High Pressure Bioscience and Biotechnology 61 Proceedings of the 4th International Conference on High Pressure Bioscience and Biotechnology, Vol. 1, 61–67, 2007

Effect of Pressure on the Prodan Fluorescence in Bilayer Membrane of Ether-Linked Lipid, Dihexadecylphosphatidylcholine

Masataka Kusube1, Masaki Goto2, Nobutake Tamai2, Hitoshi Matsuki2 and Shoji Kaneshina*2 1Department of Material Science, Wakayama National College of Technology, 77 Noshima, Nada, Gobo, Wakayama 644-0023, Japan 2Department of Biological Science and Technology, Graduate School of Advanced Technology and Science, and Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770- 8506, Japan *E-mail: [email protected]

Received 8 December 2006/Accepted 15 December 2006

Abstract The fluorescence spectra of 6-propionyl-2-(dimethyamino)- (Prodan) in the bilayer membrane of an ether-linked phospholipid, dihexadecylphosphatidylcholine (DHPC), were observed as a function of temperature and pressure. Previous results for Prodan in the bilayer membrane of an ester-linked lipid, dipalmitoylphosphatidyl- choline (DPPC), were compared with the present results. A great difference in the fluorescence spectrum shape was found in the ripple gel phase of the DHPC and DPPC bilayers. The location of the Prodan molecule in the bilayer was speculated on the basis of the maximum emission wavelength. The ratio of fluorescence intensity of Prodan at two wavelengths was useful for the determination of bilayer phase transition, especially the pressure-induced interdigitation.

Keywords: bilayer membrane, ether-linked lipid, pressure-induced interdigitation, phase transition, Prodan-fluorescence

1. Introduction

Dipalmitoyl-(DPPC) and dihexadecylphosphatidylcholine (DHPC) are representative phospholipids with ester- and ether-linkage, respectively. DPPC has been widely found in mammalian cells and one of the most investigated phospholipids with regard to structural and thermodynamic properties [1-3], while the ether-linked phospholipids exist in the cellular membrane of the archaebacteriums, and a few studies have been reported on the properties of bilayer membranes [4-8]. A difference in linkage between hydrophobic chains and backbone of phospholipid molecules is responsible for the different phase transitions of lipid bilayers. DPPC bilayer undergoes the pretransition from the lamellar gel (Lβ’) phase to the ripple gel (Pβ’) phase, and the main transition from the Pβ’ phase to the liquid crystal (Lα) phase at ambient pressure, moreover the interdigitated gel (LβI) phase is induced by pressure above 100 MPa. On the other hand, DHPC bilayer already forms the LβI phase by fully hydration at ambient pressure, and undergoes the pretransition from the LβI phase to the Pβ’ phase and the main transition. The temperature (T)-pressure (p) phase diagrams of DHPC and DPPC bilayers were discussed in our previous studies [9, 10]: the T-p phase diagram of DHPC bilayer corresponds to the high-pressure region of DPPC bilayer phase diagram. In this study, we observe the pressure-induced phase transitions of DHPC and DPPC bilayer membranes by the method of high-pressure Prodan fluorescence. We focus our 62

attention on the differences of Prodan fluorescence in between ether-linked DHPC and ester- linked DPPC bilayer membranes. Then, we speculate the location of Prodan in the region of bilayer interface of both lipids from the maximum emission wavelength. Finally, we demonstrate that the ratio of fluorescence intensity of Prodan at two wavelengths is available to observation of bilayer phase transition, especially the bilayer interdigitation.

2. Materials and methods

DHPC and DPPC were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and Sigma-Aldrich Co. (St. Louis, MO), respectively. These lipids were used without further purification after dried under vacuum. The fluorescence probe Prodan was obtained from Molecular Probes, Inc. (Eugene, OR). Water was distilled twice from dilute alkaline permanganate solution. The vesicles containing fluorescence probe Prodan were prepared by Bangham’s method [11]. The chloroform solution of a phospholipid was mixed with the stock solution of Prodan. The mixed solution was dried under vacuum to remove all residual solvent and finally to get a dry film. Distilled water was added to the dry film of phospholipid and the suspension was hydrated by treatment of vortex and sonication for a short time at a temperature several degrees above the main transition temperature of the lipid bilayer. In all the experiments the total concentration of the phospholipid was 1.0 mM and the molar ratio of Prodan to the phospholipid was 1:500. The sample solutions were protected from the light until measurements. The fluorescence measurements under high pressure were carried out with Hitachi fluorescence spectrophotometer, Model F-2500. A high-pressure cell assembly with quartz windows was supplied by Teramecs Co. (Kyoto). Pressures were generated by a hand- operated hydraulic pump KP-3B (Hikari High Pressure Instruments, Hiroshima) and measured within an accuracy of ± 0.2 MPa by a Heise gauge. Temperature of the high- pressure cell was controlled within an accuracy of ± 0.1 °C by circulating water from a water bath through the jacket enclosing the pressure cell. The excitation wavelength was 361 nm, and the emission spectra were obtained at wavelengths from 390 to 550 nm.

3. Results and discussion

In our previous study [12, 13], a good correlation between the solvent polarity (namely solvent dielectric constant) and the maximum emission wavelengths (λmax) of Prodan was confirmed. The values of λmax were 528 nm in water, 502 nm in , 484 nm in 1- pentanol, 428 nm in benzene and so on. A higher value of λmax (namely red shift of λmax) is attributed to probe in polar or hydrophilic environment, while a lower value of λmax (namely blue shift of λmax) is attributed to probe molecule in less polar or hydrophobic environment. The emission spectra of Prodan in various phases of DPPC and DHPC bilayers under ambient pressure are shown in Figs. 1-A and 1-B, respectively. These spectra show an emission maximum characteristic of bilayer phases. The λmax values of Prodan in DPPC and DHPC bilayers are shown in Fig. 1-C as a function of temperature. The value of λmax varied clearly with bilayer phase-transitions. In the Lα phase of bilayer for both lipids, the values of λmax resemble each other. On the other hand, in the Pβ’ phase, the value of λmax for both lipid bilayers is different from each other: 440 nm for DPPC bilayer and 478 nm for DHPC bilayer. This means that the microenvironment around Prodan molecule in the DPPC bilayer is less polar in comparison with the DHPC bilayer. Blue shift in DPPC bilayer may be attributable to the strong interaction between Prodan and carbonyl group of DPPC molecules 63

Fig. 1 Fluorescence spectra of Prodan in various phases of (A) DPPC and (B) DHPC bilayer membranes. (C) The maximum emission wavelengths of Prodan in DPPC and DHPC bilayers as a function of temperature.

because an ether-linked DHPC molecule lacks carbonyl group. It is noted that the λmax in the LβI phase shows the largest value in the present study. The values of λmax characteristic of various bilayer phases are summarized in Table 1. Fluorescence spectra of Prodan in the DPPC bilayer were observed as a function of pressure at 52.3 °C and 37.5 °C, which was shown in Figs. 2-A and 2-B, respectively. The pressurizing process is shown in the T-p phase diagram (Fig. 2-C) [3] as broken lines. The DPPC bilayer undergoes two phase transitions with increasing pressure at 52.3 °C: one is the main transition from the Lα phase to the Pβ’ phase and the other is the transition from the Pβ’ phase to the LβI phase. As is seen in Fig. 2-A, the shape of spectrum varied significantly with increasing pressure. In the Lα phase, the value of λmax was about 480 nm, and in the Pβ’ phase the λmax was about 440 nm. When the LβI phase appeared at higher pressure than 150 MPa, the emission spectrum became a broad peak at about 500 nm with shoulder at about 430 nm. At 37.5 °C, the state of DPPC bilayer varies with increasing pressure, in turn, the Pβ’, Lβ’ and LβI phase. The emission spectra of Prodan at 37.5 °C show the resemblance among the λmax in three phases, but in the LβI phase it has a shoulder around 500 nm. Fig. 3 shows the emission spectra of Prodan in the DHPC bilayer and the T-p phase diagram of DHPC bilayer. The fluorescence spectra of Prodan in the DHPC bilayer were measured at two temperatures of 50.0 and Table 1. Maximum emission wavelengths of 59.0 °C as a function of pressure. At these two Prodan in various phases of lipid bilayers. temperatures, as the pressure increases the DHPC Lipid Bilayer phase λmax bilayer undergoes the main transition from the Lα phase to the Pβ’ phase and then the transition DPPC Lα 482 from the Pβ’ phase to the LβI phase [9], which Pβ’ 440 are the same transitions as in the DPPC bilayer at L’ 433 52.3 °C. As is seen from Figs. 3-A and 3-B, the β values of λmax in these three phases of DHPC DHPC Lα 484 bilayer varied only slightly with increasing Pβ’ 478 pressure. In the Pβ’ phase of DHPC bilayer, the emission spectrum has a peak at 478 nm and a LβI 486 64

Fig. 2 Fluorescence spectra of Prodan in DPPC bilayer membrane at (A) 52.3 °C and (B) 37.5 °C as a function of pressure. (C) Temperature-pressure phase diagram of DPPC bilayer, which is from Ichimori et al (1998) [3]. Broken lines in the phase diagram mean pressurizing process. shoulder at about 430 nm. It is noted that the emission spectrum in the Pβ’ phase of DHPC bilayer is different from that in the DPPC bilayer, that is, the value of λmax is 478 nm in the DHPC bilayer while the λmax in the DPPC bilayer is 440 nm. This means that the microenvironment of Prodan molecule in the Pβ’ phase of DHPC bilayer is more hydrophilic than that in the DPPC bilayer, which is probably attributable to a weak interaction between Prodan molecule and glycerol backbone of lipid molecule because of a lack of carbonyl group in the DHPC molecule. Next we consider the Prodan location in the lipid bilayers from the shift of emission maximum on the basis of a correlation between the solvent polarity and the λmax of Prodan in

Fig. 3 Fluorescence spectra of Prodan in DHPC bilayer membrane at (A) 59.0 °C and (B) 50.0 °C as a function of pressure. (C) Temperature-pressure phase diagram of DHPC bilayer, which is from Maruyama et al (1996) [9]. Broken lines in the phase diagram mean pressurizing process. 65

bilayers. Location of the probe molecule Prodan in DPPC bilayer has been discussed by several researchers [14-16]. Zeng and Chong [15] revealed that the emission spectrum of the Prodan in DPPC bilayer changes dramatically with the ethanol-induced phase transition from the noninterdigitated gel state to the fully interdigitated gel state. Spectral changes are attributed to the probe relocation from a less polar environment to a more polar environment due to the bilayer interdigitation. Bernik et al. [16] examined the fluorescence properties of some probes inserted at different levels of bilayer interface and suggested that Prodan locates in the region between the ester carbonyls at glycerol backbone and the phosphate group. With regard to a polar environment in the lipid bilayers, Okamura and Nakahara [17] demonstrated by NMR study that hydrated lipid bilayers could be divided into three zones. Zone I is the hydrophilic part of bilayer, which includes the polar head groups consisting of choline and phosphate groups. Zone II is the interfacial region between the hydrophilic and the hydrophobic parts of the lipid. Glycerol backbone and ester carbonyl group belong this region. Zone III is the hydrocarbon chain region and composed by the bilayer core. They described how the dielectric constant or the water density distributes along the lipid molecule in the bilayer. A significant decrease in the dielectric constant or the water density with a large gradient is characteristic of the zone II. In the previous study on the Prodan fluorescence in the DPPC bilayer [12], we demonstrated that positioning of Prodan’s fluorophore along the phospholipids molecule in bilayer can be determinable from the dramatic shift of λmax. The 430 or 440 nm peak of fluorescence spectra means that the Prodan molecules exist in less polar region of the lipid bilayers around glycerol backbone (zone II), the 480 nm peak suggests that they exist in between zone I and II such as around phosphate, and the 500 nm peak implies that they exist in hydrophilic region around lipid head group (zone I). In the present study, there exists a distinguished difference in the fluorescence spectra of Prodan in the Pβ’ phase between DHPC and DPPC bilayers: the λmax is 478 nm in DHPC bilayer and 440 nm in DPPC bilayer. A comparison of Prodan location in the Pβ’ phase between DHPC and DPPC bilayers is schematically depicted in Fig. 4. The fluorophore of Prodan molecule in the Pβ’ phase of DPPC bilayer distributes around glycerol backbone (namely zone II). On the other hand, in

Fig. 4 Schematic drawing for the Prodan location in the Pβ’ phase of (A) DHPC and (B) DPPC bilayer membranes. 66

the DHPC bilayer the Prodan’s fluorophore distributes around phosphate group (namely intermediate region between zone I and II). Because of the lack of carbonyl groups in the DHPC lipid molecule, the interaction between Prodan and zone II of lipid molecule is weakened. Therefore, Prodan molecules exist in less hydrophobic region around phosphate groups of lipids. Finally, we demonstrate how the Prodan fluorescence method is useful for determining the bilayer phase transitions. The emission spectra of Prodan in lipid bilayers during the phase transformation may contain two peaks of similar intensities, making the selection of the emission maximum difficult. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, is useful for the determination of bilayer phase transition. The values of F480/F430 were plotted as a function of pressure in Fig. 5. In the figure are also shown the phase transition pressures by arrows, which were taken from the T-p phase diagram shown in Figs. 2-C and 3-C. The values of F480/F430 show an abrupt change at a certain pressure, which corresponds to the phase transitions. In the previous studies, the pressure-induced interdigitation has been detected from the bilayer thickness determined by the X-ray diffraction [18, 19] or the neutron diffraction methods [20, 21]. Present results using Prodan fluorescence propose an easy and convenient recognition method for the pressure-induced interdigitation of lipid bilayers instead of the previous elaborate time-consuming methods.

Fig. 5 Ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430 versus pressure. Fluorescence intensity ratio of Prodan in (A) DPPC and (B) DHPC bilayer membranes. The phase transition pressures, which were taken from the T-p phase diagram, are shown by arrows.

4. Conclusions

The λmax of Prodan in the ether-linked DHPC bilayer was found to be 484, 478 and 486 nm in the Lα, Pβ’ and LβI phases, respectively. These results were compared with the emission spectra of Prodan in the ester-linked DPPC bilayer. There exists a great difference in the spectra shape in the Pβ’ phase between DHPC and DPPC bilayers: a main peak at 478 nm with a small shoulder at about 430 nm for the DHPC bilayer, on the other hand, a single peak at 440 nm for the DPPC bilayer. Since the λmax reflects the environment around the probe molecules, we could speculate on the location of the Prodan molecule in the bilayer 67

membranes. The fluorophore of Prodan molecule in the Pβ’ phase of DHPC bilayer distributes around phosphate group in polar environment, whereas in the DPPC bilayer the Prodan molecule distributes near glycerol backbone in less polar environment. Since the values of F480/F430 showed a sharp change at the phase-transition pressure, the plot of F480/F430 vs. pressure is useful for the determination of bilayer phase transition, especially the pressure-induced interdigitation.

5. References

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