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Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Paper ID Volume ID Publish Year Pages File Format Full-Text
34752 45042 2013 9 PDF Available
Title
Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata
Abstract

•Three novel serine proteases were purified from a marine annelid worm species.•Purified enzymes were characterized biochemically in terms of enzymatic kinetics.•Purified enzymes cleaved efficiently fibrin polymer as well as cross-linked fibrin.•The three enzymes actively dissolved the fibrin clot even in blood plasma milieu.

Three distinct alkaline serine proteases (named CTSP-1, -2, and -3) were purified from the polychaete Cirriformia tentaculata and characterized in terms of their enzymatic properties and kinetics. The estimated molecular masses of CTSP-1, -2, and -3 enzymes were found to be 28.8, 30.9, and 28.4 kDa, respectively. The enzymes were active at the temperature range of 50–60 °C under pH 8.5–9.0 and completely inactivated by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, but not by 1,10-phenanthroline and bestatin, suggesting that they are all typical serine proteases and not metalloproteases or cysteine proteases. CTSP-1 and -2 cleaved arginine, whereas CTSP-3 digested tyrosine residue at the carboxyl sides in their peptide substrates. A typical hepta-sequence (I-X-X-G-X-X-A) conserved in serine proteases from annelid species was found in N-termini of all CTSPs. CTSP-2 was the most active enzyme among the proteases purified as shown by kinetic values. The enzymes cleaved all chains of fibrinogen within 20 min and also hydrolyzed actively fibrin polymer as well as cross-linked fibrin. In addition, the enzymes could actively digest the fibrin clot in blood plasma milieu. Taken together, the results obtained demonstrate that CTSP enzymes have a potential of becoming therapeutic agents for thrombus dissolution.

Keywords
1,10-PT, 1,10-phenanthroline; BSA, bovine serum albumin; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; DFP, diisopropyl fluorophosphate; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline; PMSF, phenylmethanesul
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Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 48, Issues 5–6, May–June 2013, Pages 979–987
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
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Any Questions? feel free to contact us