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Langmuir 1998, 14, 2256-2262
Phase Transitions in Phospholipid Vesicles: Excited State Prototropism of 1-Naphthol as a Novel Probe Concept J. Sujatha and A. K. Mishra* Department of Chemistry, Indian Institute of Technology, Madras, Chennai-600 036, India Received March 10, 1997. In Final Form: January 14, 1998 1-Naphthol (1-ROH), an excited state acid, undergoes partial dissociation in the excited state in liposome membrane, giving two fluorescence peaks as against a single peak in water. 1-Naphthol distributes between two different sites of different excited state reactivity in liposome, and the relative population in the two different sites changes with any perturbations in the membrane fluidity. This property is made use of in studying the thermotropic phase changes and cholesterol effect on the transition temperature of DMPC (dimyristoylphosphatidylcholine) and DPPC (dipalmitoylphosphatidylchloline) liposomes. The change in the permeability of the membrane due to thermotropic phase changes and the phase transition temperature of liposomes could be easily monitored by fluorescence spectroscopy from a conspicuous change in the emission of the neutral form of 1-naphthol. The applicability of 1-naphthol probe to mixed lipid sytems has also been examined.
Introduction A variety of physical techniques have been applied for the past decade to study phase transitions of phospholipids.1-10 The well-studied phospholipids in this respect are DMPC (dimyristoylphosphatidylcholine) and DPPC (dipalmitoylphosphatidylcholine), which undergo phase transition at approximately 23 and 42 °C, respectively, when in equilibrium with water.1 The endothermic reaction observed by differential scanning calorimetry2 at this temperature (Tc) marks the onset of liquidity of the hydrocarbon region of the lamellar phase. The loss of crystallinity at Tc has been documented by X-ray diffraction.3 This transition is accompanied by a decrease in the thickness of the lipid membranes,3 a change in bilayer volume as observed by dilatometry,4 a marked decrease in the proton NMR line width,5 a decrease in the order parameter of ESR probes,6 a decrease in the fluorescence polarization of various probes,7-9 and an increase in the number of ANS (8-anilinonaphthalenesulfonate) and TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxy) binding sites.6,10 The solid to liquid crystalline phase transition has a marked effect on the permeability properties of phospholipid membranes, which are of physiological interest.11 Although a variety of sophisticated physical methods have been used, extensive work is still going on in this field in search of simple techniques. The effects of phase transition on both the physical state and the permeability (1) Hinz, H. J.; Sturtevant, J. M. J. Biol. Chem. 1974, 247, 60716075. (2) Oldfield, E.; Chapman, D. FEBS Lett. 1972, 23, 285-297. (3) Chapman, E.; Williams, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids 1967, 1, 445-475. (4) Trauble, H.; Haynes D. H. Chem. Phys. Lipids 1971, 7, 324-335. (5) Lee, A. G.; Birdsall, N. J. M.; Levine, Y. K.; Metcaye, J. C. Biochim. Biophys. Acta. 1972, 255, 43-56. (6) Hubbell, W. L.; McConnell, H. M. J. Am. Chem. Soc. 1971, 93, 314-326. (7) Lussan, C.; Faucon, J. F. FEBS Lett. 1971, 19, 186-188. (8) Vanderkooi, J. M.; Chance, B. FEBS Lett. 1972, 22, 23-26. (9) Cogan, U.; Shinitzky, M.; Weber, G.; Nishida, T. Biochemistry 1973, 12, 521-528. (10) Sackmann, E.; Trauble, H. J. Am. Chem. Soc. 1972, 94, 44824491. (11) Raison, J. K.; Lyons, J. M.; Mehlhorn, R. J.; Keith, A. D. J. Biol. Chem. 1971, 246, 4036-4040.
properties of DMPC and DPPC lipid membranes have been well studied.2 The present work is aimed at making use of excited-state proton transfer (ESPT) as a simple steady-state technique to probe the properties of these phospholipid membranes. Aromatic hydroxy compounds are known to be excitedstate acids.12 For 1-naphthol in water, the light-absorbing species is the neutral form (1-ROH), but rapid proton transfer follows excitation and fluorescence is usually observed from the anionic species (1-RO-) formed in the excited state. The kinetics of 1-ROH emission and ESPT processes have been extensively investigated using timeresolved studies12 which lead to two different views. Robinson and Lee13 have argued that the ESPT rates of aromatic alcohols in aqueous solutions are limited by the time water takes to wrap itself around the charge and also the proton dissociation requires a common (H2O)4 cluster as the proton acceptor. An alternative model is proposed by Huppert, Pines, and Agmon14 according to which a reversible formation of a (RO-*‚‚‚H+) geminate ion pair and subsequent separation to free ions under the Debye-Smoluchowski equation conditions can successfully explain ESPT processes. Whatever the mechanism, the general feature of ESPT is that the equilibrium is sensitive to the environment and any change in the equilibrium leads to a change in the ratio of anionic to neutral peak intensities. These systems are easy to monitor fluorometrically since the bands due to the neutral (360 nm) and anionic (478 nm) forms are well separated. As the size of these aromatic hydroxy compounds are small, they are expected to perturb the membrane system minimally. Since Weller’s15 initial work, excited state proton transfers have been intensively (12) (a) Nome, F.; Reed, W.; Politi, M.; Tundo, P.; Fendler, J. H. J. Am. Chem. Soc. 1984, 106, 8086-8093. (b) Shizuka, H. Acc. Chem. Res. 1985, 18, 141. (c) Arnaut, L. G.; Formosinho S. J. J. Photochem. Photobiol., A: 1993, 75, 120. (d) Tolbert, L. M.; Haubrich, J. E. J. Am. Chem. Soc. 1994, 116, 10593-10600. (e) Behera, P. K.; Mukherjee, T.; Mishra, A. K. J. Lumin. 1995, 65, 137. (13) Robinson, G. W.; Thistlethwaite, P. J.; Lee, J. J. Phys. Chem. 1986, 90, 4224. (14) Agmon, N.; Pines, E.; Huppert, D. J. Chem. Phys. 1988, 88, 5631. (15) Weller, A. Naturwissenschaften 1955, 42, 175.
S0743-7463(97)00274-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/03/1998
Phase Transitions in Phospholipid Vesicles
studied. Gutman,16 discussed the application of ESPT of aromatic alcohols as probes for mcacromolecules and solutions, emphasizing the potential role of pH jump experiments in biochemistry. A strong retardation of excited-state protolytic dissociation was observed in the case of naphthols in microemulsions,17 micelles,18 and liposome suspensions.19 In an earlier work by Kuzmin et al.,19 from an extensive kinetic analysis, it was found that neutral from of 1-naphthol in liposome showed two different lifetimes. The anionic form exists as a single lifetime component in liposome, which is different from that in bulk water. On the basis of these results, we recently derived an expression to calculate the partition coefficient (Kp) of 1-naphthol in liposome.20 Kp was found to be 5 × 106 at 30 °C, which is comparable to that of the standard probes such as ANS (8-anilinonaphthalenesulfonate), DPH (1,6-diphenylhexatriene), etc.21 As 1-naphthol partitions well into liposomes, an attempt is made here to explore the applicability of ESPT-induced fluorescence changes as a probe to study the thermotropic phase behavior of DMPC, DPPC lipid-bilayers and also lipid-cholesterol interactions, using 1-naphthol as probe. Comparison has been made for results obtained from the conceptually different techniques involving ESPT (of 1-naphthol) and polarization (of DPH) and also selfdiffusion studies using radioisotopes (22Na+).22 Experimental Section 1-Naphthol was vacuum sublimed and used after checking its purity. DMPC and DPPC were purchased from Sigma Chemical Co. (USA) and used as such after checking for a single spot in thin-layer chromatography. Cholesterol was also purchased from Sigma and its purity confirmed by melting point. Double distilled water was used for experiments. All solvents were distilled. Liposome Preparation.23 The lipids (DMPC and DPPC) were dissolved in chloroform-methanol 2:1 (v/v) at the desired molar ratio. The solution was evaporated to dryness under nitrogen. The solvent was removed by using a rotary evaporator, and residual solvent if any was removed by leaving the roundbottomed flask in a vacuum. Multilamellar vesicles (MLV) were prepared by adding the appropriate volume of phosphate-buffered saline (PBS, 10-4 M) at pH 7, to the lipid film with vigorous vortexing and then warmed at 35-40 °C (for DMPC) or 50-55 °C (DPPC) to yield a final lipid concentration of 1 mM. DMPCcholesterol liposomes were prepared by adding to the same volume from a stock solution of DMPC lipid (ca. 1 mM) different volumes of a stock solution of cholesterol (ca. 0.1 mM) such that concentration of chloesterol was varied from 0% to 50% of lipid concentration. Labeling. A stock solution of 1-naphthol was prepared in phosphate-buffered saline (PBS) at pH 7. For most of the experiments the lipid/probe ratio was kept at 100. After the probe was added, the solutions were allowed to equilibriate for 1 h at 30 °C (for DMPC) and 50 °C (for DPPC), before experiment. For all the experiments, a control solution containing the same concentration of liposome but no probe was prepared and used as a blank. (16) (a) Gutman, M.; Nachliel, E. Biochim. Biophys. Acta 1990, 1015, 391. (b) Gutman, M.; Huppert, D.; Pines, E. J. Am. Chem. Soc. 1981, 103, 3709-13. (17) Mario, J. P.; Ogden, B.; Fendler, J. H. J. Phys. Chem. 1985, 89, 2345. (18) Ill’ichev, Yu. V.; Demyashkevich, A. B.; Kuzmin, M. G. J. Phys. Chem. 1991, 95, 3438-3444. (19) Ill’ichev, Yu. V.; Demyashkevich, A. B.; Kuzmin, M. G. Photochem. Photobiol., A 1993, 74, 51-63. (20) Sujatha, J.; Mishra, A. K. J. Photochem. Photobiol., A 1996, 101, 215-219. (21) Zhijian, H.; Richard, P. Biochim. Biophys. Res. Commun. 1993, 181, (1), 166-171. (22) Papahadjopoulos, D.; Jacobson, K.; Nir, S.; Isac T. Biochim. Biophys. Acta 1973, 311, 330-8. (23) Huang C. Biochemistry 1969, 8, 344.
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Figure 1. Emission spectrum of 1-naphthol in DMPC liposome at 25 °C for concentration of lipid varying from 7 × 10-5 to 35 × 10-5 M. Concentration of 1-naphthol ) 1.25 × 10-6 M. Fluorescence Measurements. Fluorescence measurements were taken in a Hitachi F-4500 spectrofluorometer. The excitation wavelength was 300 nm and the corrected emission spectrum was recorded in a wavelength range of 300-600 nm with the excitation and emission slit widths having a band-pass of 5 nm. The fluorescence intensity of the blank was subtracted from that of the experimental solution value (contribution of blank intensity value is
