Summary
A simple method for preparation of plasminogen-free human and bovine thrombin is described.
Crude thrombin was prepared in the usual manner from oxalated plasma by means of adsorption on BaSO4, elution with trisodium citrate and activating the eluate from BaSO4 with tissue thromboplastin.
This crude thrombin was purified by means of gel-filtration and chromatography on CM-Sephadex A-50.
The gel-filtration was performed on three types of Sephadex, G-75, G-50, and G--25. By means of Sephadex G-75 the thrombin was well separated from the main part of inert protein and this type of Sephadex was used for the purification in large-scale. Separation of thrombin from protein of higher molecular weight was also obtained with Sephadex G-50 but not with Sephadex G-25 indicating a molecular weight of thrombin between 4000 and 10,000.
The importance of using an elution buffer of sufficient high ionic strength for gel-filtration is shown. A great deal of the thrombin was adsorbed to the Sephadex if the gel-filtration was performed at a too low ionic strength.
The final preparation contained 30,000 NIH units of thrombin per mg tyrosin and no detectable plasminogen.
The commercial preparation “Topostasine” was also purified in the same manner, but the plasminogen content in “Topostasine” was high and could not be completely separated from thrombin.
