Journal of Biochemical and Biophysical Methods, 20 (1990) 137-142
137
Elsevier JBBM 00787
A new electrochemical method for the production of stable ascorbate free radicals Ahmet Onal t, Ay O~,fi§ 2 and Duygu Kasakiirek 3 i Department of Science, Middle-East Technical University, 06531, Ankara, Turkey, " Department of Biology, Hacettepe University, Ankara, Turkey and ~ Department of Chemistry, Middle.East Technical University, Ankara, 7~rkey
(Received 7 June 1989) (Accepted 4 October 1~89)
Summary A new method for the production of ascorbate free radicals is established. The radical is produced from ascorbate in deionized water by applying constant potential electrolysis under a nitrogen atmosphere. Prior to electrolysis, a cyclic voltammogram (CV) of the ascorbic acid was obtained. Electrolysis potentials were selected as the oxidation peak potential of the ascorbic acid obtained by CV. The detection of the radical was done by electron spin resonance (esr) and uv spectroscopies. Key words: Ascorbate free radical; Electrolysis; Esr determination
Introduction The participation of vitamin C in electron transport reactions was posited 58 years ago [1]. It has been shown that ascorbic acid is a bivalent oxidation-reduction molecule and can reduce electron acceptors by two successive, one-electron steps. Thus ascorbic acid acts via a free radical intermediate called 'ascorbate free radical' (AFR) [1-3]. Dehydroascorbate (DHA) is the final product in this system. The involvement of AFR in the physiological role of ascorbate, especially in the DNA-scission activities of ascorbate in the presence of metal chelates and in the inactivation of proteins [4-6] should not be overlooked. In this view, it is important Correspondence address: A. Onal, Department of Science, Middle-East Technical University, 06531, Ankara, Turkey. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
138
to produce and study the characteristics of AFR in a simple system devoid of several chemical compounds and radicals. Several methods for the production of AFR have been reported in the literature. In some studies AFR have been generated by the enzymatic oxidation of ascorbic acid by ascorbate oxidase and dopamine-[~-hydroxylase [7]. Also, chemical oxidation of ascorbic acid by trace metals [8] and by several electron accepters [2] has been reported. The generation of AFR by pulse radiolysis has been studied thoroughly by Bielski et al. [1]. AFR produced by comproportionation of ascorbate and dehydroascorbate has been used as a substrate in the semidehydroascorbate assay [9]. However, the production of the radical in this way has been reported to be thermodynamically unfeasable [10]. In this study, we produced AFR in deionized water by constant potential electrolysis under a nitrogen atmosphere. The electrochemical behavior of ascorbic acid was studied by using a cyclic voltammogram (CV) technique to determine the oxidation potential at which the ascorbic acid radical was produced. Spectra of ascorbic acid free radicals generated in deionized water by electrolysis were determined by esr and confirmed by uv spectroscopies. The advantage of producing the AFR by electrolysis is that the intervening oxygen radicals, especially .OH produced by pulse radiolysis and chemical methods, are absent in this system. The effect of a buffer is also eliminated and no oxidizing chemical agent is used. Another advantage is that one can produce the radical continously. Hence, the high intensity esr signal obtained from the stable AFR is very convenient for studying the biological factors enhancing and/or decreasing the intensity of the radical.
Materials and Methods
Electrochemical method A potentiostat-function generator couple was used to programme the potential. The recordings were made with a YEW XY/YZ recorder during CV studies. The electrolysis cell consisted of a Pt-bead working electrode, Pt-wire counter electrode and a saturated calomel electrode (SCE) as reference electrode (Fig. la). Cyclic voltammograms were taken under a nitrogen atmosphere in deionized water at room temperature. Reagent-grade sodium perchlorate (10 mM) was used as the supporting electrolyte both for the CV and for the constant potential electrolysis of ascorbic acid (5 mM) with a measured pH of 3.4. A varian-E band esr spectrometer was used to detect AFR. For this purpose an in situ electrolysis flat cell was fitted with two Pt-wires, one from the top as the working electrode and the other from the bottom as the counter electrode. Silver wire, used as a reference electrode, was also inserted from the top (Fig. l b). Uv spectra were taken by a Shimadsu UV-160 UV-VIS recording spectrophotometer.
139
Results and Discussion
The ascorbic acid radical was produced electrochemically via direct electron transfer at constant potential in a deionized water-sodium perchlorate solventelectrolyte couple. The oxidation peak potentials were determined prior to electrolysis in the same solvent-electrolyte couple at room temperature under a nitrogen atmosphere by cyclic voltammetry. The CV spectrum of the ascorbic acid is given in (Fig. 2). As can be seen in Fig. 2, the ascorbic acid has two oxidation and one reduction peak potentials at +0.40, - 0 . 4 0 and -0.55 V vs. SCE. The chosen solvent-electrolyte couple was inert between - 0 . 8 0 and + 1.5 V at room temperature. The scanning in either direction did not change the number and the shapes of oxidation and reduction peaks. It has been reported [3] that ascorbic acid/ascorbate could undergo a reversible Michaelis two-step oxidation-reduction process with a free radical as the inter.. mediate, as given below:
-le
AH 2 ~ A F R - + 2H +
(1)
-le
AFR- ~ DHA
(2)
The first oxidation peak at +0.4 V can be assigned to step 1 in the above~ reaction. The absence of a reversible reduction peak of +0.4 V, however, indicates that AFR produced at this potential were converted either to ascorbic acid or to DHA at a rate higher than the scan rate. The reversible peak observed at - 0 . 4 V can be assigned to reaction step 2.
Counter .~;#- Working Electrode j ~ Electrode /: Moteriol ~1~ ~
co..
','i
~'
Ref~ence E~e~ir~ i ~ ''~ Electrode
Reference Electrode
Countf.r Electrode
,--.
WorkingEleclrode (o)
(b)
Fig. 1. (a) Cyclic voltammetry cell; (b) flat cell used for in situ electrolysis.
140
i
1.00
,
- iO
Fig. 2. Cyclic voltammogram of ascorbic acid in deionized water (0.01 M NaCIO4) at 25°C with scan rate of 200 mV/s.
The effect of the voltage scan rate on the peak currents was also studied, and current function (Ip/cV I/2) was plotted vs. log V, where Ip is the peak current, c is the concentration and V is the voltage scan rate. A negative slope was obtained (Fig. 3a) for the first oxidation peak at + 0.4 V vs. SCE, which indicates a reversible one-electron transfer followed by a chemical reaction according to the Nichelson-Shain criteria [11]. This result also confirms the lack of a reversible peak at + 0.40 V in Fig. 2. The current function did not change with the voltage scan rate for the reduction peak at -0.55 V vs. SCE, which indicates a reversible one-electron transfer according to the Nichelson-Shain criteria (Fig. 3b). The esr spectrum (Fig. 4) is produced by the electrolysis of ascorbic acid at + 0.40 V by constant potential electrolysis in a fiat quartz cell fitted into the esr cavity. For esr studies, the initial concentration of ascorbic acid was always 5 mM. Radical spectra appeared immediately after application of a + 0.40 V potential. The signal intensity stayed constant during the electrolysis. The same hyperfine parameter (1.76) of the esr signal was observed, as reported previously [2-3]. However, the signal intensity was increased after the electrolysis was stopped. Then the rapid decay of the enhanced signal took place in a few seconds and it was followed by a slow decay (30 min). The increase in radical intensity, therefore, could be related to the previously reported mechanism [12]. Conclusive evidence for the generation of AFR by constant potential electrolysis was confirmed by recording the signal with uv spectrometry.
141
._: :~ 7
~ d
6
5 (b)
I
1,4
i
1.6
I
1,8
i
i
I
I
2.0
22
2,4
26
Io9 (V roWS)
Fig, 3. Variation of current function with respect to voltage scan rate: (a) at Ep, a +0.40 V VS. SCE; (b) at Ep,c -0.55 V vs. SCE.
In view of these observations: (a) this method eliminates the presence of reactive primary radicals produced during the pulse radiolysis studies; (b) this method eliminates the effect of buffers and ascorbate oxidizing chemicals; (c) the AFR formed is stable and can be produced continously with high intensity; (d) the system is more suitable for studying the effects of biological factors that influence the intensity of the radicals.
2 Gouss Fig. 4. Esr spectrum of AFR obtained during the electrolysis of 5 mM ascorbic acid at +0.4 V vs. Ag/Ag +.
142 Simplified description of the method Ascorbate free radical is produced from ascorbate in deionized water by constant potential electrolysis and detected by esr and uv spectroscopies. The radical produced in this simple chemical environment is convenient for further chemical and biological characterizations.
Acknowledgement We are grateful to the M E T U R e s e a r c h F u n d for its p a r t i a l s u p p o r t of this work.
References 1 Bielski, B.H.J., Comstock, D.A. and Bown, A.R. (1971) Ascorbic acid free radicals. J. Am. Chem. Soc. 93, 5624-5629. 2 lyanagi, T., Yamaz.',ld, I. and Anan, K.F. (1985) One-electron oxidation-reduction properties of ascorbic acid. Biochim. Biophys. Acta. 806, 255-261. 3 Bielski, B.H.J. (1982) Chemistry of ascorbate radicals, in: Seib P.A. and Tolbert B.M. (Eds.), Ascorbic Acid: Chemistry, Metabolism and Uses, American Chem. Soc., Washington DC, pp 81-100. 4 Shinart, E., Navok, T. and Chevion, M. (1983) The analogous mechanisms of enzymatic inactivation induced by ascorbate and superoxide in the presence of copper J. Biol. Chem. 258, 14778-14783. 5 Chiou, S.H. (1983) DNA and protein.scission acti,,ities of copper ion and a copper-peptide complex. J. Biochem. 94, 1259-1267. 6 Chiou, S.H. (1984) DNA-scission activities of ascorbate in the presence of metal chelates. J. Biochem. 96, 1307-1310. 7 Skotland, T. and Jones, L. (1980) Direct spectrophotometri¢ detection of ascorbate free radical formed by dopamine-beta-monooxygenase and by ascorbate oxidase. Biochim. Biophys. Acta. 630, 30-35. 8 HaUiwell, B. and Foyer, C.H. (1976) Ascorbic acid, metal ions and the superoxide radical. Biochem. J. 155, 697-700. 9 Diliberto, E.J., Grace, D., Charles, C., Allen, P.A. and Pamela, L. (1982) Tissue subcellular and submitochondrial distributions of semidehydroascorbate reductase. J. Neurochem. 39, 563-568. 10 Kakes, W.H. (1981) Is there an equilibrium between ascorbic acid and dehydroascorbic acid? Z. Naturforsch. 36, 1088-1090. 11 Fry, A.J. (1972) Synthetic Organic Electrochemistry. Harper and Row Publishers, New York, NY, pp. 88-93. 12 Bielski, B.H.C., Allen, O.A. and Schwan, HA. (1981) Mechanism of disproportination of ascorbate radicals. J. Am. Chem. Soc. 103, 3516-3518.
137
Elsevier JBBM 00787
A new electrochemical method for the production of stable ascorbate free radicals Ahmet Onal t, Ay O~,fi§ 2 and Duygu Kasakiirek 3 i Department of Science, Middle-East Technical University, 06531, Ankara, Turkey, " Department of Biology, Hacettepe University, Ankara, Turkey and ~ Department of Chemistry, Middle.East Technical University, Ankara, 7~rkey
(Received 7 June 1989) (Accepted 4 October 1~89)
Summary A new method for the production of ascorbate free radicals is established. The radical is produced from ascorbate in deionized water by applying constant potential electrolysis under a nitrogen atmosphere. Prior to electrolysis, a cyclic voltammogram (CV) of the ascorbic acid was obtained. Electrolysis potentials were selected as the oxidation peak potential of the ascorbic acid obtained by CV. The detection of the radical was done by electron spin resonance (esr) and uv spectroscopies. Key words: Ascorbate free radical; Electrolysis; Esr determination
Introduction The participation of vitamin C in electron transport reactions was posited 58 years ago [1]. It has been shown that ascorbic acid is a bivalent oxidation-reduction molecule and can reduce electron acceptors by two successive, one-electron steps. Thus ascorbic acid acts via a free radical intermediate called 'ascorbate free radical' (AFR) [1-3]. Dehydroascorbate (DHA) is the final product in this system. The involvement of AFR in the physiological role of ascorbate, especially in the DNA-scission activities of ascorbate in the presence of metal chelates and in the inactivation of proteins [4-6] should not be overlooked. In this view, it is important Correspondence address: A. Onal, Department of Science, Middle-East Technical University, 06531, Ankara, Turkey. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
138
to produce and study the characteristics of AFR in a simple system devoid of several chemical compounds and radicals. Several methods for the production of AFR have been reported in the literature. In some studies AFR have been generated by the enzymatic oxidation of ascorbic acid by ascorbate oxidase and dopamine-[~-hydroxylase [7]. Also, chemical oxidation of ascorbic acid by trace metals [8] and by several electron accepters [2] has been reported. The generation of AFR by pulse radiolysis has been studied thoroughly by Bielski et al. [1]. AFR produced by comproportionation of ascorbate and dehydroascorbate has been used as a substrate in the semidehydroascorbate assay [9]. However, the production of the radical in this way has been reported to be thermodynamically unfeasable [10]. In this study, we produced AFR in deionized water by constant potential electrolysis under a nitrogen atmosphere. The electrochemical behavior of ascorbic acid was studied by using a cyclic voltammogram (CV) technique to determine the oxidation potential at which the ascorbic acid radical was produced. Spectra of ascorbic acid free radicals generated in deionized water by electrolysis were determined by esr and confirmed by uv spectroscopies. The advantage of producing the AFR by electrolysis is that the intervening oxygen radicals, especially .OH produced by pulse radiolysis and chemical methods, are absent in this system. The effect of a buffer is also eliminated and no oxidizing chemical agent is used. Another advantage is that one can produce the radical continously. Hence, the high intensity esr signal obtained from the stable AFR is very convenient for studying the biological factors enhancing and/or decreasing the intensity of the radical.
Materials and Methods
Electrochemical method A potentiostat-function generator couple was used to programme the potential. The recordings were made with a YEW XY/YZ recorder during CV studies. The electrolysis cell consisted of a Pt-bead working electrode, Pt-wire counter electrode and a saturated calomel electrode (SCE) as reference electrode (Fig. la). Cyclic voltammograms were taken under a nitrogen atmosphere in deionized water at room temperature. Reagent-grade sodium perchlorate (10 mM) was used as the supporting electrolyte both for the CV and for the constant potential electrolysis of ascorbic acid (5 mM) with a measured pH of 3.4. A varian-E band esr spectrometer was used to detect AFR. For this purpose an in situ electrolysis flat cell was fitted with two Pt-wires, one from the top as the working electrode and the other from the bottom as the counter electrode. Silver wire, used as a reference electrode, was also inserted from the top (Fig. l b). Uv spectra were taken by a Shimadsu UV-160 UV-VIS recording spectrophotometer.
139
Results and Discussion
The ascorbic acid radical was produced electrochemically via direct electron transfer at constant potential in a deionized water-sodium perchlorate solventelectrolyte couple. The oxidation peak potentials were determined prior to electrolysis in the same solvent-electrolyte couple at room temperature under a nitrogen atmosphere by cyclic voltammetry. The CV spectrum of the ascorbic acid is given in (Fig. 2). As can be seen in Fig. 2, the ascorbic acid has two oxidation and one reduction peak potentials at +0.40, - 0 . 4 0 and -0.55 V vs. SCE. The chosen solvent-electrolyte couple was inert between - 0 . 8 0 and + 1.5 V at room temperature. The scanning in either direction did not change the number and the shapes of oxidation and reduction peaks. It has been reported [3] that ascorbic acid/ascorbate could undergo a reversible Michaelis two-step oxidation-reduction process with a free radical as the inter.. mediate, as given below:
-le
AH 2 ~ A F R - + 2H +
(1)
-le
AFR- ~ DHA
(2)
The first oxidation peak at +0.4 V can be assigned to step 1 in the above~ reaction. The absence of a reversible reduction peak of +0.4 V, however, indicates that AFR produced at this potential were converted either to ascorbic acid or to DHA at a rate higher than the scan rate. The reversible peak observed at - 0 . 4 V can be assigned to reaction step 2.
Counter .~;#- Working Electrode j ~ Electrode /: Moteriol ~1~ ~
co..
','i
~'
Ref~ence E~e~ir~ i ~ ''~ Electrode
Reference Electrode
Countf.r Electrode
,--.
WorkingEleclrode (o)
(b)
Fig. 1. (a) Cyclic voltammetry cell; (b) flat cell used for in situ electrolysis.
140
i
1.00
,
- iO
Fig. 2. Cyclic voltammogram of ascorbic acid in deionized water (0.01 M NaCIO4) at 25°C with scan rate of 200 mV/s.
The effect of the voltage scan rate on the peak currents was also studied, and current function (Ip/cV I/2) was plotted vs. log V, where Ip is the peak current, c is the concentration and V is the voltage scan rate. A negative slope was obtained (Fig. 3a) for the first oxidation peak at + 0.4 V vs. SCE, which indicates a reversible one-electron transfer followed by a chemical reaction according to the Nichelson-Shain criteria [11]. This result also confirms the lack of a reversible peak at + 0.40 V in Fig. 2. The current function did not change with the voltage scan rate for the reduction peak at -0.55 V vs. SCE, which indicates a reversible one-electron transfer according to the Nichelson-Shain criteria (Fig. 3b). The esr spectrum (Fig. 4) is produced by the electrolysis of ascorbic acid at + 0.40 V by constant potential electrolysis in a fiat quartz cell fitted into the esr cavity. For esr studies, the initial concentration of ascorbic acid was always 5 mM. Radical spectra appeared immediately after application of a + 0.40 V potential. The signal intensity stayed constant during the electrolysis. The same hyperfine parameter (1.76) of the esr signal was observed, as reported previously [2-3]. However, the signal intensity was increased after the electrolysis was stopped. Then the rapid decay of the enhanced signal took place in a few seconds and it was followed by a slow decay (30 min). The increase in radical intensity, therefore, could be related to the previously reported mechanism [12]. Conclusive evidence for the generation of AFR by constant potential electrolysis was confirmed by recording the signal with uv spectrometry.
141
._: :~ 7
~ d
6
5 (b)
I
1,4
i
1.6
I
1,8
i
i
I
I
2.0
22
2,4
26
Io9 (V roWS)
Fig, 3. Variation of current function with respect to voltage scan rate: (a) at Ep, a +0.40 V VS. SCE; (b) at Ep,c -0.55 V vs. SCE.
In view of these observations: (a) this method eliminates the presence of reactive primary radicals produced during the pulse radiolysis studies; (b) this method eliminates the effect of buffers and ascorbate oxidizing chemicals; (c) the AFR formed is stable and can be produced continously with high intensity; (d) the system is more suitable for studying the effects of biological factors that influence the intensity of the radicals.
2 Gouss Fig. 4. Esr spectrum of AFR obtained during the electrolysis of 5 mM ascorbic acid at +0.4 V vs. Ag/Ag +.
142 Simplified description of the method Ascorbate free radical is produced from ascorbate in deionized water by constant potential electrolysis and detected by esr and uv spectroscopies. The radical produced in this simple chemical environment is convenient for further chemical and biological characterizations.
Acknowledgement We are grateful to the M E T U R e s e a r c h F u n d for its p a r t i a l s u p p o r t of this work.
References 1 Bielski, B.H.J., Comstock, D.A. and Bown, A.R. (1971) Ascorbic acid free radicals. J. Am. Chem. Soc. 93, 5624-5629. 2 lyanagi, T., Yamaz.',ld, I. and Anan, K.F. (1985) One-electron oxidation-reduction properties of ascorbic acid. Biochim. Biophys. Acta. 806, 255-261. 3 Bielski, B.H.J. (1982) Chemistry of ascorbate radicals, in: Seib P.A. and Tolbert B.M. (Eds.), Ascorbic Acid: Chemistry, Metabolism and Uses, American Chem. Soc., Washington DC, pp 81-100. 4 Shinart, E., Navok, T. and Chevion, M. (1983) The analogous mechanisms of enzymatic inactivation induced by ascorbate and superoxide in the presence of copper J. Biol. Chem. 258, 14778-14783. 5 Chiou, S.H. (1983) DNA and protein.scission acti,,ities of copper ion and a copper-peptide complex. J. Biochem. 94, 1259-1267. 6 Chiou, S.H. (1984) DNA-scission activities of ascorbate in the presence of metal chelates. J. Biochem. 96, 1307-1310. 7 Skotland, T. and Jones, L. (1980) Direct spectrophotometri¢ detection of ascorbate free radical formed by dopamine-beta-monooxygenase and by ascorbate oxidase. Biochim. Biophys. Acta. 630, 30-35. 8 HaUiwell, B. and Foyer, C.H. (1976) Ascorbic acid, metal ions and the superoxide radical. Biochem. J. 155, 697-700. 9 Diliberto, E.J., Grace, D., Charles, C., Allen, P.A. and Pamela, L. (1982) Tissue subcellular and submitochondrial distributions of semidehydroascorbate reductase. J. Neurochem. 39, 563-568. 10 Kakes, W.H. (1981) Is there an equilibrium between ascorbic acid and dehydroascorbic acid? Z. Naturforsch. 36, 1088-1090. 11 Fry, A.J. (1972) Synthetic Organic Electrochemistry. Harper and Row Publishers, New York, NY, pp. 88-93. 12 Bielski, B.H.C., Allen, O.A. and Schwan, HA. (1981) Mechanism of disproportination of ascorbate radicals. J. Am. Chem. Soc. 103, 3516-3518.
