbon segment C-2, and a position of the headgroup of plasmalogen is more perpendicular to the membrane surface than that of diacyl phospholipids. This differences in glycerol conformation between SOPC and Plg-SOPC may cause the existence of a very shallow 430 nm minimum in the Plg-SOPC bilayer below the main-transition temperature observed in our experiments. Considering the above-mentioned data and analyzing the temperature dependence of the second derivative spectra in Figure 4A, we speculate that the peculiar distribution of the Prodan molecules in the bilayer gel phase during the main transition results from the conformational change of glycerol backbone of the Plg-SOPC molecule induced by applying pressure. When the main transition occurs, the Prodan molecules start to move toward the phosphate-group region (lower-temperature transition). Then the conformational change of the backbone is induced, and the Prodan molecules are again redistributed in the newly formed backbone region (higher-temperature transition). Light transmitance data observed in the high-pressure region support this hypothesis. In summary, for the first time we described the comprehensive phase behavior of the Plg-SOPC bilayer. Introduction of a vinyl-ether bond in the sn-1 position of the glycerol backbone in the lipid molecule produces a peculiar phase transition under high pressure and dramatically reduces the thermodynamic quantities of the main transition, while the effect on the transition temperature is rather small. The Prodan fluorescence spectra suggest that the peculiar phase transition is caused by conformational changes at the glycerol backbone induced by pressure during the transition. Further experiments concerning the effect of this special bond on bilayer properties will be required to elucidate the meaning of the existence of plasmalogens in cell membranes.
The growth of minima at 430 nm indicates that the Prodan molecules go back to the vicinity of the glycerol backbone region again during the main transition of the Plg-SOPC bilayer membrane. Han and Gross have demonstrated from NMR studies13
Acknowledgment. The work was supported by a Japan Society for the Promotion of Science fellowship and in part by the Polish Ministry of Science and Higher Education (Grant 2040/B/P01/2007/33).
11268 DOI: 10.1021/la902503n
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