Decarboxylation of bovine prothrombin fragment 1 and prothrombin

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Abstract
Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their .gamma.-carboxyglutamic acid residues when the lyophilized proteins were heated in vacuo at 110.degree. C for several hours. The fully decarboxylated fragment 1 product had lost its Ba-binding ability and the Ca-binding function which caused fluorescence quenching in the presence of 2 mM Ca2+. There was no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter time periods showed that the initial decrease in Ca-binding ability occurred almost twice as rapidly as the loss of .gamma.-carboxyglutamic acid. Differential functions could apparently be ascribed to the 10 .gamma.-carboxyglutamic acid residues in fragment 1, including high- and low-affinity metal ion binding sites. Prothrombin itself also underwent total decarboxylation without any apparent alteration in secondary structure. In this case the latent thrombin activity was progressively diminished during the heating process in terms of clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. In vitro decarboxylation of .gamma.-carboxyglutamic acid in dried proteins was apparently useful for analyzing the detailed Ca-binding properties of vitamin K dependent coagulation factors.