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Langmuir 2007, 23, 9416-9422
Phosphatidylcholine Vesicle-Mediated Decomposition of Hydrogen Peroxide Makoto Yoshimoto,†,‡,* Yuya Miyazaki,‡ Ayumi Umemoto,‡ Peter Walde,§ Ryoichi Kuboi,| and Katsumi Nakao‡ Departments of Applied Molecular Bioscience and Applied Chemistry and Chemical Engineering, Yamaguchi UniVersity, 2-16-1 Tokiwadai, Ube 755-8611, Japan, Department of Materials, ETH-Zu¨rich, Wolfgang-Pauli-Strasse 10, CH-8093 Zu¨rich, Switzerland, and Department of Chemical Science and Engineering, Osaka UniVersity, 1-3 Machikaneyama-cho, Toyonaka 560-8531, Japan ReceiVed May 2, 2007. In Final Form: June 7, 2007 The decomposition of hydrogen peroxide (H2O2) was examined in aqueous solution (50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl) at 25 °C in pure buffer or in the presence of either vesicles or micelles formed from various phosphatidylcholines (PCs). In the absence of PCs, more than 90% of the initially added H2O2 (1.0 mM) remained intact after incubation for 120 h. The effect of the PCs on the decomposition of H2O2 was studied by using different PCs that varied in terms of number of carbon atoms in the two acyl chains n as well as in terms of the degree of unsaturation. PCs with short hydrocarbon chains (n ) 4, 6-8) were dissolved in the buffer solution in the form of nonassociated monomers or as micelles in equilibrium with monomers at a fixed PC concentration of 10 mM. The presence of these short-chain PCs slightly enhanced the H2O2 decomposition rate. Micelles formed by non-lipid detergents (sodium cholate, Triton X-100, and sodium dodecylsulfate) had a similar effect. In marked contrast, PCs with long hydrocarbon chains (n g 10) dispersed in buffer solution as vesicles (liposomes) significantly enhanced the rate of H2O2 decomposition, with the most effective PC being 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at 25 °C. This indicates that the packing density of the PC molecules influences the reactivity, presumably through the direct interaction of the PC assemblies with H2O2 molecules. Furthermore, in the case of vesicles formed from PCs with unsaturated acyl chains (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC; 1,2-dioleoyl-sn-glycero3-phosphocholine, DOPC), carbon-carbon double bond oxidation did not occur extensively under the conditions used. This indicates that the observed effect of PCs on the decomposition of H2O2 is indeed related to the assembly structure (vesicle Vs micelles Vs monomers) and is clearly not related to the presence of unsaturated hydrocarbon chains. Fluorescence polarization measurements of two fluorescent probes embedded either in the acyl chain region of the vesicles (DPH, 1,6-diphenyl-1,3,5-hexatriene) or on the surface of the vesicles (TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide) show that the presence of H2O2 leads to a decrease in the fluidity of the lipid-water surface and not to a change in the fluidity of the hydrophobic region of the vesicle bilayer. This indicates that the decomposition of H2O2 is triggered through interactions between H2O2 and the polar head group area of PC vesicles.
Introduction Hydrogen peroxide (H2O2) is a reactive oxygen species. It contains two oxygen atoms in a formal oxidation state of -I. Excess amounts of H2O2 cause significant damage to biological systems, for example, the peroxidation of unsaturated lipids.1 H2O2 is decomposed effectively with redox enzymes such as catalase, glutathione peroxidase, and superoxide dismutase.2 These enzymes are highly specific in their structures and functions for eliminating under physiological conditions reactive oxygen species without causing self-inactivation, similar to the enzymes that catalyze oxidation reactions.3 However, H2O2 molecules play a significant role in controlling certain cellular functions1 through the formation of adducts with various biological molecules to regulate their location and chemical stability.4 * To whom correspondence should be addressed. E-mail: yosimoto@ yamaguchi-u.ac.jp. Phone: +81-836-85-9271. Fax: +81-836-85-9201. † Department of Applied Molecular Bioscience, Yamaguchi University. ‡ Department of Applied Chemistry and Chemical Engineering, Yamaguchi University. § ETH-Zu ¨ rich. | Osaka University. (1) Chen, Z.-H.; Yoshida, Y.; Saito, Y.; Niki, E. Neurosci. Lett. 2005, 383, 256-259. (2) Maritim, A. C.; Sanders, R. A.; Watkins, J. B., III. J. Biochem. Mol. Toxicol. 2003, 17, 24-38. (3) Klinman, J. P. J. Biol. Inorg. Chem. 2001, 6, 1-13. (4) Schubert, J.; Wilmer, J. W. Free Radical Biol. Med. 1991, 11, 545-555.
In industry, H2O2 is very useful, for example, in textile bleaching and other oxidation reactions in which the concentration of H2O2 needs to be adequately controlled.5 Several heterogeneous catalysts, such as flocculated titanium dioxide (TiO2) nanocrystallites6 and other oxide particles (e.g., ZrO2 (zirconium dioxide) and CeO2 (cerium dioxide)),7 were recently reported to catalyze the decomposition of H2O2. However, the mechanism for the H2O2 interaction and decomposition at the interface of any catalyst is not yet fully understood. Recently, we reported on a liposomal catalase system, which is an in Vitro antioxidant system prepared from phospholipid vesicles (liposomes) containing encapsulated catalase.8 This system could efficiently decompose H2O2 that was produced during prolonged air oxidation of glucose, catalyzed by liposomal glucose oxidase.8 In examining the system more closely, we found in preliminary experiments that not only liposomal catalase but also enzyme-free, intact phospholipid membranes accelerated the decomposition of H2O2. It may well be that this observation was related to the binding of H2O2 to the phospholipid membrane. It has been reported previously that the interfacial region of the (5) Petlicki, J.; Palusova, D.; van de Ven, T. G. M. Ind. Eng. Chem. Res. 2005, 44, 2002-2010. (6) Du, Y.; Rabani, J. J. Phys. Chem. B 2006, 110, 6123-6128. (7) Hiroki, A.; LaVerne, A. J. Phys. Chem. B 2005, 109, 3364-3370. (8) Yoshimoto, M.; Miyazaki, Y.; Sato, M.; Fukunaga, K.; Kuboi, R.; Nakao, K. Bioconjugate Chem. 2004, 15, 1055-1061.
10.1021/la701277f CCC: $37.00 © 2007 American Chemical Society Published on Web 07/27/2007
Decomposition of Hydrogen Peroxide
membrane can indeed interact with various molecules, including not only water,9 ethanol,10 and other small moleucles11 but also proteins,12 via electrostatic and hydrophobic interactions and/or hydrogen bonding.13 In the work presented here, we examine the decomposition of H2O2 in the presence of various phosphatidylcholines (PCs) or non-phospholipid micelle-forming detergents. The PCs had an identical hydrophilic head group (phosphocholine) but varied in their acyl chain length and in the degree of unsaturation so that different types of molecular assemblies formed depending on the chemical structure and the concentration of the PC. A comparison was made among nonassociated PCs, measured at a concentration below the corresponding critical micellization concentration (cmc), PC micelles (at a concentration above the cmc), and PC vesicles. Experimental Section Materials. 1,2-Dihexanoyl-sn-glycero-3-phosphocholine (DiC6PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DiC8PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC), and chicken egg yolk phosphatidylcholine (EggPC) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). 1,2-Dibutanoyl-sn-glycero-3-phosphocholine (DiC4PC), 1.2-diheptanoyl-sn-glycero-3-phosphocholine (DiC7PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DiC10PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and Triton X-100 (mean molecular mass taken as 625 g/mol) were obtained from Sigma (St. Louis, MO). 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), sodium cholate, sodium dodecylsulfate (SDS), hydrogen peroxide (H2O2) solution (35.1%), bovine liver catalase, and horseradish peroxidase (HRP) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 1,6,-Diphenyl-1,3,5hexatriene (DPH) was obtained from Aldrich (Milwaukee, WI). 1-(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide (TMA-DPH) was obtained from Dojindo Laboratories (Kumamoto, Japan). All reagents were used as received. Preparation of Lipid Monomer Solutions, Lipid Micelle Solutions, Lipid Vesicle Suspensions, and Non-Lipid Detergent Micelle Solutions. Each phospholipid was dissolved in chloroform in a round-bottomed flask, and the solvent was removed using a rotary evaporator. Then the lipid film that formed was dried for 2 h under reduced pressure (
