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)-naphthalene (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 glycerol 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 ethanol 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 methanol, 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.