EPO (eosinophil peroxidase) and MPO (myeloperoxidase) are highly basic haem enzymes that are released extracellularly by activated eosinophils (in the case of EPO), and activated neutrophils, monocytes and some macrophages (in the case of MPO). These enzymes catalyse the reaction of H₂O₂ with Cl⁻, Br⁻ and SCN⁻ (thiocyanate), to produce (pseudo)hypohalous acids [HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid)]. These species exist as a mixture of the acid and anion forms at physiological pH values; HOCl, HOBr and HOSCN are used herein to designate these mixtures [1,2]. At physiological halide concentrations, HOBr is a significant product of EPO [1], especially when SCN⁻ is low, and HOBr is formed in approx. 25% yield from H₂O₂ by MPO at physiological pH [3]. HOBr, and other brominating interhalogen species, may also be generated via the reaction of MPO-derived HOCl with Br⁻ [4].

The extracellular matrix is a complex material composed principally of proteoglycans (proteins bearing glycosaminoglycan chains), proteins (e.g. collagens, elastin) and the free glycosaminoglycan, hyaluronan. These species play a key role in regulating cellular adhesion, migration and proliferation, and impart important physicochemical properties to tissues. For example, basement membranes provide a scaffold for the attachment of endothelial and epithelial cells and regulate vascular permeability; these functions have been shown to depend critically on the presence of specific components, such as heparan sulfate proteoglycans [5,6]. Binding of the highly cationic EPO and MPO proteins by the polyanionic glycosaminoglycan components of extracellular matrix (both proteoglycans and hyaluronan) is likely to localize the production of hypohalous acids to these materials in vivo. The degradation of extracellular matrix by hypohalous acids may have profound effects on the function and integrity of cells and tissues, and contribute to the pathophysiology of inflammatory diseases. It has been shown that co-injection of MPO and H₂O₂ into rats induces glomerular injury and proteinuria, consistent with degradation of the glomerular basement membrane by MPO-derived oxidants [7]. Heparan sulfate proteoglycans, which are critical components of the glomerular filtration barrier [6], are degraded by the MPO–H₂O₂–Cl⁻ system in vitro [8] and HOCl can induce the release of protein and carbohydrate fragments from smooth muscle cell extracellular matrix in vitro [9]. Degradation of extracellular matrix by MPO-derived hypohalous acids may be an important process in human inflammatory lesions, such as atherosclerotic plaques and the glomerular basement membrane in human membranous glomerulonephritis, where co-localization of MPO and HOCl-modified proteins has been observed (reviewed in [10,11]). Asthma is characterized by the infiltration of eosinophils and the release of EPO and other cytotoxic granule proteins within bronchial tissue. Elevated levels of EPO, and HOBr-modified proteins, have been detected in bronchioalveolar lavage and sputum of asthmatics compared with controls [12,13]. Furthermore, EPO-generated oxidants can simulate the pathological features of asthma, such as increased vascular permeability [14]. These observations implicate EPO-derived HOBr in the pathophysiology of this disease. Although there is good evidence for the extracellular matrix, and its components, being an important target for HOBr in vivo, the mechanisms of these reactions and their consequences are poorly understood. The reaction of HOBr with protein components is very rapid [15] and results in the modification of amino acid side-chains, unfolding, aggregation and fragmentation of the backbone [16,17]; N-bromo derivatives are important intermediates in these processes [16,17]. The susceptibility of glycosaminoglycans and intact extracellular matrix to degradation by HOBr has not been determined, although the related oxidant HOCl has been shown to react with glycosaminoglycans to generate polymer-bound N-chloro derivatives that decompose spontaneously, or via one-electron reduction by transition metal ions, to give polymer fragmentation [18–20]. Thus it was hypothesized that HOBr may degrade glycosaminoglycans, via N-bromo intermediates, and induce extracellular matrix fragmentation. The occurrence of these processes has been examined in the present study.

Figure 1 Structure of glycosaminoglycan N-bromo derivatives

The reaction of hyaluronan [2 mg·ml⁻¹; 5 mM in GlcNAc (N-acetylglucosamine) residues] with HOBr (1 mM) resulted in an increase in absorbance in the low UV region (i.e. λ_max<220 nm; Figure 2A), consistent with the formation of bromamides [R-NBr-C(O)CH₃]. This increase in absorbance was complete within approx. 2 h, and spectra obtained within this period had an isosbestic point at approx. 260 nm. Similar data (results not shown) was obtained with chondroitin sulfate [2.5 mg·ml⁻¹; 5 mM in GalNAc (N-acetylgalactosamine) residues]. These changes were not observed upon the reaction of polygalacturonic acid (2 mg·ml⁻¹; 10 mM in galacturonic acid residues) with HOBr (1 mM), where only a slow loss of the HOBr/OBr⁻ absorbance was detected; this was complete within approx. 8 h. These data are consistent with the reaction of HOBr with hyaluronan and chondroitin sulfate occurring predominantly at their N-acetyl functions to generate bromamides, and not with the uronic acid residues. Elution of HOBr-treated hyaluronan and chondroitin sulfate from PD10 size-exclusion columns, and subsequent assay of the fractions, showed that the TNB-reactive material co-eluted with the parent polymer, confirming the formation of polymer-bound N-bromo derivatives. The yield of these species was 20–45% (after correction for dilution during chromatography), under the conditions detailed in Table 1. These values are likely to be underestimates as a result of incomplete consumption of the HOBr, and decay of the bromamides (see below) prior to their assay.

Figure 2 Formation of glycosaminoglycan N-bromo derivatives on reaction of glycosaminoglycans with HOBr

Table 1 Preparation of glycosaminoglycan N-bromo derivatives

The reaction of heparin [3.2 mg·ml⁻¹; 4.61 mM in GlcNSO₃ (glucosamine N-sulfate) residues, 0.59 mM in GlcNAc residues] with HOBr (1 mM) resulted in the formation of an absorbance peak with λ_max 307 nm (Figure 2B), assigned to N-bromosulfonamides (R-NBr-SO₃⁻). This conclusion is supported by the detection of a peak with λ_max 302 nm on reaction of the model compound N-cyclohexylsulfamate (R-NH-SO₃⁻, R=cyclohexyl) with HOBr (results not shown). Formation of the heparin-derived N-bromosulfonamides was complete within approx. 0.5 h and occurred with a yield of approx. 64% with respect to HOBr, as determined by an assay with TNB; this is likely to be an underestimate of the true yield, for the reasons outlined above. The more rapid consumption of HOBr by heparin than hyaluronan and chondroitin sulfate (at approximately equal ratios of glycosamine residues to HOBr) indicates that the rate constant for formation of N-bromosulfonamides (from GlcNSO₃ residues) is much higher than that for the formation of bromamides (from GlcNAc or GalNAc residues).

The reaction of heparan sulfate [3.2 mg·ml⁻¹; 0.113 mM in GlcNH₂ (glucosamine) residues, 2.96 mM in GlcNSO₃ residues and 3.74 mM in GlcNAc residues] or partially de-N-sulfated heparin (3.2 mg·ml⁻¹; 0.908 mM in GlcNH₂ residues, 3.79 mM in GlcNSO₃ residues and 0.60 mM in GlcNAc residues) with an equimolar concentration of oxidant with respect to GlcNH₂ residues (i.e. 0.908 mM HOBr), or less, resulted in the formation of an absorbance peak with λ_max approx. 291–293 nm (as determined by difference spectra relative to the parent polymer; Figures 2C and 2D). With a 2-fold excess of HOBr (with respect to GlcNH₂ residues), a pair of absorbance peaks with λ_max approx. 246–251 nm and approx. 379 nm were detected in difference spectra. The peak with λ_max 291–293 nm is characteristic of (mono)bromamines (R-NBr-H) (cf. a λ_max of 288 nm for taurine bromamine [25]), and the pair of peaks with λ_max approx. 246–251 nm (major) and approx. 379 nm (minor) are characteristic of dibromamines (R-NBr₂) (cf. absorbances at λ_max 241 nm and 336 nm for taurine dibromamine [25]). Formation of these chromophores, assigned to bromamine and dibromamine species, was complete within 0.5 min and the yields of N-bromo derivatives were 72–94% with respect to added HOBr, as quantified using TNB.

The absence of spectral features from a dibromamine when equimolar concentrations of HOBr and GlcNH₂ residues were employed is consistent with the rate constant for bromamine formation from the parent amine being significantly higher than that for dibromamine formation from the bromamine. This is consistent with the corresponding HOCl rate constants which differ by several orders of magnitude [19]. The more rapid consumption of HOBr by heparan sulfate and partially de-N-sulfated heparin than by the other polymers (at approx. equal ratios of glycosamine residues to HOBr), indicates that the rate constants for formation of bromamines and dibromamines are considerably higher than those for formation of N-bromosulfonamides or bromamides.

Glycosaminoglycan N-bromo derivatives were prepared for subsequent studies by reaction of the parent polymers with HOBr under the reaction conditions summarized in Table 1. Preparations of the N-bromo derivatives of heparin, heparan sulfate and partially de-N-sulfated heparin are herein identified by their most abundant N-bromo derivative. The absence of residual HOBr in these preparations was confirmed spectrophotometrically (for bromamines, dibromamines and N-bromosulfonamides) or by passage of the HOBr-treated polymer through PD10 size-exclusion columns (for bromamides).

Figure 3 Decomposition of glycosaminoglycan N-bromo derivatives at 37 °C

All of the N-bromo derivatives also decomposed rapidly on addition of Cu⁺ (generated in situ by the reduction of Cu²⁺ by Ti³⁺) and Fe²⁺ at 22 °C (Figure 4); anoxic reaction conditions were employed for these studies to prevent metal ion autoxidation. Cu²⁺ at similar concentrations was an ineffective catalyst under these conditions (Figure 4).

Figure 4 Decomposition of glycosaminoglycan N-bromo derivatives by transition metal ions

Figure 5 Polymer fragmentation upon decomposition of glycosaminoglycan N-bromo derivatives

The reaction of Cu⁺ with hyaluronan- and chondroitin sulfate-derived bromamides, heparin-derived N-bromosulfonamides and partially de-N-sulfated heparin-derived bromamines and dibromamines resulted in significant polymer fragmentation (Figures 5E–5G). Fe²⁺ gave similar extents of fragmentation (results not shown). The fragments generated via metal ion-dependent decomposition of the hyaluronan- and chondroitin sulfate-derived bromamides gave discrete bands on high-resolution (30%) polyacrylamide gels (Figures 5H and 5I). The spacing of these bands is equidistant to those produced by hydrolytic cleavage of the parent polymers by testicular hyaluronidase, which occurs at the GlcNAc β-(1→4) glycosidic bonds [27] (i.e. at disaccharide intervals). This indicates that the fragmentation induced by metal ion-dependent bromamide decomposition occurs, like enzymatic cleavage, in a site-selective manner at disaccharide intervals along the polymer backbone.

Decomposition of hyaluronan- and chondroitin sulfate-derived bromamides by Cu⁺ at 22 °C in the presence of MNP (20.4 mM) or DMPO (102 mM) at pH 7.4 resulted in the detection of broad signals due to MNP and DMPO adducts of polymer-derived carbon-centred radicals [MNP, a(N, NO)=1.55 mT, a(β-H)=0.31 mT; DMPO, a(N, NO)=approx. 1.5–1.7 mT, a(β-H)=approx. 2.2–2.4 mT] and sharp features due to the DMPO adduct of CO₂^•− [a(N, NO)=1.57 mT, a(β-H)=1.86 mT] (Figures 6A and 6B; DMPO results not shown). The signals due to MNP adducts of carbon-centred radicals, which possessed a substrate-derived hydrogen splitting, are assigned to species with the partial structure ·CHRR'. With heparin-derived N-bromosulfonamides, broad signals due to adducts of polymer-derived carbon-centred radicals were detected with both MNP and DMPO [MNP, a(N, NO)=approx. 1.50 mT; DMPO, a(N, NO)=approx. 1.57 mT, a(β-H)=approx. 2.34 mT] as well as the MNP adduct of the SO₃^•− radical [a(N, NO)=1.47 mT] and the DMPO adduct of the CO₂^•− radical (parameters as above) (Figures 6C and 6D). With bromamines derived from partially de-N-sulfated heparin, weak signals due to adducts of polymer-derived radicals were detected with both MNP and DMPO (Figures 6E and 6F). With the corresponding dibromamines, signals due to adducts of polymer-derived radicals were detected with both MNP and DMPO, as well as the MNP adduct of SO₃^•− (parameters as above) (Figures 6G and 6H). These signals were similar to those detected with the N-bromosulfonamides, particularly those detected with MNP (Figure 6G compared with Figure 6C), and it is likely that decomposition of low levels of N-bromosulfonamides present in the dibromamine preparation gave rise to these species.

Figure 6 Radical formation upon decomposition of glycosaminoglycan N-bromo derivatives detected by EPR spin trapping

Figure 7 Release of radioactivity from 3H- and 14C-radiolabelled VSMC extracellular matrix upon reaction with HOBr

Activation of leucocytes results in the extracellular release of the highly basic haem enzymes EPO and MPO from cytoplasmic storage granules. Concurrently these cells undergo an oxidative burst with the formation of superoxide radicals, and hence H₂O₂, via an NADPH oxidase complex. Both EPO and MPO utilize H₂O₂, generated from this and other sources, to catalyse the oxidation of halide and pseudohalide ions to powerful oxidants. With EPO, HOBr is a major product especially in the absence of high levels of SCN⁻ [1]; with MPO, HOBr formation accounts for approx. 25% of the H₂O₂ consumed at pH 7.4 [3]. As these proteins associate with components of the extracellular matrix as a result of ionic interactions, matrix components are likely to be major targets for these oxidants. Recent data have implicated excess, or inappropriate, HOBr generation in a number of human pathologies, and particularly asthma [12–14]. In fact, elevated levels of HOBr-modified proteins have been detected in bronchioalveolar lavage fluid and sputum.

In the present study it has been shown that HOBr can fragment heparan sulfate, heparin, hyaluronan and chondroitin sulfate via metal ion-dependent and independent processes, and induce the release of carbohydrate and protein fragments from extracellular matrix in vitro. HOBr has been shown to react selectively with the glycosamine residues (GlcNH₂, GlcNSO₃, GlcNAc and GalNAc) of glycosaminoglycans to generate N-bromo derivatives.

The reaction of HOBr with GlcNH₂ residues in these polymers generates bromamines (R-NBr-H) and dibromamines (R-NBr₂). Slower reactions occur with GlcNSO₃ residues to generate N-bromosulfonamides (R-NBr-SO₃⁻) and with GlcNAc and GalNAc residues to generate bromamides [R-NBr-C(O)CH₃]. The reaction of HOCl with these species generates analogous N-chloro derivatives (chloramines, dichloramines, N-chlorosulfonamides and chloramides) and absolute rate constants for these reactions have been determined [18,19]. Rate constants for the reaction of HOBr with these targets have yet to be determined, however the data obtained indicate that the reaction of HOBr to generate dibromamines, N-bromosulfonamides and bromamides occurs at much higher rates than the corresponding reactions induced by HOCl. These data are consistent with previous studies with proteins, where the rate constants for N-bromination by HOBr are, typically, 10–100-fold higher than for N-chlorination by HOCl [15].

All of the glycosaminoglycan N-bromo derivatives decomposed readily in the absence of added agents at physiological pH and temperature and, with the exception of the bromamides, this resulted in polymer fragmentation. It has previously been shown that glycosaminoglycan dichloramines (but not other N-chloro derivatives) decompose readily under these conditions, resulting in polymer fragmentation [18,19]. Cleavage of heparan sulfate at its GlcNH₂ residues, via the formation of dibromamines, resulted in only small changes in molecular mass, consistent with the low abundance of GlcNH₂ residues in this polymer (0.5 per chain) and their location near the chain termini [26]. Much greater changes in mass were detected with de-N-sulfated heparin (an analogue of heparan sulfate) dibromamines, which have a higher abundance of GlcNH₂ residues (approx. 4 per chain, cf. data in [19]) that are located randomly along the backbone. More extensive fragmentation would also be expected with other heparan sulfates that possess GlcNH₂ residues in greater abundance, such as that from human aortae (which also has approx. 4 per chain [26]).

Cu⁺ and Fe²⁺ induced rapid decomposition of all the N-bromo derivatives, with this resulting in polymer fragmentation. These reactions are attributed to one-electron reduction of the N-bromo derivatives to nitrogen-centred radicals (Reaction 1):

(1)

It has been shown that N-bromo derivatives are important intermediates in HOBr-mediated protein oxidation, with the formation of these species resulting in structural alterations and protein fragmentation [16,17]. A key route to fragmentation is believed to involve the decomposition of N-bromo derivatives, formed either on the backbone or on amino acid side chains, to nitrogen-centred radicals. In the case of the side-chain species, subsequent (intramolecular) hydrogen atom abstraction reactions (1,2- or 1,5-hydrogen shifts) of the initial nitrogen-centred radicals, generate peptide α-carbon-centred radicals [17]. Transition metal ions have been shown to promote damage via reduction of protein N-bromo derivatives to nitrogen-centred radicals [17].

Fragmentation of the glycosaminoglycans and proteins of extracellular matrix, via the reactions outlined above, can account for the observed release of these materials from extracellular matrix, synthesized by VSMCs in vitro, upon exposure to HOBr. The generation and subsequent decomposition of N-bromo intermediates appear to be crucial intermediates in matrix damage, as there is a time-dependent release of fragments that is inconsistent with direct damage by HOBr. Thus fragmentation occurs for a considerable period (hours) after the removal, or complete consumption, of HOBr. This is consistent with the lifetime of the N-bromo intermediates detected on the glycosaminoglycans in the present study, and those formed on proteins reported previously [16,17].

The higher (fractional) release of carbohydrate, compared with protein, components observed in the present study needs to be interpreted with caution. These data may indicate that the glycosaminoglycans react more rapidly with HOBr than the proteins (cf. the high rate constant reported for the reaction of HOCl with GlcNH₂ residues [19]) and/or that damage to the glycosamine residues is translated more efficiently into backbone cleavage (and hence release from the matrix) than with proteins. There is considerable evidence that much of the damage induced by HOBr on proteins occurs on the side chains [15–17], rather than on the backbone, and that these modifications do not necessarily lead to protein fragmentation (and hence release of low molecular mass material). Kinetic studies with glycosaminoglycans may shed light on this point. Degradation of extracellular matrix by HOBr may be of considerable importance at inflammatory foci where EPO and MPO are released. Transition metal ions may exacerbate damage by stimulating the decomposition of matrix-bound N-bromo derivatives to nitrogen-centred radicals, with subsequent formation of carbon-centred radicals and polymer fragmentation. Recent studies have provided evidence for elevated levels of metal ions at some inflammatory sites, such as in atherosclerotic plaques [29], and for a correlation between metal ions levels and the extent of protein oxidation at such sites [30]. The degradation of extracellular matrix components by HOBr might be expected to have profound effects on both the structural integrity of tissues, and cellular and tissue function. Further investigation of the consequences of these reactions may provide further insights into the role of HOBr-mediated damage in the pathogenesis of inflammatory diseases.

The authors are grateful to the Australian Research Council (through the ARC Centres of Excellence and Discovery programmes) and the National Health and Medical Research Council for financial support.