Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus 英文参考文献.docVIP

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Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus 英文参考文献.doc

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Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus 英文参考文献

Phanetal.AMBExpress2011,1:26 /content/1/1/26 ORIGINAL OpenAccess Purificationandcharacterizationofnovel fibrinolyticproteasesaspotentialantithrombotic agentsfromearthwormPerionyxexcavatus TramThiBichPhan1,TienDuyTa2*,DungThiXuanNguyen3,LambertusAMVanDenBroek4and GiangThiHuongDuong3 Abstract Sixproteasefractions,namelyFI,FII,FIII-1,FIII-2,FIII-3andFIV,wereisolatedfromPerionyxexcavatusearthworm biomassbyacetoneprecipitation,followedbyserialchromatographyusinganionexchange,hydrophobicinteraction andsizeexclusionchromatography.Allfractionsexhibitedstronghydrolyticactivitytowardscasein.Theactivityofsix fractionstowardsfibrin,determinedbyfibrinplateassay,rangedfrom44to831plasminunit.mg-1andrankedasFIII- 3FIII-2FIFIII-1FIVFII.CaseindegradationwasoptimalatpH7and11,andat45-60°C.Allfractionswere considerablystableathightemperatureandwidepHrange.Theywerecompletelyinhibitedby phenylmethylsulfonylfluoride(PMSF).Themolecularweights(MW)andisoelectricpoints(pI)determinedby 2D-electrophoresiswere27.5-34.5kDa,and4.3-5.2,respectively.Tandemmassspectrometry(MS)analysiswasused todeducetheaminoacidsequencesofsomepeptidesfromFIII-1andFIII-2.Thesequencesshared16.9%and13.2% similarity,respectively,withthefibrinolyticenzymesfromtworelatedearthwormspecies,Lumbricusrubellusand Eiseniafetida.TheP.excavatusproteaseswereclassifiedasserineproteases.Theycouldperformrapidhydrolysison bothcoagulatedfibrousfibrinandsolublefibrinogenmonomerswithoutthepresenceofactivatorssuchastPAor urokinase. Keywords:chromatography,fibrinolysis,Perionyxexcavatus,PMSF,serineprotease,tandemMSanalysis Introduction hemostasis. Thefibrinogen monomerisa304kDagly- Cardiovascular diseaseshavebecomeoneofthebiggest coprotein containing twosets ofthree different chains: concernsallovertheworld(Grundyetal.1999).Among Aa,Bbandg(Mosesson2005).Theconversionoffibri- these, thrombosis is the most widespread within the nogen into fibrin requires the presence of thrombin, a elderly population. The disease results from sever