外文文献.docx

外文文献.docx

《外文文献.docx》由会员分享，可在线阅读，更多相关《外文文献.docx（17页珍藏版）》请在冰豆网上搜索。

外文文献

One-steppurification

of

metallothionein

extractedfromtwodifferentsources

RubensT. Hondaa,RozieteMendes Araújoa,BrunoBrasil Hortab,AdalbertoL. Vala,Marilene Demasia, 

a

InstitutoNacionaldePesquisasdaAmazônia,INPA,Manaus,AM,Brazil

b

InstitutodeBiociênciasdaUniversidadedeSãoPaulo,SãoPaulo,SP,Brazil

Received1October2004;Accepted14March2005.Availableonline25April2005.

Abstract

Wedescribeaone-step

purification

ofhepatic

metallothionein

fromtheAmazonfishColossomamacropomuminjectedwithcadmiumandfromthecopper-loaded

metallothionein

fromtheyeastSaccharomycescerevisiae,performedbyaffinitychromatographythroughmetal-chelatingcolumns.Yeast

metallothionein

waspurifiedfromCu2+-loadedresinandelutedbyacontinuousEDTAgradientwhereashepatic

metallothionein

extractedfromfisheswaspurifiedbyNi2+-loadedresinandelutedbyacontinuousimidazolgradient.PurifiedmetallothioneinswereevaluatedbySDS–PAGEandcharacterizedbyUVspectraoftheapo-andCd2+-loadedprotein.Thismethodallowedhighpurityandyieldaswellasrapidone-stepextractionofbothmetal-loadedandapoprotein.

Keywords:

Metallothionein

;Environmentalsciences;NiandCdbinding;Metal-chellatingaffinitychromatography

ArticleOutline

1.Introduction

2.Conditions

2.1.Reagents

2.2.Animalsandcadmiumtreatment

2.3.Yeastgrowthandmetallothioneinextraction

2.4.Purificationofmetallothionein

2.5.PreparationandcharacterizationofApoMT

2.6.Ni2+andCd2+quantificationbygraphitefurnaceatomicabsorption

2.7.Polyacrylamidegelelectrophoresis（PAGE）

2.8.Determinationofproteinconcentration

3.Results

4.Discussion

Acknowledgements

References

1.Introduction

Metallothioneins（MT）areasuperfamilyoflowmolecularweightcysteine-richproteinsfirstlydescribedinequinekidneybyMargoshesandVallee[1].Theyhaveawidespreadexpressionthroughoutalleukaryotesaswellasinsomeprokaryoticspecies.AllvertebratescontaintwoormoreMTisoforms[2].Theirfunctionisassociatedtothehighd10metalionsbindingcapacitythoughaprimaryrolehasnotbeenidentified.Thetwomostwidelyexpressedisoformsinvertebrates,MT-1andMT-2,arerapidlyinducedintheliverbyawiderangeofmetals,drugsandinflammatorymediators.ThethiolateclusterstructureoftheseproteinsisinvolvedinthemetabolicregulationviaZn2+donation,protectionagainstoxidativedamage,andsequestrationand/orredoxcontrol[3].AnimportantpropertyassignedforMTsreferstotheirroleintransitionmetaldetoxification（especiallyCd2+）.Itmaybenotedthatinhigherorganisms,MTsarethesoleproteinsinwhichcadmiumaccumulatesnaturally.OnthatregardingMTshavebeenproposedasbiomarkersforenvironmentalcontrolandoccupationaldiseases[4].

Itisnoteworthybyliteraturethatmethodsfor

metallothionein

purification

describedthusfargenerallyincludeseveralcombinedchromatographicsteps,suchas:

gelfiltration,ionexchangeandHPLC[5],[6]and[7].Thosemethodstaketimefor

purification

besidesthehighlossofproteinmassalongtheentireprocedurewhencomparedtomostproceduresbyaffinitychromatography.Indeed,theextractionand

purification

ofnativeproteinsusuallyinvolveseveralstepsunlesstheproteinpossessessomepeculiarstructuralcharacteristicorphysical–chemicalpropertythatallowsaspecialprocedurefor

purification

.Inthatregard,metallothioneinspresentacysteine-richprimarystructure.Aswellknown,cysteineispronetochelatetransitionmetals.Basedonthatproperty,inthepresentwork,wedescribeasimplemethodfor

purification

ofmetallothioneinsfromtwodifferentsourcesbyaffinitychromatographythroughmetal-chelatingcolumns.

Ourgoaltoconducttheexperimentsdescribedinthepresentreportwastodevelopasimplemethodtoisolate

metallothionein

fromtissuesoffishessinceourmajorinterestwillbethestudyupontheroleof

metallothionein

fromsomeAmazonfishspeciesasbiomarkersofmetal-pollutedwaters,asalreadydescribedforotherspeciesfromdifferentenvironments[12],[13],[14]and[15].

2.Conditions

2.1.Reagents

BovineSerumAlbumin,BradfordReagentandcadmium,nickelorcoppersaltswerepurchasedfromSigma（SaintLouis,LO,USA）.The1-mLHiTrap™ChelatingHPcolumnandproteinmolecularmassmarkers,Rainbow™andBroadRangePMWS™,werepurchasedfromAmershamPharmaciaBiotech（Uppsala,Sweden）.AllotherreagentsutilizedwereofanalyticalgradeandthewaterwaspurifiedwiththeMilli-QsystemfromMillipore.

2.2.Animalsandcadmiumtreatment

Specimensoftambaqui（Colossomamacropomum）werecollectedfromfloodplainsinMarchantariaisland（3°15′S59°58′W）.Theanimalsweremaintainedin2000-Ltanksat25°Cwithaeratedfreshwaterandfedadlibitumforoneweek.Afterthisperiod,theanimals（n=6）wereweighted（319.38±38.98g）,measured（21.13±0.62）andinjectedintra-peritoneallywithCdNO3·4H2Odissolvedin50mMsodiumphosphatebuffer,pH7.4,toafinalconcentrationof3μgCdNO3·4H2O/gfish.Sixanimalswereutilizedforthecontrolgroupinjectedwithsimilarvolumesofbuffersolutionwithoutcadmium.Theanimalsweremaintainedindividuallyintoaerated20-Lfishbowlsfor48h.Afterwards,theliverwasextractedandhomogenized（35%w/v）in50mMTris–HCl,pH7.4;containing0.1mMPMSF,0.5mMDTTand150mMNaClinaTeflonhomogenizer.Thehomogenatewascentrifugedat15,000×gfor90minat4°C.ThesupernatantwasimmediatelyappliedtoaHiTrap™ChelatingHPcolumnpreviouslyloadedwithNiSO4·6H2O,asdescribedbelow.

2.3.Yeastgrowthand

metallothionein

extraction

WeutilizedastrainofSaccharomycescerevisiaebelongingtotheExClone™collection（yeastORFexpressionclones;ResGen,InvitrogenCorp.,UK）,transformedwithaplasmidcarryinganopenreadingframefortheyeast

metallothionein

CUP1A.Cellswereculturedinsyntheticminimummedium（2%glucoseand2%peptone,supplementedwithaminoacids,exceptleucineanduracylbase）at30°Cwithreciprocalshakingtoanopticaldensityofthecultureat600nmof0.8.Atthatpoint,0.5mMCuSO4·5H2Owasaddedtotheculturemediumandcellswereincubatedforfurther2hinthesameconditionsdescribedabove.Afterwards,cellswereharvestedandpelletedbycentrifugation.Thepelletwaswashedtwicewithextractionbuffer（50mMTris–HCl,pH7.4;containing0.1mMPMSF,0.5mMDTTand150mMNaCl）.YeastcellsweredisruptedaccordingtotheprotocoldescribedbyVermaetal.[8].Aftercentrifugedat15,000×gfor90min,thecellextractwasappliedtoaHiTrap™ChelatingHPcolumnpreviouslyloadedwithCuSO4·5H2O,asdescribedbelow.

2.4.Purificationofmetallothionein

Affinitychromatographywasperformedusinga1-mLHiTrap™ChelatingHPcolumnattachedtoaP1peristalticpump（AmershamPharmaciaBiotech）.SupernatantsfromyeastcellsandfromtheliveroffishesobtainedasdescribedabovewereappliedtothecolumnpreviouslyloadedwithCuSO4·5H2OorNiSO4·6H2O,respectively.Aftermetalloading,thecolumnwasequilibratedwith10volumes50mMTris–HCl,pH7.4addedof0.15MNaCl,herecalledequilibratingbuffer.Afterapplyingthesupernatants,10volumesofequilibratingbufferwerepassedthroughthecolumn.Theelutionwasperformednext,eitherby5mLofcontinuousgradientofEDTA（0–0.1mM）bufferedin50mMTris/HCl,pH7.4（MTextractedfromyeasthomogenates）orby10mLofcontinuousgradientofimidazol（0–500mM）fortheextractionofMTfromliverhomogenatesoffish.Imidazolwasdissolvedin50mMTris–HCl,pH7.4addedof0.15MNaCl.Inbothextractions（yeastorliverhomogenates）,fractions（1mL）werecollectedataflowrateof1mL/minthroughtheentireprocedure.The

purification

wasperformedatroomtemperature.Oncefinishedtheelutionprocedure,thecolumnwaswashedwith10volumesof1mMEDTA（thisstepiscalledwashing-step）,followedby20volumesofMilli-Qfreshwatertorecoverthecolumn,accordingtotheprotocolenclosedtothemanufacturer'stechnicaldatasheet.Aliquots（20μL）ofeachfractionobtainedfromtheelutionstepwereappliedto15%SDS–PAGE.

Metallothionein

-positivefractionswerededucedonthegelbythemolecularmass,spectralcharacterizationandmetaldetermination,asdescribedbelowandshowninthefigures.

Metallothionein

-positiveandpurestfractionswerecombinedtopreparetheapoprotein（apoMT）.

2.5.PreparationandcharacterizationofApoMT

ApoMTwasobtainedbytheincubationof

metallothionein

-positivefractions,obtainedasdescribedaboveanddeducedfromSDS–PAGE,with50mMDTTand10mMHClsolution,accordingtoDallingeretal.[9].ThepreparationwasappliedtoaG-25（Sigma）column（20cm×1cm）pre-equilibratedwith10mMHCland10mMNaCl.One-millilierfractionswerecollectedstartingatsampleapplication.TheentireproceduretoobtainapoMTwasperformedunderheliumatmosphereat4°C.Fractionsobtainedwereappliedto15%SDS–PAGEunderreducingconditions.MT-spectrawereobtainedfromapoMTsoleorbyaddingCdNO3·4H2Oatincreasingconcentrations（from8μMupto80μM）totheapoMTaliquotsobtainedfromfishlivers[10].

2.6.Ni2+andCd2+quantificationbygraphitefurnaceatomicabsorption

NickelandcadmiumquantificationwasperformedbyatomicabsorptionspectrometryinaPerkin-ElmerAnalyst800equipmentaccordingtothemanufacturer'stechnicalprotocolunderAratmosphere.Samplesweredigestedby10%HNO3for12hat70°C.Twentymicrolitersaliquotsweretakenfromeachsampleandanalyzedat232nm（Ni2+）or228.8nm（Cd2+）.StandardcurvesofNiSO4·6H2OandCdNO3·4H2Owere