title: A disulfide bond in antithrombin is required for heparin-accelerated thrombin inactivation.
abstract: Heparin accelerates the rate of reaction of antithrombin with thrombin, an effect which is abolished by mild reduction of the antithrombin with dithiothreitol. Reduced antithrombin incorporates 1.7 mol of [14C]acetamide/mol of protein, with cysteine as the only amino acid modified. Tryptic digestion of the reduced and alkylated antithrombin results in the formation of only two labeled peptides. In the absence of heparin, the second order rate constant for the reaction of thrombin with both reduced and native antithrombin is 5.9 to 9.6 x 10(5) M-1 min-1. In the presence of heparin, the rate constant for the reaction between reduced antithrombin and thrombin is 8.3 to 12.2 x 10(5) M-1 min-1, while the rate of reaction between native antithrombin and thrombin is too fast to follow under the conditions used. Reduced antithrombin elutes from a heparin-Sepharose column at 0.5 M NaCl, contrast to 10 M NaCl required for elution of the native protein. The intrinsic tryptophan fluorescence enhancement caused by heparin binding to native antithrombin is not observed with reduced antithrombin. These data indicate that cleavage of one of the three antithrombin disulfide bonds results in reduced affinity for heparin and the loss of heparin-accelerated antithrombin activity and imply that heparin and thrombin bind at different sites on the antithrombin molecule.
mesh_terms: D000991:Antithrombins / Q000494:pharmacology*; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000502:physiology
chemical_list: D000991:Antithrombins; D004220:Disulfides; D006493:Heparin; D013917:Thrombin; D004229:Dithiothreitol; D007460:Iodoacetamide