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Biochem. J. (2005) 391 (125–134) (Printed in Great Britain)

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

Martin D. REES, David I. PATTISON and Michael J. DAVIES1

Free Radical Group, Heart Research Institute, Camperdown, Sydney, NSW 2050, Australia

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, k₂~3.1×10⁵ and 9 M^-1·s^-1 (at 37 °C) respectively. Reaction at GlcNSO₃ and GlcNAc residues yields N-chlorosulphonamides and chloramides with k₂~0.05 and 0.01 M^-1·s^-1 (at 37 °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 °C and does not result in major structural changes. In contrast, dichloramine decay is rapid at 37 °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.

Key words: glycosaminoglycan, heparan sulphate, heparin, hypochlorite, myeloperoxidase, oxidation.

Abbreviations used: b-HP, bovine lung heparin; dnsp-HP, partially de-N-sulphated porcine intestine heparin; GlcNAc, N-acetylglucosamine; GlcNH₂, (N-unsubstituted) glucosamine; GlcNSO₃, glucosamine-N-sulphate; HA, hyaluronan; MPO, myeloperoxidase; p-HP, porcine intestine heparin; p-HS, porcine intestine heparan sulphate; TNB, 5-thio-2-nitrobenzoic acid.

¹To whom correspondence should be addressed (email m.davies@hri.org.au).

Received 18 April 2005/1 June 2005; accepted 3 June 2005

Published as BJ Immediate Publication 3 June 2005, doi:10.1042/BJ20050630

The Biochemical Society, London ©2005

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