©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Oxidation of Bromide by the Human Leukocyte Enzymes Myeloperoxidase and Eosinophil Peroxidase
FORMATION OF BROMAMINES (*)

(Received for publication, July 26, 1994; and in revised form, November 29, 1994)

Edwin L. Thomas (§) Paula M. Bozeman M. Margaret Jefferson Charles C. King

From the Dental Research Center and Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163 and the Division of Critical Care and Pulmonary Medicine, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Myeloperoxidase and eosinophil peroxidase catalyzed the oxidation of bromide ion by hydrogen peroxide (H(2)O(2)) 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(2)O(2), indicating that bromamine formation prevented the reduction of HOBr/OBr by H(2)O(2) and the loss of oxidizing and brominating activity. Bromamines differed from HOBr/OBr in that bromamines reacted slowly with H(2)O(2), 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(2)) of the betaamino 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.


INTRODUCTION

MPO (^1)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(2)O(2), producing oxidizing and halogenating agents. Oxidation of a halide (X) yields the halogen (X(2)), 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 H(2)O(2) 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(2)O(2), 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(2)O(2) to 2 mol of water and one of molecular oxygen (O(2)), which is the same result that would be obtained by adding catalase.

Reduction of HOBr/OBr by H(2)O(2) is accompanied by formation of singlet oxygen, which rapidly releases energy as light and forms O(2)(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(2)O(2).

Little or no singlet oxygen is detected when MPO (15) or EPO (16) is incubated with H(2)O(2) 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(2)O(2) 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(2)O(2) 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(2)O(2). Unlike HOCl/OCl, chloramines are not reduced by H(2)O(2)(11, 23) .

Because chloramine derivatives of alpha-amino acids are unstable, the beta-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 alpha-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(2)O(2)(28) . Extinction coefficients of 415-420 Mbulletcm were reported for mono-bromamines at 289 nm(29) , similar to the value of 429 Mbulletcm 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 H(2)O(2) 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(2)O(2) when the enzyme, bromide, and taurine concentrations were sufficiently high. Bromamine formation prevented the reduction of HOBr/OBr by H(2)O(2), and all of the H(2)O(2) was consumed in bromide oxidation before reduction of bromamines by H(2)O(2) 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.


EXPERIMENTAL PROCEDURES

MPO and EPO were purified (30) from cytoplasmic granules of human granulocytes and stored at -70 °C in 0.1 M Na(2)SO(4) 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 bullet 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(2)), sodium hypochlorite (4-6%), H(2)O(2) (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(2) 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(2)SO(4) 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(2)O(2) was diluted into autoclaved water and the concentration determined from the extinction coefficient of 70 M bullet cm at 230 nm. To prepare Nbs, 100 ml of a 1 mM Nbs(2) solution in 0.1 M Na(2)SO(4) 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 bullet 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 bullet 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(2)(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(2)SO(4) 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 bullet 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.


RESULTS

Properties of Mono-and Di-bromamines

Absorption spectra of mono- and di-bromamine derivatives of the amine compound taurine are compared in Fig. 1. Identical spectra were obtained with other primary amines. The mono-bromamine had a single peak at 288 nm. The di-bromamine had major and minor peaks at 241 and 336 nm, each 47-48 nm distant from the mono-bromamine peak. Extinction coefficients of mono- and di-bromamines are compared with values for the analogous chloramine derivatives (23) in the legend. Bromamine spectra were similar to chloramine spectra but shifted 36 nm toward higher wavelengths.


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 bullet 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(2): 2720*, 190, and 370* at 205, 252, and 300 nm. RNBr(2): 2713*, 491, and 371* at 241, 288, and 336 nm. (* = (max) values.) Extinction coefficients for HOCl/OCl at 205, 252, and 300 nm were 234, 64, and 134 M bullet cm. Values for HOBr/OBr at 241, 288, and 336 nm were 100, 65, and 36 M bullet 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 (circle) and two times D (box).



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 (Delta) and the mono-bromamine (circle) and di-bromamine (box). 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(3)SOCH(3)) to the sulfone (CH(3)SO(2)CH(3)). 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 (Delta, ) and the mono-bromamine (bullet) and di-bromamine (). Dimethyl sulfoxide was added, and the amount of oxidant remaining was measured with Nbs after 5 min on ice (Delta) or 1 h at 37 °C (, bullet, ).



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(2)O(2). As shown in Fig. 5(left), HOBr/OBr was rapidly reduced by H(2)O(2) with a 1:1 stoichiometry, consistent with reduction of HOBr/OBr to bromide and oxidation of H(2)O(2) to water and O(2). The reaction was complete within the time required for mixing on ice. HOCl/OCl was also instantly reduced by H(2)O(2) (not shown). Bromamines were reduced with a 1:1 stoichiometry when incubated with H(2)O(2) 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(2)O(2) within 1 h at 37 °C (not shown).


Figure 5: Reduction of HOBr/OBr and bromamines by H(2)O(2). HOBr/OBr (0.3 mM) was added to buffer and to 100 mM and 0.15 mM taurine to obtain solutions of HOBr/OBr (Delta) and the mono-bromamine (circle) and di-bromamine (box). Left, after 5 min on ice (Delta) or 1 h at 37 °C (circle, box), catalase (15 µg/ml) was added, and oxidants were measured with Nbs. Right, following incubations at 37 °C with 0.5 mM H(2)O(2), catalase (15 µg/ml) was added, and oxidants were measured with Nbs.



Reduction of bromamines could be the result of direct attack of H(2)O(2) on bromamines, or the bromamines could hydrolyze to yield HOBr/OBr, which would then react with H(2)O(2). The results obtained with dimethyl sulfoxide and H(2)O(2) indicate that hydrolysis was undetectable and that bromamines reacted directly with H(2)O(2).

Differences in the reactivity of chloramines and bromamines with H(2)O(2) provided a method to measure amounts of chloramines and bromamines in mixtures of the two. When mixtures were incubated with excess H(2)O(2) for 30 min at 37 °C, only chloramines remained (Fig. 6). The amount of oxidant that was reduced by H(2)O(2) 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(2)O(2) (Delta) or with excess H(2)O(2) (0.5 mM) (). Catalase (15 µg/ml) was added, and the oxidant concentration was measured with Nbs.



Non-enzymatic Oxidation of Bromide

Oxidation of bromide by HOCl/OCl and chloramines was compared (Table 1). First, HOBr/OBr and HOCl/OCl were added to solutions containing a 200-fold excess of amine to obtain the mono-bromamine and mono-chloramine. The 288 to 252 nm absorbance ratios were 2.4 and 0.21 for the bromamine and chloramine, and there was no significant change in the spectra within 1 h at 37 °C.



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.

Bromide Oxidation by MPO and EPO in Chloride-free Media

Rates of bromide oxidation by MPO and EPO were compared with 100 mM taurine as a trap for HOBr/OBr (Fig. 7). With both enzymes, an oxidant accumulated in the medium that could be measured at the end of the incubation by adding catalase to remove H(2)O(2) and then adding Nbs and measuring the oxidation of 2 Nbs to Nbs(2). As shown below, the oxidant was identified as the mono-bromamine.


Figure 7: Bromide oxidation in chloride-free medium. Left, MPO. Right, EPO. Top, mixtures contained 0.1 µM MPO or EPO, 0.1 mM H(2)O(2), and 0.1 mM (, Delta), 1 mM (bullet, circle), or 10 mM (, box) bromide, with 100 mM taurine. Bottom, mixtures contained 0.1 µM (bullet) or 0.2 µM () MPO, 0.005 µM (circle) or 0.01 µM (box) EPO, 0.1 mM H(2)O(2), 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 H(2)O(2). Therefore, all of the H(2)O(2) 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 H(2)O(2), 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 H(2)O(2), 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 H(2)O(2), 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(2)O(2). Inset, concentrations of mono-bromamine (M) and di-bromamine (D) were calculated from the absorbance at 241 and 288 nm. Values plotted are M (circle), two times D (box), and the total oxidant, M + two times D (Delta).



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 H(2)O(2) when the amine concentration was less than 10 mM. With 0.1 mM taurine, which was equal to the amount of H(2)O(2) 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(2)O(2).

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). H(2)O(2) (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(2)O(2).



When MPO was incubated with low bromide (0.2 mM), 0.1 mM H(2)O(2), 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(2)O(2). The same results were obtained in the absence of bromide (not shown), indicating that H(2)O(2) rather than products of bromide oxidation destroyed the heme group. When catalase was added to remove the excess H(2)O(2), 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 H(2)O(2), 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(2)O(2) rather than inactivation of the enzymes.

Reduction of HOBr/OBr and bromamines by H(2)O(2) was confirmed by measuring the production of O(2) by the MPO-H(2)O(2)-bromide system, using an O(2) electrode. One mol of O(2) was produced per 2 mol of H(2)O(2) within 2 min at 37 °C by the mixture of 0.1 µM MPO, 0.1-0.3 mM H(2)O(2), and 10 mM bromide. The yield of O(2) was the same as when catalase rather than MPO was added. Production of O(2) by the MPO-H(2)O(2)-bromide system with 0.1 mM H(2)O(2) was cut in half by 0.1 mM taurine, and no O(2) 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(2)O(2).

Chloride and Bromide Oxidation

The yield of stable oxidants obtained with and without chloride was compared (Fig. 10). In the absence of chloride, EPO produced a higher yield of bromamine than MPO at low bromide concentrations. At high bromide (1-10 mM), both MPO and EPO produced 1 mol of bromamine/mol of H(2)O(2).


Figure 10: Effect of chloride on the yield of oxidants. Left, without chloride. Right, with 100 mM chloride. Mixtures contained 0.1 µM MPO (, box) or EPO (bullet, circle), 0.1 mM H(2)O(2), 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 H(2)O(2) 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(2)O(2) 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 H(2)O(2), 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 H(2)O(2), 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(2)O(2). 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 H(2)O(2) 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(2)O(2), 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(2)O(2) (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.


DISCUSSION

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 H(2)O(2) in chloride oxidation and 0.03 mM H(2)O(2) in bromide oxidation, indicating a 40-fold preference for bromide. EPO used 0.01 mM H(2)O(2) in chloride oxidation and 0.09 mM H(2)O(2) 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 H(2)O(2) 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(2)O(2).

Trapping oxidized forms of bromide with high levels of amines also prevented the reduction of HOBr/OBr by H(2)O(2). The reaction of HOBr/OBr with amines to form bromamines was much faster than the reduction of HOBr/OBr by H(2)O(2) when the amine/bromide ratio was 100:1 or higher. Although bromamines were reduced by H(2)O(2), reduction of bromamines was much slower than reduction of HOBr/OBr. All of the H(2)O(2) was consumed in bromide oxidation before reduction of bromamines could occur, and 1 mol of bromamine was produced per mol of H(2)O(2).

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(2)O(2). 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(2)O(2). No oxidant accumulated in the medium, and 1 mol of O(2) was produced per 2 mol of H(2)O(2). 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(2)O(2) 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 H(2)O(2), 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(2)O(2). 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(2)O(2) 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(2)O(2) 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(2)O(2). However, the amount of nitrogen compounds released by isolated leukocytes may not be adequate. Even when excess amine is added, H(2)O(2) may reduce HOBr/OBr and bromamines if the activity of secreted peroxidase enzymes in the medium is low and the rate of H(2)O(2) production is greater than the rate at which H(2)O(2) is consumed in bromide oxidation. Similarly, if the amount of H(2)O(2) produced exceeds the amount of bromide, oxidation of bromide will stop as bromide is depleted, and the excess H(2)O(2) 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(2)O(2) may account for these results. If reduction of HOBr/OBr and bromamines does occur, then bromide should appear to inhibit leukocyte O(2) uptake because the peroxidase and bromide catalyze the formation of up to 1 mol of O(2)/2 mol of H(2)O(2).


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL02373, AI16795, and DE04235 and the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 210 Nash Bldg., 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-4879; Fax: 901-448-6517.

(^1)
The abbreviations used are: MPO, myeloperoxidase; EPO, eosinophil peroxidase; Nbs, 5-thio-2-nitrobenzoic acid or TNB; Nbs(2), 5,5-dithiobis(2-nitrobenzoic acid) or DTNB; DETAPAC, diethylenetriaminepentaacetic acid.


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