Research On Purification And Characteristics Of Fibrinolytic Enzymes UFEⅠ From Urechis Unicinctus

Thrombotic disease is a frequently-occurring disease which do harm to people seriously.A lagre proportion of cardio-cerebrovascular diseases are actually of this kind.It mainly damages the blood systems of heart,brain and lung,resulting in death or disability in adult population.In China,over 2,600,000 people die of cardio-cerebrovascular disease each year,and more than 3,000,000 people need thrombolytic therapies.And,the number of patients with thrombotic disease has been increased fast in recent years.Thrombolytic therapy is one of the most important pathway in treatment of thrombotic disease.Although there have been three generations of thrombolytic agents developed so far,more or less side effects appear in each of them.For this reason,it's important to research and develop new ones currently.In the light of the unique advantage of marine lives,they are becoming more and more precious in the development.For years of research in the lab,a novel fibrinolytic enzyme is investigated and isolated from a marine invertebrate,Urechis unicinctus,in this paper.The main achievements in the investigations are shown as follows.1.The fibrinolytic enzyme abounds in the haemocoelomic fluid and gut tissues of Urechis unicinctus,which can be isolated and purified by several steps,Maybe there are a group of isoenzymes.By trying two procedures,he made UFEâ… .the recovery yield is 27.0-30.0%,and the specific activity increases by 11-12 folds over the extracted crude.2.UFEâ… showes only one band by using the SDS-PAGE and Native-PAGE, which implies that purity of the enzyme is above 99%,and can be effectively prepared in large scale.By using SDS-PAGE and MS analyses,the molecular weight of UFEâ… is about 23,800 Da.The results determined by spectrum colorimetric assay of anthrone-sulfuric acid method show that UFEâ… hardly contains sugar,and is not a glycoprotein.3.UFEâ… is considerably stable.When the enzyme is stored in the temperature below 45℃for 6 hours,its activity remains almost unchanged.And also when the enzyme is stored in 10 kinds of different buffer solutions with pH value ranging from 3-11 respectively at room temperature for 24 h,its activity is quite stable.4.The optimum temperature of UFEâ… is about 45℃.The optimum pH is slight acidic of 6.0-6.5.5.During investigation,it is discovered that Ag⁺ ions is inhibitor of UFEâ… ,but Mg²⁺ and Mn²⁺ ions are activators of the enzyme.6.Using casein as substrate,the enzyme dynamic parameters calculated according to Lineweaver-Burk plot are shown as follows:K_m=3.48×10^-3 mol·L^-1, V_max=1.20×10^-2 mol·L^-1·min^-1.7.UFEâ… can not only directly degrade fibrin but also indirectly hydrolyze fibrin through transforming plasminogen into plasmin.For this reason,its fibrin selectivity and enzyme activity are considerably high.8.Like lumbrokinase,the results of anticoagulation and thrombolytic test in vitro show that there are apparent anticoagulant effect and thrombolytic effect.Besides, UFEâ… is mild and less harmful to erythrocytes in comparison with Iumbrokinase which can damage erythrocytes in some extent.In summary,Urechis unicinctus fibrinolytic enzyme takes on potential applied values in the future due to its obvious advantages such as low molecular weight, high stability,strong potent anticoagulant and thrombolytic activities,and little damage to the erythrocytes.