Construction of the Active Site of Glutathione Peroxidase on Polymer

Apr 8, 2008 - A new nanoenzyme model with glutathione peroxidase-like active site was constructed on polystyrene nanoparticle. (PN1) via microemulsion...
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Biomacromolecules 2008, 9, 1467–1473

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Construction of the Active Site of Glutathione Peroxidase on Polymer-Based Nanoparticles Xin Huang, Yang Liu, Kai Liang, Yong Tang, and Junqiu Liu* State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, People’s Republic of China Received December 14, 2007; Revised Manuscript Received February 18, 2008

A new nanoenzyme model with glutathione peroxidase-like active site was constructed on polystyrene nanoparticle (PN1) via microemulsion polymerization. In this model system, two functional monomers were designed: one is a tellurium-containing compound that was introduced on the surface of the nanoparticle and acts as a catalytic center, and the other one is an arginine-containing compound designed as a binding site for the complexation of the carboxyl group of substrate 3-carboxy-4-nitrobenzenethiol (ArSH, 1). As a new glutathione peroxidase (GPx) mimic, it demonstrated excellent catalytic activity and substrate specificity. In ArSH assay system, it was at least 316000-fold more efficient than PhSeSePh for the reduction of cumene hydroperoxide (CUOOH) by ArSH. In contrast to model PN2, which lacks of substrate binding site, PN1 exhibits an obvious enhancement in catalytic activity. To further promote the catalytic efficiency, a substrate ArSH surface-imprinted nanoenzyme model (IPN) was developed. By correctly incorporating and positioning the catalytic center tellurium and functional binding factor guanidinium, a continuative activity enhancement of 596000-fold for the reduction of CUOOH by catalyst I-PN compared with diphenyl diselenide (PhSeSePh) was observed. The results clearly show that polymeric nanoparticle can be developed as an excellent model for combining most of catalytic factors of enzyme into one scaffold.

Introduction Reactive oxygen species (ROS) such as superoxide anion, H2O2, organic peroxide, and hydroxyl radical are generated as byproducts of cellular metabolism and are mainly controlled by antioxidative defense system, especially by antioxidative enzyme system.1,2 Once the overproduct of ROS is present in the human body, it would result in a variety of human disease. Examples of such oxidative stress-related disease include reperfusion injury, inflammatory process, neuronal apoptosis, cancer, and so on. In biological organisms, superoxide dismutase (SOD), catalyst (CAT), glutathione peroxidase (GPx), and other antioxidative enzymes contribute dominatingly to enhance cellular antioxidative defense against oxidative stress. Among them glutathione peroxidase (GPx, EC.1.11.1.9), an important selenium-containing enzyme, functions to protect various living organism from aerobic oxidative stresses by catalyzing the reduction of hydroperoxides (ROOHs), using glutathione (GSH) as a reducing substrate.3 The catalytic moiety of GPx, selenocysteine, is in a depression on the protein’s surface in which some charge and hydrophobic amino acid residues form a hydrophobic cavity for thiol substrate binding. The selenium undergoes a redox cycle involving the selenol as the active form. The selenol is first oxidized to selenenic acid, which reacts with reduced glutathione to form selenenylsulfide. A second glutathione then regenerates the active form of the enzyme by attacking the selenenylsulfide moiety of the enzyme to form the oxidized glutathione (GSSG).3,4 Owing to its biologically crucial role, considerable efforts have been devoted to produce organoselenium/tellurium compounds that mimic the properties of GPx in recent years.5–10 In our group, based on the understanding of GPx structure, some artificial GPx models have * To whom correspondence should be addressed. Fax: +86-43185193421. E-mail: [email protected].

been designed in which a catalytic center, selenium/tellurium, was introduced into existing or artificially generated substrate binding scaffolds by chemical or genetic strategies, for example, 2,2′-ditellurobis(2-deoxy-β-cyclodextrin) (2-Te-CD), tellurosubtilisin, seleno-GST, telluro-micelle, catalytic antibody, and so on.11–14 However, taking a nanoparticle as a scaffold to mimic the properties of this important selenoenzyme has not been explored yet. Recent development in nanotechnology has provided a variety of nanostructure materials with highly controlled and exceptional properties. Among these materials, nanoparticles sized between 1 and 100 nm elicit an intense interest because of their unique optical, electronic, magnetic, catalytic, and other physical properties.15,16 Apart from the properties arising from the core and its nanometer dimensions, the control of surface functionalities of nanoparticles is equally important. Accordingly, tailoring the properties of nanostructure in a very predictable manner can form more complex nanoarchitectures on surfaces and can meet the needs of specific applications, thus, this field attracts scientists a great deal of interest and has been widely used in the design of molecular electronics, biosensors, medical diagnostics, coating, drug delivery, and so on.17–27 Among these particles, the system based on polystyrene attracts people interest for their low cost, availability, and easy preparation. Recently, a number of functional polystyrene latex particles bearing various groups, such as, carboxyl, hydroxyl, thymine, amine, silanol, and so forth, have been reported.28–41 However, employing the functional polymer nanoparticle as an artificial enzyme scaffold is rarely reported. Herein, we chose polystyrene nanoparticle as a scaffold, and a glutathione peroxidase-like active site was designed on the surface of nanoparticles via microemulsion polymerization and further by surface molecularly imprinting strategy. The nanoen-

10.1021/bm701386b CCC: $40.75  2008 American Chemical Society Published on Web 04/08/2008

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Figure 1. SEM images of the (a) I-PN and (b) PN1. Chart 1.

Structure of Designed Monomers

zyme system exhibited significant GPx activity. The nanoparticles and their catalytic behaviors have been characterized in detail.

Experimental Section Sodium borohydride, 3-bromo-1-propanol, acryloyl chloride, and diphenyl diselenide were purchased from Fluka and were used without further purification. Hexadecyltrimethylammonium bromide (CTAB) was purchased from Tianjinfuchen and purified by recrystallizing from methanol and ether. Potassium persulfate (KPS) was purchased from Shanghai Chem. Reagent Co. Styrene (St) was purified upon distillation under reduced pressure and kept refrigerated until use. The characterization of the structure of the mimics were performed with Bruker Advance 500 (500 MHz) 1H NMR spectrometer using a TMS proton signal as the internal standard. UV–vis spectra were obtained using a Shimadzu 2450 UV–vis-NIR spectrophotometer. Scanning electron microscopy (SEM) observations were carried out on a JEOL FESEM 6700F scanning electron microscope with primary electron energy of 3 kV. Synthesis of Bis(3-hydroxypropyl) Telluride (5).13 Water (30 mL) was added to 1.27 g (10.0 mmol) of tellurium powder and 1.89 g (50.0 mmol) of sodium borohydride. The mixture was heated and the tellurium was dissolved under nitrogen until it formed a pale pink solution (ca. 30 min). The solution was cooled to room temperature, and 1.88 mL (20.0 mmol) of 3-bromo-1-propanol was added. The mixture turned yellow within 5 min and was stirred for an additional 30 min. The product was extracted with ether, dried, concentrated in vacuo, and chromatographed (elution with ethyl acetate) to give 0.81 g (33.1%) of 5 as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 3.78 (t, 4 H, -CH2OH), 3.21 (t, 4 H, -TeCH2), 2.02 (m, 4 H, -CH2-), 1.82 (br s, 2 H, -OH). Synthesis of Acryloyloxypropyl 3-Hydroxypropyl Telluride (2). Acryloyl chloride (93.3 µL, 1.0 mmol, Fluka, 97% purity) was dissolved in 2 mL of anhydrous 1,4-dioxane and added dropwise to a stirred solution of bis(3-hydroxypropyl) telluride (5; 245 mg, 1 mmol) and anhydrous triethylamine (138 µL, 1 mmol) in anhydrous 1,4dioxane (20 mL). The mixture was stirred for 2.5 h at 60 °C under a nitrogen atmosphere. After the mixture was cooled, the precipitated triethylamine-hydrochloride was filtered. The solvent in the filtrate was removed by vacuum. The crude product was purified by column chromatography (silica gel, ethyl acetate). Yield: 28.5%. 1H NMR (500 MHz, CDCl3) δ 6.39-5.82 (3 H, CH2dCH), 4.21 (t, 2 H, COOCH2), 3.58 (t, 2 H, -CH2OH), 3.14 (t, 2 H, -TeCH2), 2.14 (m, 2 H, -CH2-), 1.55 (s, 1 H, -OH). Synthesis of Allyl Arginine (3). Arginine (2.0 g, 11.5 mmol) in 10 mL of allyl alcohol was purged with hydrogen chloride at 60 °C for 10 h. The solution was concentrated in vacuo and precipitated with ethyl ether; buff solid was obtained. Yield: 58.5%. 1H NMR (500 MHz, CDCl3) δ 5.85–5.02 (3 H, CH2dCH), 4.06 (d, 2 H, COOCH2), 3.14

(t, 1 H, -CCH-NH2), 1.93–1.60 (m, 4 H, -CH2-CH2-), 8.32 (br s, 2 H, -NH2), 7.09 (s, 1 H, CdNH). Synthesis of Nanoenzyme Model (PN1). Briefly, surfactant CTAB (0.2 g, 0.55 mmol), compound 2 (3.1 mg, 0.01 mmol), compound 3 (40 mg, 0.19 mmol), acrylamide (60 mg, 0.85 mmol), and potassium peroxydisulfate (15 mg, 0.056 mmol) in 10 mL of water in a flask equipped with a stirrer. The mixture was stirred vigorously, while styrene (0.55 g, 5.29 mmol) was added dropwise. Then the mixture was purged with nitrogen for 30 min and heated in an oil bath to 80 °C for 10 h. The products were purified by centrifugation at 12000 rpm to remove the deposit, and the solution was dialyzed against water for three days to remove the other small molecular weight compound. White powder was obtained by lyophilized. Synthesis of Nanoenzyme Model (PN2). The procedure was the same as mentioned above, except the compound 3 was replaced by acrylic acid. Synthesis of Surface-Imprinted Nanoenzyme Model (I-PN). Compound 2 (3.1 mg, 0.01 mmol) and substrate ArSH (2 mg, 0.01 mmol) were added into 10 mL of water in a flask equipped with a stirrer, and the mixture was stirred for 10 min. Then surfactant hexadecyltrimethylammonium bromide (CTAB; 0.2 g, 0.55 mmol), compound 3 (40 mg, 0.19 mmol), acrylamide (60 mg, 0.85 mmol), and potassium peroxydisulfate (15 mg, 0.056mmol) were added into the mixture. Finally, the mixture was stirred vigorously and styrene (0.55 g, 5.29 mmol) was added dropwise. The mixture was purged with nitrogen for 30 min and heated in an oil bath to 80 °C for 10 h. The products were purified by centrifugation at 12000 rpm to remove the deposit, and the solution was dialyzed against water for three days to remove other small molecular weight compounds. Finally, white powder was obtained by lyophilized. Characterization. The morphology of the I-PN and PN1 was well characterized via scanning electron microscopy (SEM; Figure 1), and the average size was about 30 and 40 nm, respectively. Static light scattering experiments were also performed at Malven ZETAS12ERNANOSERIES instrument in water solution. The average diameters of I-PN 47.6 nm and PDI is about 0.27. The functional monomers that were introduced to nanoenzyme models were detected by FTIR spectrum and 1H NMR spectrum. For the FTIR spectrum of I-PN, the styrene C-H stretching peaks at approximately 3000 cm-1 to 3100 cm-1, and the methyl (2975 cm-1, 2800 cm-1) and amide of allyl arginine (3200 cm-1 and 1730 cm-1

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Scheme 1. Polymerization Process of the Surface-Imprinted Nanoenzyme Model (I-PN)

for the CdO) indicate that the functional monomers have been polymerized in nanoparticles. In the 1H NMR spectrum of I-PN, the signal of the double carbon bond disappears, and this indicates no monomers left in I-PN. Furthermore, Optima 3300DV inductively coupled plasma instrument (ICP) was used to check tellurium in polymeric nanoparticles, the obviously characteristic spectra of tellurium at 214.28 nm was observed, indicating that the functional monomer 2 has been linked in the enzyme model. The content of the functional 2 in I-PN (about 0.3% w/w) was also obtained. Elemental analysis for I-PN are found as follows: C, 87.97; H, 7.88; N, 1.97. Furthermore, the content of guanidinium on the surface was estimated according to the method reported by Suh et al.17 by determining the degree of the complexation of guanidinium and ArSH. ArSH was added to the solution of I-PN, then the mixture solution was dialyzed against water for 1 day, and the concentration of ArSH, which did not complex to the nanoparticle, was measured by UV spectra. Analysis of the data, provided that the content of guanidinium on the surface was estimated to be about 1.27% relative to total mass. Thus, the ratio of the functional monomer 3 and 2 in the nanoparticle was about 6:1.

Results and Discussion Design and Preparation of Nanoenzyme Models. The crystal structure of bovine erythrocyte glutathione peroxidase was reported by Epp et al. twenty years ago.4 The active center of glutathione peroxidase is in a flat depression on the molecular surface. The catalytically active selenocysteine residues located at the N-terminal ends of R helices forming βRβ substructures

together with two adjacent parallel β strands. In the vicinity of the reactive group, some aromatic amino acid side chains are located. Functional residues such as Arg-40, Arg-167, and Gln130 take an important role for binding substrate; they could form salt bridges and a hydrogen bond with substrate GSH molecule. Based on the structure of GPx, corresponding monomers (Chart 1) acryloyloxypropyl 3-hydroxypropyl telluride (2) with a similar function of selenocysteine was prepared as a catalytic center, allyl arginine (3), which is responsible for the complexation of carboxyl group of substrate was designed to mimic the binding site of native GPx, and acryl amide was employed to enhance the hydrophilicity of nanoenzymes. Via microemulsion polymerization, the nanoenzyme model (PN1) with well-distributed catalytic center tellurium and functional binding factor guanidinium moiety of arginine on the surface was obtained. As a control, another nanoenzyme model PN2, which had a negative charged surface by using acrylic acid instead of allyl arginine monomer, was also synthesized in the same way. Moreover, to further enhance the catalytic activity, a surfaceimprinted nanoenzyme model (I-PN) was designed, and the polymerization process was depicted in Scheme 1.42–51 First, the formation of the template molecule telluro-sulfide compound was detected by UV spectrum. As shown in Figure 2A, the characteristic absorbance peak of ArSH at 410 nm (curve a) disappears after adding tellurium-containing compound (2), and the new characteristic peak of telluro-sulfide at 330 nm (curve b) evidently

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Figure 2. (A) UV spectra of ArSH in water (curve a), and after adding tellurium-containing compound 2 (curve b). (B) Spectra of ArSH with varying concentration of 3 in water: the concentration of ArSH is 95 µM, and from a to g the concentrations of 3 are 0, 0.82, 8.2, 16.3, 24.5, 48.9, and 73.4 mM, respectively. Scheme 2. Proposed Catalytic Cycle of Diorganotellurides52–54

indicated the formation of the template molecule telluro-sulfide compound. Then, the prepolymerization complex formed between imprinted molecule and functional monomers 3 via noncovalent interactions (self-assemble), this complex was also observed by UV spectrum (Figure 2B). The similar complexation between the guanidinium group and the carboxyl group was also reported by other groups.18 After the microemulsion formed in the presence of CTAB as a surfactant in water, I-PN was obtained via polymerization by using potassium persulfate (KPS) as an initiator. The nanocatalyst was finally purified by centrifugation and dialysis against water for three days. Being different from other imprinting process, in our study, the designed template molecule telluro-sulfide compound was an important intermediate during the catalytic cycle, this intermediate was demonstrated in the reaction mechanism of diorganotellurides, which mimic the properties of GPx reported by Engman and Detty (Scheme 2),52–54 thus, it does not need to remove from the catalyst I-PN. Normally, to construct a GPx mimic by the imprinting method, the process is as following. First a binding site was constructed by imprinting, then the imprinted molecule is removed, and finally the catalytic center is incorporated into the binding site. Being the difference of this process, here the imprinted molecule ArSH makes binding functional groups and the catalytic center locate into proper sites at the same time. Then, after imprinting, the catalytic center need not be incorporated anymore. Furthermore, to investigate the imprinting effects by substrate, another substrate, benzenethiol, which is lacking a carboxyl functional group compared with ArSH, was employed as an alternative of ArSH. After imprinting, the catalytic activity of polymer was very close to that of nonimprinting polymer (PN1) and about 2-fold less than that of I-PN. This is mainly due to when using benzenethiol as a substrate, it can not form the prepolymerization complex; thus, it could not make the catalytic center and binding functional group locate in a proper position and resulted in a relative low activity. Catalytic Behavior. The catalytic activities of these nanoenzyme models were investigated in an ArSH assay system

Figure 3. Plots of absorbance vs time during the catalytic reduction of CUOOH (250 µM) with ArSH (150 µM) at pH 7.0 and 37 °C. (a) No catalyst, (b) nanoenzyme model without catalytic center, and (c) PN1 (3.2 µM catalytic center). Table 1. Initial Rates (ν0) and Activities for the Reduction of CUOOH (250 µM) by Thiol ArSH (150 µM)a v0 (µM min-1)c catalysts PhSeSePhb PN1 PN2 I-PN

thiol

H2O2

CUOOH

activity H2O2 CUOOH

ArSH 0.012 ( 0.001 0.011 ( 0.001 1 1 ArSH 0.84 ( 0.02 24.1 ( 1.8 10000 316000 ArSH 0.02 ( 0.001 3.2 ( 0.2 240 42000 ArSH 1.9 ( 0.1 45.4 ( 4.3 23000 596000

a In the presence of various catalysts at pH 7.0 (50 mM PBS) and 37 °C. b The initial rate of reaction was corrected for the spontaneous oxidation. c The concentration of catalyst: PhSeSePh (462 µM), I-PN, PN1, and PN2 (3.2 µM catalytic center) in the assay systems and assuming one molecule catalytic center (tellurium monomer) as one active site of enzyme.

according to a modified method reported by Bill and Hilvert et al.55–57 using ArSH (1) as a GSH alternative. The assay mixture contained 50 mM phosphate buffer, pH 7.0, 150 µM TNB, 250 µM ROOH, and a moderate amount of test compound at 37 °C. The activities were given (vide infra) assuming one molecule catalytic center (Te-monomer) in the nanoenzyme models as one active site of enzyme, and the content of tellurium in the models were determined by ICP. Reaction was initiated by the subsequent addition of ROOH and the absorbance at 410 nm (ε ) 13600 M-1 cm-1, pH 7.0) was recorded for a few minutes to calculate the reaction rate (Figure 3). The relative activities were summarized in Table 1. The initial rate of the background (nonenzymic) reaction between cumene hydroperoxide (CUOOH) and ArSH is very slow (ν0 ) 0.49 µM min-1). A slight enhancement in the rate is observed (ν0 ) 0.012 µM min-1) when PhSeSePh (462 × 10-6 M) is added. Under the identical conditions, the PN1 exhibits a remarkable rate enhancement

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Figure 4. Plots of initial rates at different concentrations of CUOOH in the presence of the I-PN. The initial concentration of ArSH was fixed to 0.15 mM. The concentration of CUOOH was 0.05, 0.10, 0.25, 0.38, 0.50, 0.65, and 0.70 mM, respectively.

Figure 5. Spectra of ArSH with varying PN1 concentration, keeping the concentration of ArSH 78 µM; from a to d, the concentration of PN1 increases step by step.

(ν0 ) 24.1 µM min-1; Figure 3). Assuming that the rate has a first-order dependence on the concentration of catalysts, this data suggests that the activity of PN1 is at least 316000-fold more efficient than that of PhSeSePh. After imprinting, the catalytic reaction rate of I-PN was observed to further increase, about 596000-fold more efficient than that of PhSeSePh and 1.8-fold more efficient than that of PN1. Moreover, saturation kinetics of I-PN catalysis for the peroxidase reaction was studied at the individual concentrations of ArSH and CUOOH, which indicates that this model shows typical saturation kinetics and exhibits as a real catalyst for peroxidase reaction (Figure 4). In the ArSH assay system, in the presence of I-PN (3.2 µM catalytic center) at pH 7.0 and 37 °C, the apparent kinetic parameters of I-PN catalysis were obtained V(max) ) 163.9 µM min-1, k(app)cat ) 51.2 min-1, KmCUOOH ) 545.9 µM, k(app)cat/ KmCUOOH ) 9.4 × 104 M-1 min-1 (the initial concentration of ArSH fixed to 0.15 mM), and the turnover number per catalytic center tellurium was calculated to be 52 min-1. The binding process is of importance in enhancing catalytic activity of enzyme mimics.58,59 For enzymes, to complete a catalytic cycle, they first recognize and bind their substrates to set up the correct geometry, then stabilize the transition state for a particular reaction. In this study, for these nanoenzyme models, their high catalytic activities also ascribe to their similarity between the native GPx and designed nanoenzyme model. This similarity between two systems can be found by comparison of the structures of the native GPx catalytic center and nanoenzyme model: first, to imitate the catalytic center selenocysteine in native GPx, a tellurium-containing compound (2) was introduced into the nanoparticles as a catalytic group; second, to mimic the binding site in which two arginines (Arg40, Arg167) form a saltbridge with GSH, a monomer with guanidinium group was designed for binding the carboxyl group of substrate ArSH. Finally, being similar to the hydrophobic cavity in the native GPx, which is formed by hydrophobic amino acid residues (Phe, Trp, Asp), there was a hydrophobic framework formed by polystyrene in nanoparticles. Thus, the high catalytic efficiency of designed nanoenzyme model should be attributed to both the orientation of the catalytic tellurium and the substrate binding ability derived from the positive charged interaction and the hydrophobic interaction. For confirming the hydrophobic binding of nanoparticles to substrate ArSH, we checked the interaction between them by UV spectrum. As we have known, ArSH has a maximum absorbance wavelength at 410 nm in aqueous solution (pH 7.0), but its maximum absorbance wavelength will appear as a red shift if it is in a hydrophobic environment. In the UV spectrum test, the concentration of ArSH was kept constant in PBS solution (pH 7.0), and when adding

various amounts of PN1, there presented an apparent red shift of maximum absorbance wavelength (Figure 5). This indicated that a strong hydrophobic interaction existed between the aryl moiety of ArSH and the polystyrene framework of PN1. Furthermore, as seen from Table 1, the dramatic increase in the catalytic activity when using hydrophobic substrate CUOOH as an alternative substrate instead of hydrogen peroxide (H2O2) and keeping all other conditions constant also confirmed this hydrophobic interaction. Considering that the hydrophobic framework of polystyrene has a strong interaction with hydrophobic substrate CUOOH, and this interaction becomes weaker when using H2O2 as a substrate, the nanoenzyme shows high catalytic activity toward substrate CUOOH. On the other hand, to study the positive charged interaction in catalysis, a negative charged nanoenzyme model (PN2) was constructed by employing acrylic acid instead of allyl arginine ester as a monomer. In contrast to positive charged PN1, the PN2 showed a large decrease in catalytic activity. It was not surprising, because each of the other repulsions between the negative charged PN2 and the negative charged carboxyl moiety of ArSH makes the substrate ArSH away from the catalytic center, resulting in low activity. To improve the enzyme model further, molecularly imprinting technology was used to generate substrate binding sites. As seen from Table 1, 596000-fold more efficient by I-PN catalysis than PhSeSePh was observed, the imprinting led to stronger binding and better match between the binding site and the catalytic center and endowed the nanoenzyme higher catalytic activity. Optimizing the Structure of Nanoenzyme Models. It was well-known that, for a native enzyme, the slight change of the structure would result in dramatic change in activity. For these nanoenzyme models, it was no doubt that the better match between the catalytic center and the binding site would result in the higher catalytic activity. Here, to get the optimum structure of these nanoenzyme models for high activity, a serial of I-PN and PN1 via altering the molar ratio of compound 2 to compound 3 were synthesized. A plot of catalytic reaction rates ν0 against molar ratio of compound 2 to compound 3 in I-PN and PN1 was given in Figure 6. For I-PN, when the ratio was 10:1, it reached the highest catalytic reaction rate (about 46 µM min-1), and for PN1, when the ratio was 6:1, it reached the highest catalytic reaction rate (about 36 µM min-1). It was not surprising that both I-PN and PN1 give the similar bell shape curves. Because at the prime stage, with the ratio of compound 2 in the nanoparticles going up, the binding ability increases accordingly, this certainly led to the increase of the catalytic activity. However, the stronger binding does not imply the higher catalytic activity, so it is for both I-PN and PN1, when the content of compound 2 increases further, the activity went down

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Figure 6. Plots of catalytic reaction rates v0 against the molar ratio of compound 2 to compound 3 in I-PN and PN1, respectively.

(15) (16) (17)

despite the binding ability is enhanced. Because some of compound 2 may result in a distribution away from the catalytic center, the substrates that bind to nanoparticles could not efficiently take part in the catalytic cycle, accordingly resulted in the decrease of the activity. In addition, by the comparison of the curves of the I-PN and PN1, all the imprinted nanoenzyme models I-PN synthesized at various ratios of compound 2 to compound 3 (shown in Figure 6) demonstrated higher catalytic activity than that of corresponding none imprinted nanoenzyme models PN1; the higher catalytic activity was mainly due to the better match between the catalytic center and the substrate and the better binding endowed by imprinting.

Conclusions A serial of nanoenzyme models with glutathione peroxidaselike active site were synthesized to mimic the properties of GPx. The major catalytic factors, including the substrate recognition and the orientation of catalytic tellurium, were constructed onto polystyrene nanoparticle (PN1) for the first time. For designing a desirable GPx mimic, the specific substrate binding is no doubt very important. However, how to adjust the positions of the catalytic center and the binding sites, which makes the substrate properly oriented to the active center, is also another important factor for obtaining high GPx activity. As new GPx mimics, considering their very high catalytic activity, good water solubility, and substrate specificity, these nanoenzymes may have potential application in medicine. We anticipated that the method for taking a nanoparticle as a scaffold and combining most of the catalytic factors of enzyme into one nanoparticle would open a new field in designing enzyme model and other biologically related functional nanoparticles. Acknowledgment. We are grateful for the financial support from the Natural Science Foundation of China (No. 20534030, 20725415), the National Basic Research Program (2007CB808006), and the Innovative Research Team in University (IRT0422).

References and Notes (1) Sies, H. Oxidative Stress: Introductory Remarks. In OxidatiVe Stress; Sies, H., Ed.; Academic Press: London, 1985, p 1. (2) Sies, H. Biochemistry of oxidative stress. Angew. Chem., Int. Ed. Engl. 1986, 25, 1058–1071. (3) Flohé, L.; Loschen, G.; Günzler, W. A.; Eichele, E. Hoppe-Seyler’s Z. Physiol. Chem. 1972, 353, 987–999. (4) Epp, O.; Ladenstein, R.; Wendel, A. Eur. J. Biochem. 1983, 133, 51– 69. (5) Mugesh, G.; du Mont, W. W.; Sies, H. Chem. ReV. 2001, 101, 2125– 2179.

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