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J Biol Chem, Vol. 273, Issue 46, 30116-30121, November 13, 1998
,From the Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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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 The "radical sink hypothesis" proposed by Winterbourn (1, 2)
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 (1,
2), this oxidizing thiyl radical is not biologically benign and can
undergo other potentially harmful reactions. The reaction of GS·
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.
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INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) 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 (3).
(Eq. 1)
(Eq. 2)
(Eq. 3)
However, the physiological relevance of GSH as an antioxidant has
been questioned (4-6). Based on known rate constants, Wardman (4) has
estimated the fraction of thiyl radicals that are not conjugated as
GSSG
(Eq. 4)
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 mM
GSH, ~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 (7, 8) 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
(9), clozapine (10), crystal violet (11), 17
-estradiol (12), and
phenolphthalein (13). 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 (9-13).
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 (5). Goldman et al. (6) 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.
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MATERIALS AND METHODS |
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Reagents--
Ascorbic acid, diethylenetriaminepentaacetic acid
(DTPA), GSH, horseradish peroxidase type VI-A (EC 1.11.1.7), and
tyrosine were purchased from Sigma and used as received. The quoted
horseradish peroxidase activity is based on the
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assay. Hydrogen
peroxide (H2O2) was purchased from Fisher. The
H2O2 concentration was verified using the UV
absorption at 240 nm (
= 43.6 M
1).
Catalase (from beef liver, 65,000 units/mg suspension in water) (EC
1.11.1.6) and SOD (from bovine erythrocytes, 5000 units/mg of
lyophilizate) (EC 1.15.1.1) were purchased from Boehringer Mannheim and
used as received. The 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap was purchased from Aldrich, vacuum-distilled twice,
and stored under nitrogen at
70 °C until needed. All reactions used a 100 mM phosphate buffer at pH 7.4, Chelexed
overnight, and 100 µM DTPA was added after Chelexing. Due
to the high concentration of GSH (8 mM) used in most
experiments, all pH values were carefully checked and adjusted as
required prior to initiation of tyrosyl radical generation. Stock
solutions of reagents were made fresh daily and stored on ice while
experiments were being performed. The stock solutions were made up as
follows: horseradish peroxidase in buffer, H2O2
in deionized water, tyrosine in buffer, GSH in deionized water,
ascorbic acid in deionized water, and SOD in buffer. Catalase was used
as received, and DMPO was used undiluted.
Oxygen Consumption Experiments-- Oxygen consumption measurements were made using a Clark-type oxygen electrode fitted to a 1.8-ml Gilson sample cell and monitored by a Yellow Springs Instrument Company model 53 oxygen monitor (Yellow Springs, OH). The reagents were added in the following order: tyrosine/buffer stock solution, GSH stock solution, H2O2 stock solution, and SOD stock solution (when used). Then, after establishing a 2-min base-line measurement, horseradish peroxidase stock solution was added to initiate tyrosyl radical generation. In experiments that contained ascorbate, ascorbic acid stock solution was added either 1 min prior to horseradish peroxidase or 1 min after horseradish peroxidase. In experiments that contained DMPO, undiluted DMPO was added either 1 min prior to horseradish peroxidase or 1 min after horseradish peroxidase.
ESR Experiments--
ESR experiments were carried out on a
Bruker ER-200D ESR spectrometer interfaced to an IBM-compatible
computer. All experiments were done at room temperature. Fast-flow ESR
experiments which detected the tyrosyl radical were done using a
TM110 resonator equipped with a 17-mm quartz, mixing flat
cell (~225-µl volume) designed for fast-flow measurements (Wilmad,
NJ) (14). This mixing flat cell had a post-mixing dead volume of
approximately 300 µl, which, when flowing at 60 ml/min, resulted in a
post-mixing observation time of approximately 300 ms. Fast-flow ESR
measurements of the ascorbate radical were done using a Bruker
dielectric mixing resonator with a 1-µl active volume. Spin-trapping
experiments were done using a standard 17-mm quartz flat cell
(~225-µl volume) mounted in the TM110 resonator.
Spin-trapping samples were prepared, mixed, and then aspirated into the
flat cell with ESR data acquisition started approximately 15 s
after mixing.
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RESULTS |
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Oxygen Consumption in the Presence of Ascorbate--
The role of
GSH in the proposed concerted action of GSH and SOD is to channel
electrons to molecular oxygen through the formation of the highly
reducing disulfide radical anion (GSSG
). As outlined in
Equations 1-3, where R· = tyrosyl radical, the formation of
GS· will consume molecular oxygen. For this reason we have made
oxygen consumption measurements in the presence and absence of
ascorbate, GSH, and SOD. As reported (15), oxygen was consumed during
the horseradish peroxidase-dependent oxidation of tyrosine
in the presence of GSH (Fig. 1,
A). Oxygen consumption was
strictly dependent on the presence of horseradish peroxidase (Fig. 1,
B), tyrosine (Fig. 1, C), and GSH (Fig. 1,
F). The omission of H2O2 decreased the rate only slightly (Fig. 1, D). Hydrogen peroxide is
obligatory to support the oxygen consumption as shown by catalase
inhibition (Fig. 1, E). Fig. 2
shows the oxygen consumption data for a system that contained
horseradish peroxidase (15 U/ml), tyrosine (2 mM), H2O2 (36 µM), GSH (8 mM), and varying concentrations of ascorbate. The
post-addition (1 min after horseradish peroxidase-initiation) of 25 µM ascorbate (Fig. 2, B) had only a minor
effect on the rate of oxygen consumption. The post-addition of 50 µM ascorbate (Fig. 2, C) inhibited the rate
and extent of oxygen consumption. The post-addition of 100 µM ascorbate (Fig. 2, D) nearly eliminated oxygen consumption. Identical results were observed when ascorbate was
added 1 min before the addition of horseradish peroxidase (data not
shown).
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If all GSH-dependent superoxide (Equation 3) underwent nonenzymatic bimolecular dismutation, then SOD would have no effect on the oxygen uptake. Fig. 3 shows that the rate of oxygen consumption in the absence of SOD (Fig. 3, A) was higher than in the presence of SOD (Fig. 3, B), indicating that the superoxide generated was involved in additional oxygen-consuming chemistry (16-18). In the presence of SOD, 25 µM ascorbate (Fig. 3, C) inhibited the rate and extent of oxygen consumption, whereas in the absence of SOD, 25 µM ascorbate had a minor effect on the rate and extent of oxygen consumption (Fig. 2, B), indicating that, in the absence of SOD, the higher rate of oxygen consumption (and radical production) probably depleted the relatively low ascorbate concentration. Under conditions of lower GSH concentrations (<2 mM), SOD actually stimulates oxygen consumption in this system (15), so the effect of SOD is quite complex.
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Direct ESR Observation of Enzymatically Generated Tyrosyl
Radical--
The enzymatically generated, free tyrosyl radical has
been directly observed for the first time using fast-flow ESR (Fig. 4). Tyrosine is oxidized by horseradish
peroxidase compound I at a rate of 5.0 × 104
M
1 s
1 (19) and compound II at a
rate of 1.1 × 103 M
1
s
1 (20). The observed tyrosyl radical ESR spectrum (Fig.
4, A) was dependent on the presence of horseradish
peroxidase (Fig. 4, C), tyrosine (Fig. 4, D), and
H2O2 (Fig. 4, E). The ESR spectrum can be explained based on rotational exchange effects related to the
hindered internal rotation of the
ArCH2-CHNH2CO2
bond
(21). Deaeration of the solutions by nitrogen bubbling prior to tyrosyl
radical generation produced ESR spectra essentially identical to those
shown in Fig. 4 (data not shown). This tyrosyl radical-generating
system did not consume oxygen (Fig. 1, F), implying that no
oxygen-centered radicals were formed. Tyrosyl radical reacts with
oxygen with a rate constant less than 103
M
1 s
1 (22, 23).
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Direct ESR Observation of Ascorbate Radical Anion--
When the
tyrosyl radical was formed in the presence of ascorbate (pH 7.4), the
ascorbate was rapidly oxidized by the tyrosyl radical to the ascorbate
radical (k = 4.4 × 108
M
1 s
1) (22). Ascorbate is
readily oxidized by a number of phenoxyl radicals (~108
M
1 s
1) (24). The fast-flow ESR
spectrum of the ascorbate radical generated from the tyrosyl
radical-generating horseradish peroxidase system is shown in Fig.
5, A. The intensity of the
ascorbate radical was decreased only slightly in the presence of 8 mM GSH (Fig. 5, B). The ESR spectrum of the
ascorbate radical was dependent on the presence of horseradish
peroxidase (Fig. 5, C), tyrosine (Fig. 5, D),
H2O2 (Fig. 5, E and F),
and ascorbate (Fig. 5, G). The ascorbate radical ESR signal observed in
the absence of tyrosine (Fig. 5, D) is a result of direct
oxidation of ascorbate by horseradish peroxidase compound I (25) and
was only slightly greater than the nonenzymatic ascorbate radical
formation (Fig. 5, C). For this reason ascorbate has been
referred to as a "sluggish" substrate of horseradish peroxidase
compound I (26). The stimulation of ascorbate radical by tyrosine (Fig.
5, A) demonstrated that tyrosine was oxidized by horseradish
peroxidase in the presence of ascorbate and that ascorbate did not
merely inhibit tyrosine oxidation.
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Because ascorbate (AH
) will react with both the tyrosyl
radical (Tyr·) (Equation 5) and GS· (Equation 6) to form
the ascorbate radical (A
), it is unclear from the results in
Fig. 5 whether ascorbate inhibited GS· formation by reacting
with all of the tyrosyl radical or if ascorbate was oxidized by
GS· and, hence, eliminated oxygen consumption (Equation 3). As
shown by oxygen consumption measurements, 100 mM DMPO
completely inhibited oxygen consumption (Fig. 2, E),
indicating that DMPO efficiently traps all GS· generated
(Equation 7).
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(Eq. 5) |
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(Eq. 6) |
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(Eq. 7) |
1 s
1) (27) and decrease the
intensity of the fast-flow ascorbate radical ESR signal by eliminating
the ascorbate radical generated in Equation 6. When DMPO (100 mM) was added to the tyrosyl radical-generating horseradish
peroxidase system with 100 µM ascorbate, there was no
change in intensity of the ascorbate radical ESR signal (data not
shown), indicating that, under fast-flow conditions, the ascorbate radical was formed only by reaction with the tyrosyl radical and not by
reaction with GS·.
Spin Trapping of GS· in the Presence of Ascorbate and the
Direct Reduction of the DMPO/·SG Adduct by Ascorbate--
The
glutathione thiyl radical (GS·) was generated as a result of the
horseradish
peroxidase/H2O2-dependent formation
of the tyrosyl radical as shown by ESR spin trapping with DMPO. DMPO was shown to completely inhibit oxygen consumption in a
GS·-forming system (Fig. 2, E), indicating high
trapping efficiency (k = 2.6 × 108
M
1 s
1) (27). The characteristic
DMPO/·SG adduct ESR spectrum is shown in Fig.
6. The DMPO/·SG adduct ESR
spectrum was simulated using hyperfine coupling constants
(aN = 15.2 G and aH = 16.2 G) similar to earlier reports (12, 13, 28, 29). The
DMPO/·SG adduct spectrum was dependent on the presence of
horseradish peroxidase, tyrosine, H2O2, GSH,
and DMPO (data not shown). The addition of ascorbate resulted in a
decrease in the DMPO/·SG adduct ESR intensity (Fig. 6,
spectra A-G). Double integration of the low field
transition (at 3459 G) of the DMPO/·SG adduct ESR spectrum is
plotted versus ascorbate concentration in Fig.
7B (
).
This decrease in the amount of DMPO/·SG adduct can be attributed
to two mechanisms. The first mechanism is the preferential oxidation of
the ascorbate (Equation 5), as opposed to GSH (Equation 1), by the
tyrosyl radical, which would result in less GS· formation and,
hence, less DMPO/·SG adduct. The second mechanism is the
reduction of the DMPO/·SG adduct by ascorbate to the
corresponding ESR-silent hydroxylamine. The reduction of the
DMPO/·SG by ascorbate complicates the interpretation of the
DMPO/·SG adduct results.
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In order to evaluate the contribution of the reduction of the
DMPO/·SG adduct by ascorbate to the overall
ascorbate-dependent decrease in DMPO/·SG adduct ESR
intensity, the DMPO/·SG adduct was allowed to form (in the
absence of ascorbate) for 90 s by the horseradish peroxidase
system with 8 mM GSH. Immediately following the 90-s
formation time, catalase was added to remove any remaining
H2O2 and, hence, eliminate any further
GS· formation and, therefore, any additional
DMPO/·SG adduct formation. Immediately following the addition of
the catalase, varying amounts of ascorbate were added. This
post-addition of ascorbate resulted in a decrease in the
DMPO/·SG radical adduct ESR spectrum due only to reduction of
DMPO/·SG adduct to the hydroxylamine form by ascorbate. The
double integration of the low field transition (at 3459 G) of the
DMPO/·SG adduct ESR spectrum is plotted versus the
post-addition ascorbate concentration in Fig. 7B (
). Due
to the decrease in the steady-state concentration of the radical adduct
over time (Fig. 7A), the decay data in Fig. 7B
are not directly comparable, but it can be seen that the rate of decay
of the DMPO/·SG adduct by the direct ascorbate (post-addition)
reduction of DMPO/·SG adduct (Fig. 7B (
)) is
significantly slower then the overall decrease observed (Fig.
7B (
)). While reduction of the
DMPO/·SG adduct by ascorbate does occur, this was not the
dominant mechanism resulting in the decrease of the DMPO/·SG
adduct. It should be noted in Fig. 7B
(
) DMPO/·SG was being generated; where
in Fig. 7B (
), the DMPO/·SG adduct was not being
generated because the H2O2 has been eliminated from the system.
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DISCUSSION |
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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. 6, D).
The formation of the tyrosyl radical and the subsequent oxidation of ascorbate or GSH are outlined in Scheme 1. 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 (1, 2) 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.
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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 (30-32) with many cells having 2-5 mM levels (32, 33). There is evidence that intracellular compartmentalization of GSH occurs (34-36). GSH levels in blood plasma are quite low (25 µM) (37). The regeneration of GSH is mainly controlled by glutathione disulfide reductase (30). The concentration of GSH used throughout these experiments represents the upper limit of the concentrations found in mammalian tissue.
Ascorbate concentrations in human tissue are quite variable, ranging
from roughly 40 µM in blood plasma (38, 39) to over 1 mM in the eye lens and the pituitary gland (38). Tissues
such as the liver, kidneys, spleen, pancreas, lungs, heart, brain, and
skin range between 100 and 800 µM (33, 38, 39). The concentration of ascorbate used in the present experiments represents the lower limit of the concentrations found in mammalian tissue. With
respect to regeneration mechanisms of ascorbate, the ascorbate radical
will undergo a pH-dependent second-order dismutation at the
rate of approximately 2 × 105
M
1 s
1 at pH 7 (40), as well as
enzymatic reduction by semidehydroascorbate reductase (26, 41). In
addition, dehydroascorbate is reduced to ascorbate by an
NAD(P)H-dependent enzymatic system (41).
The ascorbate/GSH concentrations and ratios used in this study
realistically represent physiological concentrations. The superoxide pathway proposed by Winterbourn could occur in a biological system which does not have sufficient ascorbate such as cultured cells, which
can be devoid of ascorbate (6). Even in systems devoid of ascorbate,
GSH may not be properly labeled an antioxidant since other reactive
radicals (GS·, GSSG
, GSOO·, and O
2) are
generated. Ascorbate is the superior reducing agent with a reduction
potential of +0.3 V versus +0.9 V for GSH (3, 42). The
ascorbate radical is a highly delocalized
radical and is quite
unreactive, which makes it a highly desirable biological antioxidant.
Unlike GS·, the ascorbate radical is not known to cause any
biological damage (40).
These results clarify the roles of ascorbate and GSH in neutralizing
the tyrosyl radical. The results are not specific for the tyrosyl
radical and should hold true for many oxidizing free radicals formed by
peroxidases (9-13).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Pharmacology and Chemistry, NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-7574; Fax: 919-541-1043; E-mail: sturgeon{at}niehs.nih.gov.
§ Permanent address: Dept. of Chemistry, Hampden-Sydney College, Hampden-Sydney, VA 23943.
The abbreviations used are:
SOD, superoxide
dismutase; GS·, glutathione thiyl radical; GSSG
, glutathione disulfide radical anion; O
2, superoxide; ESR, electron spin resonance; DTPA, diethylenetriaminepentaacetic acid; DMPO, 5,5-dimethyl-1-pyrroline N-oxide.
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