Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida

Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis–Menten constant (K_m) for Chromozym ELA (245 μM) is much higher than those for the thrombin (90 μM) and trypsin (60 μM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1′-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55–60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys₇₇–Arg₇₈, Arg₃₄₂–Met₃₄₃, Ala₄₄₄–Ala₄₄₅ and Arg₅₅₇–Ile₅₅₈. The site Arg₅₅₇–Ile₅₅₈ is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala₄₄₄–Ala₄₄₅ released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the A chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val₂₁–Glu₂₂ site.