Improved procedures for the purification of selected vitamin K-dependent proteins.
Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4,340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8,200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III thrombin leads to Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin, and furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was also not converted to the active enzymes when the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The "activation peptide" released by RVV-X from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same "actid of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.
D000818:Animals; D001665:Binding Sites; D001780:Blood Coagulation Tests; D002097:C-Reactive Protein / Q000302:isolation & purification*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D005164:Factor IX / Q000302:isolation & purification*; D005170:Factor X / Q000302:isolation & purification*; D007122:Immunoelectrophoresis; D011485:Protein Binding; D011516:Prothrombin / Q000302:isolation & purification* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014462:Ultrafiltration; D014812:Vitamin K / Q000494:pharmacology*
D014812:Vitamin K; D011516:Prothrombin; D005164:Factor IX; D005170:Factor X; D002097:C-Reactive Protein; D013917:Thrombin