Purification and characterization of the fibrinolytic enzyme (eFE-D) from earthworm Eisenia foelide

The fibrinolytic enzyme eFE-D was isolated and purified from earthworm Eisenia foelide by gel-filtration on Sephacryle S-200, ion-exchange chromatography on DEAE-Sepharose Fast Flow and hydrophobic chromatography on Phenyle-Sepharose Fast Flow as detected by the fibrinolytic activity with a standard fibrin plate method. The most strong fibrinolytic component eFE-D not only hydrolyzed fibrin directly,but also activated the plasminogen to plasmin. Its apparent fibrinolytic value was equal to 2 800 UK IU per mg. Its molecular weight as estimated by SDS-PAGE and MS analysis was 29 kD and 24.849 kD respectively and its isoelectric point (pi) was 4.0. Fibrinolytic enzyme eFE-D was very thermostable with a single polypeptide chain. Studies with protease inhibitors indicated that eFE-D was a kind of serine protease. Its N-terminal amino acid sequence is M-I-G-G-T-N-A-S-P-G-E-F-P-W-Q-L-S-Q-Q-R, The result of amino acid composition analysis showed that the enzyme contained abundant amino acids of low molecular weight, but few aromatic and alkaline amino acids.