Thrombin partially purified from bovine plasma can be inactivated at 60°C. In the presence of 10 units of heparin the extent of inactivation decreases. When thrombin and heparin are mixed and incubated for 5 min at 0°C before gel filtration on Sephadex G-200, thrombin with heparin is eluted prior to either thrombin or heparin alone. These data suggest a complex formation between thrombin and heparin. Immobilized heparin binds thrombin. The enzyme can be eluted with 0.05 M Tris-HCl buffer, pH 7.3, containing an ion mixture of Na+, K+ and Ca2+ at 73, 3 and 11 mM, respectively, at 0°C and with 0.05 M Tris-HCl buffer, pH 7.3, containing 0.5 M NaCl at 20°C. During the same chromatographic procedure, antithrombin-III (heparin cofactor) partially purified from human plasma is eluted with 0.05 M Tris-HCl buffer, pH 7.3, at 0°C as well as 20°C. Although, as described in the literature, heparin binds to antithrombin, our findings suggest another possibility, i.e. that the binding of heparin to thrombin induces a conformational change in the enzyme facilitating a complex formation between thrombin and antithrombin-III.
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