Hydrogen Peroxide Formation by Reaction of Peroxynitrite with
HEPES and Related Tertiary Amines
IMPLICATIONS FOR A GENERAL MECHANISM*
Michael
Kirsch
,
Elena E.
Lomonosova
,
Hans-Gert
Korth§,
Reiner
Sustmann§, and
Herbert
de Groot
¶
From the
Institut für Physiologische Chemie,
Universitätsklinikum, Hufelandstrasse 55, D-45122 Essen and
§ Institut für Organische Chemie,
Universität-GH Essen,
Universitätsstrasse 5, D-45117 Essen, Germany
 |
ABSTRACT |
Organic amine-based buffer compounds such as
HEPES (Good's buffers) are commonly applied in experimental systems,
including those where the biological effects of peroxynitrite are
studied. In such studies 3-morpholinosydnonimine
N-ethylcarbamide (SIN-1), a compound that simultaneously
releases nitric oxide (·NO) and superoxide (O
2), is
often used as a source for peroxynitrite. Whereas in mere phosphate
buffer H2O2 formation from 1.5 mM
SIN-1 was low (~15 µM), incubation of SIN-1 with
Good's buffer compounds resulted in continuous
H2O2 formation. After 2 h of incubation of
1.5 mM SIN-1 with 20 mM HEPES about 190 µM H2O2 were formed. The same
amount of H2O2 could be achieved from 1.5 mM SIN-1 by action of superoxide dismutase in the absence
of HEPES. The increased H2O2 level, however,
could not be related to a superoxide dismutase or to a NO scavenger
activity of HEPES. On the other hand, SIN-1-mediated oxidation of both
dihydrorhodamine 123 and deoxyribose as well as
peroxynitrite-dependent nitration of
p-hydroxyphenylacetic acid were strongly inhibited by 20 mM HEPES. Furthermore, the peroxynitrite scavenger
tryptophan significantly reduced H2O2 formation
from SIN-1-HEPES interactions. These observations suggest that
peroxynitrite is the initiator for the enhanced formation of
H2O2. Likewise, authentic peroxynitrite (1 mM) also induced the formation of both O
2 and
H2O2 upon addition to HEPES (400 mM)-containing solutions in a pH
(4.5-7.5)-dependent manner. In accordance with previous
reports it was found that at pH
5 oxygen is released in the decay of
peroxynitrite. As a consequence, peroxynitrite(1 mM)-induced H2O2 formation (~80
µM at pH 7.5) also occurred under hypoxic conditions. In
the presence of bicarbonate/carbon dioxide (20 mM/5%) the
production of H2O2 from the reaction of HEPES
with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good's buffers resulted in the
formation of piperazine-derived radical cations as detected by ESR
spectroscopy. These findings suggest a mechanism for
H2O2 formation in which peroxynitrite (or any
strong oxidant derived from it) initially oxidizes the tertiary amine
buffer compounds in a one-electron step. Subsequent deprotonation and
reaction of the intermediate
-amino alkyl radicals with molecular
oxygen leads to the formation of O
2, from which
H2O2 is produced by dismutation. Hence, HEPES
and similar organic buffers should be avoided in studies of oxidative
compounds. Furthermore, this mechanism of H2O2
formation must be regarded to be a rather general one for biological
systems where sufficiently strong oxidants may interact with various
biologically relevant amino-type molecules, such as ATP, creatine, or
nucleic acids.
 |
INTRODUCTION |
The term "peroxynitrite" is commonly used to describe the
equilibrium mixture of oxoperoxonitrate(1
) (ONOO
) and
its conjugated acid, hydrogen oxoperoxonitrate(1
) (peroxynitrous acid, ONOOH). Peroxynitrite is a strong oxidant formed in the diffusion-controlled reaction of superoxide (O
2) and nitric
oxide (nitrogen monoxide, ·NO) (k = 3.9-6.7 × 109 M
1
s
1) (1, 2). Peroxynitrite has been suggested to play a
major role in many pathological processes like atherosclerosis (3) and
stroke (4). The pathological activity of ONOO
/ONOOH is
assumed to result from its ability to attack various biological
targets, including protein- and non-protein sulfhydryls (5), DNA (6),
low density lipoproteins (3), or membrane phospholipids (7). A favored
method to generate peroxynitrite for experimental purposes is to use
SIN-11 (8). This compound
decays in solution in the presence of oxygen with simultaneous release
of ·NO and O
2 in a 1:1 stoichiometry (9).
Consequently, SIN-1 has been shown to attack many biological targets in
nearly the same manner as authentic peroxynitrite (3). The formation of ONOO
from SIN-1 can be suppressed by the enzyme
superoxide dismutase (SOD), resulting in the formation of ·NO
and hydrogen peroxide (H2O2) as major products
(10).
In obvious contradiction to the assumption of a central role of
ONOO
/ONOOH in cell injuring processes, almost complete
protection from SIN-1 cytotoxicity in experiments with rat liver
endothelial cells and Fu5 rat hepatoma cells was provided by catalase
but not by SOD (11, 12). Since catalase does not effectively react with
peroxynitrite (13), these results strongly suggest a participation of
H2O2 in SIN-1-mediated cytotoxicity rather than
a participation of ONOO
/ONOOH. Indeed, formation of
H2O2 from SIN-1 was observed under certain
experimental conditions (12). We (14) have recently demonstrated that
the formation of H2O2, and consequently the protection exerted by catalase, decisively depends on the presence of
the organic buffer compound HEPES in the incubation medium. In the
absence of this "Good's buffer" neither
H2O2 was formed nor was catalase protective.
The question, however, how HEPES and similar Good's buffers (15, 16)
mediate the formation of H2O2 from SIN-1
remained open. The present study aims at the elucidation of the
underlying chemical mechanism.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Catalase from beef liver (EC 1.11.1.6),
peroxidase from horseradish (EC 1.11.1.7), cytochrome c from
horse heart, and copper-zinc superoxide dismutase from bovine
erythrocytes (EC 1.15.1.1) were obtained from Boehringer Mannheim
(Mannheim, Germany). Dihydrorhodamine 123 (DHR123) and spermine NONOate
were purchased from Molecular Probes (Leiden, The Netherlands).
Diethylenetriaminepentaacetic acid (DTPA), manganese dioxide, hydrogen
peroxide (H2O2), bovine hemoglobin, HEPES, and
other Good's buffers were obtained from Sigma (Deisenhofen, Germany).
SIN-1 and its decomposition product, SIN-1C, were generously provided
by Dr. K. Schönafinger (Hoechst Marion Roussel, Frankfurt/Main,
Germany). Nitrogen 5.0, commercially available mixtures of oxygen 5.0 and nitrogen 5.0 (20.5% O2, 79.5% N2,
"synthetic air"), and commercially available mixtures of oxygen 5.0 and nitrogen 5.0 and carbon dioxide 4.6 (20.5% O2, 74.5%
N2, 5% CO2) were purchased from
Messer-Griessheim (Oberhausen, Germany). Oxoperoxonitrate(1
) (0.62 M) was prepared by isoamyl nitrite-induced nitrosation of
hydrogen peroxide (0.12 mol of isoamyl nitrite, 100 ml of
H2O2 (1 M) plus DTPA (2 mM)) and purified (e.g. solvent extraction,
removal of excess H2O2, N2-purging,
storage) as described by Uppu and Pryor (17). Oxyhemoglobin was
prepared as described previously (12). All other chemicals were of the
highest purity commercially available.
Solutions--
Care was taken to exclude possible contamination
by bicarbonate/carbon dioxide. Doubly distilled water was bubbled (2 liters/min) with synthetic air at room temperature for 20 min. This
water was used for synthesis of oxoperoxonitrate(1
), NaOH (0.01-0.5 N), and for all other solutions. Potassium phosphate buffer
(50 mM) containing DTPA (0.1 mM) was prepared
freshly each day. The pH was adjusted to 7.5 at 37 °C, and the
solution was again bubbled (2 liters/min) with synthetic air (normoxia)
or with nitrogen (hypoxia) or with the carbon dioxide mixture for 20 min. In the case of bubbling with the CO2 mixture, the pH
must be readjusted to 7.5. SIN-1 and spermine NONOate solutions were
prepared as 100× stock solutions at 4 °C in 50 mM
KH2PO4 and 10 mM NaOH,
respectively, and used within 15 min. DHR123 200× stock solution was
prepared in water-free, nitrogen-purged dimethylformamide and stored in the dark at
20 °C.
Experimental Conditions--
SIN-1 and spermine NONOate (final
pH 7.5) were added to 1 ml of phosphate buffer and incubated in 12-well
cell culture plates (volume of each well 7 ml, Falcon, Heidelberg,
Germany). For the detection of H2O2 with the
catalase assay, SIN-1 was added to 10 ml of buffer and incubated in
tissue culture dishes (75 ml, Falcon, Heidelberg, Germany). Under
normoxic conditions these plates/dishes were placed in an air-tight
anaerobic vessel (10 liters). During the first 15 min of each
experiment these vessels were flushed (5 liters/min) with synthetic air
in a warming incubator (Heraeus, Hanau, Germany). In the presence of
HCO3
/CO2 the plates/dishes
were placed in an incubator for cell culture (37 °C, humidified
atmosphere of 95% authentic air and 5% CO2, Labotect,
Göttingen, Germany). The experiments with authentic peroxynitrite
were performed in reaction tubes (1.4 ml, Eppendorf, Hamburg, Germany)
by using the drop-tube Vortex mixer technique as described by Sampson
et al. (18). In brief, 1 ml of buffer (50 mM
potassium phosphate, 0.1 mM DTPA, pH 7.5, 37 °C, in few experiments also in the presence of
HCO3
/CO2 (20 mM/5%)), containing a known amount of target molecules (Good's buffer, p-HPA), was carefully placed into a
reaction tube. A drop of peroxynitrite solution (2 µl, 0.5 M ONOO
in 0.5 N NaOH) was layered
over the buffer at the dry wall. The reaction was started by vortexing.
Thereby the pH increased to 7.56 ± 0.01. Under hypoxic conditions
both the experiments with authentic peroxynitrite and the treatment of
the solutions were performed in a glove bag (Roth, Karlsruhe, Germany)
under nitrogen.
Determination of H2O2, O2,
and O
2--
Hydrogen peroxide was quantified by two
techniques. First, H2O2 was determined by the
horseradish peroxidase-catalyzed reaction of
H2O2 with 4-aminoantipyrine and
3,5-dichloro-2-hydroxybenzenesulfonic acid to a quinoneimine dye that
was measured spectrophotometrically at 546 nm (12) (peroxidase assay).
Alternatively, H2O2 was quantified by the
amount of O2 released upon addition of catalase (1000 units/ml) (catalase assay). O2 was determined
polarographically with a Clark-type oxygen electrode (Eschweiler, Kiel,
Germany). This electrode was also used to detect the production of
O2 from decomposed peroxynitrite.
Superoxide radicals were determined by using the modified
ferricytochrome c3+ reduction technique of
McCord and Fridovich (19). Peroxynitrite was vortexed to the reaction
solution in the absence and presence of various concentrations of
HEPES. Cytochrome c (20 µM) or cytochrome c plus SOD (100 units/ml) were added 2 min after
peroxynitrite addition. This time lag guaranteed that neither
cytochrome c nor SOD reacted directly with peroxynitrite.
The resulting mixture was stored for 40 min at 37 °C. The amount of
cytochrome c reduction was determined by reading the
absorbance at 550 nm (
550 = 21,000 M
1 s
1) (20). The amount of
reduced cytochrome c in the presence of SOD was subtracted
from the amount of reduced cytochrome c in the absence of
SOD for each HEPES concentration. This difference was used to calculate
the amount of trapped O
2.
Capillary Zone Electrophoresis Measurements--
SIN-1 and
SIN-1C were quantified on a Beckman P/ACE 5000 apparatus. Separation
conditions for SIN-1 and SIN-1C were as follows: fused silica capillary
(50-cm effective length, 75 µM internal diameter),
hydrodynamic injection for 5 s, temperature 23 °C, voltage 20 kV, normal polarity, UV detection at 280 nm, and 100 mM
potassium phosphate, pH 6.3, as electrolyte system.
ESR Measurements--
ESR spectra were recorded at ambient
temperature on a Bruker ESP300E X-band spectrometer (Bruker,
Rheinstetten, Germany) equipped with a TM110 wide bore
cavity. Solutions were prepared from 1 ml of the buffer solution (pH
7.5) containing HEPES or PIPES (both 400 mM) and SIN-1 (10 mM). Alternatively, 200 µl of authentic peroxynitrite
(500 mM) and 1.8 ml of the buffer solution (pH 7.5) containing HEPES or PIPES (both 1 M) were vortexed. The
reaction solutions were quickly transferred to an aqueous solution
quartz cell (Willmad, Buena, NY). The first spectra were run as fast as
possible, i.e. within 1 min, and then in 5-min intervals.
Recording conditions were as follows: microwave frequency, 9.8 GHz;
modulation, 0.04 mT; signal gain, 5 × 105; sweep
range, 20 mT; sweep time, 4 min. Spectrum simulation was performed
using the WinSim program (21).
Determination of SIN-1-driven Reactions--
The
peroxynitrite-forming properties of 25 µM SIN-1 were
detected with 50 µM DHR123. Formation of rhodamine 123 (RH123) was quantified spectrophotometrically at 500 nm
(
M = 78,000 M
1
cm
1) (22).
Thiobarbituric acid-reactive substances (TBARS) produced from the SIN-1
(1.5 mM)-induced oxidation of deoxyribose (1.5 mM) were detected spectrophotometrically at 532 nm using
the TBARS assay (23). A standard calibration curve was prepared from
known amounts of malondialdehyde generated by acid hydrolysis of
1,1,3,3-tetramethoxypropane.
Determination of Peroxynitrite-driven Nitration
Reactions--
The peroxynitrite (1 mM)-dependent nitration of
para-hydroxyphenylacetic acid (p-HPA, 1 mM) was employed. After vortexing, the sample was allowed
to stand for 2 min. Then 0.5-1 N NaOH was added (4:1 v/v,
final pH 11-11.5), and the formed 3-nitro-4-hydroxyphenylacetic acid
(3-NO2-4-HPA) was detected spectrophotometrically at 430 nm
(
M = 4400 M
1
cm
1) (17).
Determination of Superoxide Dismutase-like
Activity--
SOD-like activity of HEPES was evaluated using both the
NADH/Mn2+/EDTA/mercaptoethanol (24) and the xanthine
oxidase/xanthine/cytochrome c3+ (19) assay.
Determination of NO Scavenger Activity--
The spermine NONOate
(25 µM)-driven reduction of oxyhemoglobin (70 µM) in the absence and presence of HEPES (1-20
mM) was used. Formation of methemoglobin was quantified
spectrophotometrically at 578 nm (
M = 12100 M
1 cm
1) (12).
 |
RESULTS |
Peroxynitrite from SIN-1 Decomposition
H2O2 Production from SIN-1 in the Presence
of Good's Buffers--
In mere phosphate buffer low levels of
H2O2 (15.2 ± 1.8 µM) were
found after 2 h of incubation with 1.5 mM SIN-1 (Fig.
1). Addition of 20 mM HEPES
to the phosphate buffer largely stimulated H2O2
formation. The H2O2 concentration increased
almost linearly within 2 h up to 183.7 ± 1.5 µM. The yield of H2O2 after
2 h of incubation increased with the concentration of HEPES to
reach a plateau value of 203.0 ± 12.6 µM
H2O2 at about 50 mM HEPES (Fig. 2A). The effect of HEPES on
the formation of H2O2 from SIN-1 was half-maximal at 7 ± 1 mM. In all experiments
described here 0.1 mM DTPA was present to inhibit the
influence of heavy metal contaminations (25). DTPA (0.1 mM)
itself did not affect H2O2 formation, neither in the presence nor in the absence of HEPES (data not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Time dependence of hydrogen peroxide
formation from SIN-1. SIN-1 (1.5 mM) was incubated in
50 mM potassium phosphate buffer (pH 7.5, 37 °C, 0.1 mM DTPA) in the absence and presence of 20 mM
HEPES or 100 units/ml SOD. Aliquots were taken at selected time points,
and H2O2 production was quantified using the
peroxidase assay. Data are means ± S.D. of three experiments
performed in duplicate.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Influence of HEPES and SOD on hydrogen
peroxide formation from SIN-1. SIN-1 (1.5 mM) was
incubated for 2 h in 50 mM potassium phosphate buffer
(pH 7.5, 37 °C, 0.1 mM DTPA). A, in the
absence and presence of various concentrations of HEPES (0-100
mM). B, in the absence and presence of various
activities of SOD (0-10000 units/ml). After the incubation,
H2O2 was quantified using the peroxidase assay.
Data are means ± S.D. of three experiments performed in
duplicate.
|
|
The rate of H2O2 generation in the presence of
20 mM HEPES was comparable to the rate of
H2O2 production in the presence of 100 units/ml
SOD (Fig. 1). 100 units/ml was already the optimal SOD activity to
induce maximal H2O2 production from SIN-1 (Fig. 2B). Both decreasing the SOD activity to 1 unit/ml or
increasing it to 10,000 units/ml strongly decreased
H2O2 formation, in full agreement with results
reported by Gergel et al. (10). Thus, 20 mM
HEPES already stimulated H2O2 formation from
SIN-1 to the maximum yield that could be obtained by SOD.
Other Good's buffers at concentrations of 20 mM were also
able to induce H2O2 formation from SIN-1 (Table
I). The highest yield of
H2O2 after 2 h was observed with EPPS
(~207 µM). HEPES, POPSO, and PIPES induced moderately
less H2O2 formation from SIN-1. The
non-piperazine-type organic buffer triethanolamine (20 mM) also stimulated some production of H2O2 from
SIN-1, about 30% of the maximum amount obtained from HEPES.
View this table:
[in this window]
[in a new window]
|
Table I
Hydrogen peroxide production from SIN-1 stimulated by Good's
buffers
SIN-1 (1.5 mM) was added to various Good's buffers (20 mM) or SOD (100 units/ml) dissolved in 50 mM
potassium phosphate buffer (0.1 mM DTPA, 37 °C, pH 7.5).
After a 2-h incubation at 37 °C, H2O2 was determined
using the peroxidase assay. Each value represents the mean ± S.D.
of three experiments performed in duplicate.
|
|
SOD-like Activity--
Due to the fact that in the presence of
HEPES H2O2 was formed at virtually the same
rate as could maximally be achieved by SOD (Fig. 1 and Fig. 2,
A and B), we first assumed that HEPES might
accelerate the dismutation of O
2. However, we were not able to
detect any SOD-like activity of HEPES neither with a
NADH/Mn2+/EDTA/mercaptoethanol assay nor with a xanthine
oxidase/xanthine/cytochrome c3+ assay (data not
shown). This result is in agreement with the work of Weiss et
al. (26) who found that 60 mM HEPES does not inhibit
O
2-mediated cytochrome c3+
reduction.
·NO Scavenger Properties of HEPES--
An alternative
explanation for the HEPES-dependent increase of
H2O2 formation from SIN-1 was that the buffer
compound might act as a ·NO scavenger, by this means preventing
peroxynitrite formation and thus allowing the released O
2 to
dismutate to H2O2. As the release of nitric
oxide from spermine NONOate is not affected by oxygen (27), we used the
spermine NONOate-driven reduction of oxyhemoglobin to verify ·NO
scavenger properties of HEPES. However, HEPES (1-20 mM)
did not inhibit methemoglobin generation (data not shown).
Involvement of Peroxynitrite--
Since the foregoing experiments
showed that the HEPES-dependent
H2O2 formation from SIN-1 is neither mediated
by a SOD-like activity of HEPES nor by ·NO-HEPES interactions,
we concluded that reaction between peroxynitrite and HEPES should be
responsible for the stimulation of H2O2
generation.
We (14) and others (28) have found that HEPES and SOD influence the
decomposition of SIN-1. To exclude possible artifacts by an altered
decomposition of this compound in the presence of HEPES or SOD, we
performed the subsequent experiments as end point determinations.
DHR123 is oxidized by peroxynitrite to RH123 in the presence of
DTPA but neither by O
2 nor ·NO alone (29), and TBARS
are formed from deoxyribose only by very strong oxidants like hydroxyl
radicals (30) and peroxynitrite (31). The influence of HEPES (20 mM) and SOD (100 units/ml) on the SIN-1-driven oxidation of
both DHR123 and deoxyribose is shown in Fig.
3. After 3 h of incubation 25 µM SIN-1 was completely converted into SIN-1C as
monitored by capillary electrophoresis. At that time SIN-1 (25 µM) had oxidized DHR123 (50 µM) with
formation of 11.4 ± 0.2 µM RH123, i.e.
with an efficiency of about 46%. In agreement, two groups have
reported that authentic peroxynitrite oxidizes DHR123 with an
efficiency close to 44% (32) or 50% (29). RH123 formation decreased
to 5.6 ± 0.6 µM in the presence of HEPES (20 mM) and to 3.7 ± 0.2 µM in the presence
of SOD (100 units/ml), indicating that both compounds were able to
either scavenge peroxynitrite or to prevent its formation. The
solubility of DHR123 in phosphate buffer is too low to allow
concentrations higher than 50 µM. For that reason the
SIN-1 concentration was limited to 25 µM. To corroborate
the reactivity of SIN-1 in the presence of HEPES, we also studied the
oxidation of deoxyribose. After 5 h of incubation of 1.5 mM SIN-1 with 1.5 mM deoxyribose, i.e. after 1.5 mM SIN-1 was completely
decomposed, 3.0 ± 0.2 µM TBARS were found. This
result agrees fairly well with data of Hogg et al. (23) who
reported the formation of about 3 µM TBARS after 5 h
incubation of 1 mM SIN-1 and 1 mM deoxyribose.
In contrast to the findings of Hogg et al. (23), the
presence of SOD (100 units/ml) did not inhibit the oxidation of
deoxyribose. Other reports support our results. Yim et al.
(33) observed a Cu,Zn-SOD-mediated production of hydroxyl radicals from
H2O2, and SOD model compounds induced the
formation of TBARS from deoxyribose in the presence of
H2O2 (34). The addition of HEPES (20 mM) to the buffer solution reduced the production of TBARS
to 1.0 ± 0.1 µM. The strong inhibition of both
TBARS and RH123 formation (Fig. 3) indicated that peroxynitrite produced from SIN-1 should have reacted with HEPES.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of HEPES and SOD on the SIN-1-induced
oxidation of DHR123 and deoxyribose. SIN-1 and DHR123 (25 µM/50 µM) or SIN-1 and deoxyribose (both
1.5 mM) were incubated for 3 or 5 h, respectively, in
50 mM potassium phosphate buffer (pH 7.5, 0.1 mM DTPA, 37 °C) in the absence and presence of HEPES (20 mM) or SOD (100 units/ml). RH123 was quantified by reading
the absorbance at 500 nm and deoxyribose oxidation by determining TBARS
formation. Data are means ± S.D. of three experiments performed
in duplicate and expressed in percent relative to control values.
Formation of 100% product indicated 11.4 ± 0.2 µM
RH123 and 3.0 ± 0.2 µM TBARS, respectively.
|
|
Influence of Tryptophan and Bicarbonate/Carbon Dioxide on
H2O2 Formation--
To support the foregoing
conclusion further, we attempted to scavenge peroxynitrite with
tryptophan, which has been reported to scavenge peroxynitrite with a
rate constant of k = 184 M
1
s
1 (35) but should not react with ·NO or
O
2 at significant rates. Accordingly, tryptophan (10 mM) inhibited the HEPES (5 and 10 mM)-stimulated H2O2 production from SIN-1 by about 50% (Fig. 4A).
The inhibitory effect of tryptophan decreased with increasing
concentrations of HEPES and was abolished at 50 mM
HEPES.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of tryptophan on the formation of
hydrogen peroxide by SIN-1. SIN-1 (1.5 mM) was
incubated for 5 h in 50 mM potassium phosphate buffer
(pH 7.5, 0.1 mM DTPA, 37 °C) in the absence and presence
of tryptophan (10 mM) and various concentrations of HEPES
(0-50 mM). A, in
HCO3 /CO2-free solutions.
B, in the presence of
HCO3 /CO2 (20 mM/5%). H2O2 production was
quantified using the catalase assay. Data are means ± S.D. of
three experiments performed in duplicate.
|
|
Because HCO3
/CO2
accelerates nitration reactions (e.g. nitration of
tryptophan (35)) but inhibits strongly the "hydroxyl radical-like"
activity of ONOOH (36), we studied the HEPES-dependent H2O2 formation from SIN-1 in the presence of
HCO3
/CO2 (20 mM, 5%) (Fig. 4B). In the presence of HEPES
concentrations typically used in experimental systems (10 and 20 mM), the addition of
HCO3
/CO2 slightly enhanced
the production of H2O2 compared with the bicarbonate-free situation. A maximum value of about 220 µM H2O2 was reached at 20 mM HEPES. Contrary to the bicarbonate-free system, however,
higher HEPES concentrations again diminished the
H2O2 level. In the presence of
HCO3
/CO2, tryptophan (10 mM) inhibited the HEPES (5, 10, and 20 mM)-mediated H2O2 production from
SIN-1 somewhat more pronounced than in its absence (Fig.
4B). These results favor the view that peroxynitrite-derived oxidants, e.g. bicarbonate radical, nitrogen dioxide, nitryl
cation, that have been postulated to be produced in the presence of
carbon dioxide (37) also might be able to induce
H2O2 production from HEPES.
Authentic Peroxynitrite
H2O2 Production from Peroxynitrite in the
Presence of HEPES--
To address peroxynitrite-HEPES interactions
more directly, we also employed solutions of authentic peroxynitrite.
As expected, reaction of authentic ONOO
(1 mM) with HEPES (0-400 mM) also resulted in the
formation of H2O2 (Fig.
5). In the absence of HEPES only 6.2 ± 0.4 µM H2O2 was found,
reflecting the hydrogen peroxide base level (0.65 mol %) of our
peroxynitrite stock solution. In the presence of a typical HEPES
concentration (20 mM) the yield of
H2O2 was about 8-fold higher. A limiting value
of 89.9 ± 3.3 µM H2O2 at pH
7.5 was reached again at a concentration of 400 mM HEPES.
The addition of 20 mM HCO3
, 5% CO2 did not
alter the base level of H2O2 (6.1 ± 0.7 µM H2O2) but increased the yield
of hydrogen peroxide by about a factor of 14 in the presence of 20 mM HEPES. A maximum value of 95.4 ± 1.9 µM H2O2 was found at 100 mM HEPES. Further increase of the HEPES concentration to
400 mM diminished the yield of H2O2
to 71.6 ± 3.7 µM. These results are in line with
the data obtained from SIN-1 (Fig. 4B).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
Peroxynitrite-dependent
H2O2 production: influence of HEPES.
Peroxynitrite (1 mM) was added by vortexing to various
concentrations of HEPES in 50 mM potassium phosphate buffer
(0.1 mM DTPA, 37 °C, pH 7.5) in the absence and presence
of HCO3 /CO2 (20 mM/5%). After the addition of peroxynitrite, the samples
were stored for 40 min in a warming or in a cell culture incubator.
H2O2 was quantified using the peroxidase assay.
Each value represents the mean ± S.D. of three experiments
performed in duplicate.
|
|
With regard to the short lifetime of peroxynitrite
(t1/2
1 s at 37 °C)
H2O2 production by action of authentic
peroxynitrite was expected to be complete within a few seconds,
provided that no chain reaction would be initiated. Accordingly, about
88% of the maximum (i.e. after 40 min) yield of
H2O2 was found already 10 s after mixing
of 400 mM HEPES with 1 mM peroxynitrite (Fig. 6). Further growth to the plateau value
of 90 µM H2O2 occurred much
slower, within 40 min.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Time evolution of
H2O2 production from peroxynitrite-HEPES
interaction under hypoxic and normoxic conditions. Peroxynitrite
(1 mM) was vortexed to HEPES (400 mM)
containing potassium phosphate buffer solution (50 mM, 0.1 mM DTPA, 37 °C, pH 7.5) under hypoxic or normoxic
conditions. Aliquots were taken at selected time points, and
H2O2 production was quantified using the
peroxidase assay. Each value represents the mean ± S.D. of three
experiments performed in duplicate.
|
|
Oxygen Dependence of H2O2
Formation--
According to our proposed mechanism (see
"Discussion") atmospheric oxygen is transformed to
H2O2; hence, H2O2
production should be critically dependent on the oxygen concentration.
However, the yield of H2O2 was found to be
largely independent on the oxygen level (Fig. 6). Somewhat
surprisingly, even under hypoxic conditions about 88% of the maximum
yield of H2O2 was produced within 10 s.
Only the minor (12%) long term production of
H2O2 appeared to be dependent on the oxygen
level.
Oxygen and H2O2 Formation under Hypoxic
Conditions--
The apparent independence of
H2O2 production on the level of dissolved
oxygen implied that either peroxynitrite is directly converted to
H2O2 or that a substantial amount of oxygen is
released in the decay of peroxynitrite. In fact, oxygen release from
peroxynitrite has been reported previously (7, 38, 39). To check on
this, we measured the release of O2 from peroxynitrite in
the presence and absence of HEPES (400 mM) (Table
II). In the absence of HEPES we observed
at pH 7.5 the formation of oxygen, in agreement with the above report
(39). In mere phosphate buffer an oxygen production of 175 ± 7.9 µM was measured within 10 s after addition of
peroxynitrite (1 mM). However, in the presence of DPTA (0.1 mM) the maximum yield of oxygen dropped to 132.8 ± 7.8 µM, in agreement with data published by Radi et
al. (7). In any case, oxygen is produced at a level sufficient to
account for the observed yield of H2O2 (78.9 ± 2.9 µM). On the other hand, release of
oxygen from peroxynitrite was abolished in the presence of 400 mM HEPES. In mere phosphate buffer at pH 5 the release of
oxygen was reduced by about 90% (17.0 ± 3.9 µM).
Consequently, H2O2 production was strongly
suppressed to 11.8 ± 0.9 µM in the presence of
HEPES under these hypoxic conditions (Table II). By way of contrast,
under normoxic conditions (see Fig. 10) peroxynitrite stimulated
formation of H2O2 at this pH to about 50 µM. Thus, atmospheric oxygen and/or oxygen donated from
peroxynitrite is necessary for H2O2
production from peroxynitrite-HEPES interactions.
View this table:
[in this window]
[in a new window]
|
Table II
Peroxynitrite-induced formation of oxygen and hydrogen peroxide under
hypoxic conditions
Peroxynitrite (1 mM) was added under hypoxic conditions in
the absence and presence of HEPES (400 mM) or DTPA (0.1 mM) in 50 mM potassium phosphate buffer
(37 °C, pH 7.5 and 5). Oxygen was measured polarographically with a
Clark-type electrode. In another set of experiments,
H2O2 was quantified after an incubation of 40 min using
the peroxidase assay. Each value represents the mean ± S.D. of
six experiments.
|
|
Influence of HEPES on Peroxynitrite-mediated Nitration of
p-Hydroxyphenylacetic Acid--
Peroxynitrite (1 mM)-mediated nitration of p-HPA (1 mM) yielded 79.6 ± 1.5 µM
3-NO2-4-HPA in the absence of
HCO3
/CO2 (20 mM/5%) and 189.2 ± 2.8 µM in its
presence (Table III). These yields (8 and
19%) are virtually identical to those found for nitration of tyrosine
(~6-7 and 19%) at the same pH (40). In the presence of SOD (100 units/ml) the yields of 3-NO2-4-HPA increased slightly by
about 10%, in line with reports by Beckman et al. (41). The
addition of HEPES strongly decreased the peroxynitrite-mediated nitration of p-HPA. At 20 mM HEPES formation of
3-NO2-4-HPA was reduced by 73% in the absence of
HCO3
/CO2 and by 64% in
its presence. Increasing the HEPES concentration to 400 mM
further inhibited 3-NO2-4-HPA formation under both
conditions by approximately 90%. The effect of HEPES on the
3-NO2-4-HPA formation from peroxynitrite in
HCO3
/CO2-free solution was
half-maximal at 6 ± 1 mM, in excellent agreement with
the half-maximally stimulation of H2O2
production (see above). In the presence of
HCO3
/CO2 the effect of
HEPES on the half-maximal yield of H2O2
formation was almost unchanged (5 ± 1 mM), whereas
nitration of p-HPA was half-maximal at 9 ± 1 mM. These results suggest that HEPES has been attacked by
the same reactive intermediates that are responsible for the nitration
of p-HPA.
View this table:
[in this window]
[in a new window]
|
Table III
Effect of HEPES and superoxide dismutase on the peroxynitrite-induced
nitration of para-hydroxyphenylacetic acid
Peroxynitrite (1 mM) was vortexed to
para-hydroxyphenylacetic acid (p-HPA, 1 mM)
in the absence and presence of HCO3 /CO2 (20 mM/5%), HEPES (0-400 mM), or SOD (0-100 units/ml) in 50 mM potassium phosphate buffer (0.1 mM DTPA,
37 °C, pH 7.5). After a 5-min incubation at 37 °C, the formation
of 3-(NO2)-4-HPA was determined by reading the absorbance at
430 nm. Each value represents the mean ± S.D. of four experiments
performed in duplicate.
|
|
Radicals from Good's Buffers--
In previous studies (42, 43)
fairly persistent ESR spectra have been observed from piperazine buffer
compounds by action of various oxidants like
Fe(II)/H2O2 or polymeric iron/O2.
The spectra were reasonably attributed to the radical cations expected to be generated by one-electron oxidation of the piperazine compounds (42), although the assignment appeared tentative as no detailed analysis of the spectra was performed. We therefore checked, by ESR
spectroscopy, the ability of peroxynitrite to mediate radical formation
from HEPES and PIPES, both by SIN-1 (10 mM) and by
authentic peroxynitrite (50 mM) solutions. After mixing of
the reactants by vortexing, we observed "instantaneously"
(i.e. within 1 min) weak, multi-line ESR spectra (shown for
authentic peroxynitrite in Fig. 7). In
some experiments a 2-4-fold increase of the signal intensity was
observed during the run. Both the HEPES- and PIPES-derived spectra were
virtually identical to those reported in the literature (42, 43). In
agreement with the previous observations the signals decayed with
half-lifes of about 10-15 min at 20 °C. The spectrum obtained from
HEPES was too weak to be evaluated in detail, but the PIPES-derived
spectrum was sufficiently intense to be completely analyzed. Elaborate
analysis by spectral simulation revealed that the ESR spectrum must be
interpreted as a superposition of the two radical cations R1
and R2 (Fig. 8).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
ESR spectrum of radical cations from
oxidation of PIPES with peroxynitrite. ESR spectrum of radical
cations R1 and R2 recorded 4 min after oxidation
of PIPES (900 mM) with peroxynitrite (50 mM),
pH 7.5, at 20 °C. Top, experimental ESR spectrum.
Bottom, simulated ESR spectrum as a 3:1 superposition of
R1 and R2. ESR parameters: R1:
[a(N) = 0.722 (2N), a(H) = 0.698 (4H),
a(H) = 0.522 (4H), a(H) = 0.493 (4H),
a(H) = 0.048 (4H) mT; g = 2.0036];
R2: [a(N) = 0.725 (2N), a(H) = 0.695 (4H), a(H) = 0.509 (4H), a(H) = 0.500 (4H),
a(H) = 0.035 (4H) mT; g = 2.0036]. The
simulated spectrum fits the experimental one with a correlation
coefficient of r = 0.999.
|
|
The presence of at least two radicals was further indicated by slight
temporal changes of the overall spectral shape, indicating somewhat
different lifetimes of both species. A similar superposition has been
observed previously for HEPES-derived ESR spectra (43). The presence of
a small amount of a possible further oxidized species, R3,
cannot be completely excluded. The identification of R1,
R2 as radical cations unambiguously follows from their ESR
spectral properties, that is the even number of interacting nuclei, the
g factor, and the magnitude of the corresponding hyperfine
splittings, all of which are strikingly similar to the data known for
other piperazine and dihydropiperazine radical cations in organic
solvents (44-46). Thus, both SIN-1-generated and authentic
peroxynitrite are able to oxidize Good's buffers by one-electron
processes.
O
2 and H2O2 Formation from HEPES
Radicals--
Based on the ESR observations we suspected that the
piperazine-derived radical cations may react with O2 to
generate O
2 which subsequently dismutates to
H2O2 and O2 (see "Discussion").
However, as shown above, about 88% of H2O2
production is already complete within the lifetime of peroxynitrite,
whereas the ESR-detected piperazine-type radicals are much more
long-lived (t1/2
10-15 min). Thus, only the
additional (12%) long term increase of H2O2
concentration should refer to the reaction of the "ESR observable"
fraction of the HEPES-derived radical cations with atmospheric oxygen. Consequently, O
2 formation in the same concentration range was expected solely by action of the HEPES-derived radical(s),
i.e. without interference by residual peroxynitrite.
In the absence of HEPES the detectable amount of O
2 was low
(
0.1 µM O
2) but increased with increasing
HEPES concentration (Fig. 9). A maximum
amount of 2.5 ± 0.3 µM O
2 was formed in
the presence 400 mM HEPES. Thus, attack of authentic
peroxynitrite on HEPES indeed generates O
2. The fact that only
micromolar amounts of O
2 were detected agrees with the low
concentrations of the HEPES-derived radical cations as deduced from the
ESR signal intensities.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 9.
Peroxynitrite-dependent
O 2 production. Influence of HEPES. Peroxynitrite (1 mM) was added by vortexing to various concentrations of
HEPES in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C, pH 7.5). After 2 min of incubation the
samples were divided in 2 aliquots. In 1 aliquot cytochrome
c3+ (20 µM) was added, and in the
other aliquot cytochrome c3+ plus SOD (100 units/ml) was added. The 2 aliquots were stored for 40 min in a warming
incubator. Cytochrome c2+ was detected by
reading the absorbance at 550 nm. For each HEPES concentration the
difference A550aliquot 1 A550aliquot 2 was used
to calculate the trapped amounts of O 2. Each value represents
the mean ± S.D. of three experiments performed in
duplicate.
|
|
pH dependence of H2O2 Formation--
Only
the amino unprotonated forms of HEPES, PIPES, etc. are amenable to
one-electron oxidation to yield radicals R1-R3 (Fig. 8). Thus, H2O2 formation was expected to
be pH-dependent. In the absence of HEPES a constant, low
amount of H2O2 (~7 µM) was
found within the pH range 4-8.5 after vortexing of peroxynitrite (1 mM) solutions (Fig. 10). At
pH 4 no change of H2O2 level was detected in
the presence of HEPES (400 mM). However, with increasing pH
H2O2 formation strongly increased to reach a
maximum value of about 90 µM at pH 7.5. Further increase
of the pH again diminished the production of
H2O2. The same pH dependence on
H2O2 formation, with maxima at pH 7.5, was also
observed for EPPS and MOPS (data not shown). The
pH-dependent half-maximal production of
H2O2 correlated well with the
pKa of the respective buffer compound (Table IV). In agreement with the proposal that
the unprotonated forms of the tertiary amines are oxidized by
peroxynitrite, the quaternary amino compound tetramethylammonium
chloride (400 mM) did not stimulate H2O2 formation (Table IV).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 10.
Influence of pH on hydrogen peroxide
formation from peroxynitrite-HEPES interactions. Peroxynitrite (1 mM) was added by vortexing to 50 mM potassium
phosphate buffer (0.1 mM DTPA, 37 °C) solutions at
various pH values (4-8.5) in the absence and presence of 400 mM HEPES. After the addition of peroxynitrite, the samples
were stored for 40 min in a warming incubator.
H2O2 was quantified using the peroxidase assay.
Data are means ± S.D. of three experiments performed in
duplicate.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Influence of pH on hydrogen peroxide production from peroxynitrite
and various amines
Peroxynitrite (1 mM) was vortexed to various amines (400 mM) in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37 °C) solutions. Maximal H2O2
formation from peroxynitrite-amine interactions were observed at pH
7.5. For pH-dependent half-maximal formation of
H2O2 from peroxynitrite-amine interactions, pH was
decreased stepwise ( pH 0.5) from 7.5 to 4.5. After the addition of
peroxynitrite, the samples were stored for 40 min in a warming
incubator. H2O2 was quantified using the peroxidase
assay. Data are means ± S.D. of three experiments performed in
duplicate.
|
|
 |
DISCUSSION |
Reactions between authentic or in situ generated
peroxynitrite and HEPES or PIPES result in the formation of HEPES- and
PIPES-derived radical cation species (R1 and R2)
(Fig. 8) as shown by ESR spectroscopy (Fig. 7). A reasonable mechanism
for the formation of R1 and R2 is displayed in
Fig. 11, where [Ox] stands for the
action of any sufficiently strong oxidant in the system,
e.g. peroxynitrite and peroxynitrite-derived oxidants formed
in the absence and presence of carbon dioxide, without specifying the
actual reactive species.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 11.
Radical cation formation from Good's
buffers by one-electron oxidants. In the first step one-electron
oxidation of the piperazine compounds produces the amine radical cation
R1 which subsequently undergoes -deprotonation, a common
decay path for such types of radical cations (47, 58). The putative
-aminoalkyl radical R4 certainly is too short-lived to be
observed under the conditions of the ESR experiment (see below).
Further oxidation of R4 to yield R2 also is
feasible, in particular in the presence of O2 (47, 58). A
further reaction of R2 to give R3 could not be
verified by the ESR spectra but cannot be ruled out.
|
|
Fig. 11 also provides the starting point for the interpretation of
O
2 formation in our system. It is common knowledge that carbon-centered radicals react rapidly (k
2 × 109 M
1 s
1) with
molecular oxygen to give peroxyl radicals (47, 48). However, as shown
by von Sonntag's group (reviewed in Ref. 47) in the case of
electron-rich alkyl radicals, i.e. those carrying electron-donating substituents, the corresponding peroxyl radicals are
highly unstable, decomposing rapidly (k
104-109 M
1
s
1) to a cationic species and O
2 (47, 49, 50).
In fact, this property of electron-rich alkyl radicals has recently
been used as the basis for the development of a thermal superoxide
source (51).
By analogy, we propose that a similar mechanism is operative in the
production of O
2 from Good's buffers by attack of authentic or SIN-1-generated peroxynitrite (Fig.
12). Thus, it would appear that the
rate of O
2 production is governed by the first two steps (electron transfer and deprotonation) of the reaction sequence. On the
other hand, R4 may also be generated directly from the
parent compound by hydrogen abstraction by suitably reactive radicals
X·, e.g. hydroxyl or peroxyl radicals. In
line with this assumption, formation of R1 and R4
has been postulated in the autoxidation of
DNA/Cu2+/H2O2 systems in the
presence of HEPES and PIPES (52).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 12.
Proposed mechanism of hydrogen peroxide
formation from piperazine-based buffer compounds and peroxynitrite or
other oxidants. As already shown in Fig. 11 the reaction sequence
is initiated by one-electron oxidation of the piperazine compound by
peroxynitrite. The -aminoalkyl radical R4 formed by
proton loss from the initial radical cation R1 would react
at a close to diffusion-controlled rate with oxygen. Although not
detected by ESR, radical R1 may likewise undergo
deprotonation at the side chains to give exocyclic -amino radicals
R4', which would react with O2 in the same
manner. Rapid fragmentation of the so-formed peroxyl radicals R5,
R5' produces O 2 and the cationic species 6.
The latter would be rapidly trapped by reaction with water or other
nucleophiles (47) and/or further oxidized to the radical cation
R2. Dismutation of O 2 finally leads to
H2O2.
|
|
Further consequences arise from the foregoing. First,
O
2/H2O2 production is expected to be
pH-dependent, decreasing with decreasing pH and reflecting
the respective pKa of the organic buffer compound.
This hypothesis was confirmed by the data of Fig. 10 and Table IV, in
agreement with the requirement of an unshared electron pair at nitrogen
for the oxidation process. Second, the efficiency of
O
2/H2O2 production among the various buffer compounds should be somehow affected by the structure and properties of the side chains attached to the piperazine rings. As
amino-derived radical cations are strong electrophiles and are normally
rapidly quenched in aqueous solution (47, 53, 54), one might
hypothesize that the negatively charged sulfonic acid side chain(s)
exert some "protective" effect on the cationic radical center in
R1, thereby inhibiting the rate of deprotonation. This would
agree with the observations that the monosulfonylated piperazines HEPES
and EPPS gave higher yields of H2O2 (Tables I
and IV) than the bis-sulfonylated POPSO and PIPES, but, vice versa, the HEPES-derived radical showed a much lower ESR signal intensity (steady-state concentration) than the PIPES-derived one.
There is no contradiction in the fact that the ESR spectra of radical
cations R1 and R2 have been detected although their reaction with oxygen is extremely fast. What we detected by ESR
is just the small "excess" amount of R1 and
R2 after all of the dissolved oxygen has been consumed. This
explains the sometimes observed short term growth of the ESR signals
immediately after mixing of the reactants.
The results presented here clearly demonstrate that the interaction of
peroxynitrite and piperazine-type buffers, regardless of the presence
of HCO3
/CO2, may lead to
serious consequences concerning the investigation of
peroxynitrite-driven actions under physiological conditions, e.g. necrosis, apoptosis, inhibition of enzymes,
formation of metabolites, etc. There are several reports in the
literature in which similar effects of the interaction of HEPES with
strong oxidants other than peroxynitrite are mentioned, although no
satisfying explanation has been given. For example, vanadyl induces
hemolysis of vitamin E-deficient erythrocytes in HEPES buffer but not
in phosphate buffer (55); HEPES stimulates hydroxyl radical generation significantly in the presence of both copper ions and
H2O2 (56), and HEPES promotes also hypochlorous
acid-induced oxidation of ferrocyanide very efficiently (57). In
conclusion, it must be emphasized that
O
2/H2O2 formation according to the
above mechanism (Fig. 12) is not restricted to peroxynitrite as an
oxidant and not to piperazine buffer compounds as targets but should be
regarded as a general pathway for compounds from which electron-rich
alkyl radicals (preferably
-amino or
,
-dialkoxyl radicals) can
be generated. Accordingly, H2O2 formation in
the range of 75 µM has been found in the reaction of
peroxynitrite (1 mM) with other tertiary amines (400 mM), viz. triethylamine and
triethanolamine.2 Generally
speaking, any oxidant strong enough to oxidize certain tertiary amines
in a one-electron step would be able to initiate the above reaction
sequence. Furthermore, any other reaction, preferably hydrogen
abstraction, that generates
-amino alkyl or
,
-dialkoxyl
radicals would induce the same process. Because tertiary amine groups
as targets for strong oxidants are present in a variety of biological
molecules, e.g. in ATP, creatine, or nucleic acids, we
propose that Fig. 12 for
O
2/H2O2 production must be
considered to be a general mechanism under in vivo
conditions. This certainly sheds a new light on a number of studies
aimed at the pathophysiological effects of various oxidative species, as actually hydrogen peroxide might have been the true operating agent.
 |
ACKNOWLEDGEMENT |
We thank Angela Wensing for excellent
technical assistance.
 |
FOOTNOTES |
*
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: Institut für
Physiologische Chemie, Universitätsklinikum Essen,
Hufelandstrasse 55, D-45122 Essen, Germany. Tel.: +201/723-4101; Fax:
+201/723-5943.
1
The abbreviations used are: SIN-1,
3-morpholinosydnonimine N-ethylcarbamide; SOD, superoxide
dismutase; EPPS,
N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid; PIPES, piperazine-N,N'-bis(2-ethanesulfonic
acid); POPSO, piperazine-N,N'-bis(2-hydroxypropanesulfonic
acid); MOPS, 3-(N-morpholino)propanesulfonic acid; DHR123,
dihydrorhodamine 123; RH123, rhodamine 123; TBARS, thiobarbituric
acid-reactive substances; 3-NO2-4-HPA,
3-nitro-4-hydroxyphenylacetic acid; p-HPA,
4-hydroxyphenylacetic acid; DTPA, diethylenetriaminepentaacetic acid; mT, millitesla.
2
M. Kirsch, H. G. Korth, R. Sustmann, and H. de Groot, unpublished observations.
 |
REFERENCES |
-
Huie, R. E.,
and Padmaja, S.
(1993)
Free Radical Res. Commun.
18,
195-199[Medline]
[Order article via Infotrieve]
-
Kobayashi, K., Miki, M., and Tagawa, A. (1995) J. Chem. Soc.
Dalton Trans. 2885-2889
-
White, C. R.,
Brock, T. A.,
Chang, L. Y.,
Crapo, J.,
Briscoe, P.,
Ku, D.,
Bradley, W. A.,
Gianturco, S. H.,
Gore, J.,
Freeman, B. A.,
and Tarpey, M. M.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1044-1048[Abstract]
-
Dawson, V. L.,
Dawson, T. M.,
London, E. D.,
Bredt, D. S.,
and Snyder, S. H.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
6368-6371[Abstract]
-
Radi, R.,
Beckman, J. S.,
Bush, K. M.,
and Freeman, B. A.
(1991)
J. Biol. Chem.
266,
4244-4250[Abstract/Free Full Text]
-
King, P. A.,
Anderson, V. E.,
Edwards, J. O.,
Gustafson, G.,
Plumb, R. C.,
and Suggs, J. W.
(1992)
J. Am. Chem. Soc.
114,
5430-5432
-
Radi, R.,
Beckman, J. S.,
Bush, K. M.,
and Freeman, B. A.
(1991)
Arch. Biochem. Biophys.
288,
481-487[Medline]
[Order article via Infotrieve]
-
Uppu, R. M.,
Squadrito, G. L.,
Cueto, R.,
and Pryor, W. A.
(1996)
Methods Enzymol.
269,
285-295[Medline]
[Order article via Infotrieve]
-
Kelm, M.,
Dahmann, R.,
Wink, D.,
and Feelisch, M.
(1997)
J. Biol. Chem.
272,
9922-9932[Abstract/Free Full Text]
-
Gergel, D.,
Misik, V.,
Ondrias, K.,
and Cederbaum, A. I.
(1995)
J. Biol. Chem.
270,
20922-20929[Free Full Text]
-
Volk, T.,
Ioannidis, I.,
Hensel, M.,
de Groot, H.,
and Kox, W. J.
(1995)
Biochem. Biophys. Res. Commun.
213,
196-203[CrossRef][Medline]
[Order article via Infotrieve]
-
Ioannidis, I.,
and de Groot, H.
(1993)
Biochem. J.
296,
341-345[Medline]
[Order article via Infotrieve]
-
Floris, R.,
Piersma, S. R.,
Yang, G.,
Jones, P.,
and Wever, R.
(1993)
Eur. J. Biochem.
215,
767-775[Abstract]
-
Lomonosova, L. L., Kirsch, M., Rauen, U., and de Groot, H. (1998)
Free Radical Biol. Med., in press
-
Good, N. E.,
Winget, G. D.,
Winter, W.,
Connolly, T. N.,
Izawa, S.,
and Singh, R. M. M.
(1966)
Biochemistry
5,
467-477[Medline]
[Order article via Infotrieve]
-
Good, N. E.,
and Izawa, S.
(1972)
Methods Enzymol.
24,
53-68[Medline]
[Order article via Infotrieve]
-
Uppu, R. M.,
and Pryor, W. A.
(1996)
Anal. Biochem.
236,
242-249[CrossRef]
-
Sampson, J. B.,
Rosen, H.,
and Beckman, J. S.
(1996)
Methods Enzymol.
269,
210-219[Medline]
[Order article via Infotrieve]
-
McCord, J. M.,
and Fridovich, I.
(1969)
J. Biol. Chem.
244,
6049-6055[Abstract/Free Full Text]
-
Massey, V.
(1959)
Biochim. Biophys. Acta
34,
255-256[CrossRef]
-
Duling, D. R.
(1994)
J. Magn. Reson.
104,
105-110[CrossRef]
-
Haddad, I. Y.,
Crow, J. P.,
Hu, P.,
Ye, Y.,
Beckman, J. S.,
and Matalon, S.
(1994)
Am. J. Physiol.
267,
L242-L249[Abstract/Free Full Text]
-
Hogg, N.,
Darley-Usmar, V. M.,
Wilson, M. T.,
and Moncada, S.
(1992)
Biochem. J.
281,
419-424[Medline]
[Order article via Infotrieve]
-
Paoletti, F.,
Aldinucci, D.,
Mocali, A.,
and Caparrini, A.
(1986)
Anal. Biochem.
154,
536-541[Medline]
[Order article via Infotrieve]
-
Graf, E.,
Mahoney, J. R.,
Bryant, R. G.,
and Eaton, J. W.
(1984)
J. Biol. Chem.
259,
3620-3624[Abstract/Free Full Text]
-
Weiss, R. H.,
Flickinger, A. G.,
Rivers, W. J.,
Hardy, M. M.,
Aston, K. W.,
Ryan, U. S.,
and Riley, D. P.
(1993)
J. Biol. Chem.
268,
23049-23054[Abstract/Free Full Text]
-
Ioannidis, I.,
Bätz, M.,
Paul, T.,
Korth, H. G.,
Sustmann, R.,
and de Groot, H.
(1996)
Biochem. J.
318,
789-795[Medline]
[Order article via Infotrieve]
-
Feelisch, M., Ostrowski, J., and Noak, E. (1989) J. Cardiovasc. Pharmacol. 14, Suppl. 11, 13-22
-
Kooy, N. W.,
Royall, J. A.,
Ischiropoulos, H.,
and Beckman, J. S.
(1994)
Free Radical Biol. Med.
16,
149-156[CrossRef][Medline]
[Order article via Infotrieve]
-
Halliwell, B.,
and Gutteridge, J. M. C.
(1981)
FEBS Lett.
128,
347-352[CrossRef][Medline]
[Order article via Infotrieve]
-
Beckman, J. S.,
Beckman, T. W.,
Chen, J.,
Marshall, P. A.,
and Freeman, B. A.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
1620-1624[Abstract]
-
Crow, J. P.
(1997)
Nitric Oxide
1,
145-157[CrossRef][Medline]
[Order article via Infotrieve]
-
Yim, M. B.,
Chock, P. B.,
and Stadtman, E. R.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
5006-5010[Abstract]
-
Ueda, J.,
Sudo, A.,
Mori, A.,
and Ozawa, T.
(1994)
Arch. Biochem. Biophys.
315,
185-189[CrossRef][Medline]
[Order article via Infotrieve]
-
Alvarez, B.,
Rubbo, H.,
Kirk, M.,
Barnes, S.,
Freeman, B. A.,
and Radi, R.
(1996)
Chem. Res. Toxicol.
9,
390-396[CrossRef][Medline]
[Order article via Infotrieve]
-
Lemercier, J. N.,
Padmaja, S.,
Cueto, R.,
Squadrito, G. L.,
Uppu, R. M.,
and Pryor, W. A.
(1997)
Arch. Biochem. Biophys.
345,
160-170[CrossRef][Medline]
[Order article via Infotrieve]
-
Lymar, S. V.,
and Hurst, J. K.
(1995)
J. Am. Chem. Soc.
117,
8867-8868
-
Alvarez, B.,
Denicola, A.,
and Radi, R.
(1995)
Chem. Res. Toxicol.
8,
859-864[Medline]
[Order article via Infotrieve]
-
Pfeiffer, S.,
Gorren, A. C. F.,
Schmidt, K.,
Werner, E. R.,
Hansert, B.,
Bohle, D. S.,
and Mayer, B.
(1997)
J. Biol. Chem.
272,
3465-3470[Abstract/Free Full Text]
-
Lymar, S. V.,
Jiang, Q.,
and Hurst, J. K.
(1996)
Biochemistry
35,
7855-7861[CrossRef][Medline]
[Order article via Infotrieve]
-
Beckman, J. S.,
Ischiropoulos, H.,
Zhu, L.,
Woerd, M. V. D.,
Smith, C.,
Chen, J.,
Harrison, J.,
Martin, J. C.,
and Tsai, M.
(1992)
Arch. Biochem. Biophys.
298,
438-445[Medline]
[Order article via Infotrieve]
-
Grady, J. K.,
Chasteen, N. D.,
and Harris, D. C.
(1988)
Anal. Biochem.
173,
111-115[Medline]
[Order article via Infotrieve]
-
Antholine, W. E.,
Kalyanaraman, B.,
Templin, J. A.,
Byrnes, R. W.,
and Petering, D. H.
(1991)
Free Radical Biol. Med.
10,
119-123[Medline]
[Order article via Infotrieve]
-
Kniep, C. (1997) Eine mechanistische Studie zur Reaktivität
aminosubstituierter Diensysteme in normalen
Diels-Alder-Reaktionen. Doctoral thesis, Universität
Essen
-
Nelsen, S. F.
(1990)
in
Landolt-Börnstein, New Series Magnetic Properties of Free Radicals (Fischer, H., ed), Vol. 17, pp. 122-245, Springer-Verlag, Berlin
-
Nelsen, S. F.
(1980)
in
Landolt-Börnstein, New Series Magnetic Properties of Free Radicals (Fischer, H., ed), Vol. 9, pp. 22-123, Springer-Verlag, Berlin
-
von Sonntag, C.,
and Schuchmann, H. P.
(1991)
Angew. Chem. Int. Ed. Engl.
30,
1229-1253[CrossRef]
-
Neta, P.,
Huie, R. E.,
and Ross, A. B.
(1990)
J. Phys. Chem. Ref. Data
19,
413-513
-
Mieden, O. J.,
and von Sonntag, C.
(1989)
J. Chem. Soc. Perkin Trans.
2,
2071-2078
-
Mieden, O. J.,
Schuchmann, M. N.,
and von Sonntag, C.
(1993)
J. Phys. Chem.
97,
3783-3790
-
Ingold, K. U.,
Paul, T.,
Young, M. J.,
and Doiron, J.
(1997)
J. Am. Chem. Soc.
119,
12364-12365[CrossRef]
-
Prütz, W. A.
(1990)
Z. Naturforsch.
45,
1197-1206
-
Bard, A. J.,
Ledwith, A.,
and Shine, H. J.
(1976)
Advances in Physical Organic Chemistry, Vol. 13, pp. 155-278, Academic Press, London
-
Hammerich, O.,
and Parker, V. D.
(1984)
Adv. Phys. Org. Chem.
20,
55-189
-
Hamada, T.
(1994)
Experientia (Basel)
50,
49-53
-
Simpson, J. A.,
Cheeseman, K. H.,
Smith, S. E.,
and Dean, R. T.
(1988)
Biochem. J.
254,
519-523[Medline]
[Order article via Infotrieve]
-
Prütz, W. A.
(1996)
Arch. Biochem. Biophys.
332,
110-120[CrossRef][Medline]
[Order article via Infotrieve]
-
Chow, Y. L.,
Dauen, W. C.,
Nelsen, S. F.,
and Rosenblatt, D. H.
(1978)
Chem. Rev.
78,
243-273
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.