Purification of Prothrombin and Thrombin: Chemical Properties of Purified Preparations

We wish to describe certain simplifications in the technique previously described (13,14) for the purification of prothrombin and thrombin.With new adaptations it is possible to process large quantities of plasma and at the same time to obtain products 2 to 5 times as potent as those previously reported from this laboratory.A brief survey of the chemical properties of these newly obtained products is also given. Preparation of ProthrombinBeef plasma is diluted lo-fold with water and brought to pH 5.3 with 1 per cent acetic acid.The precipitate is redissolved and Mg(OH)z suspension is added.In our original technique (14) the prothrombin adsorbed on the Mg(OH)z was set free with CO2 at atmospheric pressure.Improvement is achieved by using CO2 at 4 to 6 atmospheres (by shaking in a metal chamber, with 8 to 10 volumes of water to 1 volume of Mg(OH)z paste).Following this elution, the CO2 is allowed to escape, and variable amounts of magnesium carbonate settle out.As previously, the dissolved salts are then removed by dialysis.The use of increased pressure reduces the working volumes at least 90 per cent, and this simplifies enormously the procedure of dialysis.A second improvement is the isoelectric fractionation of the dialyzed prothrombin-containing eluate; this is accomplished in two steps (at pH 5.6 and at 5.3).The first fraction (pH 5.6)