(Received for publication, July 26, 1994; and in revised form, November 29, 1994)
From the
Myeloperoxidase and eosinophil peroxidase catalyzed the
oxidation of bromide ion by hydrogen peroxide
(HO
) and produced a brominating agent that
reacted with amine compounds to form bromamines, which are long-lived
oxidants containing covalent nitrogen-bromine bonds. Results were
consistent with oxidation of bromide to an equilibrium mixture of
hypobromous acid (HOBr) and hypobromite ion (OBr
). Up
to 1 mol of bromamine was produced per mole of
H
O
, indicating that bromamine formation
prevented the reduction of HOBr/OBr
by
H
O
and the loss of oxidizing and brominating
activity. Bromamines differed from HOBr/OBr
in that
bromamines reacted slowly with H
O
, were not
reduced by dimethyl sulfoxide, and had absorption spectra similar to
those of chloramines, but shifted 36 nm toward higher wavelengths.
Mono- and di-bromo derivatives (RNHBr and RNHBr
) of the
amino acid taurine were relatively stable with half-lives of 70
and 16 h at pH 7, 37 °C. The mono-bromamine was obtained with a
200-fold excess of amine over the amount of HOBr/OBr
and the di-bromamine at a 2:1 ratio of HOBr/OBr
to the amine. In the presence of physiologic levels of both
bromide (0.1 mM) and chloride (0.1 M),
myeloperoxidase and eosinophil peroxidase produced mixtures of
bromamines and chloramines containing 6 ± 4% and 88 ± 4%
bromamine. In contrast, only the mono-chloramine derivative (RNHCl) was
formed when a mixture of hypochlorous acid (HOCl) and hypochlorite ion
(OCl
) was added to solutions containing bromide and
excess amine. The rapid formation of the chloramine prevented the
oxidation of bromide by HOCl/OCl
, and the chloramine
did not react with bromide within 1 h at 37 °C. The results
indicate that when enzyme-catalyzed bromide or chloride oxidation took
place in the presence of an amine compound at 10 mM or higher,
bromamines were not produced in secondary reactions such as the
oxidation of bromide by HOCl/OCl
and the exchange of
bromide with chlorine atoms of chloramines. Therefore, the amount of
bromamine produced by myeloperoxidase or eosinophil peroxidase was
equal to the amount of bromide oxidized by the enzyme. Bromide was
preferred over chloride as the substrate for both enzymes.
MPO ()and EPO have a role in the killing of
micro-organisms and inactivation of viruses by leukocytes. The enzymes
catalyze the oxidation of halide ions and the pseudo-halide thiocyanate
ion by H
O
, producing oxidizing and halogenating
agents. Oxidation of a halide (X
) yields the halogen
(X
), hypohalous acid (HOX), or hypohalite ion
(OX
). In aqueous media, products of halide oxidation
are in rapid equilibrium, and the principal form is a
HOX/OX
mixture. These agents have antimicrobial and
antiviral activity but may also damage host tissues and contribute to
inflammatory tissue injury.
Concentrations of substrates in blood
are 0.1 M chloride, 0.02-0.1 mM bromide,
0.1-0.6 µM iodide, and 0.02-0.12 mM thiocyanate(1, 2, 3, 4) .
Because chloride is high, chloride is assumed to be the physiologic
substrate for MPO. However, other substrates compete well with
chloride, particularly when chloride levels are low, such as in
secreted fluids(5) . Moreover, the concentrations of halides
and other substrates in leukocyte phagolysosomes are unknown. EPO has
antimicrobial activity in the presence of HO
and chloride(6) , but many studies have shown that EPO is
less active than MPO with chloride as the substrate. Evidence has been
presented that bromide (4, 7, 8, 9) or thiocyanate (5, 10) may be the physiologic substrate for EPO.
Two problems make it difficult to measure bromide oxidation by
isolated leukocytes or the purified peroxidase enzymes. First, MPO and
EPO catalyze the oxidation of chloride, and products of chloride
oxidation can oxidize other halides in secondary, non-enzymatic
reactions. For example, HOCl/OCl oxidizes bromide to
HOBr/OBr
.
HOCl/OCl and other chlorinating agents also
react with ammonia and amines to produce chloramines, which contain
covalent nitrogen-chlorine bonds(11) . Chloramines retain the
two oxidizing equivalents of HOCl/OCl
and might
oxidize bromide to HOBr/OBr
or undergo exchange with
bromide and be converted to bromamines(12) .
Therefore, the formation of bromamines or other brominated products does not necessarily indicate that bromide was oxidized by MPO or EPO.
The second problem is that HOBr/OBr is
reduced by H
O
, resulting in the loss of
oxidizing and brominating activity. The net result of bromide oxidation
and HOBr/OBr
reduction is the conversion of 2 mol of
H
O
to 2 mol of water and one of molecular
oxygen (O
), which is the same result that would be obtained
by adding catalase.
Reduction of HOBr/OBr by
H
O
is accompanied by formation of singlet
oxygen, which rapidly releases energy as light and forms
O
(13) . Under some conditions, the light emitted by
singlet oxygen provides an indirect measure of the amount of bromide
that was oxidized to HOBr/OBr
(14) . However,
the detection of singlet oxygen would not indicate whether bromide was
oxidized by MPO or EPO or by products of chloride oxidation. Moreover,
singlet oxygen may also be produced in the reduction of
HOCl/OCl
by H
O
.
Little or
no singlet oxygen is detected when MPO (15) or EPO (16) is incubated with HO
and chloride,
probably as a result of inactivation of the enzyme. Similarly,
HOCl/OCl
does not accumulate in the
medium(17) . MPO is rapidly inactivated by
HOCl/OCl
, bringing chloride oxidation to a halt and
leaving H
O
in the medium.
The production of
chlorinating agents is observed only when chloride oxidation takes
place in the presence of a trap for HOCl/OCl. The
trap may be an aromatic compound that undergoes rapid chlorination to
yield a stable product with a carbon-chlorine bond (18, 19, 20) or an amine compound that yields
a stable chloramine derivative(11, 21, 22) .
One mol of chloramine is produced per mole of H
O
when the enzyme, chloride, and amine concentrations are
sufficiently high(11) . The rapid reaction of
HOCl/OCl
with the amine prevents inactivation of the
enzyme by HOCl/OCl
and the reduction of
HOCl/OCl
by H
O
. Unlike
HOCl/OCl
, chloramines are not reduced by
H
O
(11, 23) .
Because
chloramine derivatives of -amino acids are unstable, the
-amino acid taurine is often used as a trap for
HOCl/OCl
. Chlorination of taurine yields relatively
non-toxic, anionic, membrane-impermeable oxidants (22, 23, 24, 25, 26) that
can be measured based on the ability of chloramines to oxidize
sulfhydryl compounds such as Nbs, thioethers such as methionine, or
iodide ion (23) . Mono- and di-chloramine derivatives of
taurine are much less reactive than HOCl/OCl
,
particularly in the oxidation of disulfide bonds and chlorination of
phenols(23) . Leukocytes contain high levels of taurine in the
cytosol(27) , probably to trap oxidized forms of chloride and
bromide and protect the cells against oxidation and halogenation.
Peroxidase-catalyzed oxidation of bromide in the presence of
-amino acids yielded unstable bromamines (7, 28) that rapidly decomposed to products without
oxidizing and brominating activity(29) . Unlike chloramines,
the bromamines were reduced by H
O
(28) .
Extinction coefficients of 415-420 M
cm
were reported
for mono-bromamines at 289 nm(29) , similar to the value of 429 M
cm
obtained for
mono-chloramines at 252 nm(23) .
The aim of this study was
to characterize the oxidants produced by incubating MPO or EPO with
HO
and bromide in the absence of chloride or
with physiologic levels of both bromide and chloride, using taurine as
a trap for oxidized forms of bromide and chloride. As described below,
1 mol of bromamine was produced per mole of H
O
when the enzyme, bromide, and taurine concentrations were
sufficiently high. Bromamine formation prevented the reduction of
HOBr/OBr
by H
O
, and all of
the H
O
was consumed in bromide oxidation before
reduction of bromamines by H
O
could take place.
In addition, trapping of HOCl/OCl
by the amine
prevented the oxidation of bromide in secondary non-enzymatic
reactions. Therefore, enzyme-catalyzed oxidation of bromide or chloride
in mixtures of the two ions could be measured by analyzing the mixture
of bromamines and chloramines that was produced.
MPO and EPO were purified (30) from cytoplasmic
granules of human granulocytes and stored at -70 °C in 0.1 M NaSO
with 10 mM sodium
acetate buffer, pH 4.7. The ratio of 430 to 280 nm absorbance was 0.86
± 0.04 (mean ± S.D.) for three preparations of MPO, and
the ratio of 415 to 280 nm absorbance was 1.00 ± .04 for three
preparations of EPO, indicating a high degree of purity and no
cross-contamination. Concentrations were calculated assuming extinction
coefficients of 89,000 and 112,000 M
cm
/iron of MPO (31) and
EPO(32, 33) . The concentration of the MPO dimer (34) was half the indicated concentration of the heme-like
chromophore(35) .
Bromine (Br), sodium
hypochlorite (4-6%), H
O
(30%), and
potassium bromide were from Fisher Chemical Co. Sodium chloride
(containing 0.005% bromide and 0.001% iodide) was from BDH Limited,
Poole, United Kingdom. Dimethyl sulfoxide (high performance liquid
chromatography grade) was from Aldrich. Taurine, DETAPAC, superoxide
dismutase, 2-mercaptoethanol, and Nbs
were from Sigma.
Catalase crystals (Boehringer Mannheim) were washed twice by
centrifugation in water and dissolved at 0.3 mg/ml in 0.1 M Na
SO
with 15 mM phosphate buffer,
pH 7.0. Taurine (0.2 M) and DETAPAC (0.1 M) were
adjusted to pH 7 with KOH. Fresh 0.1 M potassium bromide
solutions were prepared daily in water. H
O
was
diluted into autoclaved water and the concentration determined from the
extinction coefficient of 70 M
cm
at 230 nm. To prepare Nbs, 100 ml of a 1 mM Nbs
solution in 0.1 M Na
SO
with 10 mM taurine, 0.3 mM DETAPAC, 1 µg of
superoxide dismutase/ml, and 1 µg of catalase/ml in 15 mM phosphate buffer, pH 7.0, was reduced with 4 µl of
2-mercaptoethanol(23) .
To prepare 5 mM HOBr/OBr, bromine (25 µl) was added to 4.5
ml of cold 1.0 M phosphate buffer, pH 7.0, and then diluted
with 87 ml of cold water. The concentration was confirmed by diluting
the HOBr/OBr
solution 10-fold into 0.1 M taurine with 0.1 M phosphate buffer, pH 7.0, measuring
absorbance at 288 nm, and calculating the concentration of the
mono-bromamine derivative assuming an extinction coefficient of 430 M
cm
. To prepare
5 mM HOCl/OCl
, sodium hypochlorite was
diluted 20-fold into 0.1 M KOH, and this solution was diluted
50-fold into 0.1 M taurine in 0.1 M phosphate buffer,
pH 7.0. Absorbance at 252 nm was measured(23) , and the
concentration of the mono-chloramine derivative was calculated assuming
an extinction coefficient of 429 M
cm
. Based on this calculation, the hypochlorite
solution in KOH was diluted to 5 mM with 0.1 M phosphate buffer, pH 7.0.
Peroxidase-catalyzed oxidation of
chloride or bromide was carried out in 1 ml total volume of 0.1 M phosphate buffer, pH 7.0, at 37 °C. The concentration of
oxidants was measured by the assay based on oxidation of two molecules
of the sulfhydryl compound Nbs to the disulfide
Nbs(23, 36) . Incubation mixtures were
placed on ice, and 0.05 ml of 0.3 mg/ml catalase was added, followed by
0.05 ml of 0.2 M taurine, 1.5 ml of the Nbs solution, and 5 ml
of 0.1 M Na
SO
with 0.3 mM DETAPAC in 30 mM phosphate buffer, pH 7.0. Absorbance at
409 nm was measured(37) , and the oxidant concentration
(mM) was calculated from the difference in absorbance between
the control and sample, times the ratio of the final and starting
volumes (7.6), divided by the extinction coefficient for Nbs (14.05
mM
cm
), all
divided by 2, because each oxidized chlorine or bromine atom oxidizes
two molecules of Nbs. Results shown are the mean of duplicate
determinations, and the mean standard error was ±2
µM.
Figure 1:
Bromamine absorption spectra and
comparison with chloramine spectra. HOBr/OBr (0.5
mM) was added to 100 mM and 0.25 mM taurine
to obtain the mono- and di-bromamine. After 1 min, spectra were
measured versus blanks without HOBr/OBr
.
Extinction coefficients (M
cm
) of alkyl chloramines and bromamines at the
indicated wavelengths are listed as follows. RNHCl: 136, 429*, and 36
at 205, 252, and 300 nm. RNHBr: 146, 430*, and 64 at 241, 288, and 336
nm. RNCl
: 2720*, 190, and 370* at 205, 252, and 300 nm.
RNBr
: 2713*, 491, and 371* at 241, 288, and 336 nm. (*
=
values.) Extinction coefficients for
HOCl/OCl
at 205, 252, and 300 nm were 234, 64, and
134 M
cm
. Values
for HOBr/OBr
at 241, 288, and 336 nm were 100, 65,
and 36 M
cm
. To
facilitate calculations, values are reported with up to four
significant figures, but the standard error of replicate determinations
was ±2%.
The absorbance at 288 nm and either 241
or 336 nm provided a method to measure amounts of mono- and
di-bromamine in mixtures of the two. Results of adding the same amount
of HOBr/OBr to solutions containing various amounts
of taurine are shown in Fig. 2. The highest ratio of 241:288 nm
absorbance was obtained when the ratio of HOBr/OBr
to
amine was 2:1, consistent with formation of the di-bromamine. The 288
nm absorbance increased sharply at higher amine concentrations. With 1
mM taurine, or a ratio of HOBr/OBr
to amine
of 1:2, half of the oxidized bromine atoms were in the mono-bromamine
and half in the di-bromamine. With 10 mM taurine, 90% of the
bromine atoms were in the mono-bromamine. The solution obtained at 100
mM taurine contained only the mono-bromamine, in that the
ratio of 288 to 241 nm absorbance did not increase at higher amine
concentrations.
Figure 2:
Formation of mono- and di-bromamine.
HOBr/OBr (0.5 mM) was added to solutions
containing various concentration of taurine. After 1 min, absorption
spectra were measured versus blanks without
HOBr/OBr
. At taurine concentrations below 0.25
mM, absorbance values were corrected for the absorbance of
HOBr/OBr
, assuming that 1 mol of taurine consumed 2
mol of HOBr/OBr
. Concentrations of the mono-bromamine (M) and di-bromamine (D) derivatives were calculated
from the equations: X = (146) M + (2713) D; Y = (430) M + (491) D, where X and Y are the corrected
absorbance at 241 and 288 nm. Values plotted are M (
) and
two times D (
).
For comparison, the same amount of
HOCl/OCl was added to solutions containing various
amounts of taurine (not shown). The yields of mono- and di-chloramine
were calculated from absorbance at 205 and 252 nm(23) . The
highest ratio of 205:252 nm absorbance was obtained at a 2:1 ratio of
HOCl/OCl
to amine, consistent with formation of the
di-chloramine. With 1 mM taurine, 90% of the oxidized chlorine
atoms were in the mono-chloramine. The ratio of 252 to 205 nm
absorbance reached a maximum at 10-30 mM amine.
The
results indicate that the reaction of HOBr/OBr with
amines tended to produce di-halo rather than mono-halo derivatives when
compared with the formation of chloramines. To obtain a solution in
which 90% of the oxidizing equivalents were in the mono-halo
derivative, a 20-fold excess of amine over HOBr/OBr
or a 2-fold excess of amine over HOCl/OCl
was
required.
In addition to the characteristic absorption spectra of
the bromamines, differences in stability and chemical reactivity made
it possible to distinguish between bromamines and
HOBr/OBr. The rate of loss of oxidizing activity of
bromamines is shown in Fig. 3. Less than 5% decomposition was
observed in the first hour. About half the amount of the mono-bromamine
was still present after 4 days. Loss of the di-bromamine was faster and
similar to the decomposition of HOBr/OBr
.
Decomposition appeared exponential with half-lives of 70 h for the
mono-bromamine and 16 h for the di-bromamine or
HOBr/OBr
.
Figure 3:
Decomposition of bromamines.
HOBr/OBr (0.2 mM) was added to buffer and to
100 mM and 0.1 mM taurine to obtain solutions of
HOBr/OBr
(
) and the mono-bromamine (
) and
di-bromamine (
). Oxidants were measured with Nbs following
incubations at 37 °C. Dotted lines indicate half-lives of
70 and 16 h, each ±10%.
As shown in Fig. 4,
HOBr/OBr was reduced rapidly by dimethyl sulfoxide
with a 1:1 stoichiometry, consistent with reduction of
HOBr/OBr
to bromide and oxidation of the sulfoxide
(CH
SOCH
) to the sulfone
(CH
SO
CH
). The reaction was complete
within the time required for mixing even on ice. In contrast,
bromamines did not react with a 2-fold excess of dimethyl sulfoxide
within 1 h at 37 °C.
Figure 4:
Reduction of HOBr/OBr by
dimethyl sulfoxide. HOBr/OBr
(0.3 mM) was
added to buffer and to 100 and 0.15 mM taurine to obtain
solutions of HOBr/OBr
(
,
) and the
mono-bromamine (
) and di-bromamine (
). Dimethyl sulfoxide
was added, and the amount of oxidant remaining was measured with Nbs
after 5 min on ice (
) or 1 h at 37 °C (
,
,
).
These results indicate that mono- and
di-bromamine solutions obtained at HOBr/OBr to amine
ratios of 1:200 and 2:1 did not contain detectable amounts of free
HOBr/OBr
, which would have been instantly reduced by
dimethyl sulfoxide. Moreover, hydrolysis of bromamines to yield
HOBr/OBr
was too slow to be measurable within 1 h at
37 °C.
The difference in reactivity of HOBr/OBr and bromamines with dimethyl sulfoxide was the same as with
HOCl/OCl
and chloramines(23) . On the other
hand, there was a difference in the reactivity of chloramines and
bromamines with H
O
. As shown in Fig. 5(left), HOBr/OBr
was rapidly
reduced by H
O
with a 1:1 stoichiometry,
consistent with reduction of HOBr/OBr
to bromide and
oxidation of H
O
to water and O
. The
reaction was complete within the time required for mixing on ice.
HOCl/OCl
was also instantly reduced by
H
O
(not shown). Bromamines were reduced with a
1:1 stoichiometry when incubated with H
O
for 30
min at 37 °C (Fig. 5, left). The di-bromamine was
reduced faster than the mono-bromamine, but both were reduced within 30
min (Fig. 5, right). In contrast, chloramines did not
react with H
O
within 1 h at 37 °C (not
shown).
Figure 5:
Reduction of HOBr/OBr and bromamines by H
O
.
HOBr/OBr
(0.3 mM) was added to buffer and to
100 mM and 0.15 mM taurine to obtain solutions of
HOBr/OBr
(
) and the mono-bromamine (
) and
di-bromamine (
). Left, after 5 min on ice (
) or 1
h at 37 °C (
,
), catalase (15 µg/ml) was added,
and oxidants were measured with Nbs. Right, following
incubations at 37 °C with 0.5 mM H
O
, catalase (15 µg/ml) was added, and
oxidants were measured with Nbs.
Reduction of bromamines could be the result of direct attack
of HO
on bromamines, or the bromamines could
hydrolyze to yield HOBr/OBr
, which would then react
with H
O
. The results obtained with dimethyl
sulfoxide and H
O
indicate that hydrolysis was
undetectable and that bromamines reacted directly with
H
O
.
Differences in the reactivity of chloramines and bromamines with
HO
provided a method to measure amounts of
chloramines and bromamines in mixtures of the two. When mixtures were
incubated with excess H
O
for 30 min at 37
°C, only chloramines remained (Fig. 6). The amount of
oxidant that was reduced by H
O
was equal to the
amount of bromamines.
Figure 6:
Reduction of bromamines in mixtures of
chloramines and bromamines. HOCl/OCl or
HOBr/OBr
was added to 100 mM taurine to
obtain the mono-chloramine or mono-bromamine. Mixtures were prepared
containing 0.3 mM total oxidant with decreasing amounts of
chloramine and increasing amounts of bromamine. The mixtures were
incubated 30 min at 37 °C without H
O
(
) or with excess H
O
(0.5
mM) (
). Catalase (15 µg/ml) was added, and the
oxidant concentration was measured with
Nbs.
When HOCl/OCl was added to a bromide solution and then the amine was added, the
mono-bromamine was the only observed product, as indicated by the high
absorbance ratio. The results indicate that HOCl/OCl
rapidly oxidized bromide to HOBr/OBr
. When the
amine was added, all of the HOCl/OCl
was gone, and
only HOBr/OBr
was available to react with the amine.
In contrast, when HOCl/OCl was added to the amine
to form the mono-chloramine and then bromide was added, the
mono-chloramine was the only product, and there was no change within 1
h at 37 °C. The mono-chloramine did not oxidize bromide to
HOBr/OBr
, and there was no exchange of bromide with
the oxidized chlorine atom of the chloramine.
Similarly, when
HOCl/OCl was added to a mixture of the amine and
bromide, only the mono-chloramine was obtained. Although the oxidation
of bromide by HOCl/OCl
was a fast reaction, the
reaction of HOCl/OCl
with the amine was much faster
when the bromide/amine ratio was 1:100. Once the mono-chloramine was
formed, it did not oxidize bromide or undergo exchange with bromide.
The results indicate that when a high concentration of amine was
present, the addition of HOCl/OCl
did not result in
the formation of bromamines. Bromamines were obtained only when
HOBr/OBr
was added.
In other experiments, 0.5
mM HOCl/OCl was added to 0.25 mM taurine to obtain the di-chloramine, and then 1 mM bromide was added. There was no change in the spectrum within 1 h
at 37 °C, indicating that the di-chloramine did not oxidize bromide
or undergo exchange with bromide. On the other hand, the di-bromamine
was the major product when 0.5 mM HOCl/OCl
was added to a mixture of 1 mM bromide and 0.25 mM taurine. Therefore, HOCl/OCl
reacted faster with
bromide than with the amine when the bromide/amine ratio was 4:1. Amine
concentrations of at least 10 mM were required to block the
oxidation of bromide by HOCl/OCl
.
Figure 7:
Bromide
oxidation in chloride-free medium. Left, MPO. Right,
EPO. Top, mixtures contained 0.1 µM MPO or EPO,
0.1 mM HO
, and 0.1 mM (
,
), 1 mM (
,
), or 10 mM (
,
) bromide, with 100 mM taurine. Bottom, mixtures contained 0.1 µM (
) or 0.2
µM (
) MPO, 0.005 µM (
) or 0.01
µM (
) EPO, 0.1 mM H
O
, 0.2 mM bromide, and 100
mM taurine. Following incubations at 37 °C, catalase (15
µg/ml) was added, and oxidants were measured with
Nbs.
At a high level of
bromide (10 mM), the yield of bromamine was stoichiometric, 1
mol of bromamine/mol of HO
. Therefore, all of
the H
O
was used in bromide oxidation, and all
of the HOBr/OBr
was trapped by the amine.
At equal enzyme concentrations (0.1 µM heme), faster bromide oxidation was obtained with EPO than with MPO. In addition, EPO produced a stoichiometric yield of bromamine at lower bromide concentrations. With MPO, the yield was much less than stoichiometric when bromide was less than 1 mM.
Under conditions that gave
less than 1 mol of bromamine/mol of HO
, the
yield as well as the rate was increased by raising the enzyme
concentration. These results suggest that the enzymes lost activity
during the incubations and that the production of bromamine stopped
when enzyme activity went to zero. As shown below, MPO was converted to
the Compound II form under these conditions.
MPO was incubated with
excess bromide (10 mM), 0.1 mM HO
, and various concentrations of taurine,
and the absorption spectra of the products were measured (Fig. 8). Similar results were obtained with EPO (not shown).
With 0.05 mM taurine, which would be enough to trap 0.1 mM HOBr/OBr
as the di-bromamine, the 241 nm peak of
the di-bromamine and the 288 nm peak of the mono-bromamine were
observed, indicating a mixture of the two. When the amount of taurine
was increased to 10 mM or higher, the spectra shifted, and
only the 288 nm peak of the mono-bromamine was observed.
Figure 8:
Identification of oxidants produced in
chloride-free medium. Mixtures contained 0.1 µM MPO, 0.1
mM HO
, and 10 mM bromide,
with taurine at 0.05, 0.1, 1, 10, or 100 mM (a-e).
After 15 min at 37 °C, absorption spectra were measured versus blanks without H
O
. Inset,
concentrations of mono-bromamine (M) and di-bromamine (D) were calculated from the absorbance at 241 and 288 nm.
Values plotted are M (
), two times D (
),
and the total oxidant, M + two times D (
).
The yields
of mono- and di-bromamine were calculated from the 241 and 288 nm
absorbance and plotted versus taurine in the inset.
The yield of stable oxidants was less than 1 mol/mol of
HO
when the amine concentration was less than
10 mM. With 0.1 mM taurine, which was equal to the
amount of H
O
added, the yield of oxidant was
50%. Half of the oxidized bromine atoms were in 0.025 mM mono-bromamine and half in 0.0125 mM di-bromamine. The
results indicate that the di-bromamine was a major product at low amine
concentrations. As described below, the low yield of oxidants at low
amine concentrations was the result of reduction of
HOBr/OBr
and the di-bromamine by
H
O
.
Absorption spectra of the enzyme (MPO) rather than the product (bromamines) are shown in Fig. 9. Absorbance of 0.1 mM bromamine was higher than that of 0.2 µM enzyme, but there was no overlap of the bromamine ultraviolet absorbance with the visible spectrum of MPO.
Figure 9:
Absorption spectra of MPO. Mixtures
contained 0.2 µM MPO in chloride-free medium with either
0.2 mM bromide and 100 mM taurine (a-d) or
10 mM bromide and no taurine (e-g).
HO
(0.1 mM) was added, and the spectra
were measured immediately (b and f) and after 30 min (c and g). In d, catalase (1 µg/ml) was
added after 30 min, and the spectrum was measured after an additional
30 min. Spectra were measured versus blanks without MPO and
H
O
.
When MPO
was incubated with low bromide (0.2 mM), 0.1 mM HO
, and excess taurine (100 mM),
a low yield of bromamine was obtained. MPO was converted to the
Compound II form under these conditions, as indicated by the shift of
absorbance from 430 to 456 nm and the increased absorbance at 625 nm (Fig. 9, left). The ratio of 625 to 456 nm absorbance
was 0.25, indicating that little or no Compound III was
present(38) . There was a substantial loss of absorbance that
increased with time, consistent with destruction of the heme group by
excess H
O
. The same results were obtained in
the absence of bromide (not shown), indicating that
H
O
rather than products of bromide oxidation
destroyed the heme group. When catalase was added to remove the excess
H
O
, what was left of the heme absorbance
shifted back to 430 nm, consistent with reversion of Compound II to
ferric MPO.
Compound II is an intermediate in the oxidation of one-electron donors such as phenols and aromatic amines by peroxidase enzymes, but Compound II does not oxidize two-electron donors such as halide ions(11, 35, 39) . Therefore, accumulation of MPO in the Compound II form accounts for the slowing of the rate of bromide oxidation and the low yield of bromamine at low bromide concentrations.
A low yield of oxidants was also obtained
when MPO was incubated with high bromide (10 mM), 0.1 mM HO
, and low taurine, but there was no
shift or loss of MPO absorbance at low taurine concentrations (Fig. 9, right). In other experiments, no loss of MPO
or EPO activity was observed when the incubation mixtures were diluted
to lower the bromide concentration, and activity was measured in an
assay that is proportional to the amount of active enzyme(30) .
The results suggest that the low yield of oxidants was the result of
reduction of HOBr/OBr
and bromamines by
H
O
rather than inactivation of the enzymes.
Reduction of HOBr/OBr and bromamines by
H
O
was confirmed by measuring the production of
O
by the MPO-H
O
-bromide system,
using an O
electrode. One mol of O
was produced
per 2 mol of H
O
within 2 min at 37 °C by
the mixture of 0.1 µM MPO, 0.1-0.3 mM H
O
, and 10 mM bromide. The yield
of O
was the same as when catalase rather than MPO was
added. Production of O
by the
MPO-H
O
-bromide system with 0.1 mM H
O
was cut in half by 0.1 mM taurine, and no O
was produced when 10 mM taurine was present. Therefore, trapping of HOBr/OBr
by high levels of amine prevented the reduction of oxidants by
H
O
.
Figure 10:
Effect of chloride on the yield of
oxidants. Left, without chloride. Right, with 100
mM chloride. Mixtures contained 0.1 µM MPO
(,
) or EPO (
,
), 0.1 mM H
O
, and the indicated concentrations of
bromide, with 100 mM taurine (closed symbols) or no
taurine (open symbols). After 30 min at 37 °C, catalase
(15 µg/ml) was added, and the yield of oxidants was measured with
Nbs.
With 100 mM chloride, MPO produced 1 mol of oxidant/mol of
HO
regardless of the bromide concentration. As
shown below, the oxidants were mixtures of chloramines and bromamines.
The yield was low with EPO, 100 mM chloride, and no bromide,
but approached 1 mol/mol of H
O
when bromide was
present. No stable oxidants were obtained in the absence of an amine to
trap HOCl/OCl
and HOBr/OBr
.
MPO
and EPO were incubated with 100 mM chloride, various
concentrations of bromide, 0.1 mM HO
,
and 100 mM taurine. Absorption spectra of the stable oxidants
were measured (Fig. 11). In the absence of bromide, only the 252
nm peak of the mono-chloramine was observed. As bromide was increased,
the spectra spread and shifted, and the 288 nm peak of the
mono-bromamine was observed. For comparison, the spectra obtained with
10 mM bromide and no chloride are shown.
Figure 11:
Identification of oxidants produced from
chloride and bromide. Left, MPO. Right, EPO. Mixtures
contained 0.1 µM MPO or EPO, 0.1 mM HO
, 100 mM chloride, and 0, 0.1,
1, or 10 mM bromide (a-d), or no chloride and 10
mM bromide (e), all with 100 mM taurine.
After 15 min at 37 °C, absorption spectra were measured versus blanks without H
O
. Absorbance values for
MPO were 0.042, 0.041, 0.035, 0.022, and 0.017 at 252 nm and 0.009,
0.011, 0.019, 0.036, and 0.042 at 288 nm. Values for EPO were 0.020,
0.020, 0.021, 0.018, and 0.018 at 252 nm and 0.004, 0.037, 0.044,
0.039, and 0.042 at 288 nm.
Concentrations of the chloramine and bromamine were calculated from the 252 and 288 nm absorbance. With MPO, the mixtures of oxidants obtained with 100 mM chloride and 0.1, 1, and 10 mM bromide contained 6, 30, and 83% bromamine. With EPO, the mixtures contained 88, 93, and 95% bromamine. Assuming a variation of 0.001 absorbance unit at each wavelength, the accuracy was ±4%.
Similar results were
obtained with an independent method, making use of the ability of
HO
to reduce bromamines but not chloramines.
MPO and EPO (0.1 µM) were incubated with 100 mM chloride, various concentrations of bromide, 0.1 mM H
O
, and 100 mM taurine for 30 min
at 37 °C. The enzymes were removed by passing the incubation
mixtures through centrifugal ultra-filters with a 30 kDa cut-off
(Amicon, Inc., Beverly, MA). Portions of the filtrates were incubated
with and without excess H
O
(0.2 mM)
for 30 min at 37 °C. Catalase was added, and then concentrations of
the oxidants were measured with Nbs. With MPO, the mixtures obtained
with 0.1, 0.3, 1, 3, and 10 mM bromide contained 6, 8, 30, 55,
and 70% bromamine. With EPO, the mixtures contained 78, 82, 82, 95, and
95% bromamine. The standard error of replicate determinations was
±5 µM, or 5% of the total oxidant.
Bromide was preferred over chloride as a substrate for both
MPO and EPO, although the preference was stronger with EPO. For
example, when chloride was 100 mM and bromide was 1
mM, MPO used 0.07 mM HO
in
chloride oxidation and 0.03 mM H
O
in
bromide oxidation, indicating a 40-fold preference for bromide. EPO
used 0.01 mM H
O
in chloride oxidation
and 0.09 mM H
O
in bromide oxidation,
indicating a 1000-fold preference for bromide. Both enzymes produced a
mixture of oxidants at physiologic levels of bromide (0.1 mM)
and chloride (100 mM).
When the enzymes were incubated with
HO
and both bromide and chloride, trapping of
HOCl/OCl
by high levels of an amine prevented the
non-enzymatic oxidation of bromide to HOBr/OBr
. The
reaction of HOCl/OCl
with the amine to form
chloramines was faster than the oxidation of bromide by
HOCl/OCl
, and chloramines did not oxidize bromide
under the conditions of these experiments, all at neutral pH.
Chloramines also did not undergo detectable exchange with bromide
within 1 h at 37 °C. Therefore, the composition of the mixtures of
chloramines and bromamines provided a direct measure of the amounts of
chloride and bromide that were oxidized by the enzymes. Chloramines and
bromamines could be quantified from their characteristic absorption
spectra and by selective reduction of the bromamines with
H
O
.
Trapping oxidized forms of bromide with
high levels of amines also prevented the reduction of
HOBr/OBr by H
O
. The reaction
of HOBr/OBr
with amines to form bromamines was much
faster than the reduction of HOBr/OBr
by
H
O
when the amine/bromide ratio was 100:1 or
higher. Although bromamines were reduced by H
O
,
reduction of bromamines was much slower than reduction of
HOBr/OBr
. All of the H
O
was
consumed in bromide oxidation before reduction of bromamines could
occur, and 1 mol of bromamine was produced per mol of
H
O
.
At low amine concentrations, trapping of
HOBr/OBr was less effective, and the major product
was the di-bromamine, which reacted faster than the mono-bromamine with
H
O
. Substantial reduction of
HOBr/OBr
and bromamines was observed when the amine
concentration was less than 10 mM.
When no amine was
present, bromide was oxidized to HOBr/OBr, and
HOBr/OBr
was reduced by H
O
.
No oxidant accumulated in the medium, and 1 mol of O
was
produced per 2 mol of H
O
. MPO and EPO were not
inactivated under these conditions, indicating that the enzymes were
not readily inactivated by HOBr/OBr
or that rapid
reduction of HOBr/OBr
by H
O
protected the enzymes against inactivation. The enzymes were also
not readily inactivated by singlet oxygen, which should have been
produced under these conditions.
When no chloride was present and
bromide was less than 1 mM, MPO produced less than 1 mol of
bromamine/mol of HO
, despite the presence of
high levels of amine. Under these conditions, MPO was converted to the
Compound II form, which underwent further irreversible inactivation by
the excess H
O
. EPO appeared to be less likely
to be converted to the Compound II form and was able to produce 1 mol
of bromamine/mol of H
O
at bromide
concentrations as low as 0.1 mM.
Trapping
HOBr/OBr with taurine had advantages compared with
other methods that have been used to detect products of bromide
oxidation. For example, proteins (8, 40, 41, 42, 43) and
aromatic compounds (9, 44) have been used as traps for
brominating agents. Proteins contain reactive groups such as the
thioether portion of methionine residues that are readily oxidized by
halogenating agents, but which do not yield stable halogenated
products. Aromatic compounds must be used at low concentrations to
avoid problems with solubility, toxicity, or interference in
peroxidase-catalyzed reactions. Therefore, these compounds may trap
only a small portion of the brominating agents that are formed, and may
not be able to prevent the non-enzymatic oxidation of bromide by
HOCl/OCl
.
Under physiologic conditions,
concentrations of ammonia, amine compounds, and protein amino groups
are very high. Trapping of HOCl/OCl by these nitrogen
compounds should prevent the non-enzymatic oxidation of bromide.
However, non-enzymatic oxidation may occur in experiments with isolated
leukocytes. Secretion of MPO by neutrophils and the release of
H
O
into the extracellular medium result in
chloride oxidation in the medium(45) . If both chloride and
bromide are present and no amine is added as a trap for
HOCl/OCl
, the nitrogen compounds released into the
medium by the cells (46) may not be adequate to prevent the
non-enzymatic oxidation of bromide by HOCl/OCl
.
Trapping of HOBr/OBr by high levels of ammonia and
amines in vivo may also prevent the reduction of
HOBr/OBr
and bromamines by
H
O
. However, the amount of nitrogen compounds
released by isolated leukocytes may not be adequate. Even when excess
amine is added, H
O
may reduce
HOBr/OBr
and bromamines if the activity of secreted
peroxidase enzymes in the medium is low and the rate of
H
O
production is greater than the rate at which
H
O
is consumed in bromide oxidation. Similarly,
if the amount of H
O
produced exceeds the amount
of bromide, oxidation of bromide will stop as bromide is depleted, and
the excess H
O
may reduce bromamines that were
produced before bromide was depleted.
In studies with isolated
leukocytes, evidence for the formation of brominating agents was
obtained(8, 9, 44) , but long-lived
bromamines were not detected in the medium(8) . Reduction of
HOBr/OBr and bromamines by H
O
may account for these results. If reduction of
HOBr/OBr
and bromamines does occur, then bromide
should appear to inhibit leukocyte O
uptake because the
peroxidase and bromide catalyze the formation of up to 1 mol of
O
/2 mol of H
O
.