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Electrochemical transformations of proteins adsorbed at carbon electrodes
581
Bioelectrochemistry and Bioenergetics, 19 (1988) 581-584 A section of J. Electroanal. Chem., and constituting Vol. 253 (1988) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Short communication
Electrochemical electrodes
transformations
V.A. Bogdanovskaya
and M.R. Tarasevich
of proteins adsorbed at carbon
Frumkin Instiiute of Electrochemistry, Academy of Sciences of the U.S.S.R.,
Moscow (U.S.S.R.)
R. Hintsche and F. Scheller Central Institute of Molecular Biology, Academy of Sciences of the G.D.R., DDR-I I15 Berlin-Bach (G. D. R.) (Received 15 June 1987; in revised form 14 September 1987)
INTRODUCTION
The direct electron transfer between an electrode and the redox-active prosthetic group of enzymes is of interest both for the development of new types of biosensors or biofuel cells and for the fundamental understanding of biological processes. In the present paper the redox conversions of protein molecules such as cytochrome c, horse radish peroxidase (POD), lactase and glucose oxidase (GOD), adsorbed at compact carbon electrodes, are investigated by means of cyclic voltammetry. EXPERIMENTAL
The investigations were performed in acetate buffer, pH 5.5. Before the experiments, the carbon materials used (spectrally pure pyrolitic carbon, spectral powder soot with epoxy composit, spectral soot with polyurethane, isotropic pyrocarbon) were polished on paper with 600 coarse corundum, washed in buffer solution and electrochemically activated by sweeping in the potential range of 1.4 to -0.2 V. Before each measurement the I, U curve was recorded to make sure that there were no impurities. After the electrochemical activition the protein was adsorbed by dipping the electrode in 1 ml of the enzyme solution for 10 min. The activities in the solution were 20 u/ml (GOD), 180 u/ml POD) and 160 u/ml (lactase). RESULTS
With all the enzymes investigated at these carbon materials, redox processes have been found in the cyclic voltammograms. The positions of the peak potentials and 0302-4598/88/$03.50
0 1988 Elsevier Sequoia S.A.
582
4
3
-10
/
3 +_______
4,
z
? 1
_-
-
/
i
oc
G
~-~;r
-04
u/v
---___
_
__
/
+lOi
J
Fig. 1. Current-voltage curves, measured at a pyrocarbon electrode (1) and at a pyrocarbon electrode with adsorbed cytochrome c (2). Acetate buffer solution, pH 5.5; u = 0.01 V/s; Potential vs. SCE. Fig. 2. I, U curves, measured at pyrolytic (2) and denatured GOD (3) respectively.
carbon Acetate
(1) and at pyrolytic carbon with adsorbed buffer solution, pH 5.5; u = 0.05 V/s.
native GOD
the peak currents depend on the type of the adsorbed protein and the experimental conditions. Figure 1 shows current-voltage curves measured at a pyrolytic graphite electrode with adsorbed cytochrome c. A distinct maximum both on the negative and the positive sweep of the current-voltage curve was observed after pretreatment in the potential range of 1.9 to - 3.1 V. It can be supposed that as a result of such treatment the number of active centres on the electrode surface of the carbon material is increased resulting in an enhanced adsorption of cytochrome c. At potential sweep rates u < 10 mV/s the redox conversions of the protein occur close to the equilibrium potential of cytochrome c. The quantity of the adsorbed cytochrome c, measured by the quantity of electricity needed for its reduction at maximum coverage, corresponds approximately to one protein monolayer. Here it was assumed that the reduction of a protein molecule needs one electron, and the molecule covers an area of 20 nm2 [l]. Since the redox transformations of cytochrome c are practically reversible and proceed close to the protein redox potential, and the value of dU/d log u amounts to 0.06 V/s, the electron transfer is presumably not the limiting step. The slowest stage of the process may be conformational changes of cytochrome c, which are manifest, for instance, in the change of the molecule volume occurring in the transition from oxidized to reduced state and vice versa [2]. Peroxidase, a heme-containing enzyme, has been adsorbed at various carbon materials. In the Z, U curves, redox processes in the potential range of 0.18 to 0.20 V and at potentials of -0.33 to -0.25 V were observed. The position of the cathodic maximum is close to the value of the redox potential (Table 1) of the Fe3+/*+ transfer in the active centre of peroxidase [3]. When the peroxidase inhibitor potassium cyanide is introduced into the buffer solution, the extrema of the current-voltage curve disappear. This fact suggests that the cathodic maximum in the potential region of - 0.33 to - 0.25 V is related to the reduction of the enzyme’s
583 TABLE
1
Positions of cathodic peaks .!I,+ (in V vs. SCE) of enzymes adsorbed at coal electrodes. pH 5.5 Carbon material
enzyme
Pyrolytic carbon
none lactase POD GOD
Isotropic pyrocarbon
Spectral soot with polyuretane
_ 0.775 -0.310 - 0.305
_
_ 0.28 0.18 _
_
none lactase POD GOD
0.775 - 0.310 - 0.305
0.23 0.18 _
none lactase POD GOD
0.775 - 0.310 - 0.305
0.20 0.15 _
_
_ 0.18 - 0.33 - 0.265
_ 0.13 - 0.27 -0.38
_ 0.15 - 0.25 -0.38
active centre. The anodic peak in the range of 0.18 V to 0.20 V, analogously to other proteins, may be due to the oxidation of aromatic amino acids of a protein molecule [4,51. Adsorbed lactase shows in the potential range from 0.28 to 0.15 V weakly pronounced cathodic and corresponding anodic maximum, the shape and position of which practically do not depend on u between 0.01 to 0.100 V/s. The position of the peak potentials is far from the redox-potential of lactase in the conversion of low-molecular substrates (Table 1) and also to the potential of 0.76 V characterizing the redox conversions of lactase immobilized on a carbon electrode [6] or on an electrode modified by 2.9-dimethylphenanthroline [7]. Figure 2 shows I, U curves of an electrode with adsorbed glucose oxidase. The redox process occur at potentials of - 0.26 to - 0.38 V, a range which is close to the redox potential of glucose oxidase and almost coincides with the potential values described by Ikeda and co-workers [8] and Miyawaki and Wingard [9] for GOD adsorbed at a carbon material and by Iainniello et al. [lo] for covalently immobilized GOD, respectively. The peak current with glucose oxidase adsorbed at the electrode increases proportionally to the bulk concentration of the enzyme during adsorption. At the DME [ll] the dissociation of the prosthetic group (FAD) leads to a positive peak shift by 0.1-0.2 V as compared to the I, U curve of native GOD. When the GOD was denatured by 5 min boiling of the enzyme solution before adsorption at the coal electrode a redox process at more positive potentials is observed in the current-voltage curve (Fig. 2, curve 3). The current in the adsorbed glucose oxidase maximum increases proportionally to the solution concentration of the enzyme under adsorption. Comparing the data obtained by cyclic voltammetry one can conclude that the enzymes are adsorbed at the carbon materials used. This is manifested by the decrease of polarization capacitance of the electrodes in the I, U curves in the
584
potential range far from the redox processes. Moreover, redox transformations observed for all the proteins.
are
DISCUSSION
It is known from the literature that most of enzymes preserve their enzymatic activity after adsorption at carbon materials [12]. Therefore the adsorption of enzymes directly at the electrode surface may be used as a simple immobilization method. For instance, the coverage of the electrode with POD leads to an apparent activity of 12 u/cm2 whereas with GOD the activity reaches the value of 200 mu/cm2 [13]. These values are comparable to those obtained by covalent fixation of the enzymes at the electrode surface [12]. The nature of the electrochemical signals of oxidoreductases found in the absence of mediators and substrates is not yet clear. Probably the protein-bound prosthetic group is involved in the heterogeneous electron transfer, since these signals differ typically from the signals of the free prosthetic group and also from that of the apoenzyme. In the case of a rapid heterogeneous electron transfer, the addition of a substrate excess should result in the formation of a catalytic current. But no such effect has been found either in the literature [8.9] or in the present work. Ikeda et al. [S] postulated that only a small fraction of the adsorbed molecules, which is structurally changed and therefore enzymatically inactive, is involved in the direct electron transfer, whilst most of the molecules must be enzymatically active, but hidden, for the electron transfer. The results obtained by us suggest, however, that the entire monolayer of the adsorbed cytochrome c is involved in the electron transfer. REFERENCES 1 F. ScheIIer, M. JXnchen and H.-J. Priimke, Biopolymers, 14 (1975) 1553. 2 I. Taniguchi, M. Iseki, T. Eto, Bioelectrochem. Bioenerg., 12 (1984) 373. 3 G. Dryhurst, K. Kadish, F. ScheIIer and R. Renneberg, Biological Electrochemistry, Vol. 1, Academic Press, New York, London, 1982, p. 409. 4 J. Reynaud, B. Malfoy and A. Bert, Bioelectrochem. Bioenerg., 7 (1980) 695. 5 V. Brabec and J., Schindlerova, Bioelectrochem. Bioenerg., 8 (1981) 451. 6 A.I. Jaropolov and S.O. Batschurin, Itogi Nauki i Tekhniki. Biotekhnologija, Moscow, 1983, p. 195 (in Russian). 7 C. Lee, H. Gray, F. Ansen and B. MaImstrom, J. Electroanal. Chem., 172 (1984) 289. 8 T. Ikeda, J. Katosho, M. Kanei and M. Senda, Agric. Biol. Chem., 48 (1984) 1969. 9 0. Miyawaki and L. Wingard, Biotechnol. Bioeng., 26 (1984) 1364. 10 R. Iainniello, T. Lindsay and A. Yacynych, Anal. Chem., 54 (1982) 1098. 11 F. ScheIIer, G. Stmad, B. Neumann, M. Kuhn and W. Ostrowski, Bioelectochem. Bioenerg., 6 (1979) 117. 12 V. Razumas, J. Jasaitis and J. Kulys, Bioelectrochem. Bioenerg., 12 (1984) 297. 13 R. Hintsche and F. Scheller, Studia Biophys., 119 (1987) 179.
Bioelectrochemistry and Bioenergetics, 19 (1988) 581-584 A section of J. Electroanal. Chem., and constituting Vol. 253 (1988) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Short communication
Electrochemical electrodes
transformations
V.A. Bogdanovskaya
and M.R. Tarasevich
of proteins adsorbed at carbon
Frumkin Instiiute of Electrochemistry, Academy of Sciences of the U.S.S.R.,
Moscow (U.S.S.R.)
R. Hintsche and F. Scheller Central Institute of Molecular Biology, Academy of Sciences of the G.D.R., DDR-I I15 Berlin-Bach (G. D. R.) (Received 15 June 1987; in revised form 14 September 1987)
INTRODUCTION
The direct electron transfer between an electrode and the redox-active prosthetic group of enzymes is of interest both for the development of new types of biosensors or biofuel cells and for the fundamental understanding of biological processes. In the present paper the redox conversions of protein molecules such as cytochrome c, horse radish peroxidase (POD), lactase and glucose oxidase (GOD), adsorbed at compact carbon electrodes, are investigated by means of cyclic voltammetry. EXPERIMENTAL
The investigations were performed in acetate buffer, pH 5.5. Before the experiments, the carbon materials used (spectrally pure pyrolitic carbon, spectral powder soot with epoxy composit, spectral soot with polyurethane, isotropic pyrocarbon) were polished on paper with 600 coarse corundum, washed in buffer solution and electrochemically activated by sweeping in the potential range of 1.4 to -0.2 V. Before each measurement the I, U curve was recorded to make sure that there were no impurities. After the electrochemical activition the protein was adsorbed by dipping the electrode in 1 ml of the enzyme solution for 10 min. The activities in the solution were 20 u/ml (GOD), 180 u/ml POD) and 160 u/ml (lactase). RESULTS
With all the enzymes investigated at these carbon materials, redox processes have been found in the cyclic voltammograms. The positions of the peak potentials and 0302-4598/88/$03.50
0 1988 Elsevier Sequoia S.A.
582
4
3
-10
/
3 +_______
4,
z
? 1
_-
-
/
i
oc
G
~-~;r
-04
u/v
---___
_
__
/
+lOi
J
Fig. 1. Current-voltage curves, measured at a pyrocarbon electrode (1) and at a pyrocarbon electrode with adsorbed cytochrome c (2). Acetate buffer solution, pH 5.5; u = 0.01 V/s; Potential vs. SCE. Fig. 2. I, U curves, measured at pyrolytic (2) and denatured GOD (3) respectively.
carbon Acetate
(1) and at pyrolytic carbon with adsorbed buffer solution, pH 5.5; u = 0.05 V/s.
native GOD
the peak currents depend on the type of the adsorbed protein and the experimental conditions. Figure 1 shows current-voltage curves measured at a pyrolytic graphite electrode with adsorbed cytochrome c. A distinct maximum both on the negative and the positive sweep of the current-voltage curve was observed after pretreatment in the potential range of 1.9 to - 3.1 V. It can be supposed that as a result of such treatment the number of active centres on the electrode surface of the carbon material is increased resulting in an enhanced adsorption of cytochrome c. At potential sweep rates u < 10 mV/s the redox conversions of the protein occur close to the equilibrium potential of cytochrome c. The quantity of the adsorbed cytochrome c, measured by the quantity of electricity needed for its reduction at maximum coverage, corresponds approximately to one protein monolayer. Here it was assumed that the reduction of a protein molecule needs one electron, and the molecule covers an area of 20 nm2 [l]. Since the redox transformations of cytochrome c are practically reversible and proceed close to the protein redox potential, and the value of dU/d log u amounts to 0.06 V/s, the electron transfer is presumably not the limiting step. The slowest stage of the process may be conformational changes of cytochrome c, which are manifest, for instance, in the change of the molecule volume occurring in the transition from oxidized to reduced state and vice versa [2]. Peroxidase, a heme-containing enzyme, has been adsorbed at various carbon materials. In the Z, U curves, redox processes in the potential range of 0.18 to 0.20 V and at potentials of -0.33 to -0.25 V were observed. The position of the cathodic maximum is close to the value of the redox potential (Table 1) of the Fe3+/*+ transfer in the active centre of peroxidase [3]. When the peroxidase inhibitor potassium cyanide is introduced into the buffer solution, the extrema of the current-voltage curve disappear. This fact suggests that the cathodic maximum in the potential region of - 0.33 to - 0.25 V is related to the reduction of the enzyme’s
583 TABLE
1
Positions of cathodic peaks .!I,+ (in V vs. SCE) of enzymes adsorbed at coal electrodes. pH 5.5 Carbon material
enzyme
Pyrolytic carbon
none lactase POD GOD
Isotropic pyrocarbon
Spectral soot with polyuretane
_ 0.775 -0.310 - 0.305
_
_ 0.28 0.18 _
_
none lactase POD GOD
0.775 - 0.310 - 0.305
0.23 0.18 _
none lactase POD GOD
0.775 - 0.310 - 0.305
0.20 0.15 _
_
_ 0.18 - 0.33 - 0.265
_ 0.13 - 0.27 -0.38
_ 0.15 - 0.25 -0.38
active centre. The anodic peak in the range of 0.18 V to 0.20 V, analogously to other proteins, may be due to the oxidation of aromatic amino acids of a protein molecule [4,51. Adsorbed lactase shows in the potential range from 0.28 to 0.15 V weakly pronounced cathodic and corresponding anodic maximum, the shape and position of which practically do not depend on u between 0.01 to 0.100 V/s. The position of the peak potentials is far from the redox-potential of lactase in the conversion of low-molecular substrates (Table 1) and also to the potential of 0.76 V characterizing the redox conversions of lactase immobilized on a carbon electrode [6] or on an electrode modified by 2.9-dimethylphenanthroline [7]. Figure 2 shows I, U curves of an electrode with adsorbed glucose oxidase. The redox process occur at potentials of - 0.26 to - 0.38 V, a range which is close to the redox potential of glucose oxidase and almost coincides with the potential values described by Ikeda and co-workers [8] and Miyawaki and Wingard [9] for GOD adsorbed at a carbon material and by Iainniello et al. [lo] for covalently immobilized GOD, respectively. The peak current with glucose oxidase adsorbed at the electrode increases proportionally to the bulk concentration of the enzyme during adsorption. At the DME [ll] the dissociation of the prosthetic group (FAD) leads to a positive peak shift by 0.1-0.2 V as compared to the I, U curve of native GOD. When the GOD was denatured by 5 min boiling of the enzyme solution before adsorption at the coal electrode a redox process at more positive potentials is observed in the current-voltage curve (Fig. 2, curve 3). The current in the adsorbed glucose oxidase maximum increases proportionally to the solution concentration of the enzyme under adsorption. Comparing the data obtained by cyclic voltammetry one can conclude that the enzymes are adsorbed at the carbon materials used. This is manifested by the decrease of polarization capacitance of the electrodes in the I, U curves in the
584
potential range far from the redox processes. Moreover, redox transformations observed for all the proteins.
are
DISCUSSION
It is known from the literature that most of enzymes preserve their enzymatic activity after adsorption at carbon materials [12]. Therefore the adsorption of enzymes directly at the electrode surface may be used as a simple immobilization method. For instance, the coverage of the electrode with POD leads to an apparent activity of 12 u/cm2 whereas with GOD the activity reaches the value of 200 mu/cm2 [13]. These values are comparable to those obtained by covalent fixation of the enzymes at the electrode surface [12]. The nature of the electrochemical signals of oxidoreductases found in the absence of mediators and substrates is not yet clear. Probably the protein-bound prosthetic group is involved in the heterogeneous electron transfer, since these signals differ typically from the signals of the free prosthetic group and also from that of the apoenzyme. In the case of a rapid heterogeneous electron transfer, the addition of a substrate excess should result in the formation of a catalytic current. But no such effect has been found either in the literature [8.9] or in the present work. Ikeda et al. [S] postulated that only a small fraction of the adsorbed molecules, which is structurally changed and therefore enzymatically inactive, is involved in the direct electron transfer, whilst most of the molecules must be enzymatically active, but hidden, for the electron transfer. The results obtained by us suggest, however, that the entire monolayer of the adsorbed cytochrome c is involved in the electron transfer. REFERENCES 1 F. ScheIIer, M. JXnchen and H.-J. Priimke, Biopolymers, 14 (1975) 1553. 2 I. Taniguchi, M. Iseki, T. Eto, Bioelectrochem. Bioenerg., 12 (1984) 373. 3 G. Dryhurst, K. Kadish, F. ScheIIer and R. Renneberg, Biological Electrochemistry, Vol. 1, Academic Press, New York, London, 1982, p. 409. 4 J. Reynaud, B. Malfoy and A. Bert, Bioelectrochem. Bioenerg., 7 (1980) 695. 5 V. Brabec and J., Schindlerova, Bioelectrochem. Bioenerg., 8 (1981) 451. 6 A.I. Jaropolov and S.O. Batschurin, Itogi Nauki i Tekhniki. Biotekhnologija, Moscow, 1983, p. 195 (in Russian). 7 C. Lee, H. Gray, F. Ansen and B. MaImstrom, J. Electroanal. Chem., 172 (1984) 289. 8 T. Ikeda, J. Katosho, M. Kanei and M. Senda, Agric. Biol. Chem., 48 (1984) 1969. 9 0. Miyawaki and L. Wingard, Biotechnol. Bioeng., 26 (1984) 1364. 10 R. Iainniello, T. Lindsay and A. Yacynych, Anal. Chem., 54 (1982) 1098. 11 F. ScheIIer, G. Stmad, B. Neumann, M. Kuhn and W. Ostrowski, Bioelectochem. Bioenerg., 6 (1979) 117. 12 V. Razumas, J. Jasaitis and J. Kulys, Bioelectrochem. Bioenerg., 12 (1984) 297. 13 R. Hintsche and F. Scheller, Studia Biophys., 119 (1987) 179.