Purification And Characterization Of Fibrinolytic Enzyme From Trichosanthes Kirilowii

Thrombotic disease is a serious hazard to human health and the lives of disease, which has been attracted a high degree of attention and widespread concern by medical profession. In recent years, research and development of specific, effective and safe thrombolytic drugs, has become a hot topic around the world. Modern pharmacological research proved that Chinese Herbal Medicines can improve the blood flow dynamics, blood flow and microcirculation change,and the treatment of thrombotic diseases have a good effect. The purpose of this article is to extract a high fibrinolytic activity of protein from the Chinese Herbal Medicines Trichosanthes kirilowii and then analysis of the purified protein.The fibrinolytic enzyme from Trichosanthes kirilowii was extracted by the ammonium sulfate fractionation and isoelectric point sedimentation. The fibrinolytic enzyme of Trichosanthes kirilowii was separated and purified by affinity chromatography on the SBTI which was immobilized on sepharose 4B beads, ion-exchange chromatography. The enzyme with molecular weight of 66.0 and 55.6 kD were purified from Trichosanthes kirilowii. The enzyme with molecular of 55.6 kD labeled TkFE was transferred into PVDF mambrane and determined the N-terminal amino acids sequence. The first 10 amino acids of the N-terminal sequence of TkFE was DVRSPRDTDE by Edman degradation. Compared N-terminal sequence of TkFE with other fibrinolytic enzymes' by blasting from NCBI, the similarity is lower than 30%, and so it is a new fibrinolytic enzyme.The results of the analysis about TkFE:TkFE was inhibited with protease inhibitors. And the fibrinolytic activity was strongly inhibited with soybean trypsin inhibitor (SBTI), indicating it may be a serine protease. The pI of TkFE is 9.5; Trypsin activity is 2.47 U·mg-1; its activity was stable 4-40℃and pH 4.0-11.0, and the optimun pH value is 9.0; its fibrinolytic activity was inhibited completely by Ag+, partly by Cu2+, residual activity 72.25 %, and Mg2+, Mn2+, Na+, K+ increased the fibrinolytic activity slightly; in casein as substrate in the hydrolysis reaction, the temperature, enzyme substrate ratio, reaction time and pH have different effect on the hydrolysis reaction:temperature>enzyme substrate ratio>reaction time > pH, Km is 1.89 mg·mL-1; trypsin, pepsin and bile salt were effect on TkFE, respectively, trypsin and bile salt had no effect, but at pH 3.0 TkFE was hydrolyzed by pepsin and then its fibrinolytic activity became higher.