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Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus

Tram Thi Bich Phan1, Tien Duy Ta2*, Dung Thi Xuan Nguyen3, Lambertus AM Van Den Broek4 and Giang Thi Huong Duong3

Author Affiliations

1 College of Agriculture and Applied Biology, Can Tho University, Can Tho, Vietnam

2 Faculty of Food Processing Technology, Can Tho University of Technology, Can Tho, Vietnam

3 Biotechnology Research and Development Institute, Can Tho University, Can Tho, Vietnam

4 Wageningen UR Food & Bio-based Research, 6708 WG, Wageningen, The Netherlands

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AMB Express 2011, 1:26  doi:10.1186/2191-0855-1-26

Published: 30 September 2011

Abstract

Six protease fractions, namely FI, FII, FIII-1, FIII-2, FIII-3 and FIV, were isolated from Perionyx excavatus earthworm biomass by acetone precipitation, followed by serial chromatography using anion exchange, hydrophobic interaction and size exclusion chromatography. All fractions exhibited strong hydrolytic activity towards casein. The activity of six fractions towards fibrin, determined by fibrin plate assay, ranged from 44 to 831 plasmin unit.mg-1 and ranked as FIII-3 > FIII-2 > FI > FIII-1 > FIV > FII. Casein degradation was optimal at pH 7 and 11, and at 45-60°C. All fractions were considerably stable at high temperature and wide pH range. They were completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The molecular weights (MW) and isoelectric points (pI) determined by 2D-electrophoresis were 27.5-34.5 kDa, and 4.3-5.2, respectively. Tandem mass spectrometry (MS) analysis was used to deduce the amino acid sequences of some peptides from FIII-1 and FIII-2. The sequences shared 16.9% and 13.2% similarity, respectively, with the fibrinolytic enzymes from two related earthworm species, Lumbricus rubellus and Eisenia fetida. The P. excavatus proteases were classified as serine proteases. They could perform rapid hydrolysis on both coagulated fibrous fibrin and soluble fibrinogen monomers without the presence of activators such as tPA or urokinase.

Keywords:
chromatography; fibrinolysis; Perionyx excavatus; PMSF; serine protease; tandem MS analysis