Research On Purification And Characteristics Of Fibrinolytic Enzyme From Sipunculus Nudus

Objective Nowadays, thrombotic disease has become the most severe disease in the world. Thrombotic disease causes not only the high incidence but also high mortality and disability. It mainly damages the blood systems of heart, brain and lung. Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. There are three generations of thrombolytic agents, but more or less side effects appear in each of them. It is important for medicinal experts to search for more safe and effective medicines to cure the patients. In the light of the unique advantage of marine lives, they are becoming more and more precious resources in the future. Sipunculus Nudus is a marine invertebrate animal living in the sediment of South China Sea. It is found that the coelomic fluid of Sipunculus Nudus dose not congeal after being separated for a long time. This suggests that it may contain anticoagulants or fibrinolytic substances. In this study, we try to purify fibrinolytic enzyme from a marine invertebrate-Sipunculus Nudus. In the sametime, we will study its thrombolytic effect and its safety. Methods We used sephacryl gel filtration chromatography and ion exchange chromatography to separate and purify Sipunculus Nudus fibrinolytic enzyme. The purity and relative molecular weight of Sipunculus Nudus fibrinolytic enzyme were identified by SDS-PAGE. The fibrinolytic activity was determined by fibrin plate. Rabbits were used to measure thrombolytic effects.Results (1) The fibrinolytic enzyme could be isolated and purified by using of DEAE Sepharose CL-6B, Sephadex G-75 and Superdex 200 respectively. SDS-PAGE showed that only a single band for the purified enzyme, and the molecular weight was estimated to be about 33.25 kDa.The recovery yield was 10.61%, and the specific activity increased by 6.48 folds over the extracted crude. (2) Sipunculus Nudus fibrinolytic enzyme could not only directly degrade fibrin but also indirectly hydrolyze fibrin through transforming plasminogen into plasmin. (3) The enzyme activity of Sipunculus Nudus fibrinolytic enzyme was stable in the temperature between 30℃and 50℃and the pH ranging from 6.5 to 8.0. The optimum temperature of enzyme was about 40℃and the optimum pH was 7.5 respectively. The enzyme activity was obviously inhibited by Ba2+, other metal ions including Mg2+,Ca2+,K+,Na+,Ag+ had no obvious effect on the enzyme activity. (4) PMSF was inhibitor of Sipunculus Nudus fibrinolytic enzyme. (5) The haemolytic test, hypodermic bleeding test and acute toxicity test were confirmed that Sipunculus Nudus fibrinolytic enzyme owned good bio-security.Conclution Sipunculus Nudus fibrinolytic enzyme could obviously enhance fibrinolytic activity in these experiments. It was safe and effective modern antithrombotic pharmaceutics with potential clinical applied values.