Characterization And Structural Analysis Of Two Kinds Of Insects'Plasmins, And Research Of The Mimic Enzyme

With the development of economic and life level, cardiovascular disease, such as acute myocardial infarction and stroke, are one of the life-threatening diseases that affect more and more people each year. The thrombolysis is a main therapeutic approach to treat thrombosis disease. Despite its tremendous clinical success, several major limitations of the therapy still need to be addressed, including bleeding and high price. Thus, identifying new therapeutic targets and developing safe and efficient thrombolytic agents are the focal points in the field of thrombolysis. In this thesis, two kinds of insects' plasmins were extracted from Tenibrio molitor (yellow mealworm beetle) and Eupolyphaga sinensis (ground beetle), and their properties of thrombolysis, anti-angiogenesis and tumor-suppression were studied. The amino acid sequences of the plasmins were analyzed, while their three-dimensional structures and active sites were determined by the method of bioinformatics. A novel of polymer (mimic enzyme) was designed and synthesized to simulate the plasmins, which would lay a necessary theory and practical foundation for the development of new thrombolytic drugs.Two kinds of insects' plasmins were purified form yellow mealworm beetles and Eupolyphaga sinensis, by DEAE-32cellulose chromatography column and SephadexG-75dextran gel column, which named YFP(fibrinolytic protein form Yellow mealworm beetle) and EFP(fibrinolytic protein form Eupolyphaga sinensis). The fibrinolytic activity were detected by the fibrin plate method; their sugar content were detected by the anthrone-sulfuric acid method, and their molecular weight were estimated by SDS-PAGE. The chick embryo chorioallantoic membrane experiment proved that the two kinds of plasmins could inhibit angiogenesis, and both of them had anti-tumor effects, through animal experiment of tumor-bearing mice. The results showed that the relative molecular mass of YFP was about56.1kD; sugar content of YFP was7.084percent; The fibrinolytic activity of YFP had no influence on Na+and K+, while the Mg2+, Ca2+, EDTA, urea and mercaptoethanol has some inhibitory effect to its vitality; the optimal reaction pH value and temperature of YFP were pH7.5and25℃-45℃, respectively. The Km of YFP were tested by the experiments of enzymatic kinetic reaction, It could be concluded that the capability of the YFP with various substrates was in the follwing order:ester bond＞amide bond＞peptide bond. The relative molecular mass of EFP was about41.3kD; sugar content of EFP was10.05percent; The fibrinolytic activity of EFP had no influence on Na+and K+ while the Mg2+, Ca2+,EDTA, urea and mercaptoethanol has some inhibitory effect to its vitality:the optimal reaction pH value and temperature of EFP were pH8.0and40℃. respectively. The EFP had anti-tumor effects on SI80and H22solid tumors, and one pathway of apoptosis of S180and H22tumor cells induced by EFP was activation of caspase-3.using the bioinformatics methods, the3D-structures of EFP and YFP were simulated by the the "biosun" computer program, while the isoelectric point, hydrophilicity, and flexibility of the sequences were analyzed by the the "goldkey" computer program. The acive sites of the YFP and EFP both were Histidine, Serine and Aspartic acid, and in the same position, while the Substrate binding sites both were Serine, Aspartic acid and Glycine. the plasmins' catalytic mechanism was the catalyzing hydrolysis of the peptide bonds between the Arginine and the Lysine.The acive sites of the insects' plasmins were Histidine, Serine and Aspartic acid, and the functional groups of the three amino acids were Imidazole, hydroxyl and carboxyl groups. So a novel of polymer (mimic enzyme) was designed and synthesized through by copolymerization with raw materials of acrylic acid, allyl alcohol and vinyl imidazole. The molecular weight of the mimic enzyme were estimated by osmotic method, and The fibrinolytic activity of the mimic enzym were detected by the fibrin plate method.The optimum reaction conditions were vinyl imidazole. acrylic acid, allyl alcohol in the ratio of1:20:1, benzene as solvent. zobisisobutylnitrile as initiator, and80℃. In the optimum reaction conditions, the molecular weight of the mimic enzyme was4.7×104. As the concentrations increased, the fibrinolytic activity of the mimic enzyme was increasing, and blood clots could be dissolved in20%mimic enzyme solution. The fibrinolytic activity of the mimic enzyme had no significant influence by metal ions, and the optimal pH of the mimic enzyme were6and9. The polymer was characterized by IR and NMR, and it's viscosity was studied also.