The heparin binding site of antithrombin III. Evidence for a critical tryptophan residue.

Abstract

Chemical modification of antithrombin III, the major plasma protease inhibitor, with the tryptophan reagent dimethy(2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl moiety per molecule of antithrombin III. The derivatized inhibitor does not exhibit the heparin-promoted enhancement in rate of thrombin inactivation which is characteristic of the native molecule. However, the rates of thrombin inactivation in the absence of heparin are identical with native and derivatized inhibitors, indicating that the site of protease . inhibitor complex formation is not altered. Unlike native antithrombin III, the modified inhibitor does not bind to a heparin-agarose affinity column. When the modification reaction was performed with added heparin, the extent of modification was decreased and the heparin-promoted enhancement of thrombin inactivation was preserved. These results indicate that the integrity of a specific tryptophan residue is critical to the binding of heparin to antithrombin III.