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Phase-Dependent Lateral Diffusion of r-Tocopherol in DPPC Liposomes Monitored by Fluorescence Quenching Roland Meyer, Andreas F.-P. Sonnen,† and Werner M. Nau* School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany. † Present address: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, 82152 Martinsried, Germany. Received May 13, 2010. Revised Manuscript Received July 19, 2010 The temperature-dependent fluorescence quenching of an amphiphilic palmitoyl derivative of 2,3-diazabicyclo[2.2.2]oct-2-ene (Fluorazophore-L) by R-tocopherol (R-Toc) has been determined in liposomes composed of a saturated lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The mutual lateral diffusion coefficients (DL) were extracted according to a laterally diffusion-controlled dynamic quenching model. Three distinct temperature regimes were identified: one between 65 and 39 °C, where the lateral diffusion coefficients were in the range of 10-7 cm2 s-1 and the lifetime of the probe was monoexponential in the absence of R-Toc, a second one between 39 and 30 °C, where the lateral diffusion coefficients were in the range of 10-8 cm2 s-1 and the lifetime of the probe was biexponential in the absence of R-Toc, and a third one below 30 °C, in which no diffusion was detectable, suggesting DL < 10-9 cm2s -1. These temperature domains were assigned, supported by differential scanning calorimetry (DSC) measurements, to the liquid-crystalline, ripple, and solid-gel phases of DPPC liposomes in the presence of the two additives. The absolute values of the individual lateral diffusion coefficients (taken as 1/2 of the DL values) of the Fluorazophore-L/R-Toc (ca. 2.5 10-7 cm2 s-1 at 52 °C) couple demonstrates that R-Toc does not diffuse at an unexpectedly high rate in comparison to the self-diffusion of DPPC (1.5 10-7 cm2 s-1 at 52 °C). However, diffusion in DPPC liposomes is distinctly slower than that in POPC ones (e.g., DL = 4.9 10-7 cm2 s-1 versus 6.4 10-7 cm2 s-1 at 50 °C), with an activation energy of 49 ( 5 kJ mol-1 (value for POPC: 47 ( 5 kJ mol-1), in the temperature range of the liquid-crystalline phase. Diffusion in the ripple phase, that is, below the main phase transition temperature, was found to be non-negligible, with an apparent activation energy of 175 ( 50 kJ mol-1.
Introduction Autoxidation of biological lipid bilayer membranes is generally prevented by lipophilic low molecular weight chain-breaking antioxidants,1,2 particularly tocopherols and tocotrienols, which are collectively referred to as vitamin E.3,4 In physiological situations known as oxidative stress, an imbalance between reactive oxygen or nitrogen species and antioxidants occurs in favor of the former. This may result, among other processes, in the peroxidation of lipids in vivo, which is thought to be linked to cardiovascular diseases, such as atherosclerosis (i.e., the formation of fatty streaks on the arterial walls).5-7 The understanding of the scavenging of reactive oxygen species by vitamin E constituents on a molecular and elementary reaction level is, consequently, of considerable interest in biology, medicine, as well as the food and dietary supplement industry. We have introduced a fluorescence-based method to monitor diffusion phenomena of lipid-soluble antioxidants in lipid bilayer membrane models and micellar structures.8-10 The method, *To whom correspondence should be addressed. E-mail: w.nau@ jacobs-university.de. Telephone: þ49 421 2003233. Fax: þ49 421 2003229.
(1) Niki, E.; Noguchi, N. Acc. Chem. Res. 2004, 37, 45. (2) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194. (3) Burton, G. W.; Joyce, A.; Ingold, K. U. Lancet 1982, 2, 327. (4) Halliwell, B.; Chirico, S. Am. J. Clin. Nutr. 1993, 57, 715S. (5) Steinberg, D. Lancet 1995, 346, 36. (6) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine, 3rd ed.; Oxford University Press: Oxford, 1999. (7) Sies, H.; Murphy, M. E. J. Photochem. Photobiol., B 1991, 8, 211. (8) Zhang, X.; Erb, C.; Flammer, J.; Nau, W. M. Photochem. Photobiol. 2000, 71, 524. (9) Nau, W. M. J. Am. Chem. Soc. 1998, 120, 12614. (10) Erb, C.; Nau-Staudt, K.; Flammer, J.; Nau, W. M. Ophthalmic Res. 2004, 36, 38.
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which has recently been expanded by others,11-13 is based on the fluorophore 2,3-diazabicyclo-[2.2.2]-oct-2-ene (DBO),14 which possesses a sufficiently long fluorescence lifetime above 100 ns to allow its fluorescence to be quenched by diffusion even in viscous environments such as liposomes. DBO has an n,π* electronic configuration in its first excited singlet state, which displays a radical-like reactivity and high propensity toward hydrogen atom abstraction from hydrogen donors such as R-tocopherol (R-Toc), presumably the most important chain-breaking antioxidant contained in vitamin E.15-19 Although the fluorescence quenching of DBO by R-Toc is chemically unproductive (because it involves a deactivation near a conical intersection and subsequent replenishment of the chemically unmodified ground-state reactants),20,21 we have demonstrated that the fluorescence quenching kinetics provides a useful tool to assess the diffusion between a reactive radical mimic and R-Toc and, when the quenching is observed in membrane models, to extract the associated mutual lateral diffusion (11) Anbazhagan, V.; Kalaiselvan, A.; Jaccob, M.; Venuvanalingam, P.; Renganathan, R. J. Photochem. Photobiol., B 2008, 91, 143. (12) Anbazhagan, V.; Kandavelu, V.; Kathiravan, A.; Renganathan, R. J. Photochem. Photobiol., A 2008, 193, 204. (13) Anbazhagan, V.; Renganathan, R. J. Lumin. 2008, 128, 1454. (14) Nau, W. M.; Wang, X. ChemPhysChem 2002, 3, 393. (15) Burton, G. W.; Hughes, L.; Ingold, K. U. J. Am. Chem. Soc. 1983, 105, 5950. (16) Burton, G. W.; Doba, T.; Gabe, E.; Hughes, L.; Lee, F. L.; Prasad, L.; Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 7053. (17) Traber, M. G.; Atkinson, J. Free Radical Biol. Med. 2007, 43, 4. (18) West, R.; Panagabko, C.; Atkinson, J. J. Org. Chem. 2010, 75, 2883. (19) Leonard, S. W.; Paterson, E.; Atkinson, J.; Ramakrishnan, R.; Cross, C. E.; Traber, M. G. Free Radical Biol. Med. 2005, 38, 857. (20) Nau, W. M.; Greiner, G.; Rau, H.; Wall, J.; Olivucci, M.; Scaiano, J. C. J. Phys. Chem. A 1999, 103, 1579. (21) Nau, W. M.; Greiner, G.; Wall, J.; Rau, H.; Olivucci, M.; Robb, M. A. Angew. Chem., Int. Ed. Engl. 1998, 37, 98.
Published on Web 08/19/2010
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Figure 1. Schematic representation of the fluorescent probe-quencher approach to monitor mutual lateral diffusion of two additives in unilamellar liposomes and chemical structures of the quencher (R-Toc), the fluorescent probe (Fluorazophore-L), and the lipid (DPPC).
coefficients. For our lipid bilayer experiments, the use of the amphiphilic DBO derivative Fluorazophore-L (Figure 1)22-24 has proven particularly useful. Our technique allows, in contrast to nuclear magnetic resonance (NMR) studies25-30 and complementary to fluorescence-recovery-after-photobleaching (FRAP)31-33 experiments, one to monitor the diffusion of molecules in real time within the confines of the lipid double layer.34 Previously, we have employed this methodology to extract lateral diffusion coefficients of R-Toc,22 as well as other vitamin E constituents35 and even of a synthetic lipid-soluble antioxidant,36 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. Later, even though our original motivation was the understanding of the diffusional behavior of different lipid-soluble antioxidants, we realized that our technique can more generally be employed to investigate diffusion and thereby probe the fluidity of different lipid structures (micelles versus liposomes),22 as well as their dependence on the presence of coadditives (cholesterol)37 and temperature.37 Herein, we investigate the dependence on the lipid phase by monitoring the fluorescence quenching of Fluorazophore-L by R-Toc in 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) liposomes. DPPC is exclusively derived from saturated fatty acids and consequently has a much higher phase transition temperature than the previously investigated, monounsaturated POPC (41 °C versus -2 °C).35,38 This allows us, for the first time, to investigate the fluorescence quenching by and lateral diffusion behavior of R-Toc in dependence on the (22) Gramlich, G.; Zhang, J.; Nau, W. M. J. Am. Chem. Soc. 2004, 126, 5482. (23) Gramlich, G.; Zhang, J.; Nau, W. M. J. Am. Chem. Soc. 2002, 124, 11252. (24) Gramlich, G.; Zhang, J.; Winterhalter, M.; Nau, W. M. Chem. Phys. Lipids 2001, 113, 1. (25) Aisenbrey, C.; Bechinger, B. J. Am. Chem. Soc. 2004, 126, 16676. (26) Ferrage, F.; Zoonens, M.; Warschawski, D. E.; Popot, J. L.; Bodenhausen, G. J. Am. Chem. Soc. 2003, 125, 2541. (27) Fukuzawa, K.; Ikebata, W.; Shibata, A.; Kumadaki, I.; Sakanaka, T.; Urano, S. Chem. Phys. Lipids 1992, 63, 69. (28) Hoff, B.; Strandberg, E.; Ulrich, A. S.; Tieleman, D. P.; Posten, C. Biophys. J. 2005, 88, 1818. (29) Lindblom, G.; Wennerstrom, H. Biophys. Chem. 1977, 6, 167. (30) Scheidt, H. A.; Pampel, A.; Nissler, L.; Gebhardt, R.; Huster, D. Biochim. Biophys. Acta, Biomembr. 2004, 1663, 97. (31) Saxton, M. J.; Jacobson, K. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373. (32) Wu, E. S.; Jacobson, K.; Papahadjopoulos, D. Biochemistry 1977, 16, 3936. (33) Yamazaki, V.; Sirenko, O.; Schafer, R. J.; Groves, J. T. J. Am. Chem. Soc. 2005, 127, 2826. (34) Melo, E.; Martins, J. Biophys. Chem. 2006, 123, 77. (35) Ruysschaert, T.; Sonnen, A. F. P.; Haefele, T.; Meier, W.; Winterhalter, M.; Fournier, D. J. Am. Chem. Soc. 2005, 127, 6242. (36) Nam, T. G.; Rector, C. L.; Kim, H. Y.; Sonnen, A. F.-P.; Meyer, R.; Nau, W. M.; Atkinson, J.; Rintoul, J.; Pratt, D. A.; Porter, N. A. J. Am. Chem. Soc. 2007, 129, 10211. (37) Sonnen, A. F.-P.; Bakirci, H.; Netscher, T.; Nau, W. M. J. Am. Chem. Soc. 2005, 127, 15575. (38) Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990.
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phase character of the lipid, and to probe the transition from the solid gel through the ripple to the fluid liquid-crystalline phase.39-45 Of particular interest were the diffusion properties of R-Toc in the ripple phase in liposomes, which have not been previously investigated.
Experimental Section Materials. Fluorazophore-L was synthesized as previously
reported.24 R-Tocopherol was used in its all-rac form (99.6%, donation from DSM Nutritional Products, Kaiseraugst, Switzerland). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was obtained from Sigma (Seelze, Germany). All solvents were of spectroscopic grade purity; all other chemicals were p.a. purity. Liposome Preparation. The liposomes were prepared according to the ethanol injection method46-49 to allow for a homogeneous distribution of R-Toc and Fluorazophore-L in the lipid double layer of the liposome. Stock solutions of R-Toc, Fluorazophore-L, and DPPC were freshly prepared for each set of experiments and stored at -20 °C in dark-brown graduated cylinders. To 84 μL of ethanolic stock solutions of DPPC and Fluorazophore-L, at concentrations of 30 and 3 mM, respectively, 16 μL of ethanol containing varying amounts of R-Toc was added. A total of 84 μL of this mixture was injected into 3 mL of 0.1% NaCl solution, for which Milipore water was used, yielding a liposome dispersion with a final lipid concentration of 0.7 mM and an overall ethanol concentration of 2.2%. It was important that the aqueous solution was vigorously stirred and heated above the main phase transition, 50 °C in our experiments, to ensure a homogeneous dispersion of the different components as well as the formation of unilamellar liposomes of uniform size. Fluorescence Spectroscopy. The fluorescence decay traces were recorded with a time-correlated single-photon counting (TCSPC) fluorimeter (FLS920, Edinburgh Instruments Ltd.) equipped with a PicoQuant diode laser LDH-P-CA375 (λexc = 373 nm, λobs = 450 nm, fwhm ca. 50 ps) for excitation. A circulation water bath (Julabo F25/HD thermostat) was used in conjunction with the EasyTemp software package to ensure constant temperature ((0.1 °C). The set temperature was varied (39) Loura, L. M. S.; Fernandes, F.; Fernandes, A. C.; Ramalho, J. P. P. Biochim. Biophys. Acta, Biomembr. 2008, 1778, 491. (40) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31, 7198. (41) Egli, U. H.; Streuli, R. A.; Dubler, E. Biochemistry 1984, 23, 148. (42) Heimburg, T. Biophys. J. 2000, 78, 1154. (43) Kaasgard, T.; Leidy, C.; Crowe, J. H.; Mouritsen, O. G.; Jorgensen, K. Biophys. J. 2003, 85, 350. (44) Janiak, M. J.; Small, D. M.; Shipley, G. G. Biochemistry 1976, 15, 4575. (45) Jacobson, K.; Papahadjopoulos, D. Biochemistry 1975, 14, 152. (46) Baranyai, P.; Gangl, S.; Grabner, G.; Knapp, M.; Koehler, G.; Vidoczy, T. Langmuir 1999, 15, 7577. (47) Batzri, S.; Korn, E. D. Biochim. Biophys. Acta, Biomembr. 1973, 298, 1015. (48) Domazou, A. S.; Luisi, P. L. J. Liposome Res. 2002, 12, 205. (49) Kremer, J. M. H.; van der Esker, M. W.; Pathmamanoharan, C.; Wiersema, P. H. Biochemistry 1977, 16, 3932.
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from 20 to 65 °C, with special emphasis on the 35-45 °C temperature region. The pulse frequency of the laser in the TCSPC experiments was always kept below 2% of the inverse lifetime while the count rate was kept at approximately 1% of the pulse rate. The experiments have been carried out with freshly prepared samples in quartz cuvettes, and an identically prepared reference sample was measured for each temperature to correct for scattered light and instrumental dark counts. The fluorescence lifetimes τ0 required for fitting were directly analyzed with the instrumental software by fitting monoexponential (above the main phase transition temperature) or biexponential (below the main phase transition temperature) decay functions to the decay traces. The traces in the presence of R-Toc were used directly in the global analysis to extract the diffusion coefficients (see Results and Discussion), which was performed analogously to our previous studies22,37 with the software ProFit.50 Liposome Characterization. The liposome size measurements were performed by dynamic light scattering with a Zetasizer Nano ZS 90 (Malvern Instruments), which employs a He-Ne laser at a 90° angle (40 mW, λ = 633 nm). The phase transitions were also followed by differential scanning calorimetry (DSC) on a VP-DSC Instrument by MicroCal (MA). The employed samples contained exactly the same amount of lipid, quencher, and probe as those measured by TCSPC.
Results and Discussion Phases of DPPC Lipids. Our present investigations focus on how the fluorescence decay kinetics of an immersed probe in lipids change as a function of temperature as well as the concentration of an added quencher. While our time-resolved spectroscopic technique, employing Fluorazophore-L as a long-lived fluorescent probe and the natural antioxidant R-Toc as quencher, allows us to extract information on the dynamics and mutual diffusion of the additives in lipids, it cannot directly provide information on the structure of the investigated lipidic assemblies and only indirectly report on changes of the lipid phase. The interpretations of the fluorescence decays in terms of diffusion are therefore model-based and rely on our present conceptual understanding of which structures and lipid phases we are working with. For example, for the previously investigated POPC assemblies, we could assume that we were dealing with unilamellar liposomes in their liquid-crystalline phase and have accordingly interpreted the fluorescence quenching of Fluorazophore-L as a laterally diffusion-controlled process in which the polar head groups of both additives collide (see representation in Figure 1). For the presently investigated DPPC structures, we can again presume the formation of unilamellar liposomes, based on the employed preparation method.49,51 We have further confirmed by dynamic light scattering experiments (this work) a uniformly average liposomal size of 80 nm with very low polydispersity ((5 nm), which turned out to be insignificantly dependent on the presence of the two additives in the investigated concentration ranges. In contrast to the case of POPC, DPPC is known to adapt two different main phases at ambient (solid-gel phase) and slightly elevated (liquid-crystalline phase) temperature, and these phases (50) QuantumSoft 6.0.3; Z€urich, Switzerland. (51) Moura, S. P.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 2007, 313, 519. (52) Onuki, Y.; Morishita, M.; Chiba, Y.; Tokiwa, S.; Takayama, K. Chem. Pharm. Bull. 2005, 54, 68. (53) The amounts of ethanol employed in all liposome preparations were kept constant throughout all experiments, they were the same as those used in our previous investigations (refs 22 and 37, to allow direct comparison), and they were kept sufficiently small, as circumstantially evidenced by the unaltered main phase transition temperature, to exclude major effects on lipid structure and fluidity; see ref 22 and Almeida, L. M.; Vaz, W. L. C.; St€umpel, J.; Madeira, V. M. C. Biochemistry 1986, 25, 4832–4839.
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were necessarily expected to have a pronounced effect on the fluorescence decay kinetics and the lateral diffusion process. The well-known main phase transition temperature in the absence of additives, namely, 41 °C, could be accurately reproduced by DSC experiments (see below). 35,52,53 With the addition of Fluorazophore-L to the mixture, it dropped to 39 °C, while adding both Fluorazophore-L and R-Toc to the lipid yielded a phase transition temperature of 37 °C. We consequently expected (and have observed) abrupt changes in the fluorescence decay around 37-39 °C, because we were dealing with DPPC liposomes which always contained Fluorazophore-L and in varying concentrations R-Toc. It could be further expected that diffusion in the nanosecond range (which is accessible to the probe-quencher technique) would be negligible in the solid-gel phase, while above the main phase transition temperature, in the liquid-crystalline phase, we could expect similar diffusional properties as in the previously investigated POPC liposomes, and accordingly applied the same analytical treatment of our fluorescence decay data. However, as an additional intricacy, we needed to consider the so-called ripple phase, a pretransition phase, which occurs slightly below the main phase transition. It is characterized by the formation of ripples that span rings around the liposome, thereby retaining liposome structure and size.42 Many ideas exist why ripples form in liposomes, and many models share the common idea that they result from a periodic local curvature within the bilayer itself. What induces this spontaneous curvature is still under debate, but that ripples form at the pretransition temperature is undeniable.42,43 The temperature for the pretransition in pure DPPC is reported to lie at 32.5 °C,54 but it was likely to be lowered by additives in a similar way as the main transition is altered,55 so we estimated it to be depressed below 30 °C, a conjecture which was supported by the earlier onset of the DSC curves in the presence of additives (see below) as well as independent observations of the fluorescence quenching kinetics documented herein. Because we observed non-negligible fluorescence quenching and consequently sizable diffusion in the ripple phase, we presumed a diffusion model which describes a liposome in its ripple phase as an assembly composed of at least two different microenvironments (alternating in the form of ripples), with one more resembling the solid-gel phase (with negligible diffusion) and the other as resembling the liquid-crystalline phase (but with a strongly hindered diffusion due to the neighboring more rigid domains). Our model further presumed that the thickness (and/or number) of the more fluid ripples increased with temperature, while the more rigid ones disappeared concomitantly and completely at the main transition to the liquid-crystalline phase. In the absence of information on the opposite, we further presumed that the concentration of both additives in both domains of the ripple phase remained the same. The way we modeled the different phases is schematically illustrated in Figure 2, keeping in mind that our experimental data do not provide any structural evidence, in particular not for the ripple phase, but that such a simplistic framework is sufficient to account for our experimental observations in the relevant temperature region. Intrinsic Fluorescence Lifetimes of Fluorazophore-L. The long fluorescence lifetime of Fluorazophore-L is the prerequisite for observing laterally diffusion-controlled fluorescence quenching by another additive, such as R-Toc. In essence, the fluorescent dye must be sufficiently “patient” in its excited state to wait for the (54) Csiszar, A.; Koglin, E.; Meier, R. J.; Klumpp, E. Chem. Phys. Lipids 2006, 139, 115. (55) Lewis, R. N. A. H.; Mak, N.; McElhaney, R. N. Biochemistry 1987, 26, 6118.
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Figure 2. Schematic representation of the phases in DPPC liposomes in dependence on temperature, where the patterned gray and blue regions signify rigid and more fluid regions with negligible or sizable diffusion, respectively. Figure 4. Representative fluorescence decay traces of Fluorazophore-L in DPPC liposomes with increasing R-Toc concentrations, [Q2D]/(molecules m-2), at 43 °C. The inset shows the logarithmic decay curves. The black lines represent the obtained global fits according to eq 1 under variation of the lateral diffusion coefficient as a fitting parameter.
Figure 3. Fluorescence lifetimes of Fluorazophore-L (average lifetimes were used below the main phase transition) in DPPC liposomes versus temperature (b). Shown for comparison are the lifetimes in POPC liposomes (O).37 The inset shows the pre-exponential factors of the short-lived (R1, 9) and long-lived (R2, 0) fluorescence decay components observed below the main phase transition temperature of DPPC.
quencher to diffuse through the viscous medium and reach the probe before it deactivates by other decay pathways. Moreover, the formula used to extract diffusion coefficients is an approximate one, optimized for fluorescence lifetimes in the range of 100 ns.56 It was therefore imperative to determine the fluorescence lifetimes as well as their dependence on temperature first (Figure 3). Above the phase transition temperature of 37 °C, that is, in the liquid-crystalline phase of DPPC, the fluorescence decays in the absence of quencher were monoexponential,57 as was found to be the case for the previously investigated POPC liposomes, which existed in the liquid-crystalline phase over the entire investigated temperature range.37,38 Interestingly, below the phase transition temperature, that is, in the ripple phase, the fluorescence decays were non-monoexponential;58 a biexponential decay kinetics with two lifetime components provided an adequate fitting. According to our model (Figure 2), and (56) Naqvi, K. R.; Martins, J.; Melo, E. J. Phys. Chem. B 2000, 104, 12035. (57) Earlier investigations on monolayers have shown an excellent miscibility of our fluorescent probe with lipids (ref 24), with no indications for nonhomogeneous distributions, demixing, or effects on structural integrity. In particular, the monoexponential fluorescence decay behavior in the absence of quencher and the fact that the lifetime was independent of the probe concentration clearly speak for a homogeneous distribution of probe molecules in the liquid-crystalline phase. (58) The fact that the fluorescence lifetimes remain apparently biexponential (with components of ca. 330 and 130 ns) in the solid-gel phase, i.e., below 30 °C, is presently not understood, but it could be due to a variety of reasons, including a different composition of the frozen-out ripples or differences between the inner and outer leaflets of the liposome.
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knowledge about the presumed structural characteristics of the ripple phase (see above), we assigned the short-lifetime component, whose pre-exponential factor decreased at lower temperature, to Fluorazophore-L immersed in the more fluid domain, and the long-lifetime component to Fluorazophore-L immersed in the more rigid domain. Fluorazophore-L is quenched to varying degrees even by collisions with solvent molecules,20 such that the longer lifetime in the less mobile phase is well accounted for in terms of a confinement effect. Recall that alkyl groups, just like hydrocarbon solvents, cause a slight fluorescence quenching of the parent chromophore by hydrogen atom abstraction.20,59 Hydrogen atom abstraction is a thermally activated process, which accounts for the decrease in fluorescence lifetime with increasing temperature in both phases (Figure 3). The photochemical hydrogen atom abstraction reactivity is also the reason for the shorter fluorescence lifetimes of Fluorazophore-L immersed in lipid bilayers (e.g., 218 ns in DPPC versus 127 ns in POPC, at 25 °C) than those of the parent DBO in aqueous solution (325 ns at 25 °C).9,60 The reason for the consistently shorter fluorescence lifetimes of Fluorazophore-L in POPC liposomes (e.g., 78 ns at 45 °C, dashed line in Figure 3) in contrast to DPPC (e.g., 97 ns at 45 °C, solid line) can in turn be related to the unsaturarated oleoyl fatty acid tails in POPC, which cause a more significant quenching due to the presence of more readily abstractable allylic C-H bonds.20,59 When the average lifetimes are plotted against temperature, the phase transition temperature for DPPC liposomes can be readily identified through a sudden jump (solid circles in Figure 3). This could not be observed for POPC liposomes (open circles), which has a phase transition outside our measurement range (-2 °C).37,38 The fluorescence lifetime of Fluorazophore-L can consequently be used as a convenient probe to signal phase transitions by time-resolved fluorescence spectroscopy, thereby complementing the use of established microviscosity-dependent fluorescent probes.45,61 Apart from the phase-dependent behavior, it should be noted that the fluorescence lifetimes of Fluorazophore-L ranged in all phases and at all temperature between 60 and 250 ns, ideally suitable for the semiempirical formulas employed for data analysis (see below). (59) Nau, W. M.; Pischel, U. Photoreactivity of n,π*-Excited Azoalkanes and Ketones. In Organic Photochemistry and Photophysics; Ramamurthy, V., Schanze, K., Eds.; CRC Press: Boca Raton, FL, 2005; Vol. 14, p 75. (60) Nau, W. M.; Zhang, X. J. Am. Chem. Soc. 1999, 121, 8022. (61) Kung, C. E.; Reed, J. K. Biochemistry 1986, 25, 6114.
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Table 1. Mutual Lateral Diffusion Coefficients of Lipid-BilayerIncorporated Fluorazophore-L and r-Toc in DPPC Liposomes at Various Temperatures, Determined by Fluorescence Quenching T/°C
DL/(10-7 cm2 s-1)a
T/°C
DL/(10-7 cm2 s-1)a
20 25 30 33 35 37 39 40 41 43
