Transient and Steady-state Kinetics of the Oxidation of
Substituted Benzoic Acid Hydrazides by Myeloperoxidase*
Ursula
Burner
,
Christian
Obinger
§,
Martina
Paumann
,
Paul G.
Furtmüller
, and
Anthony J.
Kettle¶
From the
Institute of Chemistry, University of
Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria and
¶ Free Radical Research Group, Department of Pathology,
Christchurch School of Medicine, P. O. Box 4345, Christchurch,
New Zealand
 |
ABSTRACT |
Myeloperoxidase is the most abundant protein in
neutrophils and catalyzes the production of hypochlorous acid. This
potent oxidant plays a central role in microbial killing and
inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a
mechanism-based inhibitor of myeloperoxidase that is oxidized to
radical intermediates that cause enzyme inactivation. We have
investigated the mechanism by which benzoic acid hydrazides (BAH) are
oxidized by myeloperoxidase, and we have determined the features that
enable them to inactivate the enzyme. BAHs readily reduced compound I
of myeloperoxidase. The rate constants for these reactions ranged from
1 to 3 × 106 M
1
s
1 (15 °C, pH 7.0) and were relatively insensitive to
the substituents on the aromatic ring. Rate constants for reduction of
compound II varied between 6.5 × 105
M
1 s
1 for ABAH and 1.3 × 103 M
1 s
1 for
4-nitrobenzoic acid hydrazide (15 °C, pH 7.0). Reduction of both
compound I and compound II by BAHs adhered to the Hammett rule, and
there were significant correlations with Brown-Okamoto substituent
constants. This indicates that the rates of these reactions were simply
determined by the ease of oxidation of the substrates and that the
incipient free radical carried a positive charge. ABAH was oxidized by
myeloperoxidase without added hydrogen peroxide because it underwent
auto-oxidation. Although BAHs generally reacted rapidly with compound
II, they should be poor peroxidase substrates because the free radicals
formed during peroxidation converted myeloperoxidase to compound III.
We found that the reduction of ferric myeloperoxidase by BAH radicals
was strongly influenced by Hansch's hydrophobicity constants. BAHs
containing more hydrophilic substituents were more effective at
converting the enzyme to compound III. This implies that BAH radicals
must hydrogen bond to residues in the distal heme pocket before they
can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs
was related to how readily they were oxidized, but there was no
correlation with their rate constants for reduction of compounds I or
II. We propose that BAHs destroy the heme prosthetic groups of the
enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex.
 |
INTRODUCTION |
Myeloperoxidase is the most abundant protein of neutrophils
(polymorphonuclear leukocytes) (1). It is stored in their azurophilic or primary granules, and they use it to kill invading pathogens (2).
These granulocytic cells ingest micro-organisms into phagosomes where
they kill them by generating an array of reactive oxidants including
superoxide and hydrogen peroxide. It is assumed that myeloperoxidase
acts by producing hypochlorous acid. The potent cytotoxic action of
hypochlorous acid (3, 4) and the demonstration that it is produced
inside phagosomes (5, 6) reinforces this assumption. Hypochlorous acid
is also likely to contribute to the tissue damage caused by neutrophils
at sites of inflammation by inactivating enzymes, cross-linking
proteins, oxidizing susceptible amino acids, and chlorinating lipids
and tyrosyl residues (7).
Myeloperoxidase is a member of the homologous mammalian peroxidase
family that also includes lactoperoxidase, eosinophil peroxidase, and
thyroid peroxidase. It is a tetrameric heme enzyme composed of two
identical dimers and has a molecular mass of approximately 145 kDa (8).
It is unique among the mammalian peroxidases in its ability to utilize
hydrogen peroxide in the oxidation of chloride to hypochlorous acid.
However, it also catalyzes the formation of hypothiocyanite at
physiological concentrations of thiocyanate and chloride (9) and
readily oxidizes numerous phenols, anilines, and
-diketones to free
radicals (7). In addition, it is capable of hydroxylating aromatic
compounds (10).
Myeloperoxidase undergoes a series of redox transformations during its
production of reactive oxidants (7). Initially, hydrogen peroxide
reacts with the ferric enzyme (MP3+) to convert it to
compound I (Reaction 1). This redox intermediate oxidizes chloride and
thiocyanate via a single two-electron reaction to produce the
respective hypohalous acids and regenerate the native enzyme (Reaction
2). Alternatively, it removes a single electron from a variety of
organic substrates (AH2) to produce free radicals and the
redox intermediate compound II (Reaction 3). The classical peroxidation
cycle is complete when compound II is reduced back to ferric
myeloperoxidase by the organic substrate (Reaction 4).
As yet there is no specific and potent inhibitor of
myeloperoxidase that can be used to unambiguously identify the role of the enzyme in microbial killing and inflammation. Such an inhibitor would also be useful in attenuating
myeloperoxidase-dependent inflammatory tissue damage. A
number of strategies have been used to inhibit the enzyme. Its activity
has been blocked by trapping it as inactive compound II (11),
preventing the binding of chloride and hydrogen peroxide (12, 13), and
destroying its heme prosthetic groups (14, 15). The most promising
inhibitors identified to date are substituted benzoic acid hydrazides,
which inhibited the peroxidation activity by 50% at concentrations
less than 10 µM (16). 4-Aminobenzoic acid hydrazide
(ABAH)1 was the best
inhibitor of both peroxidation and production of hypochlorous acid. It
had no effect on other neutrophil enzymes or on their production of
superoxide (16). Thus, ABAH has considerable potential for identifying
reactions of neutrophils that are dependent on myeloperoxidase.
ABAH was shown to be a suicide substrate of myeloperoxidase (15). The
enzyme oxidizes it to free radical intermediates that reduce ferric
myeloperoxidase to the ferrous enzyme (MP2+; Reaction 5).
In the presence of hydrogen peroxide and ABAH, the heme groups of
myeloperoxidase are destroyed. Oxygen protects the enzyme by converting
ferrous myeloperoxidase to oxymyeloperoxidase or compound III (Reaction
6). To appreciate fully how benzoic acid hydrazides inactivate
myeloperoxidase, it will be necessary to understand how these
substrates are oxidized by the enzyme. In this investigation we have
carried out kinetic studies to determine what features of benzoic acid
hydrazides make them good substrates for compound I and compound II of
myeloperoxidase. We have also investigated the mechanism by which
myeloperoxidase oxidizes ABAH, and we have assessed the structural
aspects of benzoic acid hydrazides that enable them to act as suicide substrates.
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EXPERIMENTAL PROCEDURES |
Materials
4-Aminobenzoic acid hydrazide (ABAH), 4-hydroxybenzoic acid
hydrazide, homovanillic acid, diethylenetriaminepentaacetic acid (DTPA), Cu,Zn-superoxide dismutase, and bovine liver catalase were
purchased from the Sigma. Fluka Chemicals supplied benzoic acid
hydrazide, 3-nitrobenzoic acid hydrazide, and 4-chlorobenzoic acid
hydrazide, and Aldrich supplied 3-methoxybenzoic acid hydrazide and
4-nitrobenzoic acid hydrazide. 4-Methoxybenzoic acid hydrazide was
obtained from Cambrian Chemicals (Croydon, Surrey, UK). Stock solutions
of these hydrazides were prepared daily in distilled water. Hydrogen
peroxide solutions were prepared by dilution of a 30% stock solution
(Merck), and their concentrations were determined by measuring their
absorbance at 240 nm (
240 39.4 M
1 cm
1) (17). CM-Sepharose
Cl-6B was purchased from Amersham Pharmacia Biotech.
Methods
Purification of Myeloperoxidase--
Myeloperoxidase was
purified from human neutrophils to a purity index
(A430/A280) of at least
0.80 as described previously (18). Its concentration was calculated by
measuring its absorbance at 430 nm (
430 91000 M
1 cm
1 per heme) (19).
Transient-state Kinetics--
Sequential stopped-flow
measurements were performed with an Applied Photophysics (UK)
instrument (model SX-18MV). When 100 µl was shot into a flow cell
having a 1-cm light path, the fastest time for mixing two solutions and
recording the first data point was approximately 1.5 ms. Reactions were
carried out in 100 mM sodium/potassium phosphate buffer.
Sequential stopped-flow (multi-mixing) analysis was used for kinetics
measurements of the conversion of compound I to compound II because
compound I is inherently unstable (20). With 0.5 µM
myeloperoxidase, 5 µM was the minimum hydrogen peroxide
concentration required for complete formation of compound I
(characterized by a 50% hypochromicity in the Soret band). Under these
conditions, compound I was completely formed within 40 ms and was
stable for at least a further 10 ms. For reduction of compound I by the
hydrazides, 2 µM ferric myeloperoxidase was premixed in
the aging loop with 20 µM hydrogen peroxide for 40 ms.
The compound I was then allowed to react with varying concentrations of
hydrazides. To ensure first order kinetics, their final concentrations were at least five times that of the enzyme. Formation of compound II
was monitored at 456 nm, which is the wavelength where compound II
absorbs maximally and the isosbestic point for the ferric enzyme and
compound I (21). At least three determinations (1000-4000 data points)
of kobs, the pseudo-first order rate constant,
were performed for each substrate concentration. Second order rate constants were calculated from the slope of the plot of the mean kobs values versus substrate concentration.
Two methods were evaluated for measuring the rate of reduction of
compound II by the hydrazides. In one method a large excess of hydrogen
peroxide over the enzyme was used to generate compound II (21). In the
other method, compound II was formed by using a 10-fold excess of
hydrogen peroxide in the presence of a sub-stoichiometric concentration
of homovanillic acid. We found that homovanillic acid readily reduces
compound I (k3 = 1.7 ± 0.15 × 105 M
1 s
1) but
reacts slowly with compound II (k4 = 230 ± 1.9 M
1 s
1, data not shown).
Both methods gave identical kobs values for reaction of compound II with electron donors. However, we chose to use
the latter method because a greater change in absorbance was achieved
when compound II was reduced, and the lower concentrations of hydrogen
peroxide in the incubation mixture guaranteed pre-steady-state conditions. For reduction of compound II, 2 µM ferric
myeloperoxidase was premixed with 20 µM hydrogen peroxide
and 1.8 µM homovanillic acid in the aging loop. Under
these conditions, compound II was stable for at least 40 s. Twenty
seconds after the initial mixing, compound II was allowed to react with
a substrate. To ensure first order kinetics, the final concentrations
of the hydrazides was at least five times that of the enzyme (2.5-50
µM). Reduction of compound II was monitored by recording
the loss in absorbance at 456 nm (2000-4000 data points). At least
three determinations of the pseudo-first order rate constants,
kobs, were performed for each substrate
concentration, and the mean value was used to calculate
k4 as described above.
Formation of compound III during oxidation of the benzoic acid
hydrazides was recorded using conventional stopped-flow. The native
enzyme (2 µM) was mixed with 20 µM hydrogen
peroxide in the presence of either 20 or 100 µM
hydrazide, and the kinetics of compound III formation were followed at
625 nm. Compound III and compound II were distinguished by the ratios
of their absorbances at 625 and 456 nm, which are 0.52 and 0.2, respectively (20).
Steady-state Kinetics--
The oxidation of ABAH and the
spectral changes of myeloperoxidase at steady state were recorded using
either a Zeiss Specord S-10 or a Beckman DU 7500 diode array
spectrophotometer. Oxidation of ABAH was determined by measuring the
increase in absorbance at 325 nm (15). It was also measured by using
HPLC with electrochemical detection. An aliquot of the reaction system
(10 µl) was injected into a Waters 600E HPLC equipped with a
Phenomenex Lunar C18 column (250 × 1.4 mm). ABAH was eluted at a
flow rate of 0.9 ml/min with 50 mM phosphate buffer, pH
3.0, containing 11% methanol and detected with an ESA Coulachem
detector (E1 100 mV and
E2 500 mV).
Measurement of Myeloperoxidase Activity and Its Inhibition by
Benzoic Acid Hydrazides--
The activity of myeloperoxidase was
determined by measuring the rate at which it oxidized
3,5,3',5'-tetramethylbenzidine (TMB) at 25 °C over the 1st min of
the reaction (22). The reaction was carried out in 100 mM
acetate buffer, pH 5.4, containing 8% dimethylformamide and
started by adding 300 µM hydrogen peroxide. The
concentration of hydrazide that inhibited
myeloperoxidase-dependent oxidation of TMB by 50%
(IC50) was determined by fitting a rectangular hyperbola to
the dose-response curve using nonlinear regression.
To measure residual activity of myeloperoxidase, the enzyme was
extracted from reaction systems under conditions that would ensure that
any compound III present would decay back to the active enzyme (23).
After incubating the hydrazides with myeloperoxidase and hydrogen
peroxide, reactions were stopped with 20 µg/ml catalase, and the
peroxidase was extracted with 100 µl of CM-Sepharose. This anionic
resin was used to separate myeloperoxidase from catalase and other
components of the assay. The solution was mixed for 15 min to allow for
complete binding of myeloperoxidase to the resin. The samples were spun
for 3 min at 2000 rpm. The supernatant was removed, and 1 ml of 10 mM phosphate buffer, pH 7.4, containing 0.1 M
sodium sulfate was added. Samples were mixed for a further 15 min to
wash the resin of unbound protein, centrifuged, and the supernatant
removed. Finally, 1 ml of 10 mM phosphate buffer, pH 7.4, containing 0.25 M sodium sulfate was added, and the samples were mixed for 15 min to elute myeloperoxidase. The samples were spun
at 10,000 rpm for 10 min, and myeloperoxidase activity in the
supernatants was measured using the 3,5,3',5'-tetramethylbenzidine assay.
Statistical Analysis--
Pearson's product moment correlation
was used to test the strength of association between two variables. The
strength of the association is given by the magnitude of the
correlation coefficient (r), and it was considered
significant when p < 0.05.
 |
RESULTS |
Reduction of Compound I by Benzoic Acid Hydrazides--
The
instability of the redox intermediates of myeloperoxidase made it
necessary to use the sequential mixing stopped-flow technique to
determine the rate constants for their reactions with reducing
substrates. Premixing of myeloperoxidase and hydrogen peroxide led to
compound I formation within several milliseconds (not shown) and
allowed the measurement of the rate of its subsequent reaction with an
electron donor after a defined delay time (40 ms). The reaction of
ferric myeloperoxidase with hydrogen peroxide to form compound I was
measured at 426 nm and its bimolecular rate constant
(k1) was derived from a plot of
kobs versus varied peroxide
concentrations (not shown). It was calculated to be (1.4 ± 0.2) × 107 M
1 s
1 at
15 °C (the temperature used in the presteady-state experiments), which is in accordance with previously published values (20, 21).
The kinetics of the reduction of compound I were followed at 456 nm,
which is the isosbestic point between the ferric enzyme and compound I
and the absorbance maximum for compound II formation (21). Kinetic
traces for the reduction of compound I by substituted benzoic acid
hydrazides displayed single exponential character. A typical trace for
ABAH is shown in the inset of Fig.
1A. The pseudo-first order
rate constants at pH 7.0 (kobs) were obtained from these traces and plotted against the concentration of the benzoic
acid hydrazide (Fig. 1A). Slopes of these secondary plots yielded the apparent second order rate constants
(k3) (see Table I). The magnitudes of these rate
constants varied by less than a factor of 3 and are some of the largest
ever measured for reduction of compound I by organic substrates.

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Fig. 1.
Reduction of compound I by substituted
benzoic acid hydrazides. A, the secondary plot of the
pseudo-first order rate constants for the reduction of compound I
(kobs) versus the concentration of
ABAH. Inset, typical sequential stopped-flow time trace of
the reaction of compound I with ABAH. The incubation mixture contained
0.5 µM myeloperoxidase and 5.0 µM ABAH in
100 mM phosphate buffer, pH 7.0, at 15 °C. B,
the correlation between the Brown-Okamoto substituent constants
( +) and the second order rate constants for reduction of
compound I (k3). The rate constant for benzoic
acid hydrazide is k3,0.
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Table I
Rate constants for the reactions of substituted benzoic acid hydrazides
with myeloperoxidase and their IC50 values for inhibition of
peroxidase activity
The rate constants for reduction of compound I (k3)
and compound II (k4) by substituted benzoic acid
hydrazides (BAH) were determined as outlined in Figs. 1 and 2. The
concentration of BAH that gave 50% inhibition (IC50) of the
oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase was
determined as shown in Fig. 8A.
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Reaction of compound I with the substituted benzoic acid hydrazides
conformed to the Hammett equation (Equation 1), where k3,0 is the rate constant for the unsubstituted
derivative;
is the constant for the reaction; and
is a constant
characteristic of each substituent (24).
|
(Eq. 1)
|
A positive value for
indicates an electron-withdrawing group,
and a negative value indicates an electron-donating group. We have
plotted Brown-Okamoto
+ values, which were devised for
electron-donating groups that can interact with a developing positive
charge in the transition state. These constants gave a better
correlation than the original
values (Fig. 1B). The
correlation coefficient r was
0.92 (p = 0.001; n = 8) for
+ and
0.84
(p = 0.009) for
. The value of
, obtained from
the slope of Fig. 1B, was
0.21, which indicates that the
electronic character of the substituents has only small effect on the
oxidation of reducing substrates by compound I.
There was a finite intercept for the plot of
kobs versus the concentration of ABAH
(Fig. 1A). This intercept was seen with all the hydrazides
and was independent of the nature of the substrate (not shown). It
indicates the rate of spontaneous reduction of compound I by excess
hydrogen peroxide. The value we obtained (4.5 ± 1.7 s
1 (n = 8)) is in good agreement with
that determined previously (2.2 ± 1.2 s
1) (25).
Reduction of Compound II by Benzoic Acid
Hydrazides--
Determination of rate constants for the reduction of
compound II by benzoic acid hydrazides is problematic. Previously, they have been measured using transient state conditions whereby compound II
is formed by adding a 50-fold excess of hydrogen peroxide to myeloperoxidase (25). However, with excess hydrogen peroxide the enzyme
is likely to cycle when the reducing substrate is added. Therefore,
steady-state methods have been employed to measure the rate of
reduction of compound II (22). This latter approach is not possible
with the benzoic acid hydrazides because they inactivate
myeloperoxidase during their oxidation. Therefore, we compared two
pre-steady-state strategies for investigating the reduction of compound
II. Either a 50-fold excess of hydrogen peroxide was added to the
native enzyme (25) or a 10-fold excess of hydrogen peroxide plus a
sub-stoichiometric concentration of homovanillic acid was added. Both
approaches gave approximately the same absorbance amplitudes at 456 nm
for the formation of compound II within 20 s of mixing. Using the
sequential mixing mode, similar bimolecular rate constants
(k4) were calculated using these two strategies.
However, the amplitude of the decrease in absorbance at 456 nm was much
smaller when a 50-fold excess of hydrogen peroxide was used. This
demonstrates that a large excess of hydrogen peroxide was less useful
in guaranteeing pre-steady-state conditions.
Consequently, we premixed a 10-fold excess of hydrogen peroxide and
sub-stoichiometric concentrations of homovanillic acid to generate
compound II, and we then followed its reactions with the various
hydrazides. In each case, the loss of absorbance at 456 nm displayed
single exponential character. A typical time trace for the reaction of
ABAH with compound II is shown in the inset of Fig.
2A. The apparent second order
rate constants for the reduction of compound II
(k4) were obtained from secondary plots of the
kobs values versus the concentration
of the hydrazides (Table I). The secondary plot for ABAH is shown in
Fig. 2A. The magnitudes of the rate constants varied by as
much as 600-fold. They differed from those obtained for compound I by
as little as a factor of 5 for ABAH and as much as 1000-fold for
4-nitrobenzoic acid hydrazide. The Hammett plot, in which
+ values were used, demonstrated that there is a larger
substituent effect for reduction of compound II by the hydrazides than
for compound I (Fig. 2B). The value of
was
1.21
(r =
0.92; p = 0.001).

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Fig. 2.
Reduction of compound II by substituted
benzoic acid hydrazides. A, the secondary plot of the
pseudo-first order rate constants for the reduction of compound II
(kobs) versus the concentration of
ABAH. Inset, typical sequential stopped-flow time trace of
the reaction of compound II with ABAH. The incubation mixture contained
0.5 µM myeloperoxidase and 25 µM ABAH in
100 mM phosphate buffer, pH 7.0, at 15 °C. B,
the correlation between the Brown-Okamoto substituent constants
( +) and the second order rate constants for reduction of
compound II (k4). The rate constant for benzoic
acid hydrazide is k4,0.
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With the exception of ABAH, the intercepts on the ordinate for the
secondary plots were small (0.13 ± 0.09 s
1, not
shown). However, koff for ABAH was 1.67 ± 0.67 s
1. This would normally suggest a reversible
reaction (25). However, it most probably reflects formation of compound
III, which was demonstrated to occur during the same time scale as
compound II reduction (see Fig. 9). Oxidation of ABAH by compound II
displayed a broad pH optimum between 5 and 7 (Fig.
3).

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Fig. 3.
The pH profile for reduction of compound II
by ABAH. Conditions were as described in Fig. 2 except that the
concentration of compound II was 1 µM heme, and ABAH was
present at 10 µM. The buffers used were sodium acetate
(100 mM, pH 4, 5, and 6) and sodium/potassium phosphate
(100 mM, pH 6-8).
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Oxidation of ABAH by Myeloperoxidase--
Previously it was
reported that myeloperoxidase requires hydrogen peroxide to catalyze
oxidation of ABAH. We confirmed this result by showing that the initial
rate of oxidation of 1 mM ABAH by 100 nM
myeloperoxidase in the absence of hydrogen peroxide was only 3% of
that in the presence of 50 µM hydrogen peroxide (results
not shown). Oxidation of ABAH was most favored at pH 4 but also had a
broad pH optimum between 7 and 8.5 (Fig.
4). In accordance with the earlier
proposal that oxygen protects the enzyme from inactivation, we found
that there was progressively more oxidation of ABAH and a longer time
until the enzyme was inactivated when the atmosphere was changed from
nitrogen to air to oxygen (not shown).

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Fig. 4.
The pH profiles for oxidation of ABAH.
Myeloperoxidase (100 nM) was added to 100 µM
ABAH with ( ) or without ( ) 50 µM hydrogen peroxide,
and initial rates of oxidation were determined by measuring the
A325 over the first 20 s of the
reaction. The buffers used were 100 mM acetate between pH 4 and 5.5, 100 mM phosphate between pH 6 and 8, and 100 mM Tris/HCl for pH 8.5 and 9. The results are means of at
least duplicate experiments.
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In the absence of added hydrogen peroxide, ABAH was still oxidized by
myeloperoxidase and, even though the reaction was slow, more ABAH was
eventually oxidized than with hydrogen peroxide present (Fig.
5). This situation arose because there
was virtually no enzyme inactivation in the absence of exogenous
hydrogen peroxide. In contrast, with 100 µM hydrogen
peroxide, the enzyme was inactivated within 5 min. The spectral changes
of the oxidation products of ABAH that were observed in the absence of
hydrogen peroxide were identical to those recorded in its presence (not
shown). This result indicates that the product of ABAH oxidation was
the same regardless of whether or not hydrogen peroxide was added to
the reaction system.

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Fig. 5.
Effects of hydrogen peroxide, catalase,
superoxide dismutase, and DTPA on the oxidation of ABAH.
Myeloperoxidase (100 nM) was added to 100 µM
ABAH in 50 mM phosphate buffer, pH 7.4, only or containing
100 µM DTPA, or 100 µM hydrogen peroxide,
100 µg/ml of catalase (CAT), or 100 µg/ml of superoxide
dismutase (SOD). Traces are typical of at least
three experiments.
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Based on the following findings, it is evident that ABAH did not react
directly with ferric myeloperoxidase. Rather it initially underwent
auto-oxidation to produce hydrogen peroxide, which was used by the
enzyme for subsequent oxidation of ABAH. First, oxidation of ABAH did
not occur under an atmosphere of nitrogen and was independent of the
concentration of ABAH between 100 µM and 1 mM
(not shown). The rate of oxidation was not linearly related to the
concentration of myeloperoxidase but reached a plateau above 200 nM enzyme (Fig. 6). Oxidation
was inhibited by the metal-chelating agent
diethylenetriaminepentaacetic acid (DTPA) and by relatively high
concentrations of superoxide dismutase and catalase (Fig. 5). In the
presence of superoxide dismutase there was a distinct lag phase for
oxidation of ABAH (Fig. 5). The length of the lag phase increased with
the concentration of superoxide dismutase, whereas the steady-state
rate of ABAH oxidation decreased as the concentration of superoxide
dismutase was increased (not shown). At least 100 ng/ml (1.6 µM/heme) catalase was required to block oxidation of ABAH
by 100 nM myeloperoxidase. Adding catalase after oxidation
of ABAH had commenced inhibited to the same extent as when it was
present from the start of the reaction. Thus hydrogen peroxide was
required for the continual oxidation of ABAH. There was minimal
oxidation of ABAH below pH 7 but at higher pH values the rate of
oxidation increased markedly (Fig. 4).

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Fig. 6.
The effect of myeloperoxidase on oxidation of
ABAH. Myeloperoxidase was added to 100 µM ABAH in 50 mM phosphate buffer, pH 7.4, and the rate of increase in
A325 was recorded over the first 5 min of the
reaction. Reactions were carried out in the absence of exogenous
hydrogen peroxide.
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Formation of Compound III during Oxidation of Benzoic Acid
Hydrazides--
When myeloperoxidase oxidizes ABAH, it is converted to
its compound III form (15). It was proposed that an ABAH radical, formed in the classical peroxidase cycle (Reactions 1, 3, and 4),
reduces the ferric enzyme to ferrous myeloperoxidase which subsequently
binds oxygen to form compound III (Reactions 5 and 6). To verify this
sequence of reactions, we monitored the inter-conversion of the redox
intermediates of myeloperoxidase during the oxidation of ABAH (Fig.
7A). Hydrogen peroxide and
ABAH were rapidly mixed with myeloperoxidase, and the absorption
spectra of the enzyme were recorded during the first 2 s of the
reaction (Fig. 7A). Within 200 ms there was a decrease in
absorbance at 430 nm and an increase in absorbance at 451 and 625 nm.
These changes are indicative of partial conversion of the enzyme from
its native form to either compound II or compound III. The contribution
these redox intermediates make to the spectrum of myeloperoxidase can be deduced by calculating the ratio of absorbances at 625 and 456 nm
(20, 26). Pure compound II has a ratio of 0.19 and that for pure
compound III is 0.52. At 200 ms
A625/A456 was 0.21 and at
2000 ms it had increased to 0.38. When the reaction was monitored
continuously it was apparent that there was a biphasicity in the
increase in absorbance at 625 nm over time (Fig. 7B, trace 1). This characteristic pattern was also apparent for the
corresponding reactions with p-hydroxybenzoic acid hydrazide
and p-nitrobenzoic acid hydrazide (Fig. 7B, traces
2 and 3). The first increase in absorbance obeyed
pseudo-first order kinetics with observed pseudo-first order rate
constants being the same order of magnitude as demonstrated for the
reduction of compound I by the hydrazides. Thus, the initial rapid
increase in A625 can be unequivocally attributed
to compound II formation. The further increase at 625 nm coupled with
the increase in A625/A456
illustrates that the enzyme is subsequently converted from compound II
to compound III. This temporal analysis demonstrates that production of
ABAH radicals must precede formation of compound III and therefore
supports the earlier proposal that ABAH radicals promote the conversion
of ferric myeloperoxidase to compound III.

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Fig. 7.
The time course for formation of compound III
during oxidation of ABAH. A, sequential formation of
compounds II and III upon mixing of myeloperoxidase (500 nM
per heme) with ABAH (5 µM) and hydrogen peroxide (5 µM). Reactions were carried out in 100 mM
phosphate buffer at pH 7.0, and serial spectra were recorded within
2 s after starting the reaction by adding hydrogen peroxide. Each
spectrum is an average of two scans taken in 140 ms. Trace
1, 0 ms; trace 2, 200 ms; trace 3, 400 ms; and trace 4, 2000 ms. Arrows indicate the
direction of maximum spectral changes. B, conventional
stopped-flow time traces of the reaction between myeloperoxidase (1 µM per heme) and a mixture of 10 µM
hydrogen peroxide and 10 µM substrate in 100 mM phosphate buffer, pH 7.0. Reactions were monitored at
625 nm as an indicator of compound II and compound III formation.
Trace 1, p-aminobenzoic acid hydrazide;
trace 2, p-hydroxybenzoic acid hydrazide; and
trace 3, p-nitrobenzoic acid hydrazide.
|
|
In control experiments we found that hydrogen peroxide (10 µM) alone converted myeloperoxidase to compound II within
20 s, but there was no subsequent accumulation of compound III.
Superoxide dismutase (50 µg/ml) had no effect on production of
compound III by any of the hydrazides. These results exclude the direct
involvement of either hydrogen peroxide or superoxide in the formation
of compound III.
Inhibition of Peroxidase Activity by Benzoic Acid
Hydrazides--
The capacity of benzoic acid hydrazides to inhibit the
peroxidation activity of myeloperoxidase has previously been determined by measuring their ability to block oxidation of TMB (16). In this
study we wanted to assess whether or not the inhibitory capacity of the
hydrazides was related to the ability of myeloperoxidase to oxidize
them. However, it has recently been shown that the method previously
used to measure oxidation of TMB by myeloperoxidase is flawed (22). We
therefore adopted a modified method proposed by Marquez and Dunford
(22) to re-evaluate the IC50 values. 3-Nitro- and
4-nitrobenzoic acid hydrazides were excluded from the analysis because
they promoted the breakdown of the oxidation product of TMB. For each
compound, the IC50 was determined from its dose-response
curve, such as that shown for ABAH (Fig.
8A). The IC50 for
ABAH was unchanged from the previous estimate, but those for the other
hydrazides increased considerably. The revised values are given in
Table I.

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Fig. 8.
The effect of benzoic acid hydrazides on the
oxidation of TMB by myeloperoxidase and hydrogen peroxide.
A, the effect of ABAH on the rate of oxidation of 1.4 mM TMB by 1 nM myeloperoxidase and 300 µM hydrogen peroxide. Reactions were carried out in 100 mM acetate buffer, pH 5.4, at 20 °C, and the rate of
increase at 655 nm due to the oxidation of TMB was determined over the
1st min of reaction. Data are means of triplicate experiments.
B, the inhibitory effect of each substituted benzoic acid
was measured by determining the concentration at which it inhibited
oxidation of TMB by 50% (IC50) under the same conditions
as described in A. These values were plotted against
Hansch's constants ( x) for the various substituents.
|
|
There were no significant correlations between IC50 or log
(1/IC50) and either
,
+, or
k4. Thus, the ability of benzoic acid hydrazides
to inhibit myeloperoxidase in the TMB assay is not related to the rate
at which they are oxidized by the enzyme. In contrast, there was a very
strong correlation between log (1/IC50) and Hansch's
hydrophobicity constants (Fig. 8B; r =
0.94; p = 0.002) (27). The best inhibitors had
substituents that make the benzoic acid hydrazide more hydrophilic (e.g. p-NH2), and the worst
inhibitors had very hydrophobic substituents (e.g.
p-Cl). Linear regression applied to the data in Fig.
8B yielded Equation 2 that can be used to predict the
IC50 of a substituted benzoic acid hydrazide.
x
is Hansch's hydrophobicity constant for substituent X.
|
(Eq. 2)
|
To determine why the IC50 values are related to
Hansch's constants rather than Hammett's constants, we probed the
mechanism by which ABAH inhibits myeloperoxidase in the TMB assay. When ABAH was present at about its IC50, the maximal inhibitory
effect had occurred within the mixing time, and there was no observable lag phase (not shown). Thus, ABAH could not act as a competitive substrate because when fully oxidized its inhibitory effect would cease. This conclusion is reinforced by the fact that the rate constants for reduction of compound I and compound II by TMB (22) are
about the same as those for ABAH (Table I). Since there was 700 times
more TMB in the assay than ABAH, only minimal oxidation of ABAH by
myeloperoxidase would be expected. Alternative explanations for the
effect of ABAH are that it either prevents hydrogen peroxide from
reacting with ferric myeloperoxidase or it inactivates the enzyme. To
check these possibilities, we incubated myeloperoxidase (2.7 nM) with TMB (1.5 mM) and hydrogen peroxide
(280 µM), plus or minus ABAH (2 µM) in 100 mM acetate buffer, pH 5.4, containing 8%
dimethylformamide. Under these conditions, oxidation of TMB was slowed
by 66% over the first 5 min of the reaction. After 5 min, the enzyme
was diluted 10-fold and its residual activity was measured. In the
absence of ABAH, myeloperoxidase lost 27 ± 2% (n = 3) of its activity, whereas 73 ± 7% (n = 3)
was lost in the presence of ABAH. If ABAH was simply binding to the
enzyme and blocking its reaction with hydrogen peroxide, there would be
no loss in activity after dilution of the enzyme. Thus, ABAH must
inactivate myeloperoxidase. As reported previously, ABAH inactivated
myeloperoxidase only in the presence of hydrogen peroxide. This
demonstrates that an oxidation product of ABAH inactivates the enzyme.
To assess whether ABAH reversibly or irreversibly inactivated
myeloperoxidase during the TMB assay, the reaction was stopped after 2 min by adding catalase (20 µg/ml), and myeloperoxidase (5 nM) was extracted with CM-Sepharose. The residual activity of the isolated enzyme was then measured. Extraction of myeloperoxidase was carried out at pH 7.4 to ensure that if any compound III was present it would decay back to the active ferric enzyme (see
"Experimental Procedures"). When the TMB assay was run in the
presence of ABAH (2 µM), 87 ± 12%
(n = 4) of the peroxidase activity was recovered after
extraction. This result demonstrates that ABAH reversibly inactivates
myeloperoxidase during the TMB assay. Presumably, inactivation occurs
through the formation of compound III.
It has been shown that ABAH inactivates myeloperoxidase by being
oxidized to radical intermediates (15). As discussed above, the direct
oxidation of ABAH by myeloperoxidase in the TMB assay is unlikely to
occur. However, myeloperoxidase may oxidize ABAH indirectly by
generating TMB radicals that interchange with ABAH. We therefore
investigated the ability of TMB to promote the oxidation of ABAH by
myeloperoxidase (Table II). Under the
reaction conditions, myeloperoxidase and hydrogen peroxide oxidized
ABAH poorly. However, in the presence of TMB, 64% of ABAH was
consumed. From these results we conclude that in the TMB assay
myeloperoxidase oxidizes ABAH indirectly through the generation of TMB
radicals. Once formed ABAH radicals would react with the ferric enzyme
to promote formation of compound III. The extent to which benzoic acid
hydrazides inhibit myeloperoxidase in the TMB assay will depend on how
readily they reduce the ferric enzyme to compound III. The strong
dependence of their IC50 values on Hansch's constants
indicates that binding of radical intermediates to the enzyme is the
rate-determining step in this process.
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Table II
The effect of TMB on the oxidation of ABAH by myeloperoxidase and
hydrogen peroxide
ABAH (2 µM) was incubated in 50 mM acetate
buffer, pH 5.4, at 20 °C either alone or in the presence of various
combinations of 27 nM myeloperoxidase (MPO), 130 µM hydrogen peroxide, and 200 µM
3,3',5,5'-tetramethylbenzidine (TMB). Two minutes after mixing the
reactants, the concentration of ABAH remaining was assayed by HPLC with
electrochemical detection. The data are means and standard deviations
of three replicates.
|
|
Irreversible Inactivation of Myeloperoxidase by Benzoic Acid
Hydrazides--
Myeloperoxidase was incubated with each of the benzoic
acid hydrazides under standard conditions at pH 7.4, and their ability to irreversibly inactivate the enzyme was determined (see Fig. 9). Three minutes after adding hydrogen
peroxide to myeloperoxidase and the benzoic acid hydrazide, the
reaction was stopped with catalase. Myeloperoxidase was extracted with
CM-Sepharose to ensure it was free of inhibitor and to allow compound
III to decay back to the native enzyme. Residual peroxidase activity
was measured in the TMB assay. All the benzoic acid hydrazides tested
promoted irreversible inactivation of myeloperoxidase. ABAH,
p-hydroxybenzoic acid hydrazide, and
p-methoxybenzoic acid hydrazide destroyed more than 90% of
the activity of the enzyme. There were good correlations between
Hammett's substituent constants (
) (r = 0.90;
p = 0.003) or the Brown-Okamoto constants
(
+) (r = 0.88; p = 0.004) and percentage of residual enzyme activity (Fig. 9). The benzoic
acid hydrazides that contained substituents that were strongly
electron-donating were the better suicide substrates. This result
indicates that oxidation of the benzoic acid hydrazides by an
intermediate of myeloperoxidase determines how effective they are at
irreversibly inactivating the enzyme. However, the correlations with
k3 (r =
0.74;
p = 0.04) and k4
(r =
0.75; p = 0.033) were poor.
There was no significant correlation with Hansch's constants. Thus
binding of an oxidized form of the inhibitors by the enzyme cannot be
involved in the rate-determining step for inactivation.

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Fig. 9.
Inactivation of myeloperoxidase by
substituted benzoic acid hydrazides. Myeloperoxidase (100 nM) was incubated with 500 µM each of a
series of benzoic acid hydrazides in 50 mM phosphate
buffer, pH 7.4, at 20 °C. Reactions were started by adding 100 µM hydrogen peroxide and stopped after 3 min with 20 µg/ml catalase. Myeloperoxidase was extracted, and its residual
activity was measured and correlated against Hammett's substituent
constants ( ). Data are means and ranges of duplicate
experiments.
|
|
 |
DISCUSSION |
4-Aminobenzoic acid hydrazide is the most potent inhibitor of
myeloperoxidase that has been identified to date. It is a
mechanism-based inhibitor and must be oxidized to radical intermediates
to promote irreversible inactivation (15). In this study we have
investigated the mechanism by which myeloperoxidase oxidizes
substituted benzoic acid hydrazides, and we have determined the
features that make them good inhibitors of the enzyme. Our findings are
pertinent to understanding how ABAH and related compounds are
metabolized by myeloperoxidase and advance the knowledge of how this
enzyme acts as a classical peroxidase. They also highlight potential strategies that may be useful in developing more effective inhibitors of myeloperoxidase.
All the benzoic acid hydrazides reacted rapidly with compound I and
compound II. They react faster than tyrosine with compound I, and only
the nitro derivatives are worse substrates than tyrosine for compound
II (25). The adherence of these reactions to the Hammett rule (Figs.
1B and 2B) indicates that the hydrazide
substituent is oxidized in preference to either the amino or hydroxyl
groups in ABAH or 4-hydroxybenzoic acid hydrazide. Furthermore, it
demonstrates that the rates of these reactions are simply related to
the ease at which the hydrazides are oxidized. The small negative value of
(
0.21) obtained for compound I (Fig. 1B) implies
that substituents that donate electrons into the ring favor oxidation
of benzoic acid hydrazides, but oxidation is relatively insensitive to
electronic effects. For comparison, reduction of compound I of
horseradish peroxidase by phenols, anilines, and indoleacetic acids
have
values of
6.9,
7.0, and
5.6, respectively (28, 29).
There was a far greater variation in the rate constants for reduction of compound II by the hydrazides, but
(
1.21) was still relatively small. The value of
for reduction of horseradish peroxidase compound II by phenols is
4.6 (30). A likely explanation for the
lesser reliance on electronic effects for oxidation by myeloperoxidase compared with horseradish peroxidase is that reduction potentials of
the redox intermediates of the mammalian enzyme are considerably higher
than for the plant enzyme (8).
Our results indicate that compound I of myeloperoxidase should be able
to oxidize a wide range of organic substrates with similar efficiency.
By contrast, reduction of compound II will be far more constrained by
the oxidation potentials of substrates. Thus, substrates that react
poorly with compound II would not be expected to be oxidized by the
enzyme. However, in the presence of suitable co-substrates that readily
reduce compound II, such as superoxide, tyrosine, and ascorbate (18,
25, 31), the high reduction potential of compound I could be
exploited to catalyze oxidation of a variety of substrates.
We found that rate constants for reduction of compound I and compound
II correlated better with Brown-Okamoto (
+) constants
than with Hammett constants (
). This result indicates that the
incipient free radical formed in the oxidation of benzoic acid
hydrazides must carry a positive charge. The positive charge will be
delocalized via resonance between the substituent on the aromatic ring
and the hydrazide group. In analogous reactions of horseradish
peroxidase with phenols and anilines the correlation with
, but not
+, was used as evidence for electron transfer between
the substrate and the enzyme with simultaneous loss of a proton (28).
This mechanism cannot apply to oxidation of benzoic acid hydrazides by
myeloperoxidase. Rather electron transfer from the hydrazide to the
enzyme must occur without transfer of a proton. In an earlier study it
was found that the mechanism by which myeloperoxidase oxidizes anilines
is quite different to that for horseradish peroxidase (11). With
myeloperoxidase, peroxidation was strongly influenced by resonance
effects. Thus, it is conceivable that electron transfer from substrate
to compound I or compound II without simultaneous loss of a proton is a
general mechanism for myeloperoxidase.
Myeloperoxidase did not need an exogenous source of hydrogen peroxide
to catalyze the oxidation of ABAH. Rather, ABAH underwent metal-catalyzed auto-oxidation to produce hydrogen peroxide that the
enzyme used for further oxidation of ABAH. The ability of DTPA,
catalase, and superoxide dismutase to inhibit oxidation of ABAH by
myeloperoxidase indicates that auto-oxidation of ABAH is likely to
follow a mechanism similar to that for phenylhydrazine (Reactions
7-R11) (32).
The transitory effect of superoxide dismutase is explained by it
initially retarding Reaction 9, which is likely to be rate-determining in the auto-oxidation of ABAH. However, once sufficient hydrogen peroxide is produced its subsequent formation would be reliant on
Reactions 3, 4, 8, and 11 and thus independent of superoxide. Generation of hydrogen peroxide via this sequence of reactions must be
essential for the continual oxidation of ABAH because catalase
inhibited even when it was added during the reaction. Presumably, ABAH
is resistant to oxidation below pH 7 (Fig. 4) because its protonated
form does not undergo Reactions 7-R9. The lack of oxidation at acidic
pH cannot reflect a slow reaction with myeloperoxidase because, in the
presence of hydrogen peroxide, ABAH was readily oxidized by the enzyme
below pH 7 (Fig. 4), and it reacted most rapidly with compound II
between pH 5 and 7 (Fig. 3). The rate of oxidation of ABAH was
dependent on myeloperoxidase only at low concentrations of the enzyme.
This phenomenon most likely reflects a change in the rate-determining
step from Reaction 1 to the generation of hydrogen peroxide as the
concentration of myeloperoxidase was increased. Other substrates,
including cysteamine, NADPH, dihydroxyfumaric acid, and indol-3-yl
acetic acid (33), are also oxidized by myeloperoxidase in the absence of added hydrogen peroxide. This oxidase activity should allow myeloperoxidase to function at inflammatory sites long after the respiratory burst of neutrophils has stopped. It is likely to be
involved in the metabolism of the anti-tuberculosis drug isoniazid (34)
and other xenobiotics (35).
Although benzoic acid hydrazides react rapidly with compound II, they
are poor peroxidase substrates because they convert the enzyme to
compound III. This explains why the pH optimum for the oxidation of
ABAH in the presence of hydrogen peroxide (Fig. 4) was markedly
different to that for reduction of compound II (Fig. 3). Thus, the pH
profile in Fig. 4 most likely reflects the turnover of compound III. In
this study we have confirmed that compound III is formed from the
reduction of the ferric enzyme by ABAH radicals (Reaction 5) generated
in the classical peroxidation cycle (Reactions 1, 3, and 4). Formation
of compound III hinders oxidation of reducing substrates and explains
how the benzoic acid hydrazides reversibly inhibited oxidation of TMB.
We found that inhibition of TMB by substituted benzoic acid hydrazides was related to Hansch's hydrophobicity constants (Fig. 8B).
From this result we conclude that binding of radicals to the ferric enzyme determines how effective they are at reducing it. The more hydrophilic substituents must favor binding by hydrogen bonding with
amino acid side chains in the distal heme cavity of myeloperoxidase as
proposed for salicylhydroxamic acid (36). Reduction of ferric myeloperoxidase by hydrazide radicals is a crucial step in its inactivation (15). Therefore, the strong influence of hydrophobicity on
this reaction could be exploited to destroy the enzyme and prevent
neutrophils from producing hypochlorous acid.
We were unable to identify the reaction responsible for turnover of
compound III. It could react with superoxide, hydrogen peroxide, ABAH,
or a reducing radical formed from the oxidation of ABAH. Superoxide can
be excluded because oxidation of ABAH occurred in the presence of
superoxide dismutase. The reaction of compound III with hydrogen
peroxide is likely to be too
slow.2 ABAH or an ABAH
radical may reduce compound III to compound I in a similar fashion to
ascorbate (37). This reaction is analogous to that proposed for the
reduction of oxyhemoglobin by phenylhydrazine (32). We did not observe
any spectral changes when ABAH was added to preformed compound III (not
shown), but this may have been due to the fact that compound III was
reformed by the reduction of ferric myeloperoxidase (Reactions 5 and 6).
Benzoic acid hydrazides inhibit myeloperoxidase reversibly by promoting
the formation of compound III and irreversibly by destroying the heme
prosthetic group (15). They inhibited myeloperoxidase in the TMB assay
because TMB promoted their oxidation to radicals that subsequently
converted the enzyme to compound III. The ability of benzoic acid
hydrazides to inhibit myeloperoxidase was not related to how easily the
enzyme oxidized them. This finding demonstrates that the TMB assay was
unsuitable for identifying their potential as mechanism-based
inhibitors. Thus, results obtained using the TMB assays should be
interpreted with caution, and additional assays should be performed to
determine how effectively a particular compound inhibits myeloperoxidase.
We determined how readily benzoic acid hydrazides irreversibly inhibit
myeloperoxidase, and we found that inactivation was strongly related to
Hammett's substituent constants (Fig. 9). However, inactivation was
poorly related to the rate at which they were oxidized by compound I or
compound II. These results indicate that oxidation of the benzoic acid
hydrazides is fundamental to inactivation, but the critical oxidation
reaction does not involve compound I or compound II. Reaction with
compound III cannot be involved either because oxygen protects the
enzyme by binding to ferrous myeloperoxidase (Reaction 6) (15).
Reaction of ferrous myeloperoxidase with hydrogen peroxide and ABAH
causes destruction of the heme prosthetic group (15). Thus, a plausible reaction that precipitates inactivation is reduction of a ferrous myeloperoxidase-hydrogen peroxide complex by ABAH. We plan to investigate this possibility by determining the structural
modifications of myeloperoxidase that occur when it is inactivated
by ABAH.
 |
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: Institute of Chemistry,
University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna,
Austria. Tel.: 43-1-36006-6073; Fax: 43-1-36006-6059; E-mail:
cobinger{at}edv2.boku.ac.at.
2
A. J. Kettle and C. C. Winterbourn,
unpublished result.
 |
ABBREVIATIONS |
The abbreviations used are:
ABAH, 4-aminobenzoic
acid hydrazide;
BAH, benzoic acid hydrazide;
TMB, 3,3'5,5'-tetramethylbenzidine;
DTPA, diethylenetriaminepentaacetic
acid;
HPLC, high performance liquid chromatography.
 |
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