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




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AMB Express

, 1:26

First Online: 30 September 2011Received: 13 September 2011Accepted: 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 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.

Keywordschromatography fibrinolysis Perionyx excavatus PMSF serine protease tandem MS analysis Electronic supplementary materialThe online version of this article doi:10.1186-2191-0855-1-26 contains supplementary material, which is available to authorized users.

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Author: Tram Thi Bich Phan - Tien Duy Ta - Dung Thi Xuan Nguyen - Lambertus AM Van Den Broek - Giang Thi Huong Duong

Source: https://link.springer.com/







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