Thermal denaturation of antithrombin III. Stabilization by heparin and lyotropic anions

Antithrombin III is of potential value for replacement therapy in patients with acquired or congenital deficiencies. Pasteurization of the purified inhibitor for 10 h at 60 degrees C can reduce the risk of transfusion hepatitis. Addition of appropriate stabilizers can largely prevent the loss of antithrombin activity which otherwise occurs during pasteurization. Studies of the mechanism of denaturation and stabilization have been facilitated by the use of 8-anilino-1-naphthalene sulfonate which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature. The extent of the increase in 8-anilino-1-naphthalene sulfonate fluorescence correlates roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure liquid chromatography. The midpoint, Td, of the thermal denaturation curve increases by 13 degrees C and 19 degrees C in the presence of 0.5 M and 1.0 M sodium citrate, respectively. Phosphate, sulfate, and EDTA are also strong stabilizers while the chaotropic anions, iodide and thiocyanate are potent destabilizers. Heparin at 10 mg/ml increases Td by 7 degrees C, presumably through a direct binding mechanism; chondroitin sulfate and hyaluronic acid have no effect. Samples pasteurized for 10 h at 60 degrees C in the presence of 0.5 M and 1.0 M citrate retain essentially full activity but exhibit evidence of minor alterations in their interaction with heparin.