Anal. Chem. 1999, 71, 1359-1365
Superoxide Dismutase Activity Measurement Using Cytochrome c-Modified Electrode F. Lisdat,*,† B. Ge,† E. Ehrentreich-Fo 1 rster,† R. Reszka,‡ and F. W. Scheller†
Institute of Biochemistry and Molecular Physiology, University of Potsdam, Im Biotechnologiepark, 14943 Luckenwalde, Germany, and Max Delbru¨ ck Centrum fu¨ r molekulare Medizin, Robert-Ro¨ ssle Strasse 10, 13122 Berlin, Germany
SOD activity was quantified by the use of a cytochrome c-modified gold electrode. The electrode responded rapidly to superoxide radicals in solution. Steady-state superoxide concentrations were established by control of the calibration conditions. On this basis very low SOD activities were detected (10-200 munits/mL). This method showed good correlation with the standard photometric test and was applied for the determination of SOD activity entrapped into liposomes. Interference by hydrogen peroxide and uric acid was characterized and minimized using long-chain thiols for the first electrode modification step. The complete modification proved to be stable for several days. Superoxide as a short-lived and reactive radical can be considered as a nonclassical messenger molecule; i.e., the signal transduction is mediated via its chemical reactivity. Acting on one hand as defense against viral or bacterial attack, it can, on the other hand, lead to oxidative damage of proteins, DNA, and lipid peroxidation.1 Under normal physiological conditions, enzymatic superoxide production by one-electron reduction is counterbalanced by catalytic and noncatalytic antioxidative-acting agents. Thus, physiological concentration is rather low (picomolar range). However, an increase in free-radical activity was observed in a number of diseases. This “radical burst” overwhelms the antioxidant defenses. Typical examples are ischemia-reperfusion injuries,2,3 septic shock,4,5 and rheumatoid arthritis.6,7 Therefore, superoxide can serve as a medical indicator of pathophysiological situations. This is particularly relevant in the pathogenesis of reperfusion injury in several organs. Along with the development of techniques for the detection of superoxide, the question of †
University of Potsdam. Max Delbru ¨ k Centrum fu ¨ r Molekulare Medizin. (1) Orrenius, S. Mechanisms of oxidative cell damage. In Free Radicals: From Basic Science to Medicine; Poli, G., Albano, E., Dianzani, M. U., Eds.; Birkha¨user Verlag: Basel, 1993; p 47. (2) Merry, P.; Grootveld, M.; Lunec J.; Blake, D. R. Am. J. Clin. Nutr. 1991, 56, 362. (3) Mayumi, T.; Schiller, H. J.; Bulkley, G. B. Pharmaceutical intervention for the prevention of post-ischaemic reperfusion injury. In Free Radicals: From Basic Science to Medicine; Poli, G., Albano, E., Dianzani, M. U., Eds.; Birkha¨user Verlag: Basel, 1993; p 438. (4) Fukuyama, N.; Takebayashi, Y.; Hida, M.; Ishida, H.; Ichimori, K.; Nakazawa, H. Free Radical Biol. Med. 1997, 22 (5), 771. (5) Kelly, K. A.; Hill, M. R.; Youkhana, K.; Wanker, F.; Gimble, J. M. Infect. Immunol. 1994, 62 (8), 3122. (6) Gambhir, J. K.; Lali, P.; Jain, A. K. Clin. Biochem. 1997, 30 (4), 351. (7) Miesel, R.; Murphy, M. P.; Kroger, H. Free Radical Res. 1996, 25 (2), 161. ‡
10.1021/ac980961k CCC: $18.00 Published on Web 02/27/1999
© 1999 American Chemical Society
therapeutic effects of antioxidants has gained considerable interest. As catalytic and very specific superoxide scavenger, superoxide dismutase (SOD)8 has the potential to ameliorate reperfusion injury. It has been shown that the inherent disadvantage of this enzymesthe limited lifetime in physiological systemsscan be overcomebySODmodificationsorliposome-entrappingtechniques.9-11 Liposome-entrapped SOD, which can penetrate cell membranes, was found to reduce significantly infarct size in cerebral ischemia in rats.10 The standard method for the activity determination of SOD is based on the spectrophotometric detection of the decreased superoxide reduction of cytochrome c in the presence of the enzyme.12 Several variations of this optical method have been developed.13-16 The basis for any sensoric SOD detection is the quantification of superoxide concentration in solution. Two approaches have been described so far. The first is based on hydrogen peroxide-sensitive electrodes in combination with the SOD-catalyzed superoxide dismutation to hydrogen peroxide and oxygen.17-19 The problem of hydrogen peroxide interference was addressed by the incorporation of a hydrogen peroxide-impermeable, hydrophobic membrane.17 However, this causes problems in miniaturized sensor fabrication. Alternatively, a second electrode that is only sensitive to hydrogen peroxide was combined with the SOD-modified electrode.18 In a second approach, superoxide directly interacts with an electrode producing the sensor signal. While a bare carbon electrode gives no specific signal for superoxide, its reaction with cytochrome c can be used for the detection of the radical.20 In the direction of sensor development, significant progress was (8) Fridovich, I. Annu. Rev. Biochem. 1995, 64, 97. (9) Takeda, Y.; Hashimoto H.; Kosaka, F.; Hirakawa, M.; Inoue, M. Am. J. Physiol. 1993, 264, H1708. (10) Imaizumi, S.; Woolwoth, V.; Fishman, R.; Chan, P. H. Stroke 1990, 21 (9), 1312. (11) Beckman, J. S.; Minor, R. L.; White, C. W.; Repine, J. E.; Rosen, G. M.; Freeman, B. A. J. Biol. Chem. 1988, 263, 6884. (12) McCord, J. M.; Fridovich I. J. Biol. Chem. 1969, 22 (25), 6049. (13) Paoletti, F.; Mocali A. In Methods in Enzymology; Packer, L., Glazer, A. N., Eds.; Academic Press: San Diego, 1990; Vol. 186, pp 209-220. (14) Martin, J. P. In Methods in Enzymology; Packer, L., Glazer, A. N., Eds.; Academic Press: San Diego, 1990; Vol. 186, pp 220-227. (15) Nakano, M. In Methods in Enzymology; Packer, L., Glazer, A. N., Eds.; Academic Press: San Diego, 1990; Vol. 186, pp 227-232. (16) Beyer, W. F.; Fridovich, I. Anal. Biochem. 1987, 161, 559-566. (17) Song, M. I.; Bier, F. F.; Scheller, F. W. Bioelectrochem. Bioenerg. 1995, 38, 419 (18) Lvovich, V.; Scheeline, A. Anal. Chem. 1997, 69, 454. (19) Mesaros, S.; Vankova, Z.; Grunfeld, S.; Mesarosova, A.; Malinski, T. Anal. Chim. Acta 1998, 358, 27.
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reached by the development of promoter-modified electrodes allowing electrical communication between the electrode and the redox center of proteins.21,22 With gold electrodes modified by cytochrome c via short thiols, various production rates of the superoxide radical have been successfully detected.23,24 The sensor is based on the reduction of immobilized cytochrome c in the presence of superoxide followed by electrochemical oxidation thus resulting in a cytochrome c again accessible to superoxide reduction and a current signal at the electrode. In this paper, we introduce a modified procedure and investigate the conditions of sensor calibration correlating the sensor signal to steady-state radical concentrations in solution. On this basis, a detection element for the determination of superoxide dismutase activity is described. EXPERIMENTAL SECTION Materials. Mercaptoundecanoic acid (MUA) was supplied by Aldrich (Steinheim, Germany) and xanthine oxidase (XOD; EC 1.1.3.22) by Boehringer/Mannheim. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), hypoxanthine, potassium superoxide, superoxide dismutase (SOD, EC 1.15.1.1) from bovine erythrocytes, superoxide dismutase-poly(ethylene glycol) (PEG-SOD) and catalase (EC 1.11.1.6) from bison liver were from Sigma (Deisenhofen, Germany). Xanthine and dimethyl sulfoxide were provided by Fluka (Deisenhofen, Germany). For preparation of the measuring buffer, sodium dihydrogen phosphate and disodium hydrogen phosphate (Fluka) were used. The pH 7.5 was adjusted by mixing 0.1 mol/L solutions of both salts and monitoring the pH with a pH glass electrode. EDTA was added to result in a concentration of 100 µmol/L. During electrode modification, a potassium dihydrogen phosphate/ dipotassium hydrogen phosphate-based buffer (pH 7.0) without EDTA was used (5-10 mmol/L). To prepare the liposomes, 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used from Sygena AG (Genzyme Pharmaceuticals), cholesterol (CH) from Roth (Karlsruhe, Germany) and stearylamine (SA) from Sigma. Apparatus. Investigations were performed in a stirred electrochemical cell with a three-electrode configuration. The cell with a volume of 1 mL comprised a Ag/AgCl/1 M KCl reference electrode and a Pt counter electrode. Cyclic voltammetry was performed using the Autolab system (Eco chemie). For SOD measurements, the modified gold electrode was polarized at +150 mV vs Ag/AgCl. The potentiostat was a “bioanalyzer” from Kreijci Engineering (Brno, Czech Republic). The data were stored in a personal computer. Impedance measurements were performed with the Autolab system applying an ac current with 10-mV amplitude in the frequency range 0.05 Hz-50 kHz under opencuircuit potential conditions. The solution was a phosphate buffer (pH 7.5, 100 mM) with 30 mmol/L K3[Fe(CN)6]/10 mmol/L K4[Fe(CN)6]. Data analysis was performed with the Zplot/Zview software from Solartron (Hampshire, UK) using an equivalent (20) McNeil, C. J.; Smith, K. A.; Bellavite, P.; Bannister, J. V. Free Radical Res. Commun. 1989, 7 (2), 89. (21) Frew, J. E.; Hill, H. A. O. Eur. J. Biochem. 1988, 172, 261. (22) Willner, I.; Katz, E.; Willner, B. Electroanalysis 1997, 9 (13), 965. (23) Cooper, J. M.; Greenough, K. R.; McNeil, C. J. J. Electroanal. Chem. 1993, 347, 267. (24) McNeil, C. J.; Greenough, K. R.; Weeks, P. A.; Self, C. H.; Cooper, J. M. Free Radical Res. Commun. 1992, 17, 399.
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circuit of a capacitance and a resistance in parallel and in series to the solution resistance.25 For photometric analysis of SOD and XOD activity the Beckman spectrophotometer DU 640 was used. Electrode Preparation. Gold wire electrodes (diameter 0.5 mm) were cleaned by boiling in 2 mol/L KOH, incubating in concentrated H2SO4 (8 h) and concentrated HNO3(10 min), and carefully rinsing with water in between. The electrodes were stored in concentrated H2SO4. Prior to use the electrode surface was controlled by cycling in 1 mol/L H2SO4. After being rinsed with water and ethanol, the electrodes were incubated in a 5 mM solution of MUA in ethanol for ∼24 h. The modified electrodes were rinsed with ethanol and 10 mM potassium phosphate buffer (pH 7) and then mounted into an electrochemical cell with the same buffer. A cytochrome c solution was added for a final concentration of 20-30 µmol/L. During cytochrome c adsorption, the electrodes were cycled in the range -0.4 to +0.4 V with a scan rate of 50 mV/s for ∼10 min. Then EDC was added to give a concentration of 5 mM, and the electrodes were incubated with this solution for about 20-30 min. After rinsing with 10 mM buffer, the electrochemical behavior of the immobilized cytochrome c was checked using cyclic voltammetry. SOD Measurements. The XOD-catalyzed oxidation of xanthine or hypoxanthine to uric acid was chosen for calibrating the modified cytochrome c electrode. The main path of electron flux from xanthine to oxygen occurs via the two-electron pathway in the flavin-oxygen complex producing hydrogen peroxide. However, a second pathway allows one-electron transfer from the reduced enzyme to oxygen to liberate superoxide. The relation of one- to two-electron transfer depends on the steady-state concentration of the reduced enzyme. Factors governing this relation, e.g., oxygen and substrate concentration, were investigated in detail.26,27 Under air saturation and physiological pH conditions, ∼20% of the electron flux occurs via a one-electron step and thus leads to the generation of superoxide radicals. Stock solutions of 20 mM were prepared for xanthine and hypoxanthine in 50 mmol/L KOH or 0.1 mol/L potassium phosphate buffer (pH 7.5), respectively. The substrate was added to the cell to result in final concentrations between 0.25 and 200 µmol/L. The reaction was started by adding XOD in the concentration range of 2-100 munits/mL. To measure SOD activity, the enzyme can be added after or before XOD addition. The current decrease compared to the measurement without SOD was evaluated as the sensor signal. A stock solution of 50-100 units/mL SOD in 0.1 M buffer (pH 7.5) was normally used. To test for possible interfering substances, superoxide was generated by the use of 100 µmol/L hypoxanthine and 50 munits/ mL XOD. A total of 5-10 µL of pure ethanol, DMSO, Triton X100 (0.1%), or a liposome suspension (see below) was added to the measuring cell. The current response was compared to the measurement without addition of potential interfering components. The standard test of McCord and Fridovich12 was used for photometric determination of SOD activity. This test is based on (25) Gabrielli, C. In Physical Electrochemistry; Rubinstein, I., Ed.; Marcel Dekker: New York, 1995; pp 243-292. (26) Olson, J. S.; Ballou, D. P.; Palmer, G.; Massey, V. J. Biol. Chem. 1974, 249 (14), 4363. (27) Olson, J. S.; Ballou, D. P.; Palmer, G.; Massey, V. J. Biol. Chem. 1974, 249 (14), 4350.
Table 1. Different Liposome Types, Compositions, and Characterization composition concn (molar ratio) (units/mL) type DPPC CH SA PEG-SOD SOD I II III
14 14 14
7 7 7
3 3 3
500
size (nm)
10 000 183 ( 55 222 ( 53 196 ( 55
ζ potential (mV) 71 92 63
the detection of reduced cytochrome c in solution. SOD activity is quantified by measuring the diminished rate of this reduction in the presence of the enzyme. XOD activity was controlled by measuring the production rate of uric acid at 293 nm during the enzymatic oxidation of xanthine.28 Therefore 3 mL of 0.1 mol/L xanthine in TRIS buffer (0.1 mol/L, pH 8.5) was mixed with 0.02 mL of 0.48 mol/L hydrogen peroxide and 0.02 mL of 500 units/mL catalase in a quartz cuvette of 1-cm thickness. The reaction was started by adding 0.05 mL of XOD solution. The increase in absorbance (∆A) was followed for 1 min. From this, XOD activity was calculated using the extinction coefficient of 12.2 L/mmol‚cm: [XOD] ) ∆A3.09/12.2 × 0.05 × 1 (units/mL sample solution). As an alternative source of superoxide radicals, potassium superoxide was used. For this purpose, a 15 mmol/L solution in DMSO was prepared, and then aliquots of 2.5-10 µL were added to the stirred electrochemical cell. SOD Liposomes. The different liposome formulations29 (see Table 1) were prepared as following: First the lipids were dissolved with 30 mL of trichloromethane. Then 15 mL of diisopropyl ether was added, followed by 4 mM HEPES buffer (pH 7.4) with or without SOD or PEG-SOD. The lipid/buffer or lipid/enzyme/buffer solution was sonicated using a Branson Sonifier 250 (Ultrasonics, USA) for 1 min. During that time, an emulsion was formed. The organic solvent was then removed for preparation of reversed-phase evaporation vesicles (REV) by rotary evaporation at 45 °C on two vacuo stages. For the reduction of particle size, width of the size distribution, and lamellarity of the REV, the dispersion was homogenized by using a filtration unit (Sartorius, Go¨ttingen, Germany) containing polycarbonate filter (Nucleopore, Pleasanton, CA) starting at 0.8, 0.6, 0.4, and 0.2 µm (all filtration steps were repeated two times). Then the nonencapsulated SOD was separated from the vesicles by centrifugation with 60 mL of HEPES buffer at 15000g for 30 min. This step was repeated three times. The liposome pellet was resuspended in buffer up to the initial volume. The size of the liposomes was measured by photon correlation spectroscopy (N4 plus). Additionally, the ζ potential was determined via laser doppler anemometry (Delsa 440 SX, Coulter Electronics, Krefeld, Germany). The liposomes of type 1 and 2 were stored as suspensions at 4 °C. On the day of SOD measurement, an aliquot of the suspension was centrifuged for 30 min at 15000g. The pellet was then unified with the same volume of pure alcohol to destroy the (28) Boehringer/Mannheim Biochemicals for the Diagnostic Industry, Clinical Chemistry 1993/94. (29) Dirnagl, U.; Lindauer, U.; Them, A.; Schreiber, St.; Pfister, H.-W.; Koedel, U.; Reska, R.; Freyer, D.; Villringer, A. J. Cereb. Blood Flow Metab. 1995, 15, 929.
Figure 1. Cyclic voltammogram of cytochrome c immobilized on a MUA-modified gold electrode in phosphate buffer (pH 7.5) with 200 µmol of hypoxanthine and catalase (1 unit/mL) (A) before and (B) after addition of XOD (200 munits/mL), scan rate 10 mV/s.
liposomes and liberate the enzyme. The mixture was diluted with buffer to result in a final SOD activity within the electrochemical cell of ∼100 munits/mL. To test the background, the liposomes of type 3 (without SOD) were also mixed with alcohol after centrifugation, 10 µL was injected into the electrochemical cell, and the current response was recorded. RESULTS AND DISCUSSION Calibration of the Cytochrome c-Modified Electrode. The antioxidative action of SOD is based on its scavenging effect on superoxide radicals. Therefore, superoxide is the primary analyte for measuring SOD activity. Consequently, sensor calibration consists of two stepssfirst, superoxide generation and detection and, second, quantification of different activities of the antioxidative-acting agent. Most desirable for such a two-step procedure is a fast-responding sensor system which allows the detection of steady-state superoxide concentrations in solution. The basis for this analysis is the reversible electrochemical behavior of immobilized cytochrome c which in the presence of superoxide shows a catalytic current in the cyclic voltammogram. For the first time we could demonstrate that the reduction of cytochrome c at the modified electrode by superoxide increases the oxidation current (Figure 1). A stable cyclic voltammogram during the enzymatic radical production by the hypoxanthine/ XOD system is only visible in the presence of catalase. The enzyme removes the coproduced hydrogen peroxide which otherwise would interfere with this measurement because of the reaction with reduced cytochrome c.30 This reaction is particularly pronounced at a negative electrode polarization but can be depressed at a more positive potential as will be demonstrated later. For superoxide measurement, the electrode was operated under constant potential in the amperometric mode. For the calibration of the electrode, the spontaneous dismutation of the radical into oxygen and hydrogen peroxide has to be considered. (30) Lo¨tzebeyer, Th.; Schuhmann, W.; Schmidt, H.-L. Sens. Actuators 1996, B33, 50.
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Figure 2. Response of the cytochrome c-modified electrode to different superoxide concentrations (100 µmol/L hypoxanthine) (A) and relationship of the steady-state electrode current and the square root of XOD activity used for superoxide production (B).
Figure 3. Dependence of the steady-state electrode current and time of the current response, respectively, on hypoxanthine concentration (100 munits/mL XOD).
Under controlled reaction conditions, the counterbalance of enzymatic generation and dismutation results in a steady-state concentration of the radical in solution which the sensor should be able to follow. In Figure 2, the response of the cytochrome c-modified electrode to different superoxide concentrations in solution is shown. The different levels result from different production rates because of increasing XOD activities in solution. According to McCord and Fridovich,31 the steady-state concentration depends on the square root of the enzyme activity. So the sensor should show the same dependence with the steady-state current level. As demonstrated in Figure 2B, this function is valid for electrode current and XOD concentration up to 100 munits/ mL. The result indicates that the modified electrode is fast enough to follow the actual concentration of this short-lived radical in solution as a necessary precondition for any scavenger measurement. Further arguments in this direction were found during investigation of the sensor response on the substrate concentration of enzymatic radical production. The dependence of the sensor current on hypoxanthine concentration given in Figure 3 reflects the kinetics of the enzyme reaction in solution. From the graph, a KM value of 1.28 µmol/L can be calculated which is identical with the literature value (1.3 µmol/L28). The same agreement was found for xanthine: 1.68 µmol/L in comparison to the literature (31) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1968, 243 (21), 5753.
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Figure 4. Response of the cytochrome c-modified electrode to repeated injections of xanthine (72 µmol/L, 100 munits/mL XOD).
value of 1.7 µmol/L.28 At higher concentrations, the reaction is limited by oxygen supply and the known substrate inhibition of XOD. During the enzymatic calibration step, a spontaneous decrease of the sensor signal toward the baseline was found after a certain time of XOD operation. If this is only caused by the consumption of the substrate, then a repeated injection of xanthine (or hypoxantine) should result in a repeated sensor response. In Figure 4, the result of such a measurement is given which confirmed this explanation. For the calibration of the sensor, it follows that the time, during which steady-state values can be obtained, has to be characterized in dependence on the substrate concentration. Only within this time can the action of SOD be quantitatively analyzed; at the end of the period, the decrease of the sensor current due to the consumption of the substrate would interfere with the measurement. The result of this investigation is plotted in Figure 3. For a given XOD concentration, a continuous increase of the signal time with the hypoxanthine concentration was found. The time decreased with increasing XOD activity, i.e., a higher conversion rate of the substrate. Oxygen is the second substrate for enzymatic calibration using xanthine or hypoxanthine. Since the concentration of oxygen influences the relation of the one- and two-electron pathway during the enzymatic action, a solution with a higher oxygen content should give a higher yield of superoxide radicals.26 This was confirmed by measuring the electrode response in air-saturated and oxygen-purged buffer. In Figure 5, the increase in the superoxide signal for the higher oxygen concentration in solution can be clearly seen. A signal increase of ∼100% was found for a 2.5 times increase in the oxygen content (which was measured by an independent oxygen electrode). In addition, it was observed that the increase in the sensor signal is proportional to the oxygen content in solution. However, because of the magnitude of the effect, it can be concluded for the calibration procedure that careful air saturation of the buffer used is sufficient for reproducible sensor response to the xanthine(hypoxanthine)/XOD system. This may be illustrated by the results of experiments with repeated superoxide generation and evaluation of the current response. The standard deviation of a series of 8-10 measurements was always
