The Study On Extraction, Purification And Stability Of Thrombin From Yak Blood

In this study, fresh yak blood as the research object, the thrombin was prepared usingsingle factor, orthogonal experimental design, response surface analysis method. Theextracting and activation process of thrombin were optimized. Thrombin was purified usingDEAE-52ion chromatography and sephadex G-75gel filtration layer analysis. Furthermore,stability of thrombin were studied. The results were as follows:(1) The optimum extracted parameters of the yak blood thrombin with isoelectricprecipitation:9-fold diluted plasma, HAc concentration3%, pH5.3. The thrombin activitywas2.19U/mg under these conditions. The rate of extraction was56.71%.(2) Extraction parameters of thrombin in the blood yak using barium citrate adsorptionmethod: additional amount of barium chloride was12%of the original plasma volumes.Under this condition, the activity of thrombin was6.34U/mg. The rate of extraction was65.15%.(3) By comparing the two extraction methods, citric acid adsorption method is superiorto isoelectric precipitation. The specific activity of enzyme was6.34U/mg and the totalactivity was1635.29U.(4) The optimal parameters of prothrombin activation process with Ca2+alone: theactivation temperature was27℃,the final concentration of Ca2+was0.01mol/L,the activationtime was1.95h. under these conditions, the specific activity of thrombin was6.34U/mg.(5) DEAE-52chromatography, the purification factor was16.52. the recovery ratio was76.21%. sephadex G-75gel filtration chromatography, The purification factor was29.21withrecovery ratio of57.75%. the purity identification results of the purified thrombin showed that:electrophoretic bands showed a single band and has reached electrophoretic homogeneity. Themolecular weight was about37kd.(6) Thrombin is relatively stable when the temperature and pH was in the range of4-37℃,6-8respectively. It was inhibited by K+, Ba2+, Mg2+, Mn2+, Fe2+and Methanol,ethanol, ethylene glycol in various degree. The enzyme is stable at low temperatures.In summary, to prepared thrombin with yak blood, the barium citrate extraction methodis superior to the isoelectric precipitation method. Thrombin can be activated by Ca2+. Usingthe exchange chromatography through sephadex G-75gel and DEAE-52ion filtrationchromatography, electrophoresis pure thrombin can be obtained. this enzyme does not acidresistant and can be inhibited by metal ions and organic solvent. The molecular weight was about37KD. Yak blood has the potential to become an important source of thrombin.