Article pubs.acs.org/Langmuir
Study on the Subgel-Phase Formation Using an Asymmetric Phospholipid Bilayer Membrane by High-Pressure Fluorometry Masaki Goto,† Agnieszka Wilk,‡,§ Kosuke Kataoka,∥ Shirish Chodankar,§ Nobutake Tamai,† Makoto Fukui,∥ Joachim Kohlbrecher,§ Hiro-O Ito,∥ and Hitoshi Matsuki*,† †
Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan ‡ Faculty of Physics, A. Mickiewicz University, Umultowska 85 61-614 Poznań, Poland § Laboratory for Neutron Scattering, ETH Zürich & Paul Scherrer Institut, CH-5232, Villigen PSI, Switzerland ∥ Department of Preventive Dentistry, Institute of Health Bioscience, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan S Supporting Information *
ABSTRACT: The myristoylpalmitoylphosphatidylcholine (MPPC) bilayer membrane shows a complicated temperature−pressure phase diagram. The large portion of the lamellar gel (Lβ′), ripple gel (Pβ′), and pressure-induced gel (LβI) phases exist as metastable phases due to the extremely stable subgel (Lc) phase. The stable Lc phase enables us to examine the properties of the Lc phase. The phases of the MPPC bilayers under atmospheric and high pressures were studied by small-angle neutron scattering (SANS) and fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan. The SANS measurements clearly demonstrated the existence of the metastable LβI phase with the smallest lamellar repeat distance. From a second-derivative analysis of the fluorescence data, the line shape for the Lc phase under high pressure was characterized by a broad peak with a minimum of ca. 460 nm. The line shapes and the minimum intensity wavelength (λ″min) values changed with pressure, indicating that the Lc phase has highly pressure-sensible structure. The λ″min values of the Lc phase spectra were split into ca. 430 and 500 nm in the LβI phase region, which corresponds to the formation of a interdigitated subgel Lc (LcI) phase. Moreover, the phase transitions related to the Lc phase were reversible transitions under high pressure. Taking into account the fluorescence behavior of Prodan for the Lc phase, we concluded that the structure of the Lc phase is highly probably a staggered structure, which can transform into the LcI phase easily.
1. INTRODUCTION Biological membranes have a highest sensitivity to high pressure among biomolecules. Pressure studies on lipid bilayer membranes have been concentrated on phosphatidylcholines (PCs) with two identical hydrocarbon chains such as dipalmitoyl-PC (DPPC) and distearoyl-PC (DSPC).1−10 We have constructed the temperature (T)−pressure (p) phase diagrams of bilayer membranes for homologues of such symmetric PCs11−13 and showed that with an increase in acyl chain lengths the phase-transition temperatures increase and the subgel (Lc) phase (i.e., hydrated crystal or lamellar crystal phase) and the gel phases (i.e., lamellar gel (Lβ′) and pressureinduced interdigitated gel (LβI) phases) become more stable. On the other hand, two acyl chains of phospholipids found in actual biological membranes are usually different from each other. In our previous studies, we revealed that bilayer membranes of asymmetric PCs show characteristic phase behavior under atmospheric and high pressures as follows.12,13 First, the bilayers comprising the asymmetric PC molecules © 2012 American Chemical Society
with aligned terminal methyl ends have relatively high thermodynamic quantities associated with the main transitions. Second, the stability of the Lβ′ phase of the asymmetric PC bilayers is much lower than that of the symmetric PC bilayers with the same total chain length. Third, the stability of the Lc and LβI phase increases with an increase in the degree of the chain alignment at the terminal methyl ends. In the studies on the asymmetric PC bilayers, we also found that the myristoylpalmitoyl-PC (MPPC) exhibited a most complicated bilayer phase behavior of all the PC bilayers. In the MPPC bilayer, the large portion of gel phases exists as metastable phases in the whole pressure range due to the extremely fast formation of the Lc phase.13 Then, it is difficult, especially under high pressure, to detect the pretransition from the Lβ′ (or LβI) phase to the ripple gel (Pβ′) phase and the Received: May 17, 2012 Revised: July 20, 2012 Published: July 24, 2012 12191
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1:500. The sample solutions were again warmed to 50 °C to prevent from transforming into the Lc phase and protected from light just before the measurements were made. 2.2. Small-Angle Neutron Scattering Measurements. Smallangle neutron scattering measurements were performed on a SANS-I instrument at Swiss Spallation Neutron Source in Paul Scherrer Institut, Switzerland. SANS diffractions from the MPPC bilayer in various phases were recorded by the method of isothermal barotropic and isobaric thermotropic phase transition measurements.15 The mean wavelength of an incident neutron beam was 0.6 nm. The scattered neutrons were detected using a two-dimensional (0.64 × 0.64 m2) detector. The experiments were performed at two sample-to-detector distances of 1.2 and 6 m to cover the data in the wave-vector-transfer Q range of 0.1−3.0 nm−1. The measured SANS data were corrected and normalized to a cross-sectional unit using BERSANS-PC dataprocessing software. 2.3. Fluorescence Measurements. Fluorescence measurements under atmospheric and high pressures were carried out using an F2500 spectrophotometer (Hitachi High-Technology Corp., Tokyo, Japan) equipped with a high-pressure cell assembly (PCI-400; Syn Corp., Ltd., Kyoto, Japan). Fluorescence spectra of Prodan in the MPPC bilayer of various phases were observed by isothermal barotropic and isobaric thermotropic phase transition measurements described previously.12,13,16,17 The excitation wavelength was 361 nm, and the emission spectra were recorded in the wavelength range from 400 to 600 nm. The pressurizing rate was 2 MPa min−1, and the heating rate was 0.5 °C min−1. Second derivative of the emission spectrum was obtained by attached software FL solutions (Hitachi High-Technology Corp., Tokyo, Japan) and Origin 7.0 (Lightstone Corp., Tokyo, Japan). By employing SigmaPlot 12 (HULINKS Inc., Tokyo, Japan), three-dimensional image plots were constructed from the spectra.
bilayer interdigitation from the Lβ′ phase to the LβI phase for the MPPC bilayer, because the formation of the Lc phase is facilitated by applying pressure. However, we succeeded in observing the bilayer interdigitation by means of a fluorescence method using a polarity-sensitive probe called Prodan combined with a quick barotropic observation technique to prevent from rapidly transforming into the Lc phase.13 Prodan molecules respond sensitively to the change in microscopic environment around the molecule in a phospholipid bilayer and exhibit characteristic fluorescence spectra depending on the phase state of the bilayer. By using the method, we confirmed the existence of the triple point at which the Lβ′, Pβ′, and LβI phases coexist despite that all these phases are metastable states. In the present study, we investigate the properties of the Lc phase in the MPPC bilayer by small-angle neutron scattering (SANS) and fluorescence spectroscopy using Prodan. It is known that it takes a long time to form the Lc phase in the bilayers of symmetric PCs with long acyl chains like DPPC.12 This is because the formation of the Lc phase results from the dehydration among head groups of PC molecules in the bilayer. Usually a special pretreatment of lipid samples, called thermal annealing, is required for the dehydration. However, this treatment also excludes the probe molecules from the bilayer irreversibly, and few of them are partitioned back into the bilayer after preparation of the vesicle suspension. Considering this, the much faster transformation in the Lc phase can be regarded as a great merit of the MPPC bilayer, and moreover, the transformation is further facilitated by applying pressure. This nature of the MPPC bilayer can reduce the precipitation accompanying the Lc phase formation and the exclusion the Prodan molecules from the outer membrane. By taking advantage of the merits, first, we performed SANS measurements to directly obtain structural information of the MPPC bilayer under atmospheric and high pressures and then observed isothermal barotropic and isobaric thermotropic phase transitions by the Prodan fluorescence. Since there has been almost no report of fluorescence studies related to the Lc phase, this is the first report on the behavior of the Lc phase in the PC bilayer membrane from the fluorescence spectroscopy.
3. RESULTS AND DISCUSSION 3.1. SANS Measurements for the MPPC Bilayer under Atmospheric and High Pressures. Figure 1 depicts the T−p
2. EXPERIMENTAL SECTION 2.1. Materials and Sample Preparation. An asymmetric phospholipid, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), and used as received. A fluorescence probe called Prodan, 6-propionyl-2(dimethylamino)naphthalene (IUPAC name: 1-[6-(dimethylamino)naphthalen-2- yl]propan-1-one), was obtained from Molecular Probes, Inc. (Eugene, OR). As a solvent, we used D2O for SANS measurements and water distilled twice from a dilute alkaline permanganate solution for fluorescence measurements. We employed two methods of vesicle preparations accompanying to the experimental techniques. For SANS measurements, the phospholipid multilamellar vesicles were prepared by suspending the MPPC in D2O solution at 2.0 mmol kg−1, and the suspensions were sonicated for a few minutes by using a sonifier at a temperature several degrees above the main transition temperature. For fluorescence measurements, the Bangham’s method was used.14 A chloroform stock solution of MPPC was mixed with an ethanol solution of Prodan. The mixed solution was dried in vacuo to remove all residual solvents and finally to get a dry film. Water was added to the dry film, and the lipid samples were hydrated by a treatment of vortex. The suspensions were sonicated at 50 °C for ca. 3 min in order to obtain the multilamellar vesicle suitable for the fluorescence measurements. The total concentration of a lipid was 1.0 mmol kg−1, and the molar ratio of Prodan to the lipid was
Figure 1. Temperature−pressure phase diagram of MPPC bilayer membrane. Bilayer phases are assigned as the subgel (Lc), lamellar gel (Lβ′), ripple gel (Pβ′), and interdigitated gel (LβI) phases. Bilayer phases in parentheses refer to metastable phases. Broken arrows in the phases diagram represent the heating and pressurizing process for SANS (closed arrow) and fluorescence spectra (open arrows) measurements in Figures 2−8.
phase diagram of the MPPC bilayer membrane, which includes a pressurizing and a heating process for the SANS (closed arrows) and fluorescence (open arrows) measurements.13 The transition temperatures of the pretransition from the Lβ′ phase to the Pβ′ phase, the subtransition from the Lc phase to the Pβ′ phase, and the main transition from the Pβ′ phase to the liquid 12192
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nm−1 (D-spacing = 6.4 nm). This manifest peak corresponds to the Lα phase. These results clearly demonstrated that the MPPC bilayer undoubtedly forms the LβI phase under the metastable condition. 3.2. Prodan Fluorescence in Noninterdigitated GelPhase Region. We have shown the Prodan fluorescence in various kinds of PC bilayer membranes and revealed that the wavelengths of the emission maximum (λmax) of Prodan in the PC bilayers are greatly dependent on the phase states. That is, the λmax values for the Lβ′ (or Pβ′), Lα, and LβI phase exhibit ca. 430, 480, and 500 nm, respectively.5,20,21 This is because the λmax value of Prodan varies with a local dielectric constant of a solvent around the Prodan molecule. The local dielectric constant or the water density along a lipid molecule in the bilayer is represented by a zone model.22 A higher value of λmax indicates that the probes exist in a polar environment, whereas a lower value of λmax does that they exist in a less polar environment. Therefore, the location of the Prodan molecules in each phase of the PC bilayer can be estimated from the λmax values: they reside in a less polar region around the glycerol backbone when the bilayer is in the gel (Lβ′ or Pβ′) phase, in an intermediate region around phosphate group for the Lα phase, and in a hydrophilic region around lipid headgroup for the nonbilayer gel (LβI) phase. Since the D-spacings for the Lβ′, Pβ′, Lα, and LβI phase are 6.3, 7.2, 6.7, and 4.8 nm, respectively, in the case of the DPPC bilayer,7,23 it can also be said that the Prodan molecules can change the location in response to the change in the membrane structure. Furthermore, we have developed a novel analysis method by using second derivatives of the fluorescence spectra, which enables us to investigate the precise location of the Prodan molecules in the PC bilayer.12,13,16,17,24−26 It is advantageous to use the secondderivative spectra to separate overlapping multiple peaks because multiple peaks including main and shoulder components in the original spectrum can be converted to individual valleys (λ″min) in its second-derivative spectrum. From temperature dependence of the second derivative spectra,16,17,25 we constructed a three-dimensional image plot (hereafter abbreviated to image plot) of the second-derivative spectra for the Prodan fluorescence of the PC bilayer membranes under atmospheric and high pressures. Here the fluorescence intensity is drawn by a color scale as an indicator: the change from low intensity to high intensity is indicated by change of color from blue to red in the order of the rainbow colors. It is a great merit of the image plot to provide the exact location of the Prodan molecules which change depending on the phase state of the bilayer. The fluorescence spectra of Prodan in the MPPC bilayer were observed at 15.3 °C as a function of pressure. The resulting image plot constructed on the basis of the secondderivative spectra is depicted in Figure 3A. Here the extractions of spectra at several pressures from each image plot are also given in Figure 3B. All the fluorescence spectra for this process are depicted in Figure SI-1 of the Supporting Information. At the temperature, no phase transitions occur in the whole pressure range. In other words, the phase remains the Lβ′ or Lc phase in the pressurizing process (see the lower open arrow along the abscissa in Figure 1). During the pressurizing process, the intensity of the second derivative of spectrum (S.D.F.I.) at ca. 430 nm increased (became shallower) at ca. 100 MPa while that at ca. 460 nm decreased, which resulted in the marked broadening of the valley as is seen in Figure 3A. With further increasing pressure, the intensity at ca. 460 nm decreased
crystal (Lα) phase are 18.2, 27.1, and 34.0 °C under atmospheric pressure, respectively. The relative order of the pretransition and subtransition temperatures of the MPPC bilayer was opposite to that of the DPPC bilayer,13,18 meaning that the Lβ′ phase and a part of the Pβ′ phase exist as metastable phases. The transition temperatures of the subtransition and main transition increased by applying pressure and another transition appeared above ca. 160 MPa. On the other hand, although the pretransition was hardly observed under high pressure, quick barotropic fluorescence measurements using Prodan enabled us to determine the bilayer interdigitation (closed squares) from the Lβ′ phase to the LβI phase and the pretransition (closed triangles) from the Pβ′ phase to the LβI phase, respectively. This provided the location of the triple point at which three phases (i.e., Lβ′, Pβ′, and LβI phases) can coexist under the metastable condition. The results of SANS measurements for the MPPC bilayer in D2O are shown in Figure 2. Here the measurements were
Figure 2. SANS data for MPPC bilayer membrane in D2O on different conditions: (1) 15 °C at 0.1 MPa, (2) 15 °C at 200 MPa, (3) 40 °C at 200 MPa, (4) 40 °C at 0.1 MPa.
performed along the pressurizing and the heating process indicated as closed arrows in Figure 1. First of all, it should be noted that the triple point of a PC bilayer moves to a higher temperature and pressure region in D2O solution than in H2O due to the enhancement of the interaction between polar head groups by the stronger hydrogen bonding in D2O than in H2O.19 In addition, it is unfortunate that the formation of the Lc phase of the MPPC bilayer decelerates in D2O as compared with that in H2O; thus, we investigated the membrane structure of the Lβ′, LβI, and Lα phase instead of the Lc phase. Curve 1 in Figure 2 shows a neutron diffraction profile of the MPPC bilayer at ca. 15 °C under atmospheric pressure. A peak around the q-value of 0.89 nm−1 (lamellar repeat distance (D-spacing) = 7.1 nm) indicates that the MPPC bilayer forms the Lβ′ phase, which was in good agreement with the T−p phase diagram in Figure 1. Even if pressure was raised up to ca. 200 MPa, the diffraction profile was almost comparable to that at ambient pressure (curve 2 in Figure 2), signifying that the phase at 15 °C, 200 MPa is still the Lβ′ phase. When temperatures were elevated to ca. 40 °C at 200 MPa, the diffraction peak shifted to higher q region (curve 3 in Figure 2). The peak around the highest q-value of 1.07 nm−1 (D-spacing = 5.9 nm) is caused by the LβI phase with a small membrane thickness. After depressurization down to atmospheric pressure at ca. 40 °C, the diffraction peak shifted to a lower value again (curve 4 in Figure 2) and became a large one around the q-value of 0.99 12193
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Figure 4. (A) Time dependence of fluorescence spectra of Prodan in MPPC bilayer at 15.3 °C and 224.9 MPa. Each spectrum was obtained at 10 min intervals. (B) Decompression effect on image plot for second-derivative spectra of Prodan in MPPC bilayer at 10.8 °C.
Figure 3. (A) Image plot for the second derivatives of fluorescence spectra of Prodan in MPPC bilayer membrane at 15.3 °C and (B) the extractions of the second derivatives of fluorescence spectra at (1) 0.1, (2) 39.7, (3) 98.6, (4) 157.3, (5) 185.9, (6) 215.3, and (7) 242.6 MPa.
distinguish from those of the Lβ′ (or Pβ′) phase. Since this spectral shift is completely reversible, it is reasonable to say that pressure is required to form the spectrum characteristic of the Lc phase. Accordingly, the MPPC bilayer highly probably forms a special structure in the Lc phase, which is easily influenced by pressure. Figures 5A and 5B show the image plots of temperature dependence on the fluorescence spectra and second derivatives of spectra at 94.3−96.8 MPa, respectively. All the fluorescence spectra obtained in the process are depicted in Figure SI-3. Here the heating process is indicated as the open arrow parallel to the ordinate. At ca. 10 °C, the spectra exhibited a broad peak (valley) characteristic of the Lc phase with a relatively low λmax (λ″min) value. With increasing temperature, the peak (valley) of the fluorescence intensity (F.I.) (S.D.F.I.) at ca. 430 nm became higher (deeper) while that at ca. 450 nm became lower (shallower), and the spectra were no longer distinguishable from the gel-phase spectra at ca. 30 °C. Comparing the results to those in Figure 4, we can say that the fluorescence spectra characteristic of the Lc phase come close to the gel-phase spectra under low pressure and/or high temperature conditions. We speculated that such conditions cause a gradual microscopic structure change from a “hard” Lc phase to a “soft” Lc phase although this change is not a macroscopic phase transition, and the latter phase has same fluorescence characteristics as the gel phase. The difference between the soft and hard Lc phases will be explained later. When further increasing temperature, since the F.I. (S.D.F.I.) values at ca. 430 nm decreased (increased) abruptly once at ca. 43 °C and they increased (decreased) from the temperature again, a definite constriction was seen in the image plots (see white arrows in the Figure 5 or Figure SI-3). This temperature agrees with the Lc/Pβ′ phase transition temperature.13 Therefore, this is first
abruptly at ca. 230 MPa. Such broadening of the valley has not been observed for other PC bilayers and means that the Prodan molecules are not fixed on specific sites in the bilayer. Therefore, we anticipated that this was the spectra characteristic of the Lc phase. In order to confirm this anticipation, we carried out the differential scanning calorimetry (DSC) and lighttransmittance measurements under atmospheric pressure for the pressure-treated suspension of the MPPC bilayer. As a result, the Lc/Pβ′ transition was clearly observed at ca. 27 °C as shown in Figure1 (data not shown).13 It is known that the formation of the Lc phase of the MPPC bilayer under atmospheric pressure is exceedingly faster than that of the DPPC bilayer, for example, only incubation at 12 °C for ca. 1 h can induce the Lc phase.27 The present results suggest that pressure further accelerates the transformation into the Lc phase in the MPPC bilayer. For confirmation of the Lc-phase spectra of Prodan in the MPPC bilayer, we investigated the time dependence of the Lc phase-spectra at 15.3 °C and 224.9 MPa. The results are shown in Figure 4A. The shape of the Lcphase spectra remained to be almost the same for more than 1 h except for the decrease in intensity of the spectra due to the slight precipitation of the sample. This indicates that the structural stability of the Lc phase in the high-pressure region is as high as those of the other phases. Figure 4B shows the decompression effect on the second-derivative spectra for the Lc phase (see also Figure SI-2). At ca. 200 MPa, the secondderivative spectra exhibited a broad valley with the λ″min values of ca. 460 nm, characteristic of the Lc phase. When the pressure decreased down to ca. 150 MPa, the λ″min value started to shift toward shorter wavelengths and became ca. 430 nm with deep blue valley at ca. 70 MPa. Interestingly, the spectra characteristic of the Lc phase at pressure below 50 MPa cannot 12194
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Figure 6. Image plot (A) for the fluorescence spectra of Prodan in MPPC bilayer membrane at 27.5 °C and (B) for their second derivatives of fluorescence spectra.
Figure 5. Image plot (A) for the fluorescence spectra of Prodan in MPPC bilayer membrane at 94.3−96.8 MPa in the temperature range 10.7−61.9 °C and (B) for their second derivatives of fluorescence spectra.
bilayer was completely in the LβI phase (see spectra 5 and 6 in Figure SI-4). We performed similar measurements at 49.1 °C in the previous study13 and obtained typical fluorescence spectra characteristic of the LβI phase. Namely, the valley at ca. 430 nm in the second derivatives of fluorescence spectra became shallower than those at ca. 500 nm without the broadening of the peak at ca. 460 nm. This spectral difference probably indicates that the structures of the LβI phases in the gel state and that in the Lc state are different. Judging from the fluorescence behavior, the latter Lc phase may be the interdigitated Lc (LcI) phase, and hence, it can be inferred from the λ″min value that the Prodan molecules are distributed both adjacent glycerol backbone and around hydrophilic region in the LcI phase. To clarify the Prodan fluorescence characteristic of the Lc phase in the metastable LβI phase region, we performed isobaric thermotropic fluorescence measurements after the Lc phase was completely formed. Here, the heating process is indicated as the open arrow parallel the ordinate at 158.8−176.6 MPa in Figure 1. Figure 7A shows the image plot of the temperature dependence of the second derivatives spectra at 158.8 MPa. The second-derivative spectra at every 5 °C, extracted from Figure 7A, and the original fluorescence spectra at the same temperatures are also given in Figures 7B and 7C, respectively. All the fluorescence spectra in the process are depicted in Figure SI-5. At 10 °C, the λ″min was ca. 460 nm and the valley was broad, which corresponds to the Prodan spectra of the complete Lc phase. In the temperature range from ca. 10 to 20 °C, the λ″min values gradually shifted to a short wavelength region in the same manner as observed in Figure 5. At ca. 27
report on the observation of so-called subtransition by fluorescence spectroscopy using a membrane probe. 3.3. Prodan Fluorescence in Interdigitated Gel-Phase Region. Although the MPPC bilayer can form the LβI phase under high pressure, as shown in Figure 1, the fast formation of the Lc phase facilitated by pressure hampers the observation of bilayer interdigitation by means of conventional isobaric thermotropic light−transmittance measurements. We have reported that measurements of Prodan fluorescence using a quick pressurization technique at a constant temperature enable us to observe the interdigitation13 and determine the triple point at which three gel phases can coexist.13 It is now interesting to investigate how the interdigitation affects the formation of the Lc phase. Figures 6A and 6B show the image plots based on the fluorescence spectra and their secondderivative spectra at 27.5 °C as a function of pressure, respectively (all the fluorescence spectra in this process are also given in Figure SI-4). In the pressurizing process, the MPPC bilayer undergoes the interdigitation from the Lβ′ phase to the LβI phase (see the upper open arrow parallel to the abscissa in Figure 1). The MPPC bilayer exhibited the Prodan fluorescence spectra characteristic of the gel phase up to at ca. 120 MPa. Around 140 MPa, the F.I. (or S.D.F.I.) at ca. 430 nm decreased (or increased) while that at ca. 500 nm increased (or decreased), which indicates that the interdigitation occurred. With further increasing pressure, peculiar behavior was observed: a broad peak around 460 nm in the LβI-phase spectra appeared at ca. 170 MPa, and the valley at ca. 430 nm was deeper than that at ca. 500 nm despite that the MPPC 12195
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phase in the PC bilayers.12,13,20 The above findings suggest that the state of the Lc phase is more susceptible to temperature and pressure than the other phases, and the Prodan fluorescence has a potential to detect the stable Lc/LβI transition. Further increases in temperature produced the valleys at 430 and subsequently that at 480 nm in response to the pre- and main transitions, respectively. To demonstrate the reversibility of the Lc-phase formation under high pressure, we carried out the quick cooling measurements at constant pressure (see the cooling process indicated as the open arrow in Figure 1). Figures 8A and 8B
Figure 8. (A) Fluorescence spectra and (B) the second derivatives of fluorescence spectra of Prodan in MPPC bilayer membrane at 205.8 MPa at (1) 78.0, (2) 63.5, (3) 54.0, (4) 24.8, and (5) 10.6 °C.
Figure 7. (A) Image plot for the second derivatives of fluorescence spectra of Prodan in MPPC bilayer membrane at 158.8 MPa in the temperature range 9.9−78.3 °C. (B) Extraction of the second derivatives of fluorescence spectra and (C) the extraction of the fluorescence spectra at 158.8 MPa: (1) 9.9, (2) 14.8, (3) 19.7, (4) 24.6, (5) 29.4, (6) 34.3, (7) 39.2, (8) 44.1, (9) 49.0, (10) 53.9, (11) 58.8, (12) 63.6, (13) 68.5, (14) 73.4, and (15) 78.3 °C.
show the fluorescence spectra and second-derivative spectra with decreasing temperature every 10 min at 205.8 MPa. The first spectrum (curve 1) at 78.0 °C was a typical result for Lα phase. With decreasing temperature to 63.5 °C (curve 2) and 54.0 °C (curve 3), the fluorescence spectra characteristics of the Pβ′ and LβI phase appeared, respectively. A remarkable feature was seen in the spectrum at 24.8 °C (curve 4); that is, a broad peak was observed, and the valley at ca. 430 nm became slightly deeper than that at ca. 500 nm (cf. curves 3 and 4). The result suggests that most of the MPPC bilayer forms the LcI phase in the LβI phase region rapidly. At ca. 10.6 °C (curve 5), the spectrum characteristic of the Lc phase was observed. From the behavior in the cooling process, we could confirm the reversibility of the Lc phase formation. 3.4. Structure of the Lc Phase. Finally, let us consider the structure of the Lc phase in the MPPC bilayer membrane. The actual structure of the Lc phase has not yet elucidated. Although there are a few reports on the Lc-phase formation of asymmetric PC bilayers, some significant reports concerning the Lc phase of the DPPC bilayer were made. The detailed DSC studies by Kodama et al.28,29 have demonstrated that the transformation of the Lβ′ phase into the Lc phase takes place by the exchange between nonfreezable and freezable interlamellar
°C, the drastic change of the spectra was observed; that is, a broad valley around 430 nm was formed (see curves 4 and 5 in Figure 7B). The minimum of S.D.F.I. at ca. 460 nm started to separate into two local minima at ca. 430 and 500 nm at ca. 26 °C. This temperature corresponds to the metastable Lβ′/LβI phase transition. The change indicates that the Prodan molecules in the Lc phase sensitively recognize the metastable LβI phase region and translocate the distribution sites quickly at the transition. It is reasonable to assume that a state of the MPPC bilayer membrane varies from the Lc phase to two kinds of Lc phases, that is, the Lc and LcI phases. With an increase in temperature, the two local minima gradually became sharp while the intensity at ca. 460 nm decreases (see curves 5−9 in Figure 7B). Beyond the phase boundary of the stable Lc/LβI transition at ca. 48 °C, the valley at ca. 460 nm vanished completely and second-derivative spectra with the two distinct local minima (see curve 10 in Figure 7B) appeared, which were often observed as a typical second-derivative spectra of the LβI 12196
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water molecules at the adjacent head groups of PC molecules. Tenchov et al.23 have performed time-resolved X-ray diffraction measurements to determine the D-spacing for all the phases observed at ambient pressure and showed that the D-spacing for the Lc phase is the thinnest among four (Lc, Lβ′, Pβ′, and Lα) phases. These results bear two possibilities with respect to the structure of the Lc phase: (1) the acyl chains of the DPPC bilayer are greatly tilted away from the bilayer normal; (2) total volume of the interlamellar water decreases. On the other hand, the Lc/Lβ′ transition (so-called subtransition) produces as a large enthalpy change as the main transition, and that large enthalpy cannot be explained by only these interpretations. Lewis and McElhaney30 have investigated the effect of the acylchain length on the Lc-phase formation by infrared spectroscopic measurements in a great detail. They showed that the Lcphase formation involves both reorientation of the acyl chains and significant changes in hydration and/or hydrogen-bonding interactions at the polar/apolar interfacial region of the lipid bilayer and suggested that the hydrogen-bonding network requires some distortion of the optimal packing of the acyl chains. Therefore, long acyl chains of the diacyl-PCs prevent optimizing of the hydrogen-bonding network. Moreover, in our previous studies of asymmetric PC bilayers,12,13 we have obtained important results which show that the terminal methyl end of the acyl chain markedly affects the stability of the Lc phase. Taking into account these facts and the results of the Prodan fluorescence characteristic of the Lc phase of the MPPC bilayer obtained in this study, we speculate that the MPPC molecules form a staggered structure in the Lc phase where the adjacent polar head groups mutually shift the position up and down as compared with the Lβ′ phase. Since the Prodan fluorescence spectrum of the Lc phase cannot be distinguished from that of the Lβ′ phase under atmospheric pressure, and the difference is enhanced under high pressure, we suppose that the deviations among the adjacent head groups in the staggered structure are facilitated enough to detect as the difference in the fluorescence spectrum under high pressure. Here we separate the Lc phase into “soft” Lc (Lcs) at low pressure or high temperature and “hard” Lc (Lch) at high pressure or low temperature in the microscopic viewpoint. On the latter condition, the MPPC bilayer in the Lc phase forms the staggered structure completely, where the Prodan molecules may be distributed widely from the adjacent glycerol-backbone region (ca. 430 nm) to the almost hydrophilic region (ca. 500 nm). This idea fits the experimental results: the wavelength characteristic of the Lch phase is ca. 460 nm, the value of which is average between 430 and 500 nm, and the fluorescence spectra exhibit a broad peak with a relatively high intensity. Moreover, the staggered structure does not contradict the fact that the Lc peak of 460 nm is enhanced by applying pressure. Considering the change in the fluorescence spectra, we propose a view, as can be schematically represented by the illustration in Figure 9, as to how the membrane states of the MPPC bilayer change depending on temperature and pressure. Under a low-pressure condition, the phase sequence of the MPPC bilayer with increasing temperature is similar to that of dimyristoyl-PC (DMPC) bilayer, where the Lβ′ phase is metastable in the whole temperature below the pretransition temperature. In contrast with this behavior, under high-pressure condition, an increase in temperature brings about the conversion from the Lch phase into the LcI + Lcs phases across the metastable Lβ′/ LβI phase boundary. With a further increase in temperature, the
Figure 9. Schematic illustration of various phase transitions and structural variations of MPPC bilayer membrane with temperature and pressure. Bilayer phases are assigned as the soft staggered subgel (Lcs), hard staggered subgel (Lch), lamellar gel (Lβ′), interdigitated subgel (LcI), ripple gel (Pβ′), interdigitated gel (LβI), and liquid crystalline (Lα) phase. Three kinds of phases are distinguished by colored head groups of the molecules: (green) subgel phase, (blue) gel phase, (red) liquid crystalline phase. Dashed arrows represent slow transitions.
MPPC membrane forms the stable LβI phase. It is interesting that these structural alterations are reversible, and such behavior of the Lc phase under high pressure was elucidated for the first time in this study.
4. CONCLUSIONS Although it is very difficult to study the Lc phase of lipid bilayers by conventional methods, we succeeded in examining the Lc phase by using an asymmetric PC that is prone to form the Lc phase and a method of high-pressure fluorescence spectroscopy. The MPPC bilayer easily formed the Lc phase by applying pressure and the second-derivative spectrum characteristic of the Lc phase under high pressure showed a minimum at ca. 460 nm with a broad shape. The line shape of the spectrum changed depending on pressure and temperature and came close to that of the gel phase under low-pressure and high-temperature conditions. This means that the Lc phase varies microscopically from the soft Lcs phase to the hard Lch phase by applying pressure. Moreover, the Lch phase separates into the LcI + Lcs phases in the metastable LβI phase region, and the alterations among the Lch, (LcI + Lcs), and LβI phases are completely reversible. Considering the fluorescence behavior of the MPPC bilayer along with the previous results of the Lc phase for PC bilayers, we concluded that the MPPC molecules form a staggered structure in the Lc phase. 12197
dx.doi.org/10.1021/la3020173 | Langmuir 2012, 28, 12191−12198
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ASSOCIATED CONTENT
S Supporting Information *
Figures SI-1−SI-5. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel +81-88-656-7513; Fax +81-88-655-3162; e-mail matsuki@ bio.tokushima-u.ac.jp. Notes
The authors declare no competing financial interest.
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