Decarboxylation of bovine prothrombin fragment 1 and prothrombin.
Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is 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 periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.
D015055:1-Carboxyglutamic Acid; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium; D002417:Cattle; D002942:Circular Dichroism; D004789:Enzyme Activation; D005971:Glutamates; D006358:Hot Temperature; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013917:Thrombin
D000596:Amino Acids; D005971:Glutamates; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D013917:Thrombin; D002118:Calcium