Efficiencies of fragmentation of glycosaminoglycan chloramides of the extracellular matrix by oxidizing and reducing radicals: potential site-specific targets in inflammation?

Hypochlorous acid and its conjugate base, hypochlorite ions, produced under inflammatory conditions, may produce chloramides of glycosaminoglycans, these being significant components of the extracellular matrix (ECM). This may occur through the binding of myeloperoxidase directly to the glycosaminoglycans. The N-Cl group in the chloramides is a potential selective target for both reducing and oxidizing radicals, leading possibly to more efficient and damaging fragmentation of these biopolymers relative to the parent glycosaminoglycans. To investigate the effect of the N-Cl group, we used ionizing radiation to produce quantifiable concentrations of the reducing radicals, hydrated electron and superoxide radical, and also of the oxidizing radicals, hydroxyl, carbonate, and nitrogen dioxide, all of which were reacted with hyaluronan and heparin and their chloramides in this study. PAGE gels calibrated for molecular weight allowed the consequent fragmentation efficiencies of these radicals to be calculated. Hydrated electrons were shown to produce fragmentation efficiencies of 100 and 25% for hyaluronan chloramide (HACl) and heparin chloramide (HepCl), respectively. The role of the sulfate group in heparin in the reduction of fragmentation can be rationalized using mechanisms proposed by M.D. Rees et al. (J. Am. Chem. Soc.125:13719-13733; 2003), in which the initial formation of an amidyl radical leads rapidly to a C-2 radical on the glucosamine moiety. This is 100% efficient at causing glycosidic bond breakage in HACl but only 25% efficient in HepCl, the role of the sulfate group being to favor the nonfragmentary routes for the C-2 radical. The weaker reducing agent, the superoxide radical, did not cause fragmentation of either HACl or HepCl although kinetic reactivity had been demonstrated in earlier studies. Experiments using the oxidizing radicals, hydroxyl and carbonate, both potential in vivo species, showed significant increases in fragmentation efficiencies for both HACl and HepCl, relative to the parent molecules. The carbonate radical was shown to be involved in site-specific reactions at the N-Cl groups, reacting via abstraction of Cl, to produce the same amidyl radical produced by one-electron reductants such as the hydrated electron. As for the hydrated electrons, the data support fragmentation efficiencies of 100 and 29% for reaction of carbonate radicals at N-Cl for HACl and HepCl, respectively. For the weaker oxidant, nitrogen dioxide, no fragmentation was observed, probably because of a low kinetic reactivity and low reduction potential. It seems likely therefore that the N-Cl group can direct damage to extracellular matrix glycosaminoglycan chloramides, which may be produced under inflammatory conditions. The in vivo species, the carbonate radical, is also much more likely to be site-specific in its reactions with such components of the ECM than the hydroxyl radical.

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

Hypochlorous acid (HOCl), produced in inflammatory conditions by the enzyme myeloperoxidase, and its anion hypochlorite (OCl(-)) exist in vivo at almost equal concentrations. Their reactions with hyaluronan and heparin (as a model for sulfated glycosaminoglycans in the extracellular matrix) have been studied as a function of pH. The major product in these reactions is the chloramide derivative of the glycosaminoglycans. Spectral, chloramide yield, and kinetic measurements show sharply contrasting behavior of heparin and hyaluronan and the data allow the calculation of second-order rate constants for the reactions of both HOCl and OCl(-) for all reaction pathways leading to the formation of chloramides and also oxidation products. By comparison with hyaluronan, it can be demonstrated that both N-sulfate and O-sulfate groups in heparin influence the proportions of these pathways in this glycosaminoglycan. Evidence is also given for further oxidation pathways involving a reaction of HOCl with the chloramide product of hyaluronan but not with heparin. The significance of these results for the mechanisms of inflammation, particularly for fragmentation of extracellular matrix glycosaminoglycans, is discussed.