A Simplified Procedure for Purification of Human Prothrombin, Factor IX and Factor X

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Abstract
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 next 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 unnecessary. The heparin-agarose column chromatographic conditions consistently resulted in the separation of human prothrombin into 2 fractions in a ratio of .apprx. 4:1. Both fractions possess similar specific activity in a 1 stage prothrombin assay and activate, at the same rate, in a Factor Xa, Ca2+ and phospholipid system. Both fractions of prothrombin comigrate in sodium dodecyl sulfate gel electrophoresis with an apparent MW 70,000.

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