Study On Activity And Protein Ingredients Of Lumbrokinase

Lumbrokinase(LK),also known as a kind of earthworm fibrinolytic enzyme,is a general term of proteases with fibrinolytic activity from earthworm.Since 1983,Japanese scientists Mihara etc.isolated six proteins with fibrinolytic activity from the powders of earthworm.Many scientists studied lumbrokinase systematically.The literatures show that main components of lumbrokinase are a group of serine-proteases from earthworm,including fibrinolytic enzyme,plasminogen activator,and tissue plasminogen activator(t-PA)etc.But the Lumbrokinase was approved as drugs for patients who have ischemic cerebrovascular disease,increased fibrinogen disease and platelet aggregation disease in China in 1992,but lumbrokinase hasn’t been approved as drugs in foreign countries till now.5 enterprises in China produce lumbrokinase APIs(Active Pharmaceutical Ingredients)and enteric coated preparation.All pharmaceutical lumbrokinase are from Eisenia fetida cultured artificially and the production process is simple and rough.Lumbrokinase as a kind of particular biochemical medicine in China,is widely used and have a good effect.But its effective ingredients is unclear and quantity control level is low till now..It is necessary to use modern analytical techniques to study deeply the products.We apply mass spectrometry,fibrinolytic zymography,affinity chromatography,anion exchange chromatography,fibrin plate to the study of the composition,chemical structure,biological activity and the quality control method of lumbrokinase.This paper focuses on three aspects below.Study on the components of lumbrokinase.We apply the enzymolysis of chymotrypsin enzyme,separation and determination of LC-MS,protein lynx global server 3.0 analysis software to the study of the components of lumbrokinase.The results show that there are 15 kinds of protein from five enterprises,including 4 kinds of fibrinolytic enzymes and 10 kinds of kinases.Fibrinolytic enzymes were detected in each sample of 5 enterprises,and the consistency of production was good.There were 5 kind of protein associated with the fibrinolytic activity from standard substance of lumbrokinase,including two kinds of fibrinolytic enzymes and 3 kinds of plasminogen kinases.The study shows that the lumbrokinase APIs include the protein with fibrinolytic activity.Study on the activity of lumbrokinase.We focus on the total activity quantitative measurement and the identification of the active components.We study the activity of lumbrokinase that hydrolyze fibrin directly and indirectly.The results show that lumbrokinase not only degrades fibrin directly,but also activates plasminogen to degrade fibrin.Based on the limitations of the biological activity of lumbrokinase and traditional determination methods of the potency,the biological statistics method---parallel line analysis method can be used to determinate the potency of lumbrokinase.Results show that lumbrokinase APIs and standard conform to requirement of the statistics method,which is more applicable to the determination of the biological activity of lumbrokinase.Based on the characteristics of lumbrokinase degrading fibrin directly,fibrinolytic zymography is established for determination of lumbrokinase active protein components.Results show that fibrinolytic zymography can clearly show the presence of lumbrokinase active protein,and can be used as a kind of method of evaluating the stability of production process.Separation and purification of Lumbrokinase activity protein.Based on the results of mass spectrometry,we try to find the active protein of lumbrokinase.We obtain a group of active protein mixture purified by DEAE anion exchange chromatography and affinity chromatography.Then identified by mass spectrometry,three kinds of active protein ingredients are fibrinolytic protease 1,lumbrokinase F238 and lumbrokinase-3、lumbrokinase-5,which are consistent with the initial result.