表没食子儿茶素EGC对溶菌酶淀粉样纤维化的抑制作用李改涛

2626520145ChemicalＲesearchandApplicationVol．26No．5May20141004-1656201405-0696-05EGC*710062。EGC。EGC。DTTEGCEGC。。O629.73AInhibitoryeffectofepigallocatechinonamyloidfibrillationoflysozymeLIGai-taoLIYuan-yuanZENGCheng-ming*CollegeofChemistryandChemicalEngineeringShaanxiNormalUniversityXi'an710062ChinaAbstractNaturalpolyphenolshadbeenrecognizedasmedicinalcandidatesfortreatingamyloiddiseases.InthepresentstudytheinhibitoryeffectofepigallocatechinEGConamyloidformationwasinvestigatedinvitrobyusinglysozymeasamodelprotein.TheresultsdemonstratedthatEGCinhibitedefficientlylysozymefibrilformationandtransformedthematurefibrilsintonon-amyloidas-semblies.ThiolcompounddithiothreitolDTTabolishedpartlythefibril-destabilizingroleofEGCindicatingthatEGCprobablya-lertedamyloidfibrillarstructurethroughcovalentlybindingtothepeptidechains.Itwassuggestedthatformationofquinoneinterme-diatesandsubsequentlymodificationoffreethiolgroupsinpeptidechainswerethemaineventsintheinhibitionoflysozymeamy-loidfibrillationbypolyphenols.Keywordsepigallocatechinlysozymeamyloidfibrillationthiolgroupcovalentlybinding12。Alzheimer’sII。β-ThTANS。34。2014-01-042014-03-161957-。[e-mail]5EGC。。567。8。EpigallocatechinEGC1EGCβ-9。10。EGC。1EGCFig.1MolecularstructureofepigallocatechinEGC11.1EGCthioflavinTThT14-14-dithiothreitolDTTMonobromobimanemBBＲSigma-Aldrich。LS-55Perkin-ElmerJEM-1400JEOL。1.2100mg10mLHCl10mmol·L－1pH2.01%w/v。65℃124。ThT。4℃。1.3ThT10mmol·L－1pH7.0ThT1mmol·L－14℃。10μL10mmol·L－1Tris-HClpH8.030μLThTThT10μmol·L－110s。440nm484nm。1.410mg·mL－10.5mg·mL－115μL10min15μL2%10min。80KV。1.5mBBＲ10μL10μLmBBr5mmol·L－1。1%SDSTris-HCl20mmol·L－1pH8.0390nm470nm。1.6EGCEGCDTT20mmol·L－1Tris-HClpH8.01.0mg·mL－1DTT1.0mmol·L－137℃1-5hThT。22.1EGCThTβ440nm482nm。12。ThT2ThT3。EGC796262EGC150-200μmol·L－112ThT。EGCEGC。12ThTEGCEGCIC5068μmol·L－1。2EGCEGC■0μmol·L－1△25μmol·L－1□75μmol·L－1▲150μmol·L－1○200μmol·L－1Fig.2Inhibitoryeffectofepigallocatechinonamyloidformationoflysozyme2.26-35nm170-4500nm3A。150μmol·L－1EGC3BEGC。3AB+150μmol·L－1EGC12Fig.3TEMimagesoflysozymeamyloidfibrils2.3EGC12914.3KDa4α。＞5mg·mL－1、pH、。5-12KDa4。mBBＲ。4。5。150μmol·L－1EGCEGC。EGC。EGC。4□■+150μmol·L－1EGCFig.4Fluorescentanalysisoffreethiolsproducedinthepresenceoflysozymefibrillaion2.4EGCEGC5hThT5AEGCβ-ThT。EGC8965EGC。EGC＞12h。DTTThT5B。150μmol·L－1EGC1.0mg·mL－137℃5hThT75%1.0mmol·L－1DTTThT32%EGC。DTT。EGCThT。DTTEGC5BEGC。5EGCDTTAEGCThT。□0μmol·L－1■75μmol·L－1▲150μmol·L－1BDTTEGC+ThT◆DTT+▲EGC+△DTT+EGC+DTT1.0mmol·L－1EGC150μmol·L－1。Fig.5effectofEGCandDTTonmatureamyloidlysozymefibrils-、、。EGC。EGC。、π－π56。11。EGCThTDTTEGC。EGCDTT12。EGC。EGC。EGCEGCDTT13。3EGC。。。EGC。EGCDTTEGC99626EGC。。EGC。1PinneyJHHawkinsPN．AmyloidosisJ．AnnClinBio-chem2012493229-241．2StefaniM．Biochemicalandbiophysicalfeaturesofbothol-igomer/fibrilandcellmembraneinamyloidcytotoxicityJ．FEBSJ．2010277224602-4613．3FndrichMMeinhardtJGrigorieffN．Structuralpolymor-phismofalzheimerabetaandotheramyloidfibrilsJ．Prion20093289-93．4HuangBHeJＲenJetal．Cellularmembranedisruptionbyamyloidfibrilsinvolvedintermoleculardisulfidecross-linkingJ．Biochemistry200948255794-5800．5PoratYAbramowitzAGazitE．Inhibitionofamyloidfi-brilformationbypolyphenolsstructuralsimilarityandaro-maticinteractionsasacommoninhibitionmechanismJ．ChemBiolDrugDes200667127-37．6AkaishiTMorimotoTShibaoMetal．Structuralrequire-mentsfortheflavonoidfisetinininhibitingfibrilformationofamyloidβproteinJ．NeurosciLett20084443280-285．7HeJXingYFHuangBetal．Teacatechinsinducedtheconversionofpreformedlysozymeamyloidfibrilstoamor-phousaggregatesJ．JAgrFoodChem2009572311391-11396．8ShovalHWeinerLGazitEetal．Polyphenol-induceddissociationofvariousamyloidfibrilsresultsinamethio-nine-independentformationofＲOSJ．BiochimBiophysActa20081784111570-1577．9FerreiraNSaraivaMJAlmeidaMＲ．Naturalpolyphenolsinhibitdifferentstepsoftheprocessoftransthyretinamy-loidfibrilformationJ．FEBSLett2011585152424-2430．10SwaminathanＲＲaviVKKumarSetal．LysozymeamodelproteinforamyloidresearchJ．AdvProteinChemStructBiol201184163-111．11FengSSongXHZengCM．Inhibitionofamyloidfibrilla-tionoflysozymebyphenoliccompoundsinvolvesquino-proteinformation．J．FEBSLett2012586223951-3955．12ChenＲWangJBZhangXQetal．Greenteapolyphenolepigallocatechin-3-gallateEGCGinducedintermolecularcross-linkingofmembraneproteinsJ．ArchBiochemBiophys20115072343-349．13．J．2013255665-669．007