Pasteurization of Antithrombin III (AT III) for 10 hours at 60°C is necessary to 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 1,8-anilinonaphthalene sulfonate (ANS) which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature as the protein unfolds. The extent of the increase in ANS fluorescence correlated roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure exclusion chromatography. The midpoint, Td, of the thermal denaturation curve increased by 13 and 19°C in the presence of 0.5 M and 1.0 M sodium citrate respectively. Phosphate, sulfate, and EDTA were also strong stabilizers while the chaotropic anions, iodidé and thiocyanate were potent destabilizers. Heparin, at 10 mg/ml, increased Td by 7°, presumbly through a direct binding mechanism. Reducing agents increased ANS fluorescence by an amount similar to that seen with thermally denatured samples, an effect which was inhibited by heparin but not by citrate. Furthermore, incorporation of 14C-iodoacetamide into AT III during thermal titration was coincident with the increase in ANS fluorescence suggesting that disulfide cleavage is the event which triggers the unfolding of the protein. Samples pasteurized for 10 hours at 60°C in the presence of 0.5 M and 1.0 M citrate retained full antithrombin activity but exhibited evidence of minor alterations in the ability to bind heparin.
Stabilizing Effect of Various Polyols on the Native and the Denatured States of Glucoamylase
