The Study Of Fibrinolytic Active Protease From Perinereis Aibuhitensis Grub

Thrombotic diseases are one of the major adult diseases that threaten human health.Patients with acute myocardial infarction?AMI?have a high rate of sudden death due to coronary artery occlusion in a short time.The blood clots of AMI can be removed by PCI or thrombolysis treatment to restore coronary blood flow.But with the imbalance of health resources distribution,the PCI treatment cannot afford timely treatment to 7.502 million AMI patients in China.Therefore,a new thrombolysis or new mechanism of thrombolysis in acute myocardial infarction patients will make a breakthrough progress in thrombolysis treatment.Clamworm?Perinereis aibuhitensis?,an ocean beaches biological bidentate animal,was taken as the research object in this paper.Through the systematical designing of purification steps with using two ways of ammonium sulfate segmented salting out method and hierarchic gel chromatography,the optimal purification process and conditions of the fibrinolytic activity protease in clamworm was confirmed.Meanwhile,its enzymology properties and the fibrinolytic activity and preliminary parsing the peptide molecular structure by LC-MS/MS-MS were studied.1.Ammonium sulfate segmented precipitation and hierarchic gel chromatography were used to purify fibrinolytic active protease of bidentate periderm.The optimal purification process of fibrinolytic active protease from Perinereis aibuhitensis Grub is as follows:crude protein of clam worms was extracted after tissue homogenate,refrigerated centrifuge and 40-60%ammonium sulfate salt out of supernatant fluid.Dialysis and lyophilization of crude protein to get Asp60,and then purification Asp60 by anion exchange chromatography?DEAE Sepharose Fast Flow?and thin layer chromatography?Phenyl Sepharose Fast Flow?,which steps can obtain a single components with high fibrinolytic activity protease Asp60-D1-P1 from crude protein in Perinereis aibuhitensis Grub,the protease activity of Asp60-D1 and Asp60-D1-P1 were as high as 49.79IU/mg and 231.94 IU/mg,respectively.And the purification multiples of protein are the 4.45 and 20.23 after two chromatography,respectively.2.Asp60-D1-P1 had excellent fibrinolytic activity.Asp60-D1 obtained by anion exchange chromatography from 40-60%ammonium sulfate salting out protease of P.aibuhitensis had the highest fibrinolytic activity in the first-order chromatography protein peak,which was 167.31 U/mg.The fibrinolytic activity of Asp60-D1-P1 was as high as 201.79 U/mg in the second-order chromatography protein peak.The fibrinolytic activity of Asp60-D1 and Asp60-D1-P1 was 0.91 times and 1.31 times higher than that before chromatography.The results of agarose-fiber plate also show that Asp60-D1-P1 had the largest transparent circle in the secondary chromatographic peaks which proved that Asp60-D1-P1 had excellent fibrinolytic activity.3.The fibrinolytic active protease Asp60-D1-P1 has excellent enzymatic characteristics.The total contents of hydrophilic amino acids and hydrophobic amino acids in Asp60-D1-P1 were 126.282mg/g and 24.182 mg/g,respectively.Combined with the retention time of first eluent peak in the Asp60 purification stage of hydrophobic chromatography,Asp60-D1-P1 was a hydrophilic protein.After comprehensive analysis by Native-PAGE and SDS-PAGE,it was found that the molecular wight of Asp60-D1-P1 is 44.5KDa,which was composed by two polypeptide chains with molecular weights of 37.8KDa and 6.5KDa,respectively.And it often existed in the form of trimer.Asp60-D1-P1 was stable at 20?-60?,the optimal temperature was 40?.,the protease is inactivated when the temperature exceeded 70?.Asp60-D1-P1 showed a double S curve in p H-gradient.The first activity peak appeared at p H8.0 and the protease is stable in the environment of ph6.0-11.0.In addition,the protease was more active in the alkaline environment.Na⁺,K⁺,Ca²⁺,Mg²⁺and Fe²⁺hardly affected the activity of Asp60-D1-P1.While infinitesimal Fe³⁺can significantly inhibit the activity of this protease.4.Asp60-D1-G1 was identified as a kind of fibrinolytic proteinase in P.aibuhitensis which belongs to serine protease family.LC-MS/MS-MS detection and uniprot?perinereis?210?20190813.fasta database matching results showed that Asp60-D1-P1 had a 95%similarity of 44.5KDa and 130KDa polypeptide fragments isolated by Native PAGE.The strip-enzymatic hydrolysis samples named as Mr?44.5and Mr?130,respectively.Mr?44.5 and Mr?130 sample both matched to fragments of fibrinolytic enzyme.The matched polypeptide fragment are r.lstdacqgdsggplvvk.d and r.taravvsgwgtlr.s.,in which,the matching degree of the secondary map of r.lstdacqgdsggplvvk.d was as high as 95%,and it contained serine?active?sites.Combined with the results of in vitro experiments in this paper,it could be inferred that Asp60-D1-P1 as a fibrinolytic active protease in Perinereis aibuhitensis Grub belongs to serine protease family.In this paper,the fibrinolytic active protease of Perinereis aibuhitensis Grub was systematically studied.The purification methods of fibrinolytic active protease by using ammonium sulfate segmented precipitation and hierarchic gel chromatography was established.The excellent enzymatic properties and the structural characteristics of serine fibrinolytic active protease were identified.In addition,the paper used ultraviolet spectrophotometry and fiber plate method to qualitatively and quantitatively purify the fibrinolytic activity of protease,which is a scientific and reasonable way of fibrinolytic activity assay.