Purification and Characterization of Six Fibrinolytic Serine-Proteases from Earthworm Lumbricus rubellus

Abstract

The six lumbrokinase fractions (F1 to F6) with fibrinolytic activities were purified from earthworm Lumbricus rubellus lysates using the procedures of autolysis, ammonium sulfate fractionation, and column chromatography. The proteolytic activities on the casein substrate of the six iso-enzymes ranged from 11.3 to 167.5 unit/mg with the rank activity orders of F2 > F1 > F5 > F6 > F3 > F4. The fibrinolytic activities of the six fractions on the fibrin plates ranged from 20.8 to 207.2 unit/mg with rank orders of F6 > F2 > F5 > F3 > F1 > F4. The molecular weights of each iso-enzyme, as estimated by SDS-PAGE, were 24.6 (F1), 26.8 (F2), 28.2 (F3), 25.4 (F4), 33.1 (F5), and 33.0 kDa (F6), respectively. The plasminogen was activated into plasmin by the enzymes. The optimal temperature of the six iso-enzymes was 50 degrees C, and the optimal pH ranged from pH 4-12. The four iso-enzymes (F1-F4) were completely inhibited by PMSF. The two enzymes (F5 and F6) were completely inhibited by aprotinin, TLCK, TPCK, SBTI, LBTI, and leupeptin. The N-terminal amino acid (aa) sequences of the first 20 to 22 residues of each fraction had high homology. All six iso-enzymes had identical aa residues 2-3 and 13-15. The N-terminal 21-22 aa sequences of the F2, F3, and F4 iso-enzymes were almost the same. The N-terminal aa sequences of F5 and F6 were identical.

Molecular Cloning, Sequencing, and Expression of a Fibrinolytic Serine-protease Gene from the Earthworm Lumbricus rubellus

“…The AFA primer was the AmpliFINDER anchor primer, and the AFAP primer was the counterpart of the AFA primer. The GSP primer, which was used for DNA amplification, was designed from the Nterminal amino acid sequences of F6 protease (Cho et al, 2004). The primer 5'-RGSP, which was used for PCR amplification, was based on the 450 bp upstream sequence of the deduced N-terminal amino acid sequence of the mature region of the F6 protease gene (Fig.…”

“…Trypsin solution (500 units/ml) was then added to the neutralized mixture, and incubated in a shaking water bath at 37 o C for 60 min. Then 10 µl of the solubilized enzyme solution was plated on the fibrin plate (Cho et al, 2004), incubated for 15 h at 37 o C, and the hydrolyzed clear zone was measured (Cho et al, 2004).…”

“…At this point, 1.0 mM of IPTG (isopropyl-α-thio-galactopyranoside) (Stratagene, Heidelbergh, Germany) was added, and the cells were re-incubated for 4 hrs at 37 o C, and centrifuged at 12,000 × g for 30 min at 4 o C. The insoluble protein pellets and supernatants were then separated, and the supernatants were concentrated with 1.0% of trichloroacetic acid and freeze-dried. The insoluble protein pellets were sonicated and centrifuged at 12,000 × g for 30 min at 4 o C. The insoluble protein pellets (LK inclusion bodies) were washed twice with 1% Triton X-100, resuspended in 150 µl of 1.0 mM Tris · HCl buffer (pH 7.6), and used for SDS-PAGE (Laemmli, 1970) and to determine fibrinolytic activity (Kumada et al, 1980;Cho et al, 2004).…”

“…Fibrinolytic proteases in the earthworm, Lumbricus rubellus have been purified and characterized by several investigators (Park et al, 1989;Mihara et al, 1991Mihara et al, , 1993Nakajima et al, 1993;Jeon et al, 1995;Cho et al, 2004) and found to hydrolyze plasmogen-rich fibrin and plasmogen-free fibrin. Earthworm protease appeared a mixture of six iso-enzyme proteins of molecular weight 25 to 32 kDa.…”

Survey Process of Autolysis from Earthworm Perionyx Excavates Obtain Protein Solution

“…1,2 The bioactive substances from earthworms so far have not been studied; the scientific evidence of the medicinal use of earthworm species has not been practically proven. 3,4 In recent years, growing earthworms has developed in the neighborhood of Ho Chi Minh City and some provinces of Mekong Delta.…”