Proton Gradients Produced by Glucose Oxidase Microcapsules

Dec 17, 2008 - biological and man-made components, it is possible to engineer ... In comparison to the previously reported system,19,20 the wall of GA...
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2009, 113, 395–399 Published on Web 12/17/2008

Proton Gradients Produced by Glucose Oxidase Microcapsules Containing Motor F0F1-ATPase for Continuous ATP Biosynthesis Li Duan,† Wei Qi,† Xuehai Yan,† Qiang He,†,‡ Yue Cui,† Kewei Wang,† Dongxiang Li,† and Junbai Li*,† Beijing National Laboratory for Molecular Science, International Joint Laboratory, CAS Key Laboratory of Colloid and Interface Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China, and Max Planck Institute of Colloids and Interfaces, D-14476 Golm/Potsdam, Germany ReceiVed: September 4, 2008; ReVised Manuscript ReceiVed: December 1, 2008

Glucose oxidase (GOD) microcapsules held together by cross-linker, glutaraldehyde (GA), are fabricated by the layer-by-layer (LbL) assembly technique. The lipid bilayer containing CF0F1-ATPase was coated on the outer shell of GOD microcapsules. Driven under the proton gradients produced by catalysis of GOD microcapsules for glucose, ATP is synthesized from ADP and inorganic phosphate catalyzed by the ATPase rotary catalysis. The results show here that ATPase reconstituted on the GOD microcapsules retains its catalytic activity. Introduction ATP synthase is a rotary machine using physical rotation of its own subunits as a step of catalysis, which is different from that of any other known enzyme.1 ATPase is composed of two motors, the membrane-bound F0 part responsible for proton translocation and the hydrophilic F1 part responsible for ATP synthesis and hydrolysis. H+-ATPase can use proton motive force to synthesize ATP from ADP and Pi by taking up protons from one side of the membrane and transpoting them to the other.2 In the past decade, the ever increasing information on the structure and function of F0F1-ATPase is providing insight into imitating life on the molecular level.3-6 By integrating biological and man-made components, it is possible to engineer life processes into micro- and nanoscale artificial devices.7 Lipid membranes have been widely used as models for biological membranes, and ATP synthase is particularly selected as a model membrane protein, since it is a major ATP supplier in the cell. It has been reported that ATPase can be successfully reconstituted into liposomes via detergent mediation.8 However, the chemical and mechanical weakness of liposomes is intrinsically limiting their many practical applications. We introduced microcapsules fabricated by the layer-by-layer (LbL) assembly technique into the biomimetic system design.9-11 The layer-by-layer (LbL) technique has been established as an effective approach to fabricate multilayer structure since it was introduced by Decher et al.12 In recent decades, the microand nanosized hollow capsules fabricated by the LbL technique have been employed in diverse application areas such as medicine, drug delivery, and catalysis due to the interior space enabled to encapsulate various materials.13-18 Furthermore, the LbL assembly strategy has control over the size, shape, composition, wall thickness, and permeability of the hollow capsules. As a biomimetic system design, we have reported * To whom correspondence should be addressed. E-mail: [email protected]. † Chinese Academy of Sciences. ‡ Max Planck Institute of Colloids and Interfaces.

10.1021/jp807883e CCC: $40.75

recently the deposition of lipid bilayers containing membranebound F0F1-ATPase onto polyelectrolyte capsules and protein capsules. We found out that the enzyme continues to function in vitro and ATP was synthesized and stored inside the capsules.19,20 Here we constructed a novel microcapsule of glucose oxidase (GOD) by covalent LbL assembly with the cross-linker glutaraldehyde (GA) and GOD as a major component was assembled in the capsule shells. We have proven that the activity of GOD was retained during the fabrication of capsules. The lipid layer containing CF0F1-ATPase was coated on the outer shell of GOD microcapsules. Glucose was added into the suspension of capsules, and protons can be generated from the oxidation and hydrolysis of glucose catalyzed by GOD. Such proton gradients formed between the exterior and interior of the microcapsules can provide driving forces for ATPase rotary catalysis, and ATP is synthesized in the microcapsule solution. In comparison to the previously reported system,19,20 the wall of GA/GOD capsules can generate protons only by adding glucose solution, which is convenient and quick for ATP synthesis. Scheme 1 shows schematically a lipid-modified GOD capsule with incorporation of CF0F1-ATPase and the catalysis process of GOD for glucose. Methods Materials. Glucose oxidase from Aspergilus niger (GOD, lyophilized powder protein, Mw ∼186 000, foreign activities: catalase e4%), peroxidase from horse radish, and 3,3dimethoxybenzidine o-dianizidine were purchased from Fluka. Phosphatidyl choline from egg yolk, phosphatidic acid from egg yolk, 8-hydroxyprene-1,3,6-trisulphonic acid (pyranine), phosphate buffered saline (PBS, pH 7.2), and manganese sulfate (MnSO4) were purchased from Sigma-Aldrich. The fluorescent probe 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) was purchased from Avanti in powder form and dissolved in chloroform  2009 American Chemical Society

396 J. Phys. Chem. B, Vol. 113, No. 2, 2009 SCHEME 1: Schematic Representation of the Arrangement of CF0F1-ATPase in Lipid-Coated GOD capsulesa

a A lipid bilayer containing CF0F1-ATPase was coated onto the outer shell of the GOD microcapsule.

before use. CF0F1-ATPase was isolated and purified from spinach chloroplasts according to procedures already described by Gra¨ber et al.21 Spherical manganese carbonate (MnCO3) particles with an average diameter of 6 µm were synthesized by mixing MnSO4 and NH4HCO3 solutions to serve as template cores. The water used in all experiments was prepared in a threestage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18.2 MΩ · cm. Capsule Preparation. MnCO3 microparticles were prepared as a template for the fabrication of LbL microcapsules. First, poly(ethylenimine) (PEI, Mw 50-100 kDa, 1 mg/mL in 0.5 M NaCl) was mixed with MnCO3 particles and the adsorption time was 20 min. After adsorption, the centrifugation technique was employed and the coated particles were washed three times by 0.5 M NaCl. Then, the particles coated with PEI were alternately dispersed into the GA (0.025%) and GOD (4 mg/mL) in pH 7.2 phosphate buffer solution (PBS) for 12 h, respectively. Thus, GA and GOD were alternately adsorbed. After the assembly of a desired number of GA/GOD layers, the coated particles were incubated in 0.1 M Na2EDTA solution (pH 7.2) to dissolve the MnCO3 templates. The hollow GA/GOD capsules obtained were stored at 4 °C. Reconstitution Procedure. We prepared liposomes by using a mixture of phosphatidyl choline and phosphatidic acid from egg yolk (9:1 by mass). The detergent-mediated procedure was used to reconstitute CF0F1 into preformed liposomes. The purified CF0F1 preparation was solubilized in the presence of Triton X-100, and the liposome solution was added. Then, the mixture was stirred gently at 4 °C for 1 h, followed by the removal of Triton X-100 with Biobeads SM-2 (Bio-Rad) as reported previously.4,19 The final concentrations of CF0F1 and lipid were 200 nM and 5 mg/mL, respectively. The bulk aqueous phase containing 20 mM tricine, 40 mM NaCl, and 5 mM MgCl2 was adjusted to pH 8.0 with 1 mM NaOH solution. Proteoliposomes containing CF0F1-ATPase were mixed with a GOD protein microcapsule dispersion and incubated for 30 min, then centrifuged at 4 °C, and then washed three times with the reconstitution buffer. Transmission Electron Microscopy (TEM). TEM measurements were performed on a Tecnai 20 microscope (Philips FEI, American) operated at 120 kV. TEM samples were prepared by dropping 3 µL of a diluted capsule suspension onto carboncoated copper grids. Sample grids were stained with uranyl acetate (UAc). Confocal Laser Scanning Microscopy (CLSM). CLSM images were taken with a Zeiss LSM 510 META confocal system (Zeiss, Germany), equipped with a 100× oil immersion

Letters objective with a numerical aperture of 1.4. 5% Texas red-labeled DHPE (w/w) was used as a fluorescent label for lipid layer visualization. Enzymatic Activity Assays. To measure the activity of the GOD in GA/GOD microcapsules, 2.5 mL of o-dianizidine buffer (pH 5.8) solution, 0.3 mL of 18% β-D-glucose solution, and 0.1 mL of 0.02% peroxidase from horse radish were mixed in a cuvette. The reaction began immediately after 0.01 mL of GA/GOD microcapsules were added, and light absorption was measured at 460 nm for 10 s with an UV-visible system (U3010 spectrophotometer, HITACHI, Japan).The catalytic activity of GOD microcapsules in different pH buffer solutions and at different temperature environments was particularly measured. For temperature experiments, the mixture must be incubated for 10 min at a certain temperature before reacting with GOD capsule solutions. The production of protons was also detected by introducing 8-hydroxyprene-1,3,6-trisulphonic acid (pyranine, in tricine buffer) with an F-4500 fluorescence spectrophotometer (HITACHI, Japan). 1 mM pyranine in tricine buffer (pH 8.0) was mixed with a suspension of capsules with stirring for several minutes followed by centrifuging and washing. Then, pyranine was encapsulated inside the capsules.The excitation spectrum of pyranine luminescence at different reaction times was scanned from 390 to 490 nm at an emission wavelength of 513 nm. Electrochemistry Activity Assay. Glassy carbon (GC, 3 mm diameter) purchased from Bioanalytical Systems Inc. (BAS, West Lafayette, IN) was polished first with emery paper and then with aqueous slurries of alumina fine powders (particle size between 1 and 0.06 µm) on a polishing microcloth. The electrodes were finally rinsed with doubly distilled water in an ultrasonic bath for 10 min. 8 µL of (GA/GOD)5 capsule suspension was dipped onto the glass carbon electrode and then dried at room temperature. Potential-controlled amperometric measurements (I-t curve) were conducted with a conventional two-compartment, three-electrode cell with a CHI660A electrochemical analyzer (CHI Inc.). The GA/GOD-capsule-modified GC electrodes were used as the working electrode, a platinum spiral wire as the counter electrode, and a Ag/AgCl electrode (saturated with KCl) as the reference electrode. The electrodes were polarized at -0.35 V. The solution was stirred with a magnetic stirrer at a rate of 800 rpm during the measurements. CF0F1-ATPase Activity Measurement. The CF0F1/lipidmodified GOD microcapsule solution was first dialyzed with measurement buffer (10 mM tricine, 30 mM NaCl, 2.5 mM MgCl2, 5 mM NaH2PO4, 0.2 mM ADP, pH 8.0). Then, the reaction was started by addition of glucose solution (the concentration is about 10%). The generation of protons during GOD catalysis was monitored using an 8-hydroxyprene-1,3,6trisulphonic acid (pyranine) encapsulated inside the microcapsule. The excitation spectrum of the luminescence of pyranine at different reaction times was scanned from 390 to 490 nm at an emission wavelength of 513 nm with an F-4500 fluorescence spectrophotometer. At various time points, aliquots of the reaction mixture were removed and vortexed with 0.4% trichloroacetic acid and analyzed for their ATP content. ATP synthesis wasmeasuredviaabioluminescenceassaykit(luciferin-luciferase) in a BPCL ultraweak chemiluminescence analyzer connected to a chart recorder. ATP production was quantified by fitting the results to a standard curve of light intensity versus ATP amount. Results In the present work, manganese carbonate (MnCO3) particles were prepared as template cores for the GOD microcapsule

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SCHEME 2: (a) Schematic Representation of the Assembled Glucose Oxidase Protein Microcapsules via Covalent Layer-by-Layer Assembly;a (b) The Biocatalytic Oxidation Process of β-D-Glucose by Glucose Oxidaseb

Figure 2. (a) Thermostability of cross-linked GOD in microcapsules. (b) Relative activity of cross-linked GOD versus pH. (c) Typical amperometric I-t responses of (GOD/GA)5 capsules/GC electrodes toward the successive addition of H2O2 with a concentration of 2, 4, 6, 10, 15, 20, and 20 µM, respectively.

a

GA and GOD were alternately used on the surface of the colloid particles followed by core removal. b GOD can catalyze the oxidation of β-D-glucose to D-glucono-1,5-lactone and the concomitant production of proton and hydrogen peroxide.

Figure 1. (a) TEM image of (GA/GOD)5 microcapsules. (b) The change of the fluorescence excitation peaks of pyranine at 404 nm (b) and 460 nm (9) as a function of time in the solution of glucose catalyzed by GA/GOD microcapsules at an emission wavelength of 513 nm.

fabrication.22 Scheme 2a shows a schematic representation of the formation process of a covalently cross-linked glucose oxidase (GOD) protein microcapsule. GOD was held together by the well-known Schiff base reaction between the amine groups of GOD molecules and aldehyde groups of glutaraldehyde (GA). After the assembly of a desired number of GA/ GOD layers, the template particles were removed. The morphology image of (GA/GOD)5 microcapsules in Figure 1a shows that hollow capsules were obtained. The wall thickness of the capsules can be controlled by the number of alternating GA/ GOD adsorption cycles. As is well-known, GOD can catalyze the oxidation of β-Dglucose to D-glucono-1,5-lactone and the concomitant reduction of molecular oxygen to hydrogen peroxide. The gluconolactone can be further hydrolyzed to gluconate, releasing H+. The biocatalytic oxidation process is described schematically in Scheme 2b. The released proton from the oxidation and hydrolysis of glucose can be detected by a pH sensitive fluorescent probe, 8-hydroxyprene-1,3,6-trisulphonic acid (py-

ranine). Pyranine was introduced into the GOD microcapsule suspension to monitor the changes of pH inside the capsules, since its excitation peak at 460 nm decreases and that at 404 nm increases as a function of pH with emission at 513 nm. In our work, a GOD protein microcapsule suspension and pyranine were first mixed, and then, β-D-glucose solution was added. It was immediately followed by continuously scanning the fluorescence excitation spectra of pyranine at an emission wavelength of 513 nm. Figure 1b shows the fluorescence intensity at 460 and 404 nm. It shows that the light intensity at 460 nm decreases with time and that at 404 nm it increases, indicating that H+ is continuously produced in the GOD microcapsule suspension. It demonstrates that GOD cross-linked by GA on MnCO3 particles keeps its catalytic activity. Additionally, according to the previous report, the quantative exploration of enzymatic activity was performed on the cross-linked GOD by GA and the free GOD, whose results showed that a larger fraction of the GOD making up the wall is available for enzymatic reaction.23 The stability of GOD cross-linked by GA on the MnCO3 particles with respect to temperature and pH is very important for its practical application, such as in biomedicine, considering the moderate GOD stability. Thus, in the current work, a known amount of GA/GOD-coated particles was exposed to different temperature environments for 10 min, and then, the catalytic activity was measured according to the previously reported method.24 Different numbers of GA/GOD-bilayer-coated particles, from 3 to 12 bilayers, were studied. The results (Figure 2a) indicate that GOD covalently cross-linked on MnCO3 particles shows good catalytic activity within a wide temperature range. These data are comparable with those observed for GOD in PEI/GOD layers immobilized on PS particles,25 also showing differences in some aspects, such as the temperature of maximum activity. The catalytic activity of GOD covalently cross-linked on particles shows a variation of activity with pH which is nearly identical for free GOD and for immobilization in layers of different thicknesses (Figure 2b). This proves GOD covalently cross-linked on MnCO3 particles still is thermostable within a wide temperature range and remains active until 60 °C. Also, GOD cross-linked by GA could hold the enzymatic activity from pH 4.0 to pH 8.0. Additionally, the increase in activity with layer number is not obvious, which means there

398 J. Phys. Chem. B, Vol. 113, No. 2, 2009

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Figure 3. CLSM image of (GA/GOD)5/lipid microcapsules with incorporation of CF0F1-ATPase.

is little effect of the number of multilayers on the enzymatic activity. Thus, in the following part, (GA/GOD)5 microcapsules were selected as the experiment system. To explore whether GOD keeps its electroactivity after assembly, potential-controlled amperometric measurements were performed. Figure 2c shows a typical amperometric response toward successive additions of H2O2, proving that GOD capsules cross-linked by GA partly keep their electroactivity. As an initial test to verify the utility of GOD microcapsules for ATP biosynthesis, the CF0F1-proteoliposomes were mixed with a GOD microcapsule suspension. The CF0F1-proteoliposomes were fabricated by reconstituting CF0F1-ATPase into liposomes based on the previously reported method.8,26 In brief, the liposomes were added into Triton X-100-solubilized CF0F1ATPase buffer solution, and then, the Triton X-100 was slowly removed with Biobeads SM-2. To prove the successful adsorption of lipids onto the capsule shells, confocal scanning fluorescence microscopy (CLSM) was applied with Texas red-labeled DHPE as the fluorescent probe. The image presented in Figure 3 shows the continuous red fluorescence over the entire surface of the capsules, which indicates the deposition of lipids onto the surface of the capsules. Thus, CF0F1-ATPase was reconstituted in the outer shell of the GOD microcapsules containing a lipid bilayer. For detecting the proton concentration changes and ATP synthesis during the catalysis of GOD, the following experiments were performed. Before the reaction, the suspensions were dialyzed with buffer solution (pH 8.0). After a few minutes of mixing of pyranine with the suspension, CF0F1-proteoliposomes were added and allowed to adsorb for 30 min, which was then followed by three times centrifugation and washing with measurement buffer solution. Thus, pyranine was encapsulated in the GOD microcapsule interior. Upon adding glucose solution, the excitation spectrum was scanned at different time intervals at an emission wavelength of 513 nm. In the excitation spectrum, the conversion from F460/F404 to pH can be performed according to the third-order equation as described in the literature,27 pH ) a + bx + c/x + dx2 + e/x2 + fx3 + g/x3, where x represents the fluorescence ratio (F460/ F404). Calculated values for the constants are the following: a, 6.13; b, 2.69; c, -0.108; d, -1.65; e, 0.013; f, 0.39; g, -0.000 82. Thus, via F460/F404, the pH can be calculated. The change of pH with time is shown in Figure 4a. The results demonstrate that protons are continuously produced in the interior of the GOD microcapsules. The pH change within 100 min is depicted in the inset image. It indicates that the glucose has penetrated into the capsules. However, in the real living system, it is known that glucose needs a sodium-glucose pump to bring the glucose into the cell. In the current experiments, it is believed that the lipid bilayer coating on the capsule surface

Figure 4. (a) pH change with time in the lipid-modified GOD microcapsule interior. The inset image shows the pH change within 100 min. (b) ATP biosynthesis as a function of reaction time in CF0F1/ lipid-modified GOD microcapsule solutions: exterior solutions (b), interior and exterior solution (O) after addition of 0.1% Triton X-100. The ATP synthesis within 50 min is particularly displayed in the inset image. Each error represents the mean of at least two experiments ((SD).

is not very highly condensed/packed along the capsule surface;9 thus, the possible pores or defects on the surface will allow the penetration of glucose from the outside of the capsule wall to realize the hydrolysis reaction. At the very short moment of the proton produced at the wall via the hydrolysis of glucose reaction catalyzed by GOD will create a proton gradient between the outside and the inside the walls, where CFoF1 ATPase is running and thus ATP synthesis from ADP and inorganic phosphate was started by adding glucose solution. The synthesis of ATP was measured by means of a luciferin-luciferase assay. As the luminescence intensity of luciferin is proportional to the amount of ATP, an ATP standard curve was plotted with the logarithm of luminescence intensity versus the logarithm of ATP concentration on the basis of the luminescence intensities from standard samples of known ATP concentration. To analyze the ATP concentration, 5 µL of the reaction solution was added into a luminometer cuvette and then mixed with 50 µL of luciferin-luciferase reagent, immediately followed by registering the luminescence on a recorder. ATP production within a 30 h reaction time was measured, as shown in Figure 4b. Additionally, we destroyed the lipid bilayer on the outer shell of the capsules by adding Triton X-100 (0.1%) to a capsule suspension to release ATP encapsulated in the interior of the capsules. As a result, the ATP production in the capsule exterior and interior at different reaction times is given in Figure 4b. The inset image concentrates on the change of ATP production

Letters within 50 min. It reveals that ATP production continuously increases with time and the ATP concentration increases after addition of Triton X-100. It can be concluded that, due to the catalysis by GOD, a continuous proton-potential difference between the exterior and interior of the microcapsules drives ATP synthesis. It is very interesting that ATP can be partly synthesized in the interior solution. It may be attributed to part of the F1 subunit extending into the capsules’ interior aqueous phase. Conclusion We report here the development of a new type of biomimetic system, in which F0F1-ATPase was assembled in glucose oxidase (GOD)-dominated microcapsules. The wall of the capsules may provide proton gradients by hydrolyzing glucose by GOD to drive the motor protein to produce ATP. A continuous protondriven force for ATP synthase rotary catalysis can be easily obtained. ATP was synthesized from ADP and inorganic phosphate driven by the ATPase rotary catalysis and stored inside the capsules. The most pronounced feature of this work is that the assembled microcapsules containing F0F1-ATPase serve as a power supply and energy storage container. It will greatly help to study the function of ATPase in biology and fabricate novel nanodevices. Acknowledgment. This work was financially supported by the National Nature Science Foundation of China (Project No. 20520130213), National Basic Research Program of China (973 program) 2007CB935900, and the German Max Planck Society collaborated project. Q.H. is grateful to the Alexander von Humboldt foundation for a research fellowship. References and Notes (1) Yoshida, M.; Muneyuki, E.; Hisabori, T. Nat. ReV. Mol. Cell Biol. 2001, 2, 669.

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