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Mild reduction of antithrombin (AT) with dithiothreitol followed by alkylation with [14C]-iodoacetamide results in the incorporation of 1.7 mol of acetamide/mol of protein with cysteine the only amino acid modified. Tryptic digestion of the reduced and alkylated AT results in the formation of only two labeled peptides indicating that mild reduction cleaves only one of the 3 disulfide bridges.  The 2nd order rate constant for the reaction of thrombin with both native and mildly reduced AT is 5.9-9.6 × 105 M-1 min-1. Addition of heparin accelerates the reaction between native AT and thrombin (k &gt;107 M-1 min-1) but has no effect on the reaction between reduced AT and thrombin (k = 8.3-12.2 ×105 M-1 min-1). Alkylation of reduced AT destroys all remaining thrombin-neutralizing activity.  Native AT elutes from a heparin-Sepharose column with 1.0 M NaCl whereas reduced AT elutes at 0.5 M NaCl. Reduced and alkylated AT is not retained by the immobilized heparin even in 0.1 M NaCl. The intrinsic tryptophan fluorescence enhancement observed following binding of heparin to native AT is not seen with reduced AT. These data indicate that reduction of a single disulfide bond in AT lowers the affinity of the protein for heparin and eliminates the heparin-mediated acceleration of thrombin neutralization.  Mildly reduced and alkylated AT was digested with CNBr and the peptides were fractionated by HPLC. Preliminary analysis of the CNBr peptides suggests that the crucial disulfide bond is between CYS239 and CYS422 (using the numbering in Magnusson’s sequence data).  Supported by grants from the USPHS. THF is a Research Career Development Awardee of the NIH (HL-00277).

The Disulfide Bond In Antithrombin Required For Interaction With Heparin