Study Of N-V Protease On Thrombolytic Mechanism And Characteristics In Enzymology

Thrombogenic disease is a threat against human's health. At present, it's anurgent task to find a safe, efficient, innocuous thrombolytic drug. N-V proteaseis a kind of protease abstracted from some marine life by the research team ofProfessor Hong Min in the department of biochemistry, Jilin University. It iscritical for the purity of all the reagents used in studying the thrombolyticmechanism of N-V protease. So we must purify N-V protease before we set outto do that. The author purified it to 97.57% by the method of electrophoresiscreatively. And the purity of N-V protease has agreed the requirement of theassay.Isoelectric focusing electrophoresis is especially suitable for the study ofthe microheterogeneity of protein because of its sensitivity. If there is one bandin SDS-PAGE but three bands in Isoelectric focusing electrophoresis gel, it ispossible that there are three types of this protein such as monophosphorylation,biphosphorylation and triphosphorylation and so on. Phosphonic group isinsig-nificant for the molecular weight of protein, so there is only one band inSDS-PAGE. But the differences of them can be determined owing to the electriccharge. Maybe only one or two amino acids are different between isoenzymes,so we can separate them very well by isoelectric focusing electrophoresis. In thisexperiment there is only one band in Isoelectric focusing electrophoresis gel,and we can conclude that N-V protease has a uniform structure, and noisoenzymes exist in the sample. We find out that the pI of N-V protease is 4.5.S2251 is a synthetic colorless specific reagent for plasmin and is insensitive toother serine protease. The carboxy group of lysine in tripeptide of valine,leucine and lysine react with p-nitroaniline and produces para nitroanilide.During the study of the N-V protease activation to bovine plasminogen, it wasfound that S2251, which was considered to be the specific substrate of plasmin,can be hydrolyzed by N-V protease. So a kind of Chromogenic substrate wasfound to assay the activation of N-V protease. The optimal temperature is 47℃or so and the optimal pH is about 8.7. But there are not apparent differences inOD405 among the pH in the range between pH8.5～pH8.9. That demonstratesthat N-V protease has a wider optimal pH extent and it can endure the changesof pH in the scope from pH8.0 to pH 9.0.Single band in SDS-PAGE gel illustrates that N-V protease is composedwith only one peptide chain. We determined the molecular weight of N-Vprotease is 30.6kD. Because the average molecular weight of 20 amino acidswhich can consist of proteins is 128, the number of amino acids composed ofN-V protease is about 273. When we sequenced the protein, we determined onlya fragment of 70 amino acids. It demonstrated that there must be groupprotection in the peptide chain. Swiss-prot presumes that there is glycosylationin the carboxyl end. That means N-V protease is a kind of glycoprotein. It isvery possible that N-V protease is a kind of glycoprotein as a protease inanimal's enteric cavity. It is very significant to determine whether it is a kind ofglycoprotein or not in researching the potential biological function. Themethod of alcian blue/silver dye is very sensitive and it can detective protein ingel in several nanograms level. We utilized it to assay N-V protease and find theenzyme is not a kind of glycoprotein. Proteases can be classified to fourmechanistic class based on their sensitivity to four 'standard' inhibitors of serine,cysteine, aspartic and metalloproteases. The activity of N-V protease was testedafter the enzyme was inhibited by the four extremes (PMSF;E64;pepstatin;1,10-phenanthroline)1h later. PSMF inhibits N-V protease, and the other threecan't act. So we draw the conclusion that N-V protease belongs to the class ofserine proteases.There are two mechanisms in fibrinolysis. One is the thrombolytic agentsact as the plasminogen activator;the other is thrombolytic agents hydrolyzefibrin directly. We assayed the mechanism of N-V protease in the two aspectsand found there was striking differences between the activating mechanism ofN-V protease and that of urokinase with plate process. And with the results ofthe SDS-PAGE electrophoresis of the products of albumin, casein, bovine fibrinand human fibrin, we drew a rudimentary conclusion that N-V protease can't bea plasminogen activator but hydrolyze proteins directly. We hydrolyzedinsulin-B chain with N-V protease, and then get rid of proteins and derivatizedthe products with 2,4-dinitrofluorobenzene before assaying amino acids byHPLC. We determined 11 kinds of amino acids-Ser, His, Gly, Arg, Thr, Ala, Val,Leu, Phe, Lys, Tyr. So we concluded that N-V protease is an exopeptidase. So Itis true that the ratiocination mentioned above. N-V protease doesn't act as aplasminogen activator but hydrolyze fibrin directly.We used Hipp-L-Phe, Hipp-L-Arg and Leu-pNA as substrates respectivelyto determine the activity of N-V protease, and found the enzyme is a kind ofaminopeptidase. Km is a characteristic constant of enzymes. We determined theKm of N-V protease is 1.97×10-3mol/L. This means there is more forcefulaffinity between the enzyme and Leu-pNA.We drew the conclusion as follows: 1. N-V protease is not a glycoprotein,and it belongs to the family of serine proteases. 2. N-V protease is a kind ofexopeptidase, further more an aminopeptidase. 3. N-V protease is not aplasminogen activator, but it can hydrolyze the fibrin in thrombous directly. 4.N-V protease can hydrolyze so many proteins thoroughly and swiftly so it canbe made as tools enzyme in protein sequencing and nucleic acid abstracting.