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Direct electron transfer reactions between immobilized cytochrome c and modified gold electrodes
267
J. Ekctroanal.
Chem., 347 (1993) 267-275
Elsevier Sequoia S.A., Lausanne
JEC 02442
Direct electron transfer reactions between immobilized cytochrome c and modified gold electrodes Jonathan M. Cooper Department
of Electronics
and Electrical Engineering,
Katharine R. Greenough
University of Glasgow, Glasgow G12 SQQ (UK)
and Calum J. McNeil
Department of Clinical Biochemktry, upon Tyne NE2 4HH &JK)
The Medical School, University of Newcastle
upon Tyne, Newcastle
(Received 2 July 1992; in revised form 25 August 1992)
Abstract In this paper we present data showing quasi-reversible electrochemistry of soluble cytochrome c using the gold electrode modifier N-ace@ cysteine (E1,2 = 2.5 mV, AE, = 60 mV, Ir,+ / vl/* = constant). Cytochrome c was subsequently immobilized at this modified electrode using a carbodiimide condensation reaction. The electron-transfer rate between the immobilized protein and the gold electrode was estimated as 3.4f 1.2 s-’ and the formal potential E”’ of the immobilized protein electrode was calculated as 2 mV/SCE. We also present data showing an application for the immobilized cytochrome c electrode as a sensor for the specific measurement of superoxide radical production.
INTRODUCTION
A study of electron transfer between proteins first published over a decade ago has provided us with an elegant model for the modification of electrodes in order to investigate the electrochemistry of proteins. In this study Poulos et al. [l] reported the detailed molecular organization between cytochrome c peroxidase (CcP> and the small soluble protein cytochrome c, and explained the enzyme-substrate interaction in terms of charge matching, spatial fitting and hydrogen bonding. Their investigations showed that a ring of basic lysines, which are positively charged at physiological pH, extend away from the surface of cytochrome c to form salt bridges with a group of complimentary negatively charged carboxylate groups of aspartate residues in CcP. The electrostatic interaction draws the two molecules together so that the two haem groups lie parallel, only 25 A apart, thus facilitating electron transfer. 0022-0728/93/$06.00
0 1993 - Elsevier Sequoia S.A. All rights
reserved
268
This biophysical model provides an explanation for the mechanism by which the direct electrochemistry of soluble cytochrome c can be measured at modified electrodes. Previously, in a series of elegant experiments Hill and coworkers [2-51 have demonstrated that electron transfer with cytochrome c can be achieved using suitably modified gold electrodes. Subsequently, a variety of electrode surfaces have been prepared with a net negative charge using several different techniques, including polishing [6] and electropolymerization [7]. Most recently, this approach has been used successfully for the study of the direct electrochemistry of adsorbed cytochrome c at self-assembled alkanethiol monolayers on gold electrodes [81. In this paper, we report on the electrochemistry of soluble cytochrome c at a modified gold electrode suitable for the covalent attachment of the protein using carbodiimide condensation. In addition, we demonstrate the direct electrochemistry of the immobilized protein. The significance of achieving direct electron transfer between an immobilized protein and an electrode is that there is potential for developing “solid-state” devices which could have applications in either molecular electronics or the fabrication of molecular sensors. To this end, we report some preliminary results which show a possible future application for this device as a sensor for the production of superoxide radicals in vivo. EXPERIMENTAL
Horse heart cytochrome c (Sigma, type VI) was repurified by CM 3ZSephadex cation exchange chromatography (Pharmacia), and was dialysed exhaustively against 10 mm01 dmv3 sodium phosphate buffer (pH 7.0). The protein concentration was estimated spectrophotometrically using a molar absorption coefficient of 106.1 cm-’ at 410 nm [9]. All aqueous solutions were prepared using double-distilled reverse osmosis water. Prior to each experiment, gold electrodes (2 mm in diameter) were cleaned using a 0.3 pm alumina slurry (BDH) and were sonicated in distilled water for 30 s. The electrodes were modified by immersion in a 2 mm01 dmm3 solution of N-acetyl cysteine (Sigma) in 10 mm01 dmm3 sodium phosphate buffer (pH 7.0) for 2 h, and were then washed in distilled water. All experiments investigating the electrochemistry of soluble cytochrome c were performed using these electrodes. Cytochrome c was immobilized at the modified electrodes using the water-soluble condensing reagent l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, (EDC) (Sigma). The N-ace@ cysteine modified electrode was first incubated in a 10% (w/v) solution of EDC in water for 2 h at 25”C, and was then washed in water. The EDC-activated electrode was then placed in a solution of cytochrome c (4.2 g dmm3) in 10 mm01 dmm3 sodium phosphate buffer (pH 7.0) at 4°C for 6 h. The electrode was finally washed exhaustively with 10 mmol dmm3 sodium phosphate buffer (pH 7.0). All electrochemical experiments investigating the electrochemistry of the soluble and the immobilized protein were carried out in triplicate at 25°C in a conventional two-compartment three-electrode system using a saturated calomel
269
reference electrode (SCE) linked to the working compartment by a Luggin capillary (ca. 0.1 rnm2). The working compartment contained a 1 cm2 platinum gauze counter-electrode and the gold working electrode. The potentiostat was built by oxford Instruments and was used with a Linseis LY1600 X-Y-f chart recorder. Electron-transfer reactions were investigated using cyclic voltammetry over the potential range - 100 to + 150 mV/SCE for the soluble protein at scan rates u between 5 and 200 mV s-l and over the range - 150 to + 100 mV/SCE for the immobilized protein at scan rates between 5 and 50 mV s-l. Tris[hydroxymethyl]aminomethane/HCl (Tris) and sodium phosphate buffers (10 mmol dme3) containing 10 mmol dme3 KC1 over a pH range 4.5-8.0 were used as the supporting electrolyte. Electroanalytical experiments for the detection of the superoxide anion 0; were performed using a purpose-built four-electrode electrochemical cell and bipotentiostat with a Goerz Metrawatt SE120 chart recorder, as described previously [lo]. The electrochemical cell contained two gold electrodes (working and background, each 1.5 mm in diameter), a platinum counter-electrode (1.5 mm diameter) and an Ag/AgCl reference electrode (2 mm in diameter). The current output from the bipotentiostat was measured as the background current subtracted from the sample current. The gold working electrode was modified with immobilized cytochrome c, as described above, whilst the background electrode was modified using the same methods but with a solution of 5 g dmv3 human serum albumin (Sigma) in place of the cytochrome c. Both the working electrode and the background electrode were poised at +200 mV vs. Ag/AgCl. Superoxide was produced using a xanthine/ xanthine oxidase generating system in 10 mm01 dme3 Tris buffer (pH 7.5) containing 10 mmol dmm3 KCI. Xanthine oxidase (XOD) (Grade III, Sigma) from buttermilk was dialysed exhaustively against the working buffer to remove the salicylate (preservative), which is a potential electroactive interferent. A stock solution of 14.4 mm01 dme3 xanthine (Sigma) was made up freshly in 2 mmol dme3 KOH and diluted-into 10 mmol dmW3 Tris buffer (pH 7.0) to give a 350 kmol dmP3 working solution. The reaction was initiated by the addition of XOD to give a final enzyme concentration in the range O-O.48 pm01 dma3. The response at the electrode was measured as the initial (linear) rate of current generated by the re-oxidation of reduced cytochrome c at the immobilized electrode. The results are expressed as rates of current produced per unit area of electrode, and are the mean of triplicate measurements. RESULTS AND DISCUSSION
The modification of the working electrode by N-ace@ cysteine occurs through the formation of a strong gold-thiol bond, leaving an exposed carboxylate group, and an ace@-protected amino group extending away from the electrode. At pH 7.0 the modified surface has a net negative charge, which leads to favourable electrostatic interactions with the positively charged lysines which surround the
270
haem group of cytochrome c. Figure l(a) shows a typical cyclic voltammogram of soluble cytochrome c in sodium phosphate buffer containing KC1 (pH 7.0), obtained at such a modified gold electrode. Figure l(b) shows the identical experiment using an unmodified gold electrode. The presence of carboxylate groups on the modified electrode surface promotes one-electron quasi-reversible electrochemistry of the soluble form of cytochrome c, with E1,2 = 25 mV/SCE and AE, = 60 mV/SCE at the modified electrode. As
-100 -50
0
II
1
E/mV
c.50 +lOO t150 t
'I
vs. SCE
Fig. 1. (a) A typical cyclic voltammogram for soluble cytochrome c (4.2 g dmm3) in 10 mm01 dme3 sodium phosphate buffer containing 10 mm01 dmm3 KC1 (pH 7.0) obtained at an N-ace@ cysteine modified gold electrode; (b) identical experiment using a bare gold electrode. In both cases the potential at the working electrode was cycled between - 100 and + 150 mV/SCE at a scan rate of 200 mV s-l.
expected, the observed peak current Zp,a was proportional to ul’* (Z&U~/* = constant). These results compare very favourably with those published elsewhere ]3,lIl. Rinsing the electrode with buffer and repeating the experiment in the same solution of cytochrome c resulted in identical current-voltage responses, demonstrating the strength of the gold-modifier thiol bond. However, in contrast with a previous study using alkanethiol gold modifiers [8], there was no evidence of persistent redox electrochemistry in the absence of the bulk solution of cytochrome c. This indicated that the protein did not interact irreversibly with the carboxylate groups of the surface modifier to become physically adsorbed to the electrode surface. Cytochrome c was immobilized at the modified gold electrode using a carbodiimide initiated condensation procedure. After covalent attachment, the electrode was thoroughly washed and the electrochemistry of the immobilized protein was investigated in sodium phosphate buffers (pH 4.5-7.0) and Tris buffers (pH 7.0-8.0) containing 10 mmol dme3 KC1 using cyclic voltammetry at scan rates of 5-50 mV s-l. It was observed that maximum peak currents Zp,a and Zpc were obtained in sodium phosphate buffer at pH 7.0. Figure 2 shows cyclic voltammograms at the cytochrome c-N-ace@ cysteine electrode in sodium phosphate buffer (pH 7.0) over the specified range of scan rates. The formal potential E”’ of cytochrome c at the electrode was calculated using Laviron’s method [12] as + 2 mV. This value was 23 mV negative of the El,* of cytochrome c at the same modified electrode in solution. Our observation of a negative shift of 23 mV is less than the negative shift of 60-70 mV which has been observed for adsorbed cytochrome c at alkanethiol monolayers [8], but is in close agreement with the value for cytochrome c bound to tin oxide electrodes [13]. Previously, electrochemical rate constants k,, have been estimated from surface voltammetric experiments for simple reversible redox systems [12]. This model has also been applied to the quasi-reversible electrochemistry of proteins, including cytochrome c 181, although in practice the analysis does not account for any departure from the ideal wave behaviour predicted at a Langmuir adsorption isotherm (as is assumed by Laviron). Tarlov and Bowden [8] made no allowance for the possibility that multiple forms of cytochrome c may exist at different orientations and distances from the electrode. The Laviron model was considered to be the best simple analysis for the calculation of electron-transfer rate constants for immobilized redox proteins. Consequently, we have applied this analysis to our results to give a best estimate for the electron-transfer rate constant. The voltammograms that we have obtained (Fig. 2) exhibited quasi-reversible electrochemistry, with an increasing AE, with increasing scan rate. The oxidation and reduction waves were characterized by broad peaks with a large background current which were attributed to either non-faradaic electrode charging or a faradaic component as a result of redox reactions within the general protein structure (e.g. C=C oxidation). In either case, without knowing the absolute size of the background current the precise determination of AE, was difficult, and the
272
VJ)
P
I 50nA
-150-100-50 11 1
0 +50 +lOO 1 'I
E/mV vs. SCE Fig. 2. Cyclic voltammograms at the immobilized cytochrome c N-acetyl cysteine electrode in 10 mmol dm-’ sodium phosphate buffer (pH 7.0) at scan rates of (a) 5, (b) 10, Cc) 20 and Cd) 50 mV s-‘. The potential at the working electrode was cycled between - 150 and + 100 mV/SCE.
result was that calculation of the transfer coefficient (which should equal 0.5 for a fully reversible system) was inaccurate. The asymmetry of the voltammetric scan, observed in Fig. 2, may have been caused by structural rearrangement in the molecule between the reduced and the oxidized forms of the protein, or more probably was caused by the fact that the immobilized cytochrome c did not form a homogeneous monolayer at the electrode surface. Given the inherent limitations of the application of Laviron’s model to bioelectrochemical systems similar to the one discussed here, as well as elsewhere [8], we have estimated an electron-transfer constant for immobilized cytochrome c as k,, 3.4 f 1.2 s-r (under the limiting case AE, 200/n mV), depending upon the range of transfer coefficients chosen. This constant was larger than that for adsorbed cytochrome c, where k,, was calculated as 0.1 s-l [81. The low electron-transfer rates at the alkanethiol-modified electrode can be expected because of the large
213
electron-transfer distance (CH,),, n = 15, in the unconjugated modifier prior to detection at the electrode. Our result obtained in this study was also compared with k,, = 30 f 5 s-l obtained using a diffusionless analysis for cytochrome c (in solution) adsorbed at a bipyridyl-modified gold electrode [3]. A more precise determination of k,, for the immobilized cytochrome c electrode will require the use of potential step methods or impedance spectroscopy. The results that we have presented suggest that cytochrome c is covalently immobilized via the bridging molecule in an orientation which enables a direct electrostatic interaction between the protein’s exposed haem edge and the N-acetyl cysteine modified electrode. N-acetyl cysteine has a dual role as both the electrode modifier and the bridging molecule. We believe that it is probable that there is
012
0:3
0:4
0:s
[X00] / umol dmm3 Fig. 3. Plot of the rate of current density measured by the immobilized cytochrome c electrode, j (PA mitt-’ cm- *) against the rate of production of superoxide. The rate of superoxide anion generation was varied by changing the concentration of xanthine oxidase [XOD] in the presence of saturating xanthine. A linear calibration curve was obtained, y = 2.2 PA mitt-’ crne2 prnol-t dm3 +0.016 PA cm-* mitt-‘, r = 0.99.
274
direct electron transport from the haem group to the electrode through the bridging molecule. The immobilized cytochrome c electrode can be used as a sensor for the amperometric detection of the superoxide radical. Superoxide is generated by the partial (one-electron) reduction of dioxygen. It is a highly reactive species which can act as both a reducing and an oxidizing agent in biological systems. In aqueous solution its redox capability has been suggested as the cause of perpetuation of inflammation and of tissue damage in disease states, including rheumatoid arthritis [14]. Therefore there is a need for such a biosensor as a research tool for in-vivo measurements in order to attribute tissue damage causally to the superoxide anion. The high reactivity of superoxide and its short life mean that ex vivo it is only possible to measure the presumed biproducts of superoxide-mediated reactions. Cytochrome c has previously been used in spectrophotometric assays for the measurement of production of superoxide radicals [15]. MoreZ recently, an electrochemical assay based upon the amperometric re-oxidation of soluble cytochrome c has been described [16]. The amperometric assay described in this paper is an adaptation of this work, but is based upon the superoxide-specific reduction of immobilized cytochrome c and its re-oxidation at the electrode surface at +200 mV/SCE. In Fig. 3 we present initial results which show a linear calibration for the rate of production of superoxide by xanthine oxidase in the presence of saturating xanthine over the range O-O.48 pmol dmm3 XOD according to the relationship y = 2.2 PA min-’ cm-’ pmol-’ dm3 + 0.01 I.LA cm-’ min-’ (r = 0.99). Work is currently in progress to develop a solid state superoxide ion sensor for the measurement of free-radical production and cellular activity in disease processes 1171. ACKNOWLEDGEMENT
The authors are grateful to the Medical Research research.
Council for funding this
REFERENCES 1 T.L. Poulos, ST. Freer, R.A. Alden, S.L. Edwards, U. Skogland, K. Takio, B. Eriksson, N. Xuong, T. Yonetani and J. Kraut, J. Biol. Chem., 255 (1980) 575. 2 M.J. Eddowes and H.A.O. Hill, J. Chem. Sot. Chem. Commun. (1977) 771. 3 W.J. Albery, M.J. Eddowes, H.A.O. Hill and A.R. Hillman J. Am. Chem. Sot., 103 (1981) 3904. 4 K. Di Gleria, H.A.O. Hill, V.J. Lowe and D.J. Page, J. Electroanal. Chem., 213 (1986) 333. 5 P.D. Barker, K. Di Gleria, H.A.O. Hill and V. Lowe, Eur. J. Biochem., 190 (1990) 171. 6 F.A. Armstrong, H.A.O. Hill and B.N. Oliver, J. Chem. Sot. Chem. Commun. (1984) 976. 7 P.N. Bartlett and J. Farington, J. Electroanal. Chem, 261 (1989) 471. 8 M.J. Tarlov and E.F. Bowden, J. Am. Chem. Sot. 113 (1991) 1847. 9 E. Margoliash and N. Frohwirt, B&hem. J., 71, (1959) 570.
275 10 C.J. McNeil, J.A. Spoors, J.M. Cooper, K.G.M.M. Alberti and W.H. Mullen, Anal. Chim. Acta, 237 (1990) 99. 11 R.E. Dickerson, R. Timkovich and P.D. Boyer in P.D. Boyer (Ed.), The Enzymes, Vol. XI A, Academic Press, New York, 1975, p. 397. 12 E. Laviron, J. Electroanal. Chem, 101 (1979) 19. 13 J.L. Willet and E.F. Bowden, J. Phys. Chem., 94 (1990) 8241. 14 P. Halliwell, J.M.C. Gutteridge and D.R. Blake, Philos. Trans. R. Sot. London, 311 (1985) 659. 15 P.L. Vandewalle and N.O. Petersen, FEBS Lett., 210 (1987)195. 16 C.J. McNeil, K.A. Smith, P. Bellavite and J.V. Bannister, Free Radical Res. Commun., 7 (1989) 89. 17 C.J. McNeil, K.R. Greenough, P.A. Weeks, C.H. Self and J.M. Cooper, Free Radical Res. Commun. In press (1992).
J. Ekctroanal.
Chem., 347 (1993) 267-275
Elsevier Sequoia S.A., Lausanne
JEC 02442
Direct electron transfer reactions between immobilized cytochrome c and modified gold electrodes Jonathan M. Cooper Department
of Electronics
and Electrical Engineering,
Katharine R. Greenough
University of Glasgow, Glasgow G12 SQQ (UK)
and Calum J. McNeil
Department of Clinical Biochemktry, upon Tyne NE2 4HH &JK)
The Medical School, University of Newcastle
upon Tyne, Newcastle
(Received 2 July 1992; in revised form 25 August 1992)
Abstract In this paper we present data showing quasi-reversible electrochemistry of soluble cytochrome c using the gold electrode modifier N-ace@ cysteine (E1,2 = 2.5 mV, AE, = 60 mV, Ir,+ / vl/* = constant). Cytochrome c was subsequently immobilized at this modified electrode using a carbodiimide condensation reaction. The electron-transfer rate between the immobilized protein and the gold electrode was estimated as 3.4f 1.2 s-’ and the formal potential E”’ of the immobilized protein electrode was calculated as 2 mV/SCE. We also present data showing an application for the immobilized cytochrome c electrode as a sensor for the specific measurement of superoxide radical production.
INTRODUCTION
A study of electron transfer between proteins first published over a decade ago has provided us with an elegant model for the modification of electrodes in order to investigate the electrochemistry of proteins. In this study Poulos et al. [l] reported the detailed molecular organization between cytochrome c peroxidase (CcP> and the small soluble protein cytochrome c, and explained the enzyme-substrate interaction in terms of charge matching, spatial fitting and hydrogen bonding. Their investigations showed that a ring of basic lysines, which are positively charged at physiological pH, extend away from the surface of cytochrome c to form salt bridges with a group of complimentary negatively charged carboxylate groups of aspartate residues in CcP. The electrostatic interaction draws the two molecules together so that the two haem groups lie parallel, only 25 A apart, thus facilitating electron transfer. 0022-0728/93/$06.00
0 1993 - Elsevier Sequoia S.A. All rights
reserved
268
This biophysical model provides an explanation for the mechanism by which the direct electrochemistry of soluble cytochrome c can be measured at modified electrodes. Previously, in a series of elegant experiments Hill and coworkers [2-51 have demonstrated that electron transfer with cytochrome c can be achieved using suitably modified gold electrodes. Subsequently, a variety of electrode surfaces have been prepared with a net negative charge using several different techniques, including polishing [6] and electropolymerization [7]. Most recently, this approach has been used successfully for the study of the direct electrochemistry of adsorbed cytochrome c at self-assembled alkanethiol monolayers on gold electrodes [81. In this paper, we report on the electrochemistry of soluble cytochrome c at a modified gold electrode suitable for the covalent attachment of the protein using carbodiimide condensation. In addition, we demonstrate the direct electrochemistry of the immobilized protein. The significance of achieving direct electron transfer between an immobilized protein and an electrode is that there is potential for developing “solid-state” devices which could have applications in either molecular electronics or the fabrication of molecular sensors. To this end, we report some preliminary results which show a possible future application for this device as a sensor for the production of superoxide radicals in vivo. EXPERIMENTAL
Horse heart cytochrome c (Sigma, type VI) was repurified by CM 3ZSephadex cation exchange chromatography (Pharmacia), and was dialysed exhaustively against 10 mm01 dmv3 sodium phosphate buffer (pH 7.0). The protein concentration was estimated spectrophotometrically using a molar absorption coefficient of 106.1 cm-’ at 410 nm [9]. All aqueous solutions were prepared using double-distilled reverse osmosis water. Prior to each experiment, gold electrodes (2 mm in diameter) were cleaned using a 0.3 pm alumina slurry (BDH) and were sonicated in distilled water for 30 s. The electrodes were modified by immersion in a 2 mm01 dmm3 solution of N-acetyl cysteine (Sigma) in 10 mm01 dmm3 sodium phosphate buffer (pH 7.0) for 2 h, and were then washed in distilled water. All experiments investigating the electrochemistry of soluble cytochrome c were performed using these electrodes. Cytochrome c was immobilized at the modified electrodes using the water-soluble condensing reagent l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, (EDC) (Sigma). The N-ace@ cysteine modified electrode was first incubated in a 10% (w/v) solution of EDC in water for 2 h at 25”C, and was then washed in water. The EDC-activated electrode was then placed in a solution of cytochrome c (4.2 g dmm3) in 10 mm01 dmm3 sodium phosphate buffer (pH 7.0) at 4°C for 6 h. The electrode was finally washed exhaustively with 10 mmol dmm3 sodium phosphate buffer (pH 7.0). All electrochemical experiments investigating the electrochemistry of the soluble and the immobilized protein were carried out in triplicate at 25°C in a conventional two-compartment three-electrode system using a saturated calomel
269
reference electrode (SCE) linked to the working compartment by a Luggin capillary (ca. 0.1 rnm2). The working compartment contained a 1 cm2 platinum gauze counter-electrode and the gold working electrode. The potentiostat was built by oxford Instruments and was used with a Linseis LY1600 X-Y-f chart recorder. Electron-transfer reactions were investigated using cyclic voltammetry over the potential range - 100 to + 150 mV/SCE for the soluble protein at scan rates u between 5 and 200 mV s-l and over the range - 150 to + 100 mV/SCE for the immobilized protein at scan rates between 5 and 50 mV s-l. Tris[hydroxymethyl]aminomethane/HCl (Tris) and sodium phosphate buffers (10 mmol dme3) containing 10 mmol dme3 KC1 over a pH range 4.5-8.0 were used as the supporting electrolyte. Electroanalytical experiments for the detection of the superoxide anion 0; were performed using a purpose-built four-electrode electrochemical cell and bipotentiostat with a Goerz Metrawatt SE120 chart recorder, as described previously [lo]. The electrochemical cell contained two gold electrodes (working and background, each 1.5 mm in diameter), a platinum counter-electrode (1.5 mm diameter) and an Ag/AgCl reference electrode (2 mm in diameter). The current output from the bipotentiostat was measured as the background current subtracted from the sample current. The gold working electrode was modified with immobilized cytochrome c, as described above, whilst the background electrode was modified using the same methods but with a solution of 5 g dmv3 human serum albumin (Sigma) in place of the cytochrome c. Both the working electrode and the background electrode were poised at +200 mV vs. Ag/AgCl. Superoxide was produced using a xanthine/ xanthine oxidase generating system in 10 mm01 dme3 Tris buffer (pH 7.5) containing 10 mmol dmm3 KCI. Xanthine oxidase (XOD) (Grade III, Sigma) from buttermilk was dialysed exhaustively against the working buffer to remove the salicylate (preservative), which is a potential electroactive interferent. A stock solution of 14.4 mm01 dme3 xanthine (Sigma) was made up freshly in 2 mmol dme3 KOH and diluted-into 10 mmol dmW3 Tris buffer (pH 7.0) to give a 350 kmol dmP3 working solution. The reaction was initiated by the addition of XOD to give a final enzyme concentration in the range O-O.48 pm01 dma3. The response at the electrode was measured as the initial (linear) rate of current generated by the re-oxidation of reduced cytochrome c at the immobilized electrode. The results are expressed as rates of current produced per unit area of electrode, and are the mean of triplicate measurements. RESULTS AND DISCUSSION
The modification of the working electrode by N-ace@ cysteine occurs through the formation of a strong gold-thiol bond, leaving an exposed carboxylate group, and an ace@-protected amino group extending away from the electrode. At pH 7.0 the modified surface has a net negative charge, which leads to favourable electrostatic interactions with the positively charged lysines which surround the
270
haem group of cytochrome c. Figure l(a) shows a typical cyclic voltammogram of soluble cytochrome c in sodium phosphate buffer containing KC1 (pH 7.0), obtained at such a modified gold electrode. Figure l(b) shows the identical experiment using an unmodified gold electrode. The presence of carboxylate groups on the modified electrode surface promotes one-electron quasi-reversible electrochemistry of the soluble form of cytochrome c, with E1,2 = 25 mV/SCE and AE, = 60 mV/SCE at the modified electrode. As
-100 -50
0
II
1
E/mV
c.50 +lOO t150 t
'I
vs. SCE
Fig. 1. (a) A typical cyclic voltammogram for soluble cytochrome c (4.2 g dmm3) in 10 mm01 dme3 sodium phosphate buffer containing 10 mm01 dmm3 KC1 (pH 7.0) obtained at an N-ace@ cysteine modified gold electrode; (b) identical experiment using a bare gold electrode. In both cases the potential at the working electrode was cycled between - 100 and + 150 mV/SCE at a scan rate of 200 mV s-l.
expected, the observed peak current Zp,a was proportional to ul’* (Z&U~/* = constant). These results compare very favourably with those published elsewhere ]3,lIl. Rinsing the electrode with buffer and repeating the experiment in the same solution of cytochrome c resulted in identical current-voltage responses, demonstrating the strength of the gold-modifier thiol bond. However, in contrast with a previous study using alkanethiol gold modifiers [8], there was no evidence of persistent redox electrochemistry in the absence of the bulk solution of cytochrome c. This indicated that the protein did not interact irreversibly with the carboxylate groups of the surface modifier to become physically adsorbed to the electrode surface. Cytochrome c was immobilized at the modified gold electrode using a carbodiimide initiated condensation procedure. After covalent attachment, the electrode was thoroughly washed and the electrochemistry of the immobilized protein was investigated in sodium phosphate buffers (pH 4.5-7.0) and Tris buffers (pH 7.0-8.0) containing 10 mmol dme3 KC1 using cyclic voltammetry at scan rates of 5-50 mV s-l. It was observed that maximum peak currents Zp,a and Zpc were obtained in sodium phosphate buffer at pH 7.0. Figure 2 shows cyclic voltammograms at the cytochrome c-N-ace@ cysteine electrode in sodium phosphate buffer (pH 7.0) over the specified range of scan rates. The formal potential E”’ of cytochrome c at the electrode was calculated using Laviron’s method [12] as + 2 mV. This value was 23 mV negative of the El,* of cytochrome c at the same modified electrode in solution. Our observation of a negative shift of 23 mV is less than the negative shift of 60-70 mV which has been observed for adsorbed cytochrome c at alkanethiol monolayers [8], but is in close agreement with the value for cytochrome c bound to tin oxide electrodes [13]. Previously, electrochemical rate constants k,, have been estimated from surface voltammetric experiments for simple reversible redox systems [12]. This model has also been applied to the quasi-reversible electrochemistry of proteins, including cytochrome c 181, although in practice the analysis does not account for any departure from the ideal wave behaviour predicted at a Langmuir adsorption isotherm (as is assumed by Laviron). Tarlov and Bowden [8] made no allowance for the possibility that multiple forms of cytochrome c may exist at different orientations and distances from the electrode. The Laviron model was considered to be the best simple analysis for the calculation of electron-transfer rate constants for immobilized redox proteins. Consequently, we have applied this analysis to our results to give a best estimate for the electron-transfer rate constant. The voltammograms that we have obtained (Fig. 2) exhibited quasi-reversible electrochemistry, with an increasing AE, with increasing scan rate. The oxidation and reduction waves were characterized by broad peaks with a large background current which were attributed to either non-faradaic electrode charging or a faradaic component as a result of redox reactions within the general protein structure (e.g. C=C oxidation). In either case, without knowing the absolute size of the background current the precise determination of AE, was difficult, and the
272
VJ)
P
I 50nA
-150-100-50 11 1
0 +50 +lOO 1 'I
E/mV vs. SCE Fig. 2. Cyclic voltammograms at the immobilized cytochrome c N-acetyl cysteine electrode in 10 mmol dm-’ sodium phosphate buffer (pH 7.0) at scan rates of (a) 5, (b) 10, Cc) 20 and Cd) 50 mV s-‘. The potential at the working electrode was cycled between - 150 and + 100 mV/SCE.
result was that calculation of the transfer coefficient (which should equal 0.5 for a fully reversible system) was inaccurate. The asymmetry of the voltammetric scan, observed in Fig. 2, may have been caused by structural rearrangement in the molecule between the reduced and the oxidized forms of the protein, or more probably was caused by the fact that the immobilized cytochrome c did not form a homogeneous monolayer at the electrode surface. Given the inherent limitations of the application of Laviron’s model to bioelectrochemical systems similar to the one discussed here, as well as elsewhere [8], we have estimated an electron-transfer constant for immobilized cytochrome c as k,, 3.4 f 1.2 s-r (under the limiting case AE, 200/n mV), depending upon the range of transfer coefficients chosen. This constant was larger than that for adsorbed cytochrome c, where k,, was calculated as 0.1 s-l [81. The low electron-transfer rates at the alkanethiol-modified electrode can be expected because of the large
213
electron-transfer distance (CH,),, n = 15, in the unconjugated modifier prior to detection at the electrode. Our result obtained in this study was also compared with k,, = 30 f 5 s-l obtained using a diffusionless analysis for cytochrome c (in solution) adsorbed at a bipyridyl-modified gold electrode [3]. A more precise determination of k,, for the immobilized cytochrome c electrode will require the use of potential step methods or impedance spectroscopy. The results that we have presented suggest that cytochrome c is covalently immobilized via the bridging molecule in an orientation which enables a direct electrostatic interaction between the protein’s exposed haem edge and the N-acetyl cysteine modified electrode. N-acetyl cysteine has a dual role as both the electrode modifier and the bridging molecule. We believe that it is probable that there is
012
0:3
0:4
0:s
[X00] / umol dmm3 Fig. 3. Plot of the rate of current density measured by the immobilized cytochrome c electrode, j (PA mitt-’ cm- *) against the rate of production of superoxide. The rate of superoxide anion generation was varied by changing the concentration of xanthine oxidase [XOD] in the presence of saturating xanthine. A linear calibration curve was obtained, y = 2.2 PA mitt-’ crne2 prnol-t dm3 +0.016 PA cm-* mitt-‘, r = 0.99.
274
direct electron transport from the haem group to the electrode through the bridging molecule. The immobilized cytochrome c electrode can be used as a sensor for the amperometric detection of the superoxide radical. Superoxide is generated by the partial (one-electron) reduction of dioxygen. It is a highly reactive species which can act as both a reducing and an oxidizing agent in biological systems. In aqueous solution its redox capability has been suggested as the cause of perpetuation of inflammation and of tissue damage in disease states, including rheumatoid arthritis [14]. Therefore there is a need for such a biosensor as a research tool for in-vivo measurements in order to attribute tissue damage causally to the superoxide anion. The high reactivity of superoxide and its short life mean that ex vivo it is only possible to measure the presumed biproducts of superoxide-mediated reactions. Cytochrome c has previously been used in spectrophotometric assays for the measurement of production of superoxide radicals [15]. MoreZ recently, an electrochemical assay based upon the amperometric re-oxidation of soluble cytochrome c has been described [16]. The amperometric assay described in this paper is an adaptation of this work, but is based upon the superoxide-specific reduction of immobilized cytochrome c and its re-oxidation at the electrode surface at +200 mV/SCE. In Fig. 3 we present initial results which show a linear calibration for the rate of production of superoxide by xanthine oxidase in the presence of saturating xanthine over the range O-O.48 pmol dmm3 XOD according to the relationship y = 2.2 PA min-’ cm-’ pmol-’ dm3 + 0.01 I.LA cm-’ min-’ (r = 0.99). Work is currently in progress to develop a solid state superoxide ion sensor for the measurement of free-radical production and cellular activity in disease processes 1171. ACKNOWLEDGEMENT
The authors are grateful to the Medical Research research.
Council for funding this
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