Oxidation of methionine residues in antithrombin - Effects on biological activity and heparin binding

Commercially available human plasma-derived preparations of the serine prot ease inhibitor antithrombin (AT) were shown to contain low levels of oxidat ion, and we sought to determine whether oxidation might be a means of regul ating the protein's inhibitory activity. A recombinant form of AT, with sim ilarly low levels of oxidation as purified, was treated with hydrogen perox ide in order to study the effect of oxidation, specifically methionine oxid ation, on the biochemical properties of this protein. AT contains two adjac ent methionine residues near the reactive site loop cleaved by thrombin (Me t(314) and Met(315)) and two exposed methionines that border on the heparin binding region of AT (Met(17) and Met(20)), In forced oxidations with hydr ogen peroxide, the methionines at 314 and 315 were found to be the most sus ceptible to oxidation, but their oxidation did not affect either thrombin-i nhibitory activity or heparin binding, Methionines at positions 17 and 20 w ere significantly oxidized only at higher concentrations of peroxide, at wh ich point heparin affinity was decreased. However at saturating heparin con centrations, activity was only marginally decreased for these highly oxidiz ed samples of AT. Structural studies indicate that highly oxidized AT is le ss able to undergo the complete conformational change induced by heparin, m ost probably due to oxidation of Met(17), Since this does not occur in less oxidized, and presumably more physiologically relevant, forms of AT such a s those found in plasma preparations, oxidation does not appear to be a mea ns of controlling AT activity.