The Fate of the Oxidizing Tyrosyl Radical in the Presence of Glutathione and Ascorbate

Abstract

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr⋅) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS⋅). GS⋅ can react with the glutathione anion (GS−) to form the disulfide radical anion (GSSG⨪). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 μm) and GSH (8 mm), the ascorbate nearly eliminated oxygen consumption and diminished GS⋅ formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases. Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr⋅) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS⋅). GS⋅ can react with the glutathione anion (GS−) to form the disulfide radical anion (GSSG⨪). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 μm) and GSH (8 mm), the ascorbate nearly eliminated oxygen consumption and diminished GS⋅ formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases. superoxide dismutase glutathione thiyl radical glutathione disulfide radical anion superoxide electron spin resonance diethylenetriaminepentaacetic acid 5,5-dimethyl-1-pyrroline N-oxide. The “radical sink hypothesis” proposed by Winterbourn (1Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (320) Google Scholar, 2Winterbourn C.C. Parker L. Cadenas E. Biothiols in Health and Disease. 2. Marcel Dekker, New York1995: 117-134Google Scholar) suggests a concerted antioxidant interaction between GSH and superoxide dismutase (SOD).1 In this proposal, biologically generated radicals (R⋅) oxidize GSH to form thiyl radicals (GS⋅) (Equation 1). As pointed out by Winterbourn (1Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (320) Google Scholar,2Winterbourn C.C. Parker L. Cadenas E. Biothiols in Health and Disease. 2. Marcel Dekker, New York1995: 117-134Google Scholar), this oxidizing thiyl radical is not biologically benign and can undergo other potentially harmful reactions. The reaction of GS⋅with the glutathione anion (GS−) produces the glutathione disulfide radical anion (GSSG⨪) (Equation 2), which, in turn, reduces molecular oxygen, forming superoxide (O⨪2) and glutathione disulfide (GSSG) (Equation 3). In a concerted effort, SOD will catalyze the dismutation of superoxide, terminating the biologically generated free radical (Equation 4). This proposal has been shown to be thermodynamically feasible (3Koppenol W.H. Free Radic. Biol. Med. 1993; 14: 91-94Crossref PubMed Scopus (56) Google Scholar). R⋅+GSH⇌RH+GS⋅(Eq. 1) GS⋅+GS−⇌GSSG⨪(Eq. 2) GSSG⨪+O2→GSSG+O⨪2(Eq. 3) 2 O⨪2+2H+ →SOD O2+H2O2(Eq. 4) However, the physiological relevance of GSH as an antioxidant has been questioned (4Wardman P. Packer L. Cadenas E. Biothiols in Health and Disease. Marcel Dekker, New York1995: 1-19Google Scholar, 5Kagan V.E. Yalowich J.C. Day B.W. Goldman R. Gantchev T.G. Stoyanovsky D.A. Biochemistry. 1994; 33: 9651-9660Crossref PubMed Scopus (70) Google Scholar, 6Goldman R. Bors W. Michel C. Day B.W. Kagan V.E. Environ. Nutr. Interact. 1997; 1: 97-118Google Scholar). Based on known rate constants, Wardman (4Wardman P. Packer L. Cadenas E. Biothiols in Health and Disease. Marcel Dekker, New York1995: 1-19Google Scholar) has estimated the fraction of thiyl radicals that are not conjugated as GSSG⨪ or GSOO⋅ (from the reaction of GS⋅ with O2). He found that in well oxygenated tissue (40 μm O2) at pH 7.4 and with 2 mmGSH, ∼80% of the thiyl radicals are not conjugated, indicating that GS⋅, not GSSG⨪, is the significant radical species under these conditions. In conjunction with this work, Wardman estimated the fraction of thiyl radicals which leads to superoxide formation in the presence of ascorbate. Based on the published rate constants (7Forni, L. G., Monig, J., Mora-Arellano, V. O., and Willson, R. L. (1983) J. Chem. Soc. Perkin Trans. 2,961–965Google Scholar, 8Tamba, M., and O'Neill, P. (1991) J. Chem. Soc. Perkin Trans. 2, 1681–1685Google Scholar) for the reaction of ascorbate with GS⋅, he found that in well oxygenated tissues (40 μm O2) at pH 7.4 with 1 mm GSH and 0.05 mm ascorbate, only 3% of the thiyl radicals follow the superoxide pathway. In addition, ascorbate abolishes O2 consumption that occurs from the reaction of GSH with enzymatically generated radical metabolites of acetaminophen (9Rao D.N.R. Fischer V. Mason R.P. J. Biol. Chem. 1990; 265: 844-847Abstract Full Text PDF PubMed Google Scholar), clozapine (10Fischer V. Haar J.A. Greiner L. Lloyd R.V. Mason R.P. Mol. Pharmacol. 1991; 40: 846-853PubMed Google Scholar), crystal violet (11Gadelha F.R. Hanna P.M. Mason R.P. Docampo R. Chem. Biol. Interact. 1992; 85: 35-48Crossref PubMed Scopus (21) Google Scholar), 17β-estradiol (12Sipe Jr., H.J. Jordan S.J. Hanna P.M. Mason R.P. Carcinogenesis. 1994; 15: 2637-2643Crossref PubMed Scopus (95) Google Scholar), and phenolphthalein (13Sipe Jr., H.J. Corbett J.T. Mason R.P. Drug Metab. Dispos. 1997; 25: 468-480PubMed Google Scholar). When these radical metabolites are generated in the presence of ascorbate, the ascorbate radical is generated via oxidation by the metabolite radical, and the concentration of the ascorbate radical is minimally affected by the presence of GSH (9Rao D.N.R. Fischer V. Mason R.P. J. Biol. Chem. 1990; 265: 844-847Abstract Full Text PDF PubMed Google Scholar, 10Fischer V. Haar J.A. Greiner L. Lloyd R.V. Mason R.P. Mol. Pharmacol. 1991; 40: 846-853PubMed Google Scholar, 11Gadelha F.R. Hanna P.M. Mason R.P. Docampo R. Chem. Biol. Interact. 1992; 85: 35-48Crossref PubMed Scopus (21) Google Scholar, 12Sipe Jr., H.J. Jordan S.J. Hanna P.M. Mason R.P. Carcinogenesis. 1994; 15: 2637-2643Crossref PubMed Scopus (95) Google Scholar, 13Sipe Jr., H.J. Corbett J.T. Mason R.P. Drug Metab. Dispos. 1997; 25: 468-480PubMed Google Scholar). Work done with the VP-16 phenoxyl radical showed that, in a system that contained ascorbate and GSH, the ascorbate is oxidized prior to the oxidation of GSH (5Kagan V.E. Yalowich J.C. Day B.W. Goldman R. Gantchev T.G. Stoyanovsky D.A. Biochemistry. 1994; 33: 9651-9660Crossref PubMed Scopus (70) Google Scholar). Goldman et al. (6Goldman R. Bors W. Michel C. Day B.W. Kagan V.E. Environ. Nutr. Interact. 1997; 1: 97-118Google Scholar) discussed the interaction of two phenoxyl radicals (from p-cresol and VP-16) and intercellular reductants (ascorbate, GSH, and NAD(P)H). Rate constants for relevant reactions led them to conclude that GSH should be well protected from oxidation by p-cresol phenoxyl radical unless the concentration of ascorbate drops below 1/600th that of GSH.In view of the absence of experimental data exploring the role of physiological concentrations of ascorbate and GSH in the fate of biologically generated free radicals, we have initiated an investigation using horseradish peroxidase in the presence of hydrogen peroxide to generate tyrosyl radicals. The fate of the tyrosyl radical was determined in the presence of varying ratios of ascorbate and GSH using oxygen consumption measurements and electron spin resonance (ESR) spectroscopy.DISCUSSIONThe results presented clearly demonstrate that the tyrosyl radical preferentially oxidizes ascorbate, as opposed to GSH. This conclusion is supported by: 1) nearly complete elimination of oxygen consumption upon the addition of 100 μm ascorbate to a complete oxygen-consuming horseradish peroxidase/H2O2/tyrosine system containing 8 mm GSH (Fig. 2, D); 2) only minor perturbation of the observed ascorbate radical ESR signal intensity when 8 mm GSH was added to a complete horseradish peroxidase/H2O2/tyrosine system with 100 μm ascorbate (Fig. 5, B); and 3) the near total elimination of the DMPO/⋅SG adduct formation upon addition of 100 μm ascorbate to a complete horseradish peroxidase/H2O2/tyrosine system containing 8 mm GSH and 100 mm DMPO (Fig. 6,D).The formation of the tyrosyl radical and the subsequent oxidation of ascorbate or GSH are outlined in Scheme FS1. In all of the experiments, tyrosine stimulated ascorbate and GSH oxidation, demonstrating that the tyrosyl radical was the major radical product of the horseradish peroxidase compound I reaction, even in the presence of ascorbate or GSH. The radical sink hypothesis put forth by Winterbourn (1Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (320) Google Scholar, 2Winterbourn C.C. Parker L. Cadenas E. Biothiols in Health and Disease. 2. Marcel Dekker, New York1995: 117-134Google Scholar) follows a reaction pathway initiated by the reaction of an oxidizing free radical with GSH. When tyrosine is the radical, this reaction does not occur in the presence of physiological concentrations of ascorbate, GSH, and O2. Instead, the tyrosyl radical reacts directly with ascorbate to form the ascorbate radical and regenerate tyrosine.In order to evaluate relative biological significance of ascorbate and GSH, one needs to consider physiological concentrations and mechanisms of regeneration. Intracellular levels of GSH in mammalian cells range from 0.5–10 mm (30Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (395) Google Scholar, 31Meister A. Anderson M.E. Annu. Rev. PubMed Scopus Google Scholar, Rev. PubMed Scopus Google Scholar) with cells mm levels Rev. PubMed Scopus Google Scholar, R. J.A. Rev. Google Scholar). is that of GSH occurs L. P. A. 1992; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar). GSH levels in are μm) M.E. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of GSH is by glutathione disulfide (30Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (395) Google Scholar). The concentration of GSH these the of the concentrations found in mammalian concentrations in tissue are from μm in PubMed Scopus Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar) to 1 mm in the and the PubMed Scopus Google Scholar). as the and range between 100 and μm R. J.A. Rev. Google Scholar, PubMed Scopus Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar). The concentration of ascorbate in the the of the concentrations found in mammalian to mechanisms of ascorbate, the ascorbate radical will undergo a dismutation at the rate of 2 at pH and Scholar), as well as by Free in New Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar). In addition, is to ascorbate by an system Cadenas E. Packer L. of Marcel Dekker, New concentrations and ratios in this physiological The superoxide pathway proposed by Winterbourn occur in a biological system which does not have ascorbate as which can be of ascorbate (6Goldman R. Bors W. Michel C. Day B.W. Kagan V.E. Environ. Nutr. Interact. 1997; 1: 97-118Google Scholar). in systems of ascorbate, GSH not be an antioxidant other reactive radicals GSSG⨪, and are is the with a of for GSH (3Koppenol W.H. Free Radic. Biol. Med. 1993; 14: 91-94Crossref PubMed Scopus (56) Google Scholar, 1993; PubMed Scopus Google Scholar). The ascorbate radical is a highly radical and is which a highly biological GS⋅, the ascorbate radical is not known to biological and results the of ascorbate and GSH in the tyrosyl radical. The results are not for the tyrosyl radical and should for oxidizing free radicals formed by (9Rao D.N.R. Fischer V. Mason R.P. J. Biol. Chem. 1990; 265: 844-847Abstract Full Text PDF PubMed Google Scholar, 10Fischer V. Haar J.A. Greiner L. Lloyd R.V. Mason R.P. Mol. Pharmacol. 1991; 40: 846-853PubMed Google Scholar, 11Gadelha F.R. Hanna P.M. Mason R.P. Docampo R. Chem. Biol. Interact. 1992; 85: 35-48Crossref PubMed Scopus (21) Google Scholar, 12Sipe Jr., H.J. Jordan S.J. Hanna P.M. Mason R.P. Carcinogenesis. 1994; 15: 2637-2643Crossref PubMed Scopus (95) Google Scholar, 13Sipe Jr., H.J. Corbett J.T. Mason R.P. Drug Metab. Dispos. 1997; 25: 468-480PubMed Google Scholar). The “radical sink hypothesis” proposed by Winterbourn (1Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (320) Google Scholar, 2Winterbourn C.C. Parker L. Cadenas E. Biothiols in Health and Disease. 2. Marcel Dekker, New York1995: 117-134Google Scholar) suggests a concerted antioxidant interaction between GSH and superoxide dismutase (SOD).1 In this proposal, biologically generated radicals (R⋅) oxidize GSH to form thiyl radicals (GS⋅) (Equation 1). As pointed out by Winterbourn (1Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (320) Google Scholar,2Winterbourn C.C. Parker L. Cadenas E. Biothiols in Health and Disease. 2. Marcel Dekker, New York1995: 117-134Google Scholar), this oxidizing thiyl radical is not biologically benign and can undergo other potentially harmful reactions. The reaction of GS⋅with the glutathione anion (GS−) produces the glutathione disulfide radical anion (GSSG⨪) (Equation 2), which, in turn, reduces molecular oxygen, forming superoxide (O⨪2) and glutathione disulfide (GSSG) (Equation 3). In a concerted effort, SOD will catalyze the dismutation of superoxide, terminating the biologically generated free radical (Equation 4). This proposal has been shown to be thermodynamically feasible (3Koppenol W.H. Free Radic. Biol. Med. 1993; 14: 91-94Crossref PubMed Scopus (56) Google Scholar). R⋅+GSH⇌RH+GS⋅(Eq. 1) GS⋅+GS−⇌GSSG⨪(Eq. 2) GSSG⨪+O2→GSSG+O⨪2(Eq. 3) 2 O⨪2+2H+ →SOD O2+H2O2(Eq. 4) However, the physiological relevance of GSH as an antioxidant has been questioned (4Wardman P. Packer L. Cadenas E. Biothiols in Health and Disease. Marcel Dekker, New York1995: 1-19Google Scholar, 5Kagan V.E. Yalowich J.C. Day B.W. Goldman R. Gantchev T.G. Stoyanovsky D.A. Biochemistry. 1994; 33: 9651-9660Crossref PubMed Scopus (70) Google Scholar, 6Goldman R. Bors W. Michel C. Day B.W. Kagan V.E. Environ. Nutr. Interact. 1997; 1: 97-118Google Scholar). Based on known rate constants, Wardman (4Wardman P. Packer L. Cadenas E. Biothiols in Health and Disease. Marcel Dekker, New York1995: 1-19Google Scholar) has estimated the fraction of thiyl radicals that are not conjugated as GSSG⨪ or GSOO⋅ (from the reaction of GS⋅ with O2). He found that in well oxygenated tissue (40 μm O2) at pH 7.4 and with 2 mmGSH, ∼80% of the thiyl radicals are not conjugated, indicating that GS⋅, not GSSG⨪, is the significant radical species under these conditions. In conjunction with this work, Wardman estimated the fraction of thiyl radicals which leads to superoxide formation in the presence of ascorbate. Based on the published rate constants (7Forni, L. G., Monig, J., Mora-Arellano, V. O., and Willson, R. L. (1983) J. Chem. Soc. Perkin Trans. 2,961–965Google Scholar, 8Tamba, M., and O'Neill, P. (1991) J. Chem. Soc. Perkin Trans. 2, 1681–1685Google Scholar) for the reaction of ascorbate with GS⋅, he found that in well oxygenated tissues (40 μm O2) at pH 7.4 with 1 mm GSH and 0.05 mm ascorbate, only 3% of the thiyl radicals follow the superoxide pathway. In addition, ascorbate abolishes O2 consumption that occurs from the reaction of GSH with enzymatically generated radical metabolites of acetaminophen (9Rao D.N.R. Fischer V. Mason R.P. J. Biol. Chem. 1990; 265: 844-847Abstract Full Text PDF PubMed Google Scholar), clozapine (10Fischer V. Haar J.A. Greiner L. Lloyd R.V. Mason R.P. Mol. Pharmacol. 1991; 40: 846-853PubMed Google Scholar), crystal violet (11Gadelha F.R. Hanna P.M. Mason R.P. Docampo R. Chem. Biol. Interact. 1992; 85: 35-48Crossref PubMed Scopus (21) Google Scholar), 17β-estradiol (12Sipe Jr., H.J. Jordan S.J. Hanna P.M. Mason R.P. Carcinogenesis. 1994; 15: 2637-2643Crossref PubMed Scopus (95) Google Scholar), and phenolphthalein (13Sipe Jr., H.J. Corbett J.T. Mason R.P. Drug Metab. Dispos. 1997; 25: 468-480PubMed Google Scholar). When these radical metabolites are generated in the presence of ascorbate, the ascorbate radical is generated via oxidation by the metabolite radical, and the concentration of the ascorbate radical is minimally affected by the presence of GSH (9Rao D.N.R. Fischer V. Mason R.P. J. Biol. Chem. 1990; 265: 844-847Abstract Full Text PDF PubMed Google Scholar, 10Fischer V. Haar J.A. Greiner L. Lloyd R.V. Mason R.P. Mol. Pharmacol. 1991; 40: 846-853PubMed Google Scholar, 11Gadelha F.R. Hanna P.M. Mason R.P. Docampo R. Chem. Biol. Interact. 1992; 85: 35-48Crossref PubMed Scopus (21) Google Scholar, 12Sipe Jr., H.J. Jordan S.J. Hanna P.M. Mason R.P. Carcinogenesis. 1994; 15: 2637-2643Crossref PubMed Scopus (95) Google Scholar, 13Sipe Jr., H.J. Corbett J.T. Mason R.P. Drug Metab. Dispos. 1997; 25: 468-480PubMed Google Scholar). Work done with the VP-16 phenoxyl radical showed that, in a system that contained ascorbate and GSH, the ascorbate is oxidized prior to the oxidation of GSH (5Kagan V.E. Yalowich J.C. Day B.W. Goldman R. Gantchev T.G. Stoyanovsky D.A. Biochemistry. 1994; 33: 9651-9660Crossref PubMed Scopus (70) Google Scholar). Goldman et al. (6Goldman R. Bors W. Michel C. Day B.W. Kagan V.E. Environ. Nutr. Interact. 1997; 1: 97-118Google Scholar) discussed the interaction of two phenoxyl radicals (from p-cresol and VP-16) and intercellular reductants (ascorbate, GSH, and NAD(P)H). Rate constants for relevant reactions led them to conclude that GSH should be well protected from oxidation by p-cresol phenoxyl radical unless the concentration of ascorbate drops below 1/600th that of GSH. In view of the absence of experimental data exploring the role of physiological concentrations of ascorbate and GSH in the fate of biologically generated free radicals, we have initiated an investigation using horseradish peroxidase in the presence of hydrogen peroxide to generate tyrosyl radicals. The fate of the tyrosyl radical was determined in the presence of varying ratios of ascorbate and GSH using oxygen consumption measurements and electron spin resonance (ESR) results presented clearly demonstrate that the tyrosyl radical preferentially oxidizes ascorbate, as opposed to GSH. This conclusion is supported by: 1) nearly complete elimination of oxygen consumption upon the addition of 100 μm ascorbate to a complete oxygen-consuming horseradish peroxidase/H2O2/tyrosine system containing 8 mm GSH (Fig. 2, D); 2) only minor perturbation of the observed ascorbate radical ESR signal intensity when 8 mm GSH was added to a complete horseradish peroxidase/H2O2/tyrosine system with 100 μm ascorbate (Fig. 5, B); and 3) the near total elimination of the DMPO/⋅SG adduct formation upon addition of 100 μm ascorbate to a complete horseradish peroxidase/H2O2/tyrosine system containing 8 mm GSH and 100 mm DMPO (Fig. 6,D).The formation of the tyrosyl radical and the subsequent oxidation of ascorbate or GSH are outlined in Scheme FS1. In all of the experiments, tyrosine stimulated ascorbate and GSH oxidation, demonstrating that the tyrosyl radical was the major radical product of the horseradish peroxidase compound I reaction, even in the presence of ascorbate or GSH. The radical sink hypothesis put forth by Winterbourn (1Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (320) Google Scholar, 2Winterbourn C.C. Parker L. Cadenas E. Biothiols in Health and Disease. 2. Marcel Dekker, New York1995: 117-134Google Scholar) follows a reaction pathway initiated by the reaction of an oxidizing free radical with GSH. When tyrosine is the radical, this reaction does not occur in the presence of physiological concentrations of ascorbate, GSH, and O2. Instead, the tyrosyl radical reacts directly with ascorbate to form the ascorbate radical and regenerate tyrosine.In order to evaluate relative biological significance of ascorbate and GSH, one needs to consider physiological concentrations and mechanisms of regeneration. Intracellular levels of GSH in mammalian cells range from 0.5–10 mm (30Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (395) Google Scholar, 31Meister A. Anderson M.E. Annu. Rev. PubMed Scopus Google Scholar, Rev. PubMed Scopus Google Scholar) with cells mm levels Rev. PubMed Scopus Google Scholar, R. J.A. Rev. Google Scholar). is that of GSH occurs L. P. A. 1992; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar). GSH levels in are μm) M.E. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of GSH is by glutathione disulfide (30Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (395) Google Scholar). The concentration of GSH these the of the concentrations found in mammalian concentrations in tissue are from μm in PubMed Scopus Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar) to 1 mm in the and the PubMed Scopus Google Scholar). as the and range between 100 and μm R. J.A. Rev. Google Scholar, PubMed Scopus Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar). The concentration of ascorbate in the the of the concentrations found in mammalian to mechanisms of ascorbate, the ascorbate radical will undergo a dismutation at the rate of 2 at pH and Scholar), as well as by Free in New Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar). In addition, is to ascorbate by an system Cadenas E. Packer L. of Marcel Dekker, New concentrations and ratios in this physiological The superoxide pathway proposed by Winterbourn occur in a biological system which does not have ascorbate as which can be of ascorbate (6Goldman R. Bors W. Michel C. Day B.W. Kagan V.E. Environ. Nutr. Interact. 1997; 1: 97-118Google Scholar). in systems of ascorbate, GSH not be an antioxidant other reactive radicals GSSG⨪, and are is the with a of for GSH (3Koppenol W.H. Free Radic. Biol. Med. 1993; 14: 91-94Crossref PubMed Scopus (56) Google Scholar, 1993; PubMed Scopus Google Scholar). The ascorbate radical is a highly radical and is which a highly biological GS⋅, the ascorbate radical is not known to biological and results the of ascorbate and GSH in the tyrosyl radical. The results are not for the tyrosyl radical and should for oxidizing free radicals formed by (9Rao D.N.R. Fischer V. Mason R.P. J. Biol. Chem. 1990; 265: 844-847Abstract Full Text PDF PubMed Google Scholar, 10Fischer V. Haar J.A. Greiner L. Lloyd R.V. Mason R.P. Mol. Pharmacol. 1991; 40: 846-853PubMed Google Scholar, 11Gadelha F.R. Hanna P.M. Mason R.P. Docampo R. Chem. Biol. Interact. 1992; 85: 35-48Crossref PubMed Scopus (21) Google Scholar, 12Sipe Jr., H.J. Jordan S.J. Hanna P.M. Mason R.P. Carcinogenesis. 1994; 15: 2637-2643Crossref PubMed Scopus (95) Google Scholar, 13Sipe Jr., H.J. Corbett J.T. Mason R.P. Drug Metab. Dispos. 1997; 25: 468-480PubMed Google Scholar). The results presented clearly demonstrate that the tyrosyl radical preferentially oxidizes ascorbate, as opposed to GSH. This conclusion is supported by: 1) nearly complete elimination of oxygen consumption upon the addition of 100 μm ascorbate to a complete oxygen-consuming horseradish peroxidase/H2O2/tyrosine system containing 8 mm GSH (Fig. 2, D); 2) only minor perturbation of the observed ascorbate radical ESR signal intensity when 8 mm GSH was added to a complete horseradish peroxidase/H2O2/tyrosine system with 100 μm ascorbate (Fig. 5, B); and 3) the near total elimination of the DMPO/⋅SG adduct formation upon addition of 100 μm ascorbate to a complete horseradish peroxidase/H2O2/tyrosine system containing 8 mm GSH and 100 mm DMPO (Fig. The formation of the tyrosyl radical and the subsequent oxidation of ascorbate or GSH are outlined in Scheme FS1. In all of the experiments, tyrosine stimulated ascorbate and GSH oxidation, demonstrating that the tyrosyl radical was the major radical product of the horseradish peroxidase compound I reaction, even in the presence of ascorbate or GSH. The radical sink hypothesis put forth by Winterbourn (1Winterbourn C.C. Free Radic. Biol. Med. 1993; 14: 85-90Crossref PubMed Scopus (320) Google Scholar, 2Winterbourn C.C. Parker L. Cadenas E. Biothiols in Health and Disease. 2. Marcel Dekker, New York1995: 117-134Google Scholar) follows a reaction pathway initiated by the reaction of an oxidizing free radical with GSH. When tyrosine is the radical, this reaction does not occur in the presence of physiological concentrations of ascorbate, GSH, and O2. Instead, the tyrosyl radical reacts directly with ascorbate to form the ascorbate radical and regenerate In order to evaluate relative biological significance of ascorbate and GSH, one needs to consider physiological concentrations and mechanisms of regeneration. Intracellular levels of GSH in mammalian cells range from 0.5–10 mm (30Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (395) Google Scholar, 31Meister A. Anderson M.E. Annu. Rev. PubMed Scopus Google Scholar, Rev. PubMed Scopus Google Scholar) with cells mm levels Rev. PubMed Scopus Google Scholar, R. J.A. Rev. Google Scholar). is that of GSH occurs L. P. A. 1992; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar). GSH levels in are μm) M.E. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of GSH is by glutathione disulfide (30Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (395) Google Scholar). The concentration of GSH these the of the concentrations found in mammalian concentrations in tissue are from μm in PubMed Scopus Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar) to 1 mm in the and the PubMed Scopus Google Scholar). as the and range between 100 and μm R. J.A. Rev. Google Scholar, PubMed Scopus Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar). The concentration of ascorbate in the the of the concentrations found in mammalian to mechanisms of ascorbate, the ascorbate radical will undergo a dismutation at the rate of 2 at pH and Scholar), as well as by Free in New Google Scholar, Cadenas E. Packer L. of Marcel Dekker, New Scholar). In addition, is to ascorbate by an system Cadenas E. Packer L. of Marcel Dekker, New Scholar). The concentrations and ratios in this physiological The superoxide pathway proposed by Winterbourn occur in a biological system which does not have ascorbate as which can be of ascorbate (6Goldman R. Bors W. Michel C. Day B.W. Kagan V.E. Environ. Nutr. Interact. 1997; 1: 97-118Google Scholar). in systems of ascorbate, GSH not be an antioxidant other reactive radicals GSSG⨪, and are is the with a of for GSH (3Koppenol W.H. Free Radic. Biol. Med. 1993; 14: 91-94Crossref PubMed Scopus (56) Google Scholar, 1993; PubMed Scopus Google Scholar). 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