Thrombin from Bovine Plasma

Extraction, Activation, and Purification of Thrombin from Bovine Plasma

Presenter: Aubrey Fox-Pardini

Co-Presenter(s):
Keth Penkian

Presenter Status: Undergraduate student

Department: Chemistry

Screenshot URL: https://drive.google.com/uc?id=1Lsq2-6UwwABFXNZLwGuGoi2AQ97POXV-

Abstract:
Thrombin is often known to be the ultimate blood coagulation protease. It has a multifunctional role in regulating platelet aggregation and blood coagulation, therefore making it a unique molecule that functions both as a procoagulant and anticoagulant. In clinical settings, thrombin has a long history of being a key to affect hemostasis, a process to prevent and arrest bleeding. The first source of thrombin studied to be used in surgical use was bovine of origin, until antibodies between human and bovine cross-reacted after surgical procedures. 1While human plasma and recombinant thrombin are safer alternatives, bovine thrombin is still used in clinical settings and for furthering research. In the current study, prothrombin was extracted from bovine plasma via barium sulfate adsorption, followed by elution of prothrombin enriched fractions using sodium citrate. Through subsequent extraction procedures, UV-Visible spectroscopy (216 nm) was used to identify the presence of proteins which would then be activated via activation buffer to detect thrombin. In order to be able to properly test the activity of thrombin, sodium phosphate dialysis followed by CM-Sephadex C-50 column chromatography was conducted to rid of impurities. The combination of Bradford assay, activity assay, and SDS-PAGE was used to quantify and confirm the success of the extraction and purification of thrombin. It was hypothesized that visual confirmation of a thrombin presence would be obtained via fluorometric assay and SDS-page bands.