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.
Highlights
X lo6 M" min".In the presence of heparin, the rate constant for the reaction between reduced antithrombin and thrombin is 8.3 to 12.2 X lo5 M" min", while the rate of reaction between native antithrombin and thrombin is too fast to follow under the conditions used
The intrinsic tryptophan fluorescence enhancement caused by heparin binding tonative antithrombin is not observed with reduced antithrombin
Porcine gastric mucosal heparin from ResearPclhus was purified by chromatography on Sephadex G-75 in 0.2 M NaC1; fractions from the central portiofnthe heparin peak (assayed with azure A (1 1)w) ere combined and the polysaccharide was precipitated with ethanol
Summary
Effect of Dithiothreitol on the Activityof AntithrombinWhen antithrombin was incubated with dithiothreitolat p H 8.3 and37°Cfor 2 min,itsabilitytoneutralizethrombin. M NaCl, while in the absence of reducing agent it eluted at M NaC1. Effect of reduction on thromhin acticity dithiothreitol had no effecton thrombin amidolytic activity, Thrombin (8units/ml) was incubated at 37'C in 0.15 M NaCl, 0.05 whether thrombin was incubated alone or with heparin or. After either 0.5 or min, an aliquot was withdrawn and added vation of thrombin by antithrombin itnhe absence of heparin to 300 p1 of assay mixture (1.8mg/ml of fibrinogen, 3.3$ gum acacia, was high as 1 examined (Fig. 1B).D mM had no apparent ithiothr effect eitol on t concent he slow r ationsas neutrali z a. 37°C and the of thrombin by antithrombin which occurs inthe absence of Dithiothreitol
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