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

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-acetyl-glucosamine), GlcNSO(3) (glucosamine-N-sulphate) and GlcNH(2) [(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 GleNH(2) residues yields chloramines and, subsequently, dichloramines with markedly slower rates, k(2) similar to 3.1 x 10(5) and 9 M-1 center dot s(-1) (at 37 degrees C) respectively. Reaction at GlcNSO(3) and GlcNAc residues yields N-chlorosulphonamides and chloramides with k(2) similar to 0.05 and 0.01 M-1 center dot 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 GlcNH(2) residues of heparan sulphate are major sites, of attack. These results suggest that HOCl may be an important mediator of damage to glycosaminoglycans and proteoglyeans at inflammatory foci.