Extraction, Activation, and Purification

Thrombin is often known to be the ultimate blood coagulation protease. It is activated from its zymogen, prothrombin. Prothrombin was prepared by barium sulfate adsorption and elution of prothrombin enriched fraction using sodium citrate. The pellets collected from mixing of barium sulfate with prothrombin would be washed with sodium citrate to remove impurities for protein identification using UV-Visible spectroscopy. Ammonium sulfate (70% saturation) is later added and mixed to the collected washing, which is centrifuged. The activation buffer is responsible for the activation of prothrombin to thrombin, which is added to the resulting pellet. The resulting thrombin is to be dialyzed with sodium phosphate buffer (pH 7) to remove the ammonium sulfate from the protein. Once purified, we perform a weak cation exchange chromatography using CM Sephadex C-50 to collect fractions of thrombin, and determine their protein activity and concentration by testing them with different assays.