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.
Highlights
Isolation ofAntithrombinZZZ-Antithrombin 111was purified from fresh bovine plasma by a modification of Method A of Thaler and Chemical modification of antithrombin111, the major Schmer [14]
After elution from the heparin-agaroseaffinity column, plasma protease inhibitor,with the tryptophanreagent thesample was concentrated with an Amicon ultrafiltration cell dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide, equipped with a PM 30 membrane, dialyzed against 20 mM imidazoleresults in the incorporationof one hydroxynitrobenzyl HC1, 150 mM NaC1, pH 7.3, and applied to a DEAE-Sephadex A-50 moiety per molecule of antithrombin III
These results indicate that the integrity of a specific tryptophan residue is critical to the binding of heparin p-nitroanilideacetateand D-phenylalanyl-L-pipecolyl-L-arginine-pnitroanilide dihydrochloride, werepurchased from Boehringer Mannheim and Ortho Diagnostics, respectively
Summary
Chemical modification.Since spectral anfdluorescence studies [8,9,10,11] suggest that the environment of 1 or more of the 6 tryptophan residues in antithrombin 111 is altered by heparin (Received for publication,October 29, 1979) binding, we utilized the active benzyl halide, dimethyl(2-h~-. Blackburn and CarolinCe. Sibley modifying reagent [12, 13]
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