不同价态金属离子对BSA结构的影响

ThisarticleappearedinajournalpublishedbyElsevier.Theattachedcopyisfurnishedtotheauthorforinternalnon-commercialresearchandeducationuse,includingforinstructionattheauthorsinstitution

andsharingwithcolleagues.Otheruses,includingreproductionanddistribution,orsellingorlicensingcopies,orpostingtopersonal,institutionalorthirdparty

websitesareprohibited.Inmostcasesauthorsarepermittedtoposttheirversionofthearticle(e.g.inWordorTexform)totheirpersonalwebsiteorinstitutionalrepository.AuthorsrequiringfurtherinformationregardingElsevier’sarchivingandmanuscriptpoliciesare

encouragedtovisit:

/copyright

SpectrochimicaActaPartA78 (2011) 523–527

ContentslistsavailableatScienceDirect

SpectrochimicaActaPartA:Molecularand

Biomolecular

Spectroscopy

journalhomepage:/locate/sa

a

Toxiceffectsofdifferentchargedmetalionsonthetarget—Bovineserumalbumin

HaoZhang,RutaoLiu ,ZhenxingChi,CanzhuGao

ShandongKeyLaboratoryofWaterPollutionControlandResourceReuse,SchoolofEnvironmentalScienceandEngineering,ShandongUniversity,Jinan250100,PRChinaAmericaCRCforEnvironment&Health,ShandongProvince,27#ShandaSouthRoad,Jinan250100,PRChina

articleinfoabstract

Inthiswork,thetoxicin uenceofmetallicions(Na+,Cu2+,Al3+)ontheserumalbuminwerestudiedby uorescence,resonancelightscattering(RLS),synchronous uorescence,UV–visabsorptionandcirculardichroism(CD)spectroscopy.Theexperimentalresultsindicatedthationelectricchargeisnotthemainfactoraffectingthestructureofbovineserumalbumin(BSA).Na+madethestructureofBSAtighterandhydrophobicityenhanced,whichimproved uorescenceintensity,whileCu2+couldreactwithsomefunctionalgroupsofBSA,makingthestructureofBSAlooser,sothattheinternalhydrophobicgroupssuchastryptophan(Trp)andotheraromaticresiduesweregraduallyexposed.Whenweobservedthemwith uorescencespectra,wefound uorescencequenchingwithincreasingCu2+dose.Al3+isshownaslittlesigni cantin uenceontheBSA,butBSAwasfoundtoaggregatewiththedoseofAl3+bymeansofRLSbecauseofthehydrolysisandionstrengtheffectofAl3+.Theresultsalsoprovednormalsalinecouldkeepliveshealthyandgood-workingasabiologicalhumour,however,heavymetalsmadeharmfuleffectstothebodywhentheyexceededtheminimaleffectlevel(MEL),suchasCu2+choseninourwork.

© 2010 Elsevier B.V. All rights reserved.

Articlehistory:

Received2September2010

Receivedinrevisedform13October2010Accepted15November2010Keywords:Metalions

Bovineserumalbumin(BSA)SpectratechniquesToxicinteraction

1.Introduction

Asisknowntoall,Proteindenaturationreferstothedestructionofthemoleculestructurebyseveralenvironmentalfactorsinclud-ingphysicalandchemicalones,suchastemperature,dehydration,ultravioletradiationandallkindsoftoxicchemicals[1,2].Gener-allyspeaking,proteindenaturationdoesnotcausethedestructionoftheprimarystructurebutthesecondaryone[3,4].Manydis-easessuchasdiabetes,cardiovasculardisease,cataractandmadcowdiseasearepositivelycorrelatedwiththedenaturationofpro-teins[5–8].Wecanalsomaketheproteinfromdenaturationtorenaturation,buttheconditionisveryharsh,weneedputuptherighttime,particulartemperature,pH,andionstrength[9,10]ifwewantthissituationhappen.

SerumAlbumin,themostabundantproteinconstituentinbloodplasma,playsavitalroleinthedispositionandtransportationofvariousmoleculesandcanreactwithmanydifferentligandsinvivoandinvitro.Weselectbovineserumalbumin(BSA)asthetargettoevaluatethetoxiceffectofmetaliononhealthbecauseofitssimilarstructuretohumanserumalbumin(HSA)[11,12]anditslowprice.

Thetoxiceffectsofmetalionstoproteinhavebeenstudiedbytoxicologistssince1990s[13,14].Moreandmoreresearchersstart

tostudytheseeffectsinmolecularlevelatpresent,especiallyana-lyzebindingsitesandparametersbetweenproteinandmetalions.ChuqiaoTuetal.[15]investigatedtheinteractionbetweenCd2+andHSAbymeansofbalanceddialysis,andhefoundthecom-plexcompoundconformationsimilartoatetrahedronafterbindingsitesbetweenCd2+andHSAwereanalyzed.Sadle[16]propoundedapatternbetweenserumalbuminandCd2+,Zn2+withsomeionscoexistinginthesolutionsbynuclearmagneticresonance.Cam-marotetal.[17]learnedtheinhibitionofmatrixmetalloproteinase(mmps)tobodytoxicitybynickelandchromium.Butnoschol-arshavestudiedthetoxicitiesbetweenionsandproteinfromthestandpointofionelectriccharge.Soourgroupsetupthisexperi-menttoinvestigatethisissuethoroughly.WeselectedthreeionswithdifferentelectricchargesNa+,Cu2+,Al3+,andtookBSAasthetargetmoleculetoanalyzethesystemswithspectroscopictech-niques.

2.Materialsandmethods2.1.Apparatus

AHitachiF-4600 uorescencespectrophotometerwasusedtomeasuretheintensityof uorescence,synchronous uores-cenceandRLS.TheUV–visabsorptionspectrumwasobtainedbyaShimadzuUV–2450spectrophotometer.AJasco-810circulardichroismspectrometerwasusedtomeasurethechangesofthesecondarystructureoftheproteinandthepHs-3CpHmeter(Peng-shun,Shanghai,People’sRepublicofChina)wasusedtomeasurethepHinalltheexperiments.

Correspondingauthor.SchoolofEnvironmentalScienceandEngineering,Shan-dongUniversity,Jinan250100PRChina.Tel.:+8653188364868;fax:+8653188364868.

E-mailaddress:rutaoliu@(R.Liu).1386-1425/$–seefrontmatter© 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.saa.2010.11.021

524

H.Zhangetal./SpectrochimicaActaPartA78 (2011) 523–527

2.2.Reagents

BSA(electrophoreticreagentgrade)purchasedfromBeijingChemicalReagentCorporationwaspreparedat10 5mol/Landpre-servedat0–4 C.

A0.2mol/LofNaH2PO4-Na2HPO4bufferwaspreparedfromNaH2PO4andNa2HPO4,whichadjustedtotheappropriatepH.

A1.0×10 3mol/LofNaClwaspreparedbydissolving0.0146gNaCl(TsingtaoSifangChemicalReagentFactory)in100mLofultrapurewater.

A1.0×10 3mol/LofCuCl2waspreparedbydissolving0.0427gCuCl2·2H2O(TianjinKermelChemicalReagentResearchInstitute)in100mLofultrapurewater.

A1.0×10 3mol/LofAlCl3waspreparedbydissolving0.0602gAlCl3·6H2O(TianjinDamaoChemicalReagentFactory)in100mLofultrapurewater.3.Methods

All uorescencespectra,RLS,andsynchronous uorescencespectrawererecordedonF-4600 uorescencespectrophotometer(HitachiJapan)ina1-cmcell.

Firstly, uorescencespectraweremeasuredwiththetoxiceffectsofNa+,Cu2+,Al3+onBSAwithdifferentconcentrationgradientsofNa+,Cu2+,Al3+.Theexcitationwavelengthwas278nm.Thescanscopewasfrom290nmto500nm.Theexci-tationandemissionslitwidthsweresetat5nm.Scanspeedwas1200nm/min.PMT(PhotoMultiplierTube)voltagewas700V.

Secondly,RLSweremeasuredduringtheNa+–BSA,Cu2+–BSA,andAl3+–BSAsystems,aswellasNa+,Cu2+andAl3+alone.Theconditionswereasfollows ex= em, ex=200–600nm.PMTweredifferentamongNa+–BSA,Cu2+–BSA,andAl3+–BSAsystems,PMT(Na+–BSA,Al3+–BSA)=700V,whilePMT(Cu2+–BSA)=600VbecausethissystemwasbeyondthedetectionlimitwhenwetookthisexperimentwithPMT700V.

Synchronous uorescencespectraofBSAinthepresenceofNa+,Cu2+,Al3+weremeasured( ex=15nm, ex=250–320nmand ex=60nm, ex=250–320nm,respectively).Theexcitationandemissionslitswereboth5nminwidth.Scanspeedwas1200nm/min.PMTvoltagewas xedat700V.

TheUV–visabsorptionspectraweremeasuredonaUV–2450spectrophotometer(Shimadzu,Japan)equippedwith1-cmquartzcells.Theslitwassetat2.0nminwidth.Thewavelengthrangewas190–320nm.

CDwasmeasuredonaJ-810Spectropolarimeter(Jasco,Tokyo,Japan)ina1-cmcellatroomtemperature.Bandwidthwas1nm.Scanningspeedwas100nm/min.Thewavelengthrangewas190–260nm.

4.Resultsanddiscussion4.1.Fluorescenceemissionspectra

FluorescenceemissionspectraofBSAwithdifferentNa+,Cu2+,Al3+concentrationsatroomtemperaturewererecorded(Fig.1).Wecanseeastrongpeaksignalat336nm.ItcanbeseenfromFig.1thatNa+didnotquenchedthe uorescenceofBSAbutincreaseit,whichindicatedthatNa+madethestructureofBSAtighterandhydrophobicityenhanced,andprovedthatphysio-logicalsalinewasprotectivetowardsourbodies.ThisresultisconsistentwithChen’sresearch[18].InCu2+–BSAsystem,wefound uorescencequenchingwiththedoseofCu2+increased,whichindicatedCu2+reactedwithseveralresiduesofBSAthatcouldemit uorescence,suchastryptophan(Trp),tyrosine(Tyr)

y

tisnetni ecnecseroulFConcentration (10-6mol/L)

Fig.1.FluorescenceintensityofBSAwithdifferentconcentrationsofionsNa+,Cu2+,Al3+.Conditions:c(BSA)=1.0×10 6mol/L,c(Na+,Cu2+,Al

3+)/(1.0×10 6mol/L):0,1,5,10,20,40,60,100respectively;pH7.4.

10000900080007000

6000S

LR50004000300020001000

Concentration (10-6

mol/L)

Fig.2.Resonancelightscattering(RLS)peaksignalsofBSAwithdifferentconcen-trationsofionsNa+,Cu2+,Al3+.Conditions:c(BSA)=1.0×10 6mol/L,c(Na+,Cu2+,Al3+)/(1.0×10 6mol/L):0,1,10,20,40,50,60,100respectively;pH7.4.

andphenylalanine(Phe).TheresultofAl3+–BSAwassimilartoNa+–BSA.4.2.RLSspectra

Fig.2showedthecomparisonofRLSspectraaffectedby

Na+–BSA,

Cu2+–BSAandAl3+–BSA.Astrongpeakwasobservedat

272nm.WefoundthatRLSofNa+–BSAremainedstable,andthepeaksignalschangedlittle,whichindicatedtheparticleofNa+–BSAmaintainedstable.ThepeaksignalsofRLSinCu2+–BSAsystemincreasedwiththedoseofCu2+,whichillustratedthatparticlesinthissystembecamelargerbecausesomeactingforcemayworkbetweenCu2+andBSA,andthisforcewasstrengthenedwithCu2+increased.ThepeaksignalsofAl3+–BSAremainedstableinlowconcentrationofAl3+,buttheywouldincreasevisiblywhentheconcentrationofAl3+exceeded4.0×10 6mol/L.WehavetestedtheRLSofAl3+aloneinordertorecognizetherealeffectprinci-plebetweenAl3+andBSA.TheresultswereshowninTable1.1.Table1.1showedpeaksignalsofAl3+–BSAwerestrongerthanthoseofAl3+alonewhenc(Al3+)≤2.0×10 5mol/L.WhiletheresultswouldgobycontrarieswithincreasingAl3+dose,andthesubtrac-

H.Zhangetal./SpectrochimicaActaPartA78 (2011) 523–527

525

Table1.1

ThecomparisonofRLSbetweenAl3+–BSAandAl3+alone.Systems01102040

BSA+Al3+

4910

4788474050055131Al3+0242633514046168 RLS

4910

2362

1389

959

2037

Ps:CmeansconcentrationofAl3+.SignalexceedsthedetectionlimitwhenC5.0×10 5mol/L.

RLSstandsforthesubtractionbetweenthepeaksignalsofBSA+Al3+andAl3+alone.

Table1.2

ThecomparisonofRLSbetweenNa+–BSAandNa+alone.SystemsC(10 6mol/L)110100BSA+Na+

464746864854Na+416529212931 RLS

482

1765

1923

tion( RLS)wasbecomingsmaller.TheresultindicatedthatBSAwouldnotreactwithAl3+inlowconcentrationofAl3+,andthepeaksignalsofRLSremainedstable;Whenc(Al3+)≥2.0×10 5mol/L,peaksignalsofthesystemraisedwithAl3+increased,butweakerthanthoseofAl3+alone,soBSAreactedwithAl3+initshighcon-centration,andformedalargeraggregationsimultaneously.Atthesametime,hydrolysisofAl3+-binedwiththeresultof uorescenceemissionspectrainAl3+–BSAsystem,wecancometoaconclusionthatthetoxiceffectofAl3+onBSAisweakenoughtoin uencethestructureofBSAvisi-bly,onlychangingthesolutionenvironmentofBSAbyhydrolysisofAl3+.

Forthesamereason,thecomparisonsofanothertwoionswereillustratedinTables1.2and1.3aswell.Wecan gureoutthatpeaksignalsofNa+–BSAwerestrongerthanthoseofNa+aloneintheconcentrationrange,andCu2+sharesthesameresultwithNa+.SowecangetaconclusionsafelythatNa+doesnotreactwithBSA,whileCu2+couldbindwithBSAbysomeactingforce,whichaggregateslargerparticles.

Theresultwhichwecangetfrom uorescenceemissionspectraandRLSgivesusanilluminationthationelectricchargeisnotthemainfactoraffectingthestructureofBSA.4.3.Synchronous uorescencespectra

Synchronous uorescencespectroscopycangiveinformationaboutthemolecularenvironmentinthevicinityofachro-mophoresuchastryptophanandtyrosine.Theshiftintheemissionmaximum( em)re ectsthechangesofpolarityaroundthechro-mophoremolecule[19].WetestedCu2+–BSAwithsynchronous uorescencespectrainorderto ndoutthequenchingmechanisminwhichCu2+playedarolewiththe uorescenceofBSAresidues,Trp,TyrandPheinparticular.When betweenexcitationandemissionwavelengthissetat15nm,thesynchronous uorescencegivescharacteristicinformationofTyr.Andwhen is xedat60nm,aspectrumcharacteristicofTrpisobtained[20,21].TheresultsdetectedbyF-4600 uorescencespectrophotometerwereillustratedinFig.3.

Table1.3

ThecomparisonofRLSbetweenCu2+–BSAandCu2+alone.SystemsC(10 6mol/L)110100BSA+Cu2+

155817419066Cu2+77111094935 RLS

787

632

4131

y

tisnetni ecnecseroulFy

tisnetni ecnecseroulFFig.3.SynchronousFluorescenceSpectraofBSAwithdifferentconcen-trationsofcopperchloride,(A) =15nm,(B) =60nm.Conditions:c(BSA)=1.0×10 6mol/L,c(Cu2+)/(1.0×10 6mol/L):0,1,5,20,50,100respectively;pH7.4.

Wecan gureoutfromFig.3thatbothTrpandTyrwerequenchedsimultaneously,andtheintensityofTrpdecreasedsharperthanthatofTyr,whichindicatedbindingsitesbetweenCu2+andBSAwasclosertoTrp[22].WiththeconcentrationofCu2+increasedgradually,aslightredshiftofTrpandTyrpeakcouldbeobserved,whichprovedmolecularconformationofBSAchangedinthetoxiceffectofCu2+,thehydrophobicityoftheTrpandTyrdecreased,andbothoftwochromophoresburiedinthenon-polarhydrophobiccavitiesweremovedtoamorehydrophilicenvironment[23,24].

Theinteractionbetweenheavymetalsandbiomoleculesisavastresearchareathatmanyresearchershavestudiedinthis eld.LiWangetal.[25]hasinvestigatedthatNi2+hadanobvioustoxiceffectonbovinehemoglobin(BHb).Zn2+,Cd2+,Mn2+,Co2+andCr2+[26–29]havealreadybeenstudiedonthetoxicityofserumalbumin.Alloftheseresultsabovewarnusthatheavymetalmakesamajorhazardtobiomolecules.4.4.UV–visabsorptionspectra

TheultravioletabsorptionspectrainFig.4wereachievedbysubtractionofNa+,Cu2+andAl3+absorption.BSAalonehastwoabsorptionpeaksatabout200and280nm,whichstandsforthestrongabsorptionofpeptidebondandaromaticresiduesrespec-tively.AswecanseeinFig.4AandB,withtheconcentrationofNa+andAl3+increased,thepeakshapeandpositionremainedstablein

526

H.Zhangetal./SpectrochimicaActaPartA78 (2011) 523–527

2.5

A

2.0

1.5

s

bA1.0

0.5

0.0

200220240260280300320

Wavelength(nm)

2.5

B

2.0

1.5

s

bA1.0

0.5

0.0

200220240260280300320

Wavelength(nm)

s

bAWavelength(nm)

Fig.4.UV–Vis.AdsorptionspectraofBSAwithdifferentconcentrationsofsodiumchloride,copperchlorideandaluminiumchlorideConditions:c(BSA)=1.0×10 7mol/L,c(Na+,Al3+)/(1.0×10 6mol/L):0,1,5,10,50,100respec-tively;c(Cu2+)/(1.0×10 6mol/L):0,1,5,50,100.pH7.4.(A)Na–BSA,(B)Al–BSA,(C)Cu–BSA.

280nm,butitchangedin210nm,thatiswhyNa+andAl3+madethechangeofskeletonstructureofBSA.Fig.4Crevealedthatbothofpeaksin200and280nmincreasedwiththeconcentrationofCu2+risen,whichindicatedthatthetoxiceffectofCu2+onBSAenhancedtheexposureofaromaticresidues,andmadethehydrophobicityoftheTrpandTyrdecreased,peptidechainsextendedsimultaneously[30].

)

gedm(θWavelength(nm)

Fig.5.CirculardichroismspectraofBSAwithdifferentconcentrationsofcopperchlorideConditions:c(BSA)=2.0×10 7mol/L,c(Cu2+)/(1.0×10 6mol/L)=0,1,10,100respectively;pH7.4.

Table2

Thecomparisonofthe -helixofBSAintheabsenceandpresenceofCu2+.System

-HelixcontentBSA

64.1%BSA+Cu2+(1.0×10 6mol/L)61.2%BSA+Cu2+(5.0×10 5mol/L)58.1%BSA+Cu2+(1.0×10 4mol/L)

53.6%

4.5.Circulardichroismspectra

TheinformationhasbeenrevealedthattheCDspectraofBSAexhibitedtwonegativebandsat208and222nm,whichischar-acteristicofthe -helixofproteins[31,32].Fig.5showedtheCDspectraofBSAintheabsenceandpresenceofCu2+,andwecan gureoutthatthe -helixofBSAdecreasedwithincreasingCu2+dose.The -helixofBSAintheabsenceandpresenceofCu2+werecalculatedfromEqs.(1)and(2)[33–35].MRE=

CD(mdeg)(1)P -Helix(%)=

MRE208 4000

33000 4000

×100

(2)

whereCPisthemolarconcentrationoftheprotein,nisthenum-berofaminoacidresiduesandlisthepathlength.MRE208istheobservedMRE(meanresidualellipticity)at208nm,4000istheMREofthe -formandrandomcoilconformationcrossat208nm,and33000istheMREvalueofapure -helixat208nm.Accord-ingtothetwoequationsabove,quantitativeanalysisresultsfortheamountof -helixinthesecondarystructureofBSAwereobtained(Table2).TheCDspectraofBSAinpresenceofNa+andAl3+areshowedinFig.6AandB,respectively.The -helixcon-tentofNa+–BSAandAl3+–BSAwascalculatedinthesamemethod(Tables3and4).Bothofthesetwoionsdidnotshowgreatin u-enceon -helixcontentsofBSA,whichrevealedthatbothNa+andAl3+didnotaffectthesecondarystructureofBSA.

Table3

Thecomparisonofthe -helixofBSAintheabsenceandpresenceofNa+.System

-HelixcontentBSA

64.1%BSA+Al3+(1.0×10 6mol/L)63.4%BSA+Al3+(1.0×10 4mol/L)

65.1%

H.Zhangetal./SpectrochimicaActaPartA78 (2011) 523–527

527

403020

)

g10edm(θ0

-10-20-30190200210220230240250260270

Wavelength(nm)

40B

3020

)

g10edm(θ0-10-20

-30

190200210220230240250260270

Wavelength(nm)

Fig.6.CirculardichroismspectraofBSAwithdifferentconcentrationsofsodiumchlorideandaluminiumchloride.Conditions:c(BSA)=2.0×10 7mol/L,c(Na+,Al3+)/(1.0×10 6mol/L):0,1,100respectively;pH7.4.

Table4

Thecomparisonofthe -helixofBSAintheabsenceandpresenceofAl3+.System

-HelixcontentBSA

64.1%BSA+Na+(1.0×10 6mol/L)61.4%BSA+Na+(1.0×10 4mol/L)

59.2%

Wecan gureoutfromtheresultsabovethatthe -helixofBSAdecreasedclearlyinthepresenceofCu2+,inotherwords,theconformationofBSAwasdestroyedharmfullyintoxicityofCu2+.However,bothNa+andAl3+didlittleharmtothesecondarystruc-tureofBSA.5.Conclusions

Inthispaper,theeffectofmetalionsNa+,Cu2+,Al3+onBSAstruc-turehasbeeninvestigatedbymultiplespectroscopictechniquesincluding uorescence,RLS,synchronous uorescence,UV–visabsorptionandCDinvitroconditions.WecangettheconclusionthatthereislittlecorrelationbetweenionelectricchargesandthestructureofBSA.Speci cally,Na+didlittlein uencewiththestruc-tureofBSA,butcouldbeprotectivetowardsit.AndCu2+couldbindwithBSAintoalargerparticle,anddenatureitirreversibly,whichtaughtusthatCu2+cancausespeci clossoffunctionof

proteintotheproductionofharmfultoxiceffectonourphysicalbodies.Therefore,heavymetalpollutionintheenvironmentanddietisamajorhazardtohumanhealth,anditisofgreatimpor-tancetoimproveandprotectthetoxicityevaluationmechanism.Theresultsof uorescence,synchronous uorescence,ultravioletabsorptionandCDspectra,Al3+didnoharmtothestructureofBSA,butRLSofAl3+–BSAindicatedthatAl3+couldchangethesolutionenvironmentofBSAbyhydrolysisofAl3+inhighconcentrations.Acknowledgments

Thisworkwasaccomplishedunder nancialsupportofNatu-ralScienceFoundationofChina(20875055),theCultivationFundoftheKeyScienti candTechnicalInnovationProject,MinistryofEducationofChina(708058),andFoundationforExcellentYoungScientistsandKeyScience-TechnologyProjectinShandongProvince(2008GG10006012).AppendixA.Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,atdoi:10.1016/j.saa.2010.11.021.References

[1]Y.Lu,X.R.Li,Prog.Chem.17(2005)905–910.[2]Schellman,Biophys.Chem.96(2002)91–101.

[3]K.S.Huang,H.Bayley,M.J.Liao,E.London,H.G.Khoran,J.Biol.Chem.256(1981)

3802–3809.

[4]J.F.Brandts,H.R.Halvorson,M.Brennan,Biol.Chem.14(1975)4953–4963.[5]C.Soto,Nat.Rev.4(2003)49.

[6]X.Cao,Q.Huang,Chem.Life19(1999)138–140.

[7]C.P.Zamber,mba,K.Yasuda,J.Farnum,K.Thummel,J.D.Schuetz,E.G.

Schuetz,Pharmacogenetics13(2003)19–28.

[8]L.Liotta,V.Espina,A.Mehta,V.Calvert,K.Rosenblatt,D.Geho,P.Munson,L.

Young,J.Wulfkuhle,E.Petricoin,CancerCell3(2003)317–325.[9]X.Wang,C.Z.Song,J.Mol.Biol.vol.25(2003)358–360.

[10]J.Liu,S.Mathias,Z.Yang,R.N.Kolesnick,J.Biol.Chem.269(1994)3047–3052.[11]X.M.He,D.C.Carter,Nature358(1992)209–215.

[12]T.Peters,in:T.Peters(Ed.),Allaboutalbumin,AcademicPress,SanDiego,1996.[13]Y.Q.Zhou,H.Liu,Y.X.Li,P.W.Shen,J.Chem.12(1991)441–443.[14]Z.X.Feng,S.G.Zhang,Q.M.Liu,J.C.Ni,Anal.Chem.12(1995)70–72.[15]C.Q.Tu,H.Z.Zhang,H.Liang,J.Chem.58(2000)229–234.[16]P.J.Sadler,J.V.Viles,Inorg.Chem.35(1996).

[17]M.Cammarota,mberti,L.Masella,P.Galletti,M.DeRosa,N.Sannolo,M.

Giuliano,Toxicol.InVitro20(2006)1125–1132.

[18]X.B.Chen,S.Li,Spectrosc.SpectralAnal.26(2006)309–312.

[19]Y.Q.Wang,H.M.Zhang,G.C.Zhang,S.X.Liu,Q.H.Zhou,Z.H.Z.H.Fei,Z.T.Liu,Int.

J.Biol.Macromol.(2007)3.

[20]Y.Q.Wang,H.M.Zhang,G.C.Zhang,Q.H.Zhou,Z.H.Fei,Z.T.Liu,Z.X.Li,J.Mol.

Struct.6(2008)77–84.

[21]L.H.Chen,L.Q.Tianqing,Int.J.Biol.Macromol.42(2008)441–446.[22]X.J.Huang,Y.Huang,Q.He,J.Chem.63(2005)223–228.[23]K.Zhu,S.Y.Shen,J.Chem.17(1996)539–542.

[24]B.Huang,G.L.Zou,T.M.Yang,J.Chem.60(2002)1867–1871.

[25]LiWang,R.T.Liu,ZhenxingChi,BingjunYang,PengjunZhang,MeijieWang,

Spectrochim.ActaPartA76(2010)155–160.

[26]E.M.L.Nadhipuram,V.Bhagavan,A.PatriciaRios,M.JinshengYang,Anna

Ortega-Lopez,HirokoShinoda,A.A.StaceyHonda,N.CarlosRios,E.CherylSugiyama,Ha.Chung-Eun,Clin.Chem.49(2003)581–585.

[27]J.H.J.Versieck,F.Barbier,H.Steyaert,J.DeRudder,H.Michels,Clin.Chem.24

(1978)303–308.

[28]C.T.HongzhiZhang,XingcanShen,Chemistry10(2000)44–45.

[29]Y.H.EmikoOhyoshi,KanaNakata,SusumuKohata,Biochemistry75(1999).[30]Y.Liu,M.X.Xie,J.Kang,J.Chem.61(2003)1305–1310.

[31]Y.Z.Zhang,B.Zhou,Y.X.Liu,C.X.Zhou,X.L.Ding,Y.Liu,J.Fluoresc.18(2008)

109–118.

[32]A.Papadopoulou,R.J.Green,R.A.Frazier,J.Agric.FoodChem.53(2005)

158–163.

[33]N.Sreerama,S.Y.Venyaminov,R.W.Woody,Anal.Biochem.287(2000)

243–251.

[34]B.A.Wallace,J.G.Lees,A.J.Orry,A.Lobley,R.W.Janes,ProteinSci.12(2003)

875–884.

[35]Y.Z.Zhang,X.X.Chen,J.Dai,X.P.Zhang,Y.X.Liu,Y.Liu,Luminescence23(2008)

150–156.

本文来源：https://www.bwwdw.com/article/3bge.html