Oxidation of heparan sulphate by hypochlorite: role of N-chloro derivatives and dichloramine-dependent fragmentation

Abstract

Activated phagocytes release the haem enzyme MPO (myeloperoxidase) and produce superoxide radicals and H2O2 via an oxidative burst. MPO uses H2O2 and Cl- to form HOCl, the physiological mixture of hypochlorous acid and its anion present at pH 7.4. As MPO binds to glycosaminoglycans, oxidation of extracellular matrix and cell surfaces by HOCl may be localized to these materials. However, the reactions of HOCl with glycosaminoglycans are poorly characterized. The GlcNAc (N-acetylglucosamine), GlcNSO3 (glucosamine-N-sulphate) and GlcNH2 [(N-unsubstituted) glucosamine] residues of heparan sulphate are potential targets for HOCl. It is shown here that HOCl reacts with each of these residues to generate N-chloro derivatives, and the absolute rate constants for these reactions have been determined. Reaction at GlcNH2 residues yields chloramines and, subsequently, dichloramines with markedly slower rates, k2 approximately 3.1x10(5) and 9 M(-1) x s(-1) (at 37 degrees C) respectively. Reaction at GlcNSO3 and GlcNAc residues yields N-chlorosulphonamides and chloramides with k2 approximately 0.05 and 0.01 M(-1) x s(-1) (at 37 degrees C) respectively. The corresponding monosaccharides display a similar pattern of reactivity. Decay of the polymer-derived chloramines, N-chlorosulphonamides and chloramides is slow at 37 degrees C and does not result in major structural changes. In contrast, dichloramine decay is rapid at 37 degrees C and results in fragmentation of the polymer backbone. Computational modelling of the reaction of HOCl with heparan sulphate proteoglycans (glypican-1 and perlecan) predicts that the GlcNH2 residues of heparan sulphate are major sites of attack. These results suggest that HOCl may be an important mediator of damage to glycosaminoglycans and proteoglycans at inflammatory foci.

Chlorination and oxidation of heparin and hyaluronan by hypochlorous acid and hypochlorite anions: effect of sulfate groups on reaction pathways and kinetics

“…The presence of an additional absorbing species is seen at 270 nm consistent with that of heparin chloramide as found in a previous study at pH 7.4 [22]. The absorbances at 270 nm show a clear effect of pH with higher values being measured at the lower pHs as shown in Figure 2b.…”

“…Using the PAGE technique, similar site-specific reactions were also observed by Davies and co-workers for oxidation of several glycosaminoglycans by peroxynitrous acid, carbonate and hydroxyl radicals [19,20]. Equally significant for the understanding of potential molecular mechanisms involved in inflammation is the demonstration in these studies that hypochlorite species form chloramides when they react with amides [21] and in particular, with glycosaminoglycans [17,18,22]. Here, a substitution reaction occurs where the N-H group becomes an N-Cl group.…”

“…Contributions to the spectra from unreacted hypochlorite and heparin have been subtracted. The characteristic maximum at 270 nm for the heparin chloramide species [22] is seen clearly at pH 6.5 and, relative to this spectrum, it is seen that increase of pH leads to a decrease in the absorption at 270 nm together with a reduction in the sharpness of the maximum. At pH 6.5, the TNB assay ( Figure 2c) showed that at least 90% of hypochlorite species had reacted to form the chloramide of heparin.…”

One-electron oxidation and reduction of glycosaminoglycan chloramides: A kinetic study

“…In vitro studies of the reactions of hypochlorite with glycosoaminooglycans have indeed demonstrated that chloramides are produced in yields and rates of reaction which are dependent upon both pH and the ratio of hypochlorite to glycosaminoglycan concentrations [22,23] and have also shown that such derivatives may accelerate the fragmentation of glycosaminoglycans within the ECM [22]. Chloramides are weak oxidising agents and are therefore, potential biological targets for reducing radicals and other reducing agents.…”

Reaction of superoxide radicals with glycosaminoglycan chloramides: a kinetic study

“…Such derivatives may accelerate the fragmentation of glycosaminoglycans within the ECM [22]. Chloramides are weak oxidising agents and are therefore potential biological targets for reducing radicals and other reducing agents.…”