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Langmuir 2007, 23, 1518-1522
Selenium-Mediated Micellar Catalyst: An Efficient Enzyme Model for Glutathione Peroxidase-like Catalysis Xin Huang, Zeyuan Dong, Junqiu Liu,* Shizhong Mao, Jiayun Xu, Guimin Luo, and Jiacong Shen Key Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin UniVersity, Changchun 130012, People’s Republic of China ReceiVed June 15, 2006. In Final Form: September 23, 2006 Mimicking the properties of the selenoenzyme glutathione peroxidase (GPx) has inspired great interest. In this report, a selenium-containing micellar catalyst was successfully constructed by the self-assembly of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) with benzeneseleninic acid (PhSeO2H) through hydrophobic and electrostatic interaction in water. The selenium-containing micellar catalyst demonstrated substrate specificity for both 3-carboxy-4-nitrobenzenethiol (ArSH, 2) and cumene hydroperoxide (CUOOH), and their complexation was confirmed by UV and fluorescence spectra. More importantly, it demonstrated high GPx activity in two assay systems. It is about 126 times more effective than the well-known GPx mimic ebselen in the classical coupled reductase assay system; however, by using hydrophobic substrate ArSH (2) as an alternative of glutathione (GSH, 1), the micellar catalyst exhibited remarkable 500-fold and 94 500-fold rate enhancements compared with that of PhSeO2H and PhSeSePh.
Introduction Since the discovery of selenium functions in a biological enzyme system,1 much attention has been devoted to elucidate the importance of selenium in selenoenzymes.2 Recently, there have been growing interests in the development of selenoenzyme biomimetic chemistry, especially in the mimicking of the antioxidant selenoenzyme, glutathione peroxidase (GPx).3 GPx is a chemically and structurally well-studied selenoenzyme that functions to catalyze the reduction of hydroperoxides (ROOH) by glutathione (GSH, 1).4,5 It therefore plays an important role in the organismal antioxidant defense mechanism in protecting cells from oxidative stress.4,5 The enzyme active site includes a selenocysteine residue in a depression on the protein’s surface, with some charged and hydrophobic amino acid residues (Phe, Trp, Asp) forming a hydrophobic cavity for substrate binding.5 For the exploration of the structural and functional importance of selenium in GPx and for the potential applications in the development of selenium-related medicine, enormous effort has been made in the development of artificial GPx models; for example, a catalytic center was introduced into existing or artificially generated substrate binding scaffolds by chemical or genetic strategies.6-8 However, the design of highly efficient GPx models by supramolecular chemistry methods remains a great challenge. * Corresponding author. E-mail address:
[email protected]. Fax: +86431-5193421. (1) Mills, G. C. J. Biol. Chem. 1957, 229, 189. (2) Stadtman, T. C. Annu. ReV. Biochem. 1990, 59, 111. (3) (a) Mugesh, G.; du Mont, W. W. Chem. ReV. 2001, 101, 2125. (b) Mugesh, G.; Singh, H. B. Acc. Chem. Res. 2002, 35, 226. (c) Mugesh, G.; Singh, H. B. Chem. Soc. ReV. 2000, 29, 347. (d) Tomoda, S.; Iwaoka, M. J. Am. Chem. Soc. 1994, 116, 2557. (e) McNaughton, M.; Engman, L.; Birmingham, A.; Powis, G.; Cotgreave, I. A. J. Med. Chem. 2004, 47, 233. (f) You, Y.; Ahsan, K.; Detty, M. R. J. Am. Chem. Soc. 2003, 125, 4918. (4) Flohe´, L.; Loschen, G.; Gu¨nzler, W. A.; Eichele, E. Hoppe-Seyler’s Z. Physiol. Chem. 1972, 353, 987. (5) Epp, O.; Ladenstein, R.; Wendel, A. Eur. J. Biochem. 1983, 133, 51. (6) Dong, Z. Y.; Liu, J. Q.; Mao, S. Z.; Huang, X.; Yang, B.; Luo, G. M.; Shen, J. C. J. Am. Chem. Soc. 2004, 126, 16395. (7) Mao, S. Z.; Dong, Z. Y.; Liu, J. Q.; Li, X. Q.; Liu, X. M.; Luo, G. M.; Shen, J. C. J. Am. Chem. Soc. 2005, 127, 11588. (8) Yu, H. J.; Liu, J. Q.; August, B.; Li, J.; Luo, G. M.; Shen, J. C. J. Biol. Chem. 2005, 280, 11930.
Among supramolecular systems, micelles represent a welldeveloped field in biomimetic chemistry due to their vital functions: (i) a three-dimensional nanoscale structure,9-13 which can be constructed in a self-assembled manner; (ii) two typologically different regions, the polar surface of which can be derived or into which functional groups for catalysis can be introduced. The interior, which can produce localized hydrophobic microenvironments, is analogous to these found at the active sites of natural enzymes. The specific structure of the micelle makes it as an excellent enzyme model.3,14-18 Although a great progress has been made on the construction of micellar models for hydrolysis, oxidation, reduction, carbon-carbon bond forming reactions, and so on;14-18 however, to the best of our knowledge, there are no reports on GPx mimics. Herein, we report a novel GPx model of a selenium-mediated, self-assembled, supramolecular micelle. Taking advantage of the microenvironment provided by the micelle, the seleniummediated micelle exhibits a remarkable GPx-like activity and is about 5 orders of magnitude more efficient than the well-studied GPx mimic, diphenyl diselenide (PhSeSePh) for the reduction of cumene hydroperoxide (CUOOH) by 3-carboxy-4-nitrobenzenethiol (ArSH, 2); interestingly, the micelle itself, without selenium, acts as an excellent catalyst for accelerating the reduction of CUOOH by ArSH. Experimental 3-Bromo-1-propanol, sodium borohydride, and diphenyl diselenide were purchased from Fluka and used without further purification. Hexadecyltrimethylammonium bromide (CTAB) was purchased from (9) Menger, F. M.; Shi, L. J. Am. Chem. Soc. 2006, 128, 9338. (10) Menger, F. M. Acc. Chem. Res. 1997, 12, 111. (11) Menger, F. M.; Dell. D. W. J. J. Am. Chem. Soc. 1984, 106, 1109. (12) Menger, F. M. Nature 1985, 313, 603. (13) Wennerstroem, H.; Lindman, B. J. Phys. Chem. 1979, 83, 2931. (14) Scrimin, P.; Tonellato, U. In Surfactants in Solution; Mittel, K. L., Shah, D. D., Eds.; Plenum Press: New York, 1991; Vol. 11. (15) Vriezema, D. M.; Comellas, A. M.; Elemans, J. A. A. W.; Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M. Chem. ReV. 2005, 105, 1445. (16) Tornssier, R.; Scrimin, P.; Tecilla, P.; Tonellato, U. J. Am. Chem. Soc. 1989, 111, 224. (17) Tang, S. S.; Chang, G. G. J. Org. Chem. 1995, 60, 6183. (18) Dwars, T.; Paetzold, E.; Oehme, G. Angew. Chem., Int. Ed. 2005, 44, 7174.
10.1021/la061727p CCC: $37.00 © 2007 American Chemical Society Published on Web 12/07/2006
Modeling GPx Using Selenium-Mediated Micelles
Langmuir, Vol. 23, No. 3, 2007 1519 vis-NIR spectrophotometer. Appropriate control of the nonenzymatic reaction was performed and subtracted from the catalyzed reaction. 2. Coupled Reductase Assay System (see following chemical equations): enzyme
2GSH + H2O2 98 GSSG + 2H2O
(1)
glutathione reductase
Tianjinfuchen and was purified by recrystallizing from methanol and ether. The characterization of the structure of the mimics was performed with a Bruker Advance 500 (500 MHz) 1H NMR spectrometer using a TMS proton signal as the internal standard. Molecular weights of the compounds were measured on an LDI1700-MALDI-TOF-MS (Linear Scientific Inc.). Elemental analyses were determined on a Perkin-Elmer 240 DS elemental analyzer. UV-vis spectra were obtained using a Shimadzu 3100 UV-vis-NIR spectrophotometer. Fluorescence spectral measurements were performed on a Shimadzu RF-5301 PC spectrofluorophotometer. Data were acquired and analyzed by using ultraviolet spectroscopy software. The temperature for UV time course studies was controlled within (() 0.5 °C by use of a LAUDA compact low-temperature thermostat RC6 CP. The buffer pH values were determined with a METTLER TOLEDO 320 pH meter. Synthesis of Benzeneseleninic Acid. According to the procedure of McCullough and Gowld,19 diphenyl diselenide (1.00 g, 3.20 mmol) was added to 5 mL of 1,4-dioxane, and the mixture was heated to about 60 °C to dissolve the solid. It was then cooled to below 5 °C, and hydrogen peroxide (C.P. 20-28% w/w) was added dropwise, stirring the mixture and cooling externally to keep the temperature under 10 °C. After the addition of a 3-fold excess of peroxide, ice (5.00 g) was added, and the white solid was filtered off and washed with 10 mL of ice water. Then the white solid benzeneseleninic acid (about 0.70 g) was obtained (benzeneselenenic acid is too unstable to obtain). 1H NMR (500 MHz, D2O): δ 7.76-7.79 (m, 2H), δ 7.57-7.60 (m, 3H). MALDI-MS: Calcd 189.07, found 189.97; Anal. Calcd for C6H5SeO2H: C, 38.11; H, 3.20. Found: C, 38.30; H 3.21. Preparation of Selenium-Mediated Micellar Catalyst. A surfactant molecule containing a long hydrophobic alkyl chain and a hydrophilic headgroup can aggregate to form a spherical micelle in water when it exceeds a certain concentration (the critical micelle concentration, or cmc). CTAB or sodium dodecane sulfonate (SDS) with PhSeO2H can spontaneously aggregate to form a comicelle in water. A selenium-mediated micellar catalyst was prepared17 by ultrasonic mixing of CTAB (502.30 mg, 5.52 mM (6 cmc) or SDS (3.50 g, 48.60 mM (6 cmc)) and benzeneseleninic acid (0.13 mg, 0.69 µM) in 250 mL of water until a clear solution was obtained. The micelle without benzeneseleninic acid was also prepared in the same way. All the solutions were used within 24 h of preparation. Determination of GPx Activity. 1. Determination of GPx ActiVity Using ArSH (2) as a Substrate (see following chemical equation): enzyme
2ArSH + H2O2 98 ArSSAr + 2H2O The catalytic activity was assayed according to a modified method reported by Hilvert et al.20 The reaction was carried out at 37 °C in 500 µL of phosphate buffer (pH 7.0) containing 1 µM seleniummediated micellar catalyst and 100 µM ArSH (2) that was prepared from the corresponding disulfide by reduction with sodium borohydride.21 The reaction was initiated by the addition of 2.5 mM CUOOH. The initial rates for the reduction of CUOOH by ArSH were determined by monitoring the disappearance of ArSH at 410 nm (410 ) 13 600 M-l cm-1, pH 7.0) with a Shimadzu 3100 UV(19) Mccullough, J. D.; Gould, E. S. J. Am. Chem. Soc. 1949, 71, 674. (20) (a) Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1990, 112, 5647. (b) Bell, I. M.; Hilvert, D. Biochemistry 1993, 32, 13969. (c) Bell, I. M.; Fisher, M. L.; Wu, Z. P.; Hilvert, D. Biochemistry 1993, 32, 3754. (21) Silver, M. Methods Enzymol. 1979, 62D, 135.
GSSG + NADPH + H+ 98 2GSH + NADP+
(2)
The GPx activity was assayed in a coupled reductase assay system described by Wilson et al.22 The reaction was carried out at 37 °C in 500 µL of 50 mM phosphate buffer (pH 7.0) containing 1 mM EDTA, 1 mM GSH, 1 unit of glutathione reductase, and 1 µM selenium-mediated micellar catalyst. The mixture was preincubated for 5 min. Then 0.25 mM NADPH solution was added, and the mixture was incubated for 3 min at 37 °C. Thereafter, the reaction was initiated by the addition of 2.5 mM CUOOH. The initial rates for the reduction of CUOOH by GSH were determined by monitoring the decrease of NADPH absorption at 340 nm (340 ) 6220 M-l cm-1).
Results and Discussion Design and Preparation of Selenium-Mediated Micellar Catalyst. The design of the selenium-mediated micellar catalyst should address some major features of a natural GPx active site.5 First, to generate substrate binding capacity, the cationic surfactant CTAB is utilized as a main unit to construct the micelle. The positively charged surface of the micelle might recognize the carboxylic group of thiol substrates by electrostatic interaction, similar to the salt-bridge formed between two arginines (Arg40, Arg167) and GSH in the binding site of natural GPx.5 Second, the selenium moiety is designated as a catalytic prosthetic group, just like the function of selenocysteine in a GPx active site, and finally, the hydrophobic interior of the micelle consisting of a long hydrophobic alkyl chain is similar to the hydrophobic pocket of GPx. Selenium-mediated micellar catalyst was prepared by ultrasonic mixing of CTAB and benzeneseleninic acid water until a clear solution was obtained.17 It was characterized by UV and fluorescence spectroscopy (see Supporting Information). The maximum UV absorption wavelength of PhSeO2H obviously shifts, and the fluorescence intensity of PhSeO2H largely increases after forming a micelle, reflecting that the hydrophobic aromatic moiety of PhSeO2H must be in the interior of the micelle. Furthermore, in 1H NMR spectra (see Supporting Information), the aromatic protons of PhSeO2H show obvious downfield shifts and remarkable cleavage in the presence of the micelle compared with the spectra of PhSeO2H alone, also suggesting that the aromatic moiety of PhSeO2H locates in the micelle. According to these results and the structure of the CTAB micelle, we gave the possible binding model for this selenium-mediated micellar catalyst (Scheme 1). Catalytic Behavior. The catalytic ability of the seleniummediated micellar catalysts were first tested according to a modified method reported by Hilvert et al.20 using ArSH (2) as a substrate. The relative activities of the mimics are summarized in Table 1. For the peroxidase activity, the enzymatic rates were corrected for the background (nonenzymic) reaction between hydroperoxide and thiol. The initial rate of the background (nonenzymic) reaction between CUOOH and ArSH was very slow, and a slight enhancement of the reaction rate was observed when PhSeSePh (462 µM) was added (υ0 ) 0.011 µM min-1). However, under identical conditions, the selenium-mediated (22) Wilson, S. R.; Zucker, P. A.; Huang, R.-R. C.; Spector, A. J. Am. Chem. Soc. 1989, 111, 5936.
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Scheme 1. Proposed Model for Selenium-Mediated Micellar Catalyst
Figure 1. Plots of absorbance vs time during the catalytic reduction of CUOOH (150 µM) by ArSH (100 µM) at pH 7.0 and 37 °C: (a) no catalyst, (b) 100 µM PhSeSePh, (c) 5.52 mM CTAB, and (d) selenium-mediated micellar catalyst (2.5 µM PhSeO2H and 5.52 mM CTAB).
Table 1. The Initial Rate (ν0)a and Activity for the Reduction of ROOH (250 µM) by Thiol ArSH (0.10 mM) and GSH (0.10 mM) in the Presence of Various Catalysts at pH 7.0 (50 mM PBS, 1 mM EDTA) and 37 °C catalyst PhSeSePh PhSeO2H CTAB+PhSeO2H SDS+PhSeO2H
hydroperoxide CUOOH CUOOH H2O2 CUOOH H2O2 CUOOH
V0a (µM‚min-1)
activity
ArSH
ArSHb GSHc
0.011 ( 0.0001 0.093 ( 0.009 0.017 ( 0.001 2.25 ( 0.24 NDd ND
1 190 714 94 500 ND ND
1 1.5 5 63 ND ND
a
The initial rate of reaction was corrected for the spontaneous oxidation in the micelle without PhSeO2H. b The concentration of catalyst: PhSeSePh (462 µM), PhSeO2H (20 µM), selenium-mediated micellar catalyst (1.00 µM PhSeO2H and 5.52 mM (6 cmc) CTAB or 48.6 mM (6 cmc) SDS) in ArSH assay system and assuming one molecule PhSeO2H as one active site of enzyme. c The concentration of catalyst: PhSeSePh (1.00 µM), selenium-mediated micellar catalyst (0.39 µM PhSeO2H and 5.52 mM (6 cmc) CTAB or 48.6 mM (6 cmc) SDS). d ND, no detectable GPx activity in this assay system. Calculated based upon GPx activity of PhSeSePh equal to 1.
micellar catalyst (1.0 µM PhSeO2H and 5.52 mM (6 cmc) CTAB) exhibits a remarkable rate enhancement (2.25 µM min-1). Assuming that the rate had a first-order dependence on the concentration of catalyst catalyzing the reduction of ROOH by ArSH (2) (Table 1), it is suggested that the selenium-mediated micellar catalyst was at least 500 and 94 500 times more efficient than PhSeO2H and PhSeSePh (Figure 1). The activities of the selenium-mediated micellar catalyst were also assessed in the classical coupled reductase assay system using GSH (1) as a substrate. The relative activities of the compounds are also listed in Table 1. Typical saturation kinetics was observed in both assay systems for the peroxidase-like reactions, indicating that this model is a real catalyst for peroxidase reaction. In the classical coupled reductase assay system, in the presence of the selenium-mediated micellar catalyst (0.78 µM benzeneseleninic acid and 5.52 mM CTAB) at pH 7.0 (50 mM PBS, 1 mM EDTA) and 37 °C, the apparent kinetic parameters of the selenium-mediated micellar catalyst were obtained V(max) ) 115.4 µM min-1, k(app) cat ) 377.07 min-1, KmGSH ) 2.05 mM, and k(app) /Km ) 1.84 × 105 M-1 GSH cat -1 min (the initial concentration of CUOOH fixed to 0.50 mM), and the turnover number per benzeneseleninic acid was calculated to be 115 min-1 (Figure 2). In the ArSH assay system, in the
Figure 2. Plots of initial rates ν0 (µM min-1) at different concentrations of GSH in the presence of the selenium-mediated micellar catalyst (0.78 µM benzeneseleninic acid and 5.52 mM CTAB) at pH 7.0 (50 mM PBS, 1 mM EDTA). The initial concentration of CUOOH was fixed to 0.50 mM with NADPH (0.25 mM) and glutathione reductase (1 unit). The concentration of GSH was 0.20, 0.50, 1.00, 2.50, 5.00, and 10.00 mM, respectively.
presence of selenium-mediated micellar catalyst (5.0 µM benzeneseleninic acid and 5.52 mM CTAB) at pH 7.0 (50 mM PBS, 1 mM EDTA) and 37 °C, the apparent kinetic parameters of the selenium-mediated micellar catalyst were obtained V(max) -1 ) 2.52 µM min-1, k(app) cat ) 2.86 min , KmArSH ) 0.027 mM, 5 M-1 min-1 (the initial and k(app) /Km ) 1.06 × 10 CUOOH cat concentration of CUOOH fixed to 0.25 mM) (Figure 3). To probe the mechanism of selenium in catalysis, some of the intermediates possibly involved in the catalytic cycle were characterized. When iodoacetic acid was added in the catalytic system, the PhSeO2H was found to lose its activity in the classical coupled reductase assay system. This clearly shows that the selenol moiety must be one of the intermediates in the catalytic cycle. In addition, when ArSH was added to PhSeO2H in PBS (pH 7.0), the ArSH quickly disappeared by monitoring the UV absorbance at 410 nm, and intermediate selenenyl sulfide appeared; this important intermediate was confirmed by MALDI-TOF-MS. For the selenium-mediated micellar catalyst, as seen from Table 1, a 500-fold activity enhancement by PhSeO2H catalysis after it formed the selenium-mediated micellar catalyst was found in the ArSH assay system; however, an only 40-fold activity enhancement of only was observed in the classical coupled reductase assay system. The large difference in the activity enhancement in the two assay systems suggests that the micelle seems to be a better scaffold for compound ArSH (2) than the hydrophilic compound GSH (1). Here, we ascribe this difference
Modeling GPx Using Selenium-Mediated Micelles
Figure 3. Plots of initial rates (ν0) at different concentrations of ArSH in the presence of a selenium-mediated micellar catalyst (5.0 µM PhSeO2H and 5.52 mM CTAB) in PBS (pH 7.0) and 37 °C. The initial concentration of CUOOH was fixed to 0.25 mM. The concentration of ArSH was 33.10 µM, 63.20 µM, 108.61 µM, 150.00 µM, and 0.20 mM, respectively.
in activity enhancement to the binding environment. The micelle allows both ArSH and CUOOH to easily enter the hydrophobic interior and prompts the reaction rate. To support this assumption, the binding of the micelle and ArSH (2) was first estimated by UV and fluorescence spectra (see Supporting Information), and an apparent red-shift of the maximum absorption wavelength of ArSH (2) at 410 nm was observed in this micelle. As we know, the maximum absorption wavelength of ArSH (2) at 410 nm shifts to a high wavelength when it is in a hydrophobic environment. For the hydrophilic substrate GSH (1), it is only on the surface of the micelle.23 Thus, it is reasonable for us to believe that the hydrophobic interior of the micelle has an important role in catalysis. Moreover, this hydrophobic role was also confirmed by the active difference when various substrates were used. The rate constants of the spontaneous reaction between hydroperoxide and thiol vary in magnitude in the order k(H2O2) > k(CUOOH);6 however, as seen in Table 1, the catalytic activity of the selenium-mediated micellar catalyst has a significant 120fold enhancement in the ArSH assay system when using CUOOH instead of H2O2 as a substrate. To confirm the electrostatic interaction in catalysis, anionic SDS instead of CTAB was employed to construct a new seleniummediated micellar catalyst; however, it does not show any GPx activity in both assay systems. Comparing with the seleniummediated micellar catalyst constructed with CTAB, the reason for this dramatic difference in activity is due to the electrostatic interaction between them. For employing SDS to construct the selenium-mediated micellar catalyst, the electrostatic repulsion prohibits the substrates from easily approaching the active center of the selenium-mediated micellar catalyst, thus resulting in undetectable activity. Moreover, to further confirm this electrostatic interaction, the UV spectra of ArSH in an SDS micelle were also measured, and no red-shift of the maximum absorption wavelength of ArSH (2) at 410 nm indicates that the negative charge surface of the SDS micelle prevents ArSH from entering the interior of the micelle. The micelle without catalytic center selenium acts a good catalyst for catalyzing the reduction of CUOOH by ArSH (2). The second-order rate constant is as high as 129 M-1 s-1, and is about 2 orders of magnitude more efficient than the spontaneous reaction. This is mainly because of the good binding ability of the micelle for ArSH (2) and CUOOH, which allows both substrates to fully take advantage of the hydrophobic microen(23) Lindkvist, B.; Weinander, F.; Engman, L.; Koetse, M.; Engberts, J. B. F. N.; Morgenstern, R. Biochem. J. 1997, 323, 39.
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Figure 4. Plots of the concentration of CTAB in selenium-mediated micellar catalyst vs GPx activity during the catalytic reduction of CUOOH (0.5 mM) by GSH (1 mM) with NADPH (0.25 mM), glutathione reductase (1 unit), and selenium-containing micellar catalyst (benzeneseleninic acid 0.78 µM and CTAB 0.92 mM (1 cmc), 1.84 mM (2 cmc), 2.76 mM (3 cmc), 3.68 mM (4 cmc), 5.52 mM (6 cmc), 7.36 mM (8 cmc), and 9.20 mM (10 cmc), respectively) at pH 7.0 (50 mM PBS, 1 mM EDTA) and 37 °C.
Figure 5. Plots of the concentration of benzeneseleninic acid in selenium-containing micellar catalyst vs activity during the catalytic reduction of CUOOH (0.50 mM) by GSH (1 mM) with NADPH (0.25 mM), glutathione reductase (1 unit) and selenium-containing micellar catalyst (CTAB 5.52 mM (6 cmc) and benzeneseleninic acid 0.078, 0.16, 0.39, 0.72, 1.56, 2.34, 3.12, and 4.16 µM, respectively) at pH 7.0 (50 mM PBS, 1 mM EDTA) and 37 °C.
vironment to react. As a control, when using hydrophilic GSH (1) which would be excluded from the hydrophobic region as an alternative of ArSH (2), only a slight enhancement of the reaction rate for catalyzing CUOOH by GSH (1) was observed. The Optimum Structure of the Micelle on Activity. It is well-known that, for a natural enzyme, a slight change of the structure will result in a dramatic change in activity. Here, for the selenium-mediated micellar catalyst, the concentration of CTAB and benzeneseleninic acid affected the activity significantly. First, with the CTAB concentration going up, the substrates are first increasingly bound to the micelle, which leads to high activity. Then the substrates start diluting in different micelles and, hence, the reactivity goes down. To obtain the optimum concentration of CTAB for this selenium-mediated micellar catalyst, we kept the concentration of benzeneseleninic acid constant and change the concentration of CTAB, and a bellshaped curve was obtained for the concentration of the surfactant against activity (Figure 4); when the concentration of the surfactant was 5.52 mM (6 cmc), the selenium-mediated micellar catalyst reached the highest reaction rate. Considering that the anionic benzeneseleniate competes with the anionic substrate GSH for
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the binding to the micelle, at higher benzeneseleniate loading GSH from the micelle, another bell-shaped curve was obtained by plotting the activity against the concentration of PhSeO2H (Figure 5) at a CTAB concentration of 5.52 mM (6 cmc). Accordingly, from Figures 4 and 5, under the conditions of 5.52 mM (6 cmc) CTAB and 1.5 mM benzeneseleninic acid, the selenium-mediated micellar catalyst demonstrated the highest GPx activity of 125 U µmol-1. In summary, we have designed a self-organized micellar GPx model by means of a supramolecular chemistry strategy. This selenium-mediated micellar catalyst acts as an excellent GPx model. Compared with other GPx models, the selenium-mediated micellar catalyst has its obvious advantages: easier control of essential catalytic factors of GPx in one nanoscaffold, the simple (24) Takebe, G.; Yarimizu, J.; Saito, Y.; Hayashi, T.; Nakamura, H.; Yodoi, J.; Nagasawa, S.; Takahashi, K. J. Biol. Chem. 2002, 227, 43, 41254. (25) Luo, G.; Ren, X.; Liu, J. Q.; Mu, Y.; Shen, J. Curr. Med. Chem. 2003, 10, 1151. (26) Flohe´, L.; Gu¨nzler, W. A.; Jung, G.; Schaich, E.; Schneider, F. HoppeSeyler’s Z. Physiol. Chem. 1971, 352, 159.
Huang et al.
preparation process, and remarkable rate enhancement. To date, GPx mimics that exhibit strong binding for both thiol and peroxide are rarely reported.24-26 Considering the double binding for both CUOOH and thiol substrates, the micelle is a good scaffold for constructing GPx mimics. It is anticipated that this work opens a new field for the design of selenium-based antioxidants and for the mechanistic and practical understanding of natural selenoenzymes. Acknowledgment. We thank the Natural Science Foundation of China (No. 20534030, 20471023), the 111 project (B06009), the Innovative Research Team in University (IRT0422), and the Ministry of Education of China for financial support. Supporting Information Available: Characterization of the selenium-mediated micellar catalyst and details of the complexations of ArSH and CuOOH with the micelle. This material is available free of charge via the Internet at http://pubs.acs.org. LA061727P
