Study On The Earthworm Fibrinolytic Enzyme Based On Proteomics Methods

Earthworm fibrinolytic enzyme (EFE) is a group of serine-protease extracted from earthworm. EFE has been widely used in clinical treatment of various thrombotic diseases due to its good effects in reducing blood viscosity and platelet aggregation, and improving microcirculation with less adverse effect. Oral capsule of EFE for the prevention and treatment of thrombosis has been produced by Institute of Biophysics, Chinese Academy of Sciences with Eisenia as the raw material. In fact, this capsule (lumbrokinase) contains a variety of active components. A well established quanlity control protocol is mandatory for relialble control of the production process and the product quanlity assurance.Literature survey showes that the physical and chemical properties of the enzymes are very close to each other, which means that further separation and purification of the EFE components are relatively chellenging. In this work, different separation methods were tried to isolate the components from the total protein of the raw extract of earthworm fibrinolytic enzyme. SDS-PAGE and strong anionic exchange chromatograph were found very effective for this task. Under optimum gel electrophoresis separation condition, up to20protein bands were recognized. The bands were identified by MALDI-TOF-MS and2D-LC-MS/MS, respectively. And under optimum chromatographic conditions,35peaks were successfully separated and were divided into26fractions. The molecular weight data of the proteins from each fraction were accurately identified by MALDI-TOF-MS and Q-TOF-MS, respectively.Based on the "shotgun" strategies, the enzymes from EFE were identified by protein database and EST (express sequence tags) database:20different proteins were identified by the protein database (Lumbricidae sequence); and57different mRNA sequences were identified by the form of EST database (Lumbricidae sequence).Proteomics methods were applied for the first time in the EFE analysis and identification, and a standard protocol was established for the qualitiy assurance of EFE production. This is very significant for the development and production of new protein drugs.