A simplified procedure for purification of human prothrombin, factor IX and factor X.
A simplified procedure is described for the purification of prothrombin, Factor X and Factor IX in overall yields of 35-40% from pooled human plasma. The initial steps, which are common to prior purification techniques, include adsorption onto and elution from barium citrate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. The procedure differs from previous techniques in that the nest step, heparin-agarose chromatography, is carried out in a (sodium) citrate buffer, pH 7.5. These chromatographic conditions permit the separation of prothrombin, Factor X and Factor IX from each other, yielding fractions with apparent homogeneity in several electrophoretic systems. The additional chromatographic steps of earlier purification procedures are therefore unnecessary. The heaprin-agrarose column chromatographic conditions consistently resulted in the separation of human prothrombin in into two fractions in a ratio of approximately 4:1. Both fractions possess similar specific activity in a one stage prothrombin assay, and also activate at the same rate in a Factor Xa, Ca2+ and phospholipid system. Both fractions of prothrombin also comigrate in sodium dodecyl sulfate gel electrophoresis with an apparent Mr integral of 70,000.
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000302:isolation & purification*; D005170:Factor X / Q000302:isolation & purification*; D006801:Humans; D008722:Methods; D008970:Molecular Weight; D011516:Prothrombin / Q000302:isolation & purification*
D011516:Prothrombin; D005164:Factor IX; D005170:Factor X