Engineered Superoxide Dismutase Monomers for Superoxide

Anal. Chem. 2006, 78, 928-935

Engineered Superoxide Dismutase Monomers for Superoxide Biosensor Applications Moritz K. Beissenhirtz,† Frieder W. Scheller,† Maria S. Viezzoli,‡ and Fred Lisdat*,§

Analytical Biochemistry, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, H. 25, 14476 Golm, Germany, Department of Chemistry and Centro Risonanze Magnetiche, University of Florence, Via Sacconi, 6, 50019 Sesto Fiorentino (FI), Italy, and Biosystems Technology, Wildau University of Applied Science, Bahnhofstrasse 1, 15745 Wildau, Germany

Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasireversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by ∼1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold. In recent years, the study of oxygen radicals has attracted considerable attention due to their harmful interaction with biological molecules and their involvement in signaling pathways. The superoxide radical O2- has been shown to damage proteins,1 DNA,2 and lipid structures3 in the human body. It is constantly produced as a byproduct of enzymatic reactions4,5 and leads to a * Corresponding author. E-mail: [email protected]. Fax: (+49)3319775053. † University of Potsdam. ‡ University of Florence. § Wildau University of Applied Science. (1) Sohal, R. S.; Sohal, B. H.; Orr, W. C. Free Radical Biol. Med. 1995, 19, 499-504. (2) Misiaszek, R.; Crean, C.; Joffe, A.; Geacintov, N. E.; Shafirovich, V. J. Biol. Chem. 2004, 279, 32106-32115. (3) Woods, J. R. Placenta 2001, 22, S38-S44. (4) Brand, M. D.; Affourtit, C.; Esteves, T. C.; Green, K.; Lambert, A. J.; Miwa, S.; Pakay, J. L.; Parker, N. Free Radical Biol. Med. 2004, 37, 755-767.

928 Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

cascade of toxic oxidative species. It plays a role in some important pathologies such as heart disease,6 cancer,7 and neuronal deterioration.8,9 Therefore, the quantitative determination of superoxide radical concentrations and the beneficial effects of antioxidant compounds is of great interest to the medical community. A major challenge of radical analysis is the short half-life of superoxide (in the ms to s range) due to its spontaneous dismutation to oxygen and hydrogen peroxide.10 Chromatography, EPR spin trapping,11,12 and several dyes13,14 have been used for the quantification of the radical, along with indirect approaches such as the study of radicalmediated damage of biomolecules.15,16 Protein electrochemistry has been shown to allow real-time, on-line quantification of radical concentrations. Mostly, the reduction of cytochrome c (cyt. c) by the superoxide radical and its subsequent reoxidation by an electrode has been used for this purpose.17,18 Such sensors have been applied for in vivo measurements19,20 as well as for the study of antioxidants in vitro.21 However, cyt. c is not a natural reaction partner of superoxide and thus has only a medium reaction rate with the radical. In addition, it was found that cyt. c can show a pseudoperoxidase activity,22 which may falsify the biosensor’s signal if not carefully controlled. (5) Fridovich, I. Science 1978, 201, 875-880. (6) Mak, S.; Newton, G. E. Chest 2001, 120, 2035-2046. (7) Kovacic, P.; Jacintho, J. D. Curr. Med. Chem. 2001, 8, 773-796. (8) Leonard, B. E. Int. J. Dev. Neurosci. 2001, 19, 305-312. (9) Liang, L. P.; Patel, M. J. Neurochem. 2004, 90, 1076-1084. (10) Fridovich, I. Acc. Chem. Res. 1972, 5, 321. (11) Inanami, O.; Yamamori, T.; Takahashi, T. A.; Nagahata, H.; Kuwabara, M. Free Radical Res. 2001, 34, 81-92. (12) Chen, R.; Warden, J. T.; Stenken, J. A. Anal. Chem. 2004, 76, 4734-4740. (13) Frahry, G.; Schopfer, P. Planta 2001, 212, 175-183. (14) Daiber, A.; Oelze, M.; August, M.; Wendt, M.; Sydow, K.; Wieboldt, H.; Kleschyov, A. L.; Munzel, T. Free Radical Res. 2004, 38, 259-269. (15) Salles, B.; Sattler, U.; Bozzato, C.; Calsou, P. Food Chem. Toxicol. 1999, 37, 1009-1014. (16) Kalka, K.; Mukhtar, H.; Turowski-Wanke, A.; Merk, H. Skin Pharmacol. Appl. Skin Physiol. 2000, 13, 143-149. (17) Lisdat, F.; Ge, B.; Ehrentreich-Forster, E.; Reszka, R.; Scheller, F. W. Anal. Chem. 1999, 71, 1359-1365. (18) Cooper, J. M.; Greenough, K. R.; McNeil, C. J. J. Electroanal. Chem. 1993, 347, 267-275. (19) Buttemeyer, R.; Philipp, A. W.; Schlenzka, L.; Mall, J. W.; Beissenhirtz, M.; Lisdat, F. Transplant. Proc. 2003, 35, 3116-3120. (20) Scheller, W.; Jin, W.; Ehrentreich-Forster, E.; Ge, B.; Lisdat, F.; Buttemeier, R.; Wollenberger, U.; Scheller, F. W. Electroanalysis 1999, 11, 703-706. (21) Ignatov, S.; Shishniashvili, D.; Ge, B.; Scheller, F. W.; Lisdat, F. Biosens. Bioelectron. 2002, 17, 191-199. 10.1021/ac051465g CCC: $33.50

© 2006 American Chemical Society Published on Web 12/22/2005

The protein superoxide dismutase (SOD) is part of the biological defense mechanism against oxygen radicals10,23 and has been shown to be vital for all organisms in contact with oxygen. For SOD-deficient mutants of eukaryotes and prokaryotes, an exposure to air has proved to be lethal.24,25 Superoxide dismutases are metalloenzymes that, through alternate oxidation and reduction of their catalytic metal ions, catalyze the dismutation of superoxide to oxygen and hydrogen peroxide,10 which, in turn, is removed by catalases and peroxidases. The reaction is performed by different metalloenzymes: Fe-, Mn-, Ni- and Cu,Zn-superoxide dismutase. In the case of Cu,Zn-SOD, the dismutation of superoxide proceeds via a two-step reaction in which the copper ion switches between the two oxidation states, copper(I) and copper (II):

O2- + Cu(II)ZnSOD f O2 + Cu(I)ZnSOD O2- + Cu (I)ZnSOD + 2H+ f H2O2 + Cu(II)ZnSOD leading to an overall reaction stoichiometry of

2O2- + 2H+ f H2O2 + O2 SODs show high rate constants, up to the order of 109 M-1 s-1, and are distinguished by a highly uncommon specificity to superoxide.26 No side reactions with other molecules have been reported to date, rendering SOD an interesting protein for biosensorial application. While sensing superoxide indirectly using H2O2 electrodes for signal generation has been successful in principle, the preparation and reproducibility of the electrode design and the influence of external H2O2 has been a problem.27,28 The direct electrochemistry of Cu,Zn-SOD has been studied on promotor-modified electrodes, and attempts at using it for biosensors have been made.29-32 SOD biosensors can make use of both reaction steps, so that two potential windows can be evaluated. Although the sensitivity was enhanced, different modes of operation have been reported.29,30 The promotor molecules used in these studies (e.g., mostly mercaptopropionic acid and cysteine), however, show redox peaks of their own in the potential range of interest.33,34 To avoid complications stemming from this (22) Kim, N. H.; Jeong, M. S.; Choi, S. Y.; Kang, J. H. Bull. Korean Chem. Soc. 2004, 25, 1889-1892. (23) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049-6055. (24) Carlioz, A.; Touati, D. Embo J. 1986, 5, 623-630. (25) Vanloon, A.; Pesoldhurt, B.; Schatz, G. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 3820-3824. (26) Bertini, I.; Mangani, S.; Viezzoli, M. S. Adv. Inorg. Chem. 1998, 45, 127250. (27) Lvovich, V.; Scheeline, A. Anal. Chem. 1997, 69, 454-462. (28) Mesaros, S.; Vankova, Z.; Grunfeld, S.; Mesarosova, A.; Malinski, T. Anal. Chim. Acta 1998, 358, 27-33. (29) Ge, B.; Scheller, F. W.; Lisdat, F. Biosens. Bioelectron. 2003, 18, 295-302. (30) Tian, Y.; Mao, L.; Okajima, T.; Ohsaka, T. Anal. Chem. 2002, 74, 24282434. (31) Ohsaka, T.; Tian, Y.; Shioda, M.; Kasahara, S.; Okajima, T. Chem. Commun. 2002, 990-991. (32) Tian, Y.; Mao, L.; Okajima, T.; Ohsaka, T. Anal. Chem. 2004, 76, 41624168. (33) Petri, M.; Kolb, D. M.; Memmert, U.; Meyer, H. Electrochim. Acta 2003, 49, 175-182. (34) Fei, S. D.; Chen, J. H.; Yao, S. Z.; Deng, G. H.; He, D. L.; Kuang, Y. F. Anal. Biochem. 2005, 339, 29-35.

and guaranteeing a proximity of active site and electrode surface, this work describes the design, production, and biosensorial application of monomeric mutants of human Cu,Zn-SOD. The mutants contain one or two additional cysteine residues with the aim of immobilizing the proteins directly onto unmodified gold electrodes via the sulfur of the amino acid introduced. This binding approach has been demonstrated on gold surfaces for other proteins such as peroxidase, azurin, and some receptor proteins.35-37 MATERIALS AND METHODS Xanthine oxidase (XOD) was purchased from Roche (Mannheim, Germany) and further purified by centrifugation (15 min at 11 000 rpm), resuspension in 20 mM phosphate buffer (pH 8), and dialysis against the same buffer (twice overnight). Bovine dimeric Cu,Zn-SOD, xanthine, H2O2 (30% solution in water), ascorbic acid, uric acid, isopropyl β-D-1-thiogalactopyranoside (IPTG), and dithiothreitol (DTT) were provided by SigmaAldrich (Taufkirchen, Germany). HEPES (>99% purity) was bought from Roth (Karlsruhe, Germany) and set to the correct pH with NaOH. TOPP1 Escherichia coli strain, used for protein expression, was purchased from Stratagene (La Jolla, CA). The plasmid pF50E/G51E/E133QSOD, a derivative of pBR322, bearing the recombinant human Cu,Zn-SOD gene, which encodes for the monomeric protein,38 was used as template to introduce the additional mutations and will be referred as wildtype (WT). The software tool ProsaII39,40 was used to select the mutations. The mutations were introduced by using the PCR-based commercial QuikChange Site-Directed Mutagenesis Kit made by Stratagene (La Jolla, USA) according to Stratagen procedures. Each mutated gene was sequenced to confirm the correct sequence. Expression and purification of the proteins were conducted according to a previously published protocol.41 Briefly, chemically competent TOPP1 E. coli cells were transformed with the plasmid. Recombinant human SOD proteins were produced after overnight induction of the tac1 promoter with 1 mM IPTG in 2× YT medium containing 200 µg/mL ampicillin. CuSO4 (0.1 mM final concentration) was also added at induction time. Cells were harvested by centrifugation. The periplasmic proteins were released by osmotic shock,42 purified by DEAE-Sepharose chromatography, and eluted with a NaCl gradient (0-150 mM). The SOD-containing fractions were identified by SDS-PAGE and collected. To avoid the formation of intermolecular sulfur bridges, 5 mM DTT was added before the solutions were stored at -20 °C. Apo-SOD was prepared by incubation (6 h) of the protein (100 µM) in a 20 mM EDTA/buffer (HEPES 50 mM, pH 7,5) solution, followed by extensive dialysis against buffer. Cyclic voltammetry was performed on an Autolab System (Metrohm) and its integrated software system GPES 4.8. Am(35) Ferapontova, E.; Schmengler, K.; Borchers, T.; Ruzgas, T.; Gorton, L. Biosens. Bioelectron. 2002, 17, 953-963. (36) Carmon, K. S.; Baltus, R. E.; Luck, L. A. Biochemistry 2004, 43, 1424914256. (37) Wang, N.; Wang, Y. L. Chem. Res. Chin. Univ. 2004, 20, 437-439. (38) Banci, L.; Bertini, I.; Chiu, C. Y.; Mullenbach, G. T.; Viezzoli, M. S. Eur. J. Biochem. 1995, 34, 855-860. (39) Sippl, M. J. Proteins: Struct., Funct., Genet. 1993, 17, 355-362. (40) Sippl, M. J. Struct. Folding Des. 1999, 7, R81-R83. (41) Getzoff, E. D.; Cabelli, D. E.; Fisher, C. L.; Parge, H. E.; Viezzoli, M. S.; Banci, L.; Hallewell, R. A. Nature 1992, 358, 347-351. (42) Koshland, D.; Botstein, D. Cell 1980, 20, 749-760.

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perometric measurements were conducted on a model 720A Potentiostate from CHI Instruments (Austin, TX) under constant stirring. Impedance spectroscopy was performed on a SI 1260 from Solartron Analytical (Farnborough, U.K.). For the electrochemical studies, a custom-made 1-mL measuring cell and a Ag/AgCl/1 M KCl reference electrode (Biometra, Go¨ttingen, Germany) and a platinum counter electrode were used. All potentials mentioned in this work refer to this reference electrode. Gold wires (>99% purity, diameter 0.5 mm) were purchased from Goodfellow (Cambridge, U.K.). Electrodes were cleaned by successive incubations in boiling KOH (5 M, 4 h), concentrated sulfuric acid (overnight), and 65% nitric acid (at least 15 min), with rinsing steps with water in between. Gold wire electrodes were modified by incubation of the cleaned electrodes in a 50-300 µM solution of the mutant overnight, followed by thorough cleansing in buffer. The electrochemical characterization of the SOD mutants was performed in 50 mM HEPES buffer pH 7.5. Cyclic voltammograms were measured between -200 and +500 mV at a scan rate of 100 mV/s. The heterogeneous electrontransfer rate constant ks was determined by a variation of the scan rate between 50 mV/s and 6 V/s and evaluation of the cyclic voltammograms according to the model of Laviron.43 Impedancy studies were performed in buffer in the presence of 5 mM ferrocyanide and 5 mM ferricyanide. The measurements were performed at open circuit potential and an ac amplitude of 10 mV in the frequency range from 0.5 to 50 t000 Hz with 10 points per decade. Amperometric experiments were conducted at fixed potentials of +220 and -130 mV, respectively. The electrode was immersed in buffer under constant stirring, and the current recording was started. After a stable baseline of a few nanoamperes was established, 2-5 µL of a 10 mM xanthine solution (50 mM HEPES, pH 9) was added, followed by addition of 1-5 µL of XOD (stock solution 20 munits/1.08 mL). A 5-µL sample of bovine Cu,Zn-SOD (15 000 units/mL in water) was then added to remove the radicals. The signal was determined by subtraction of the baseline value from the signal after the start of radical generation. For the evaluation of the influence of potentially interfering substances, a threshold value of 1nA was defined. The concentrations of the typical interfering substances H2O2, uric acid, and ascorbic acid were increased stepwise until a current signal of 1 nA was recorded at the SOD-modified electrode (+220 mV vs Ag/AgCl). Surface plasmon resonance studies were conducted on a Biacore 2000 (Biacore AB, Uppsala, Sweden) with a clean Biacore Pioneer Au sensor chip at a flow rate of 1 µL/min buffer. After 260 s, a solution of the respective SOD-mutant (100 µM) was pumped over the chip for 3 h. Then, the buffer was again used for another 260 s in order to remove unspecifically bound protein. RESULTS AND DISCUSSION Human Cu,Zn-SOD is a homodimeric protein. The copper ion, in the active site, is responsible for the catalytic reaction, while the zinc ion has been shown to be important for the stability and the correct folding of the protein.44 To design the cysteine(43) Laviron, E. In J. Electroanal. Chem. 1978, 101, 19-28.

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Table 1. Amino Acids Selected for Change to Cysteine and New Nomenclature mutation

no. of Cys residues

name

Ala60 f Cys Ser68 f Cys Ser142 f Cys Gly61/Gly141 f Cys Gly61/Ser142 f Cys Ala60/Ser142 f Cys

1 1 1 2 2 2

Mut1A Mut1B Mut1C Mut2A Mut2B Mut2C

containing mutants, a monomeric recombinant form of this enzyme was used. It was obtained by substitution of two residues (F50E, G51E) responsible for hydrophobic interactions at the dimer interface and which also contains a mutation in the active site (E133Q) to increase the activity of the monomeric species.38 By using the software tool Prosa II, it was possible to select the residues that, if mutated into cysteines, do not cause, by an energetic point of view, destabilization of the protein structure. Five mutation sites were chosen that suggested no significant change in structure and function of the enzyme, while located in proximity to the active site and the protein surface. The positions of the mutations and the combinations chosen are listed in Table 1. All the mutants were expressed in E. coli with a good yield and purified by one-step chromatography on an anion exchange column, according to a protocol published previously.39 Their activity was verified by an in vitro superoxide assay. A cyt. c-based sensor electrode described in previous work45 was used to quantify steady-state superoxide concentrations in the lower micromolar range, generated by a known enzymatic system. Addition of small aliquots of the SOD mutants decreased the cyt. c sensor signal to its background value, thus showing that the mutants in solution removed all radicals from the system. However, the activity of the mutants was found to be ∼2 orders of magnitude lower than that of the monomeric WT-SOD. Electrochemical Studies. SOD electrodes were prepared by incubation of gold wires in the respective protein solution. Cyclic voltammograms of the immobilized mutants in protein-free solution are depicted in Figure 1. All mutants showed a well pronounced redox wave in oxidation and reduction, with very similar peak areas. Control experiments were conducted to show that these peaks were caused by the copper in the active site of the protein and that the mutants indeed were immobilized on the gold via the engineered cysteine residues. For two mutants (Mut1A, Mut2C), apoforms were prepared by removing the metal ions as reported in Materials and Methods. The apo-SODs were incubated with gold wires in a manner similar to that of the holoforms. Both apomutants showed a binding to gold surfaces in SPR studies (see Figure 2). However, they caused no peaks in cyclic voltammetry, thus indicating that the mutants’ redox behavior is due to the copper in the protein’s active site and not to some other influence, such as the cysteine itself. (44) Banci, L.; Bertini, I.; Cantini, F.; D’Onofrio, M.; Viezzoli, M. S. Protein Sci. 2002, 11, 2479-2492. (45) Beissenhirtz, M. K.; Scheller, F. W.; Lisdat, F. Anal. Chem. 2004, 76, 46654671.

Figure 1. Cyclic voltammograms of SOD mutants containing one (left) or two additional cysteine residues (right) chemisorbed onto gold electrodes. Conditions: 100 mV/s, reference Ag/AgCl/1 M KCl, 50 mM HEPES pH 7,5 (protein-free).

Figure 2. SPR sensograms of Mut1C (a) and apo-Mut1c (b) binding to a clean, unmodified gold chip. 50 mM HEPES pH 7,5, 100 µM SOD, flow rate 1 µL/min.

This was verified by impedance measurements in the presence of ferri-/ferrocyanide, which showed a much increased chargetransfer resistance of the SOD-mutant and apo-SOD-mutant electrodes in comparison to a bare gold electrode, thus proving the immobilization of the mutant proteins on the gold electrode (see Figure 3). In addition, gold electrodes incubated with the electroactive cysteine-free monomeric WT protein showed no redox peaks either. This leads to the conclusion that the mutants are immobilized via the cysteines and that their redox behavior is due to the interaction of their active site copper with the electrode. The kinetics of redox conversion of the surface-fixed mutants could be described by the model of Laviron, which allowed a determination of the heterogeneous electron-transfer rate constant ks (see Figure 4). For all mutants , an R value of 0.5 was found. The formal potentials (E0) and ks were determined for all mutants and are summed up in Table 2. The peak width at half peak height was found to be between 160 and 180 mV. While such values can be regarded as relatively high, they nevertheless

Figure 3. Bode plot of the impedance measurement for Mut1C (dashed line), apo-Mut1C (solid line) immobilized on gold, and an unmodified gold electrode (dotted line) in a ferri-/ferrocyanide solution (each 5 mM). Buffer: 50 mM HEPES pH 7,5.

appear consistent with the rather heterogeneous behavior of immobilized redox proteins. The surface coverage of the redoxactive protein mutants (which can be deduced from cyclic voltammetry) varied slightly from electrode preparation to electrode preparation and was found to be in the range 28 ( 6 pmol/ cm2. This is in correspondence with SPR measurements where a Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

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Figure 4. Determination of ks according to the model of Laviron for a gold electrode modified with Mut1A (one example, average data given in Table 2). R was determined as 0.5. Table 2. Formal Potential and Heterogeneous Electron-Transfer Rate Constant for the SOD Mutant-Modified Electrodes mutant

E° (mV) (vs Ag/AgCl/ 1 M KCl; n ) 6)

ks (s-1) (n ) 4)

Mut1A Mut1B Mut1C Mut2A Mut2B Mut2C

158 ( 5 146 ( 9 146 ( 6 154 ( 6 149 ( 4 151 ( 3

7.8 ( 1.7 9.4 ( 1.5 6.4 ( 2.7 7.3 ( 2.1 10.5 ( 4.1 5.8 ( 1.3

protein loading of ∼18 pmol/cm2 was estimated. For the explanation of the somewhat lower amount in the latter case, one has to consider the different gold surfaces used (wire electrode and gold chips) and the different immobilization conditions (unstirred solution and solution flow). The distance of the mutation site to the copper was calculated for every mutant. Although these distances range from 6 to 18 Å, this is not reflected in the kinetics of the heterogeneous electron transfer. For the immobilized protein, the interaction with the gold surface modifies the protein structure, thus resulting in very similar kinetic and thermodynamic properties of the immobilized mutants. To compare the electrochemical properties of the mutated proteins with WT SOD, both the WT SOD and SODMut1C were immobilized on promotor-modified electrodes. Mercaptopropionic acid (MPA) was used since it had been successfully applied for the study of bovine SOD in previous works.29 Both electrodes showed quasi-reversible electrochemistry with formal potentials of 94 ( 5 (WT) and 116 ( 8 mV (Mut1C) and ks values of 6.4 ( 0.8 (WT) and 4.8 ( 2.2 s-1 (Mut1C), respectively (n ) 3). In comparison, Tian and co-workers30 found a formal potential of +73 mV (vs Ag/AgCl/1 M KCl) for the dimeric bovine WT SOD at a cysteine-modified gold electrode. Clearly, the introduction of the cysteine shifts the potential slightly toward more positive values. When the enzyme is bound with its cysteine to the gold surface, as compared to electrostatic adsorption onto the promotor layer, this shift is even more pronounced. Such an influence of the binding modus on a protein’s 932 Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

Figure 5. Cyclic voltammograms of Mut1C electrode in the presence of superoxide (solid line) and its absence (dotted line). Conditions: 30 mV/s, reference Ag/AgCl/1 M KCl, 50 mM HEPES pH 7,5, 10 munits/mL XOD, 50 µM xanthine; removal of the radical was reached by addition of dimeric bovine Cu,Zn-SOD to the solution.

formal potential is not unusual. For cyt. c, similar differences have been reported between the adsorbed and the covalently bound state on the same promotor, while the use of different promotors can also change the formal potential of cyt. c.18,46,47 The electron-transfer rate of the mutants on gold is only slightly higher than for the MPA-adsorbed SODs. Since MPA is a rather short-chain promotor, the decrease in electron-transfer distance is not drastic. Also, more tightly bound proteins have been shown to yield a slower electron transfer; for example, covalent fixation of bovine SOD reduces its ks value by a factor of 3 as compared to the adsorbed form.29 Therefore, the loss in flexibility compared to the promotor-adsorbed protein molecules partially offsets the smaller distance between the copper site and the electrode for the bound mutants. In conclusion, the immobilized mutants allow for their application as superoxide biosensors. Superoxide Detection. The interaction between superoxide radicals and the SOD mutants was first studied using cyclic voltammetry. Cyclic voltammograms were recorded in the presence of a superoxide-producing enzyme (XOD) and xanthine as substrate. Figure 5 shows the results of such an experiment with Mut1C. Both currents in the oxidation and reduction region were increased in the presence of the radical (underlined by the arrows in Figure 5). When (nonimmobilized) bovine Cu,Zn-SOD was added to the solution, the radical was instantly removed from solution, and both peaks shrank. In addition, it should be mentioned that xanthine oxidase alone did not cause any electrochemical response at the gold electrode. This leads to the conclusion that this difference in peak height is due to an interaction between superoxide and the SOD electrode. Since both peaks are concerned, it seems that both partial reactions (oxidation and reduction of the radical) can indeed be investigated with the SOD-mutant modified electrodes. Therefore, both reactions were studied using amperometry. (46) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847-1849. (47) Taniguchi, I.; Toyosawa, K.; Yamaguchi, H.; Yasukouchi, K. J. Chem. Soc., Chem. Commun. 1982, 1032-1033.

Figure 6. Amperometric detection of superoxide with a Mut1Cmodified electrode. (1) Start of radical generation; (2) removal of radical from the solution by addition of dimeric bovine Cu,Zn-SOD. Conditions: +220 mV, reference Ag/AgCl/1 M KCl, 50 mM HEPES pH 7,5, 20 munits/mL XOD, 50 µM xanthine.

Figure 7. Amperometric detection of superoxide with a Mut1Cmodified electrode. (1) Start of radical generation; (2) removal of radical from the solution by addition of dimeric, bovine Cu,Z-SOD. Conditions: -130 mV, reference Ag/AgCl/1 M KCl, 50 mM HEPES pH 7,5, 20 munits/mL XOD, 50 µM xanthine.

The reduction of the radical by SOD and the subsequent reoxidation of the protein by the electrode were investigated at a constant potential of +220 mV. The resulting curve for Mut1C is shown in Figure 6. Prior to the start of superoxide generation, after 10-30 s, a constant background current of a few nanoamperes was recorded. When XOD was added to the xanthinecontaining solution, the current rose sharply (1), until a constant level was reached. Addition of bovine SOD to the solution (2) immediately decreased the signal back to its background level. This is in keeping with previous radical studies.48 A few seconds after the start of the radical generation, a steady-state concentration is established by the simultaneous production and spontaneous dismutation of superoxide.10 The addition of SOD to the cell removes all radical molecules. These results show that the sensor can follow the change in radical concentration. Its signal is caused by the reaction between superoxide and the immobilized SOD. In the potential region where the protein is oxidized the following reactions take place

removal of superoxide from the solution. Again, neither uric acid nor H2O2 influenced the sensor signal. At potentials where the protein is reduced, the reaction can be written as follows:

O2- + Cu(II)ZnSOD f O2 + Cu(I)ZnSOD electrode

Cu(I)ZnSOD 98 Cu(II)ZnSOD + 1eAfter removal of the radical (by addition of SOD to the solution), the further generation of the main reaction products (uric acid and H2O2) did not influence the sensor’s response since the basic current was stably reached. Similar experiments were conducted at a potential of -130 mV. The resulting curve is shown in Figure 7. A few seconds after current recording was begun, a steady baseline was established. At this potential, the generation of superoxide caused a reduction current, which was again reset to its background value by the (48) Ge, B.; Lisdat, F. Anal. Chim. Acta 2002, 454, 53-64.

O2- + Cu(I)ZnSOD + 2H+ f H2O2 + Cu(II)ZnSOD electrode

Cu(II)ZnSOD + 1e- 98 Cu(I)ZnSOD

Therefore, both potential windows allow a specific superoxide detection with the mutant-modified electrodes. These results with the mutant-modified electrodes correspond to the results by Tian et al.30,32 at a promotor-modified electrode. Since the resulting current signals are much larger in the positive potential range, this was chosen for further studies. The sensitivity of the new biosensor was determined by varying the XOD activity used for the radical generation. This approach has previously been shown to result in steady-state superoxide concentrations in the range of a few hundred nanomolar to micromolar.48 The sensor signal at different superoxide levels is shown in Figure 8. From this, the sensitivity was calculated as 0.23 A M-1cm-2 with the calibration procedure described previously.48 Compared to the cyt. c monolayer electrode, this is an increase of ∼1 order of magnitude. A comparison to the works of Tian and co-workers30-32 is difficult since they used a different calibration procedure. However, the sensitivity of the SODmutant sensor seems to be lower than the setup employing the bovine WT SOD. Since the noise level of the electrode in a stirred solution was rather small ((0.2 nA), rather low superoxide concentrations can be analyzed. The error bars in Figure 8 also illustrate the reproducibility of the electrode preparation. While for a manual preparation the result is satisfying, more precise data can be obtained using an automatized incubation procedure. From the current-concentration dependence, it can also be estimated that the reaction rate constant of the immobilized SODAnalytical Chemistry, Vol. 78, No. 3, February 1, 2006

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Figure 8. Calibration of Mut1C sensor at an electrode potential of +220 mV. (Current change after the addition of varying amounts of XOD (activity from 5 to 120 munits/mL), 50 µM xanthine; error bars indicate the variation from three independently prepared electrodes). Table 3. Interference Threshold Concentrations for Potentially Electroactive Substancesa

substance H2O2 uric acid ascorbic acid

threshold threshold concn concn (+220 mV) (-130 mV) 1.8 mM 450 µM 80 µM