不同价态金属离子对BSA结构的影响

第第页不同价态金属离子对BSA结构的影响

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SpectrochimicaActaPartA78(2022)523–527

ContentslistsavailableatScienceDirect

SpectrochimicaActaPartA:Molecularand

Biomolecular

Spectroscopy

journalhomepage:/locate/sa

a

To*iceffectsofdifferentchargedmetalionsonthetarget—Bovineserumalbumin

HaoZhang,RutaoLiu,Zhen*ingChi,CanzhuGao

ShandongKeyLaboratoryofWaterPollutionControlandResourceReuse,SchoolofEnvironmentalScienceandEngineering,ShandongUniversity,Jinan250100,PRChinaAmericaCRCforEnvironmentHealth,ShandongProvince,27#ShandaSouthRoad,Jinan250100,PRChina

articleinfoabstract

Inthiswork,theto*icinuenceofmetallicions(Na+,Cu2+,Al3+)ontheserumalbuminwerestudiedbyuorescence,resonancelightscattering(RLS),synchronousuorescence,UV–visabsorptionandcirculardichroism(CD)spectroscopy.Thee*perimentalresultsindicatedthationelectricchargeisnotthemainfactoraffectingthestructureofbovineserumalbumin(BSA).Na+madethestructureofBSAtighterandhydrophobicityenhanced,whichimproveduorescenceintensity,whileCu2+couldreactwithsomefunctionalgroupsofBSA,makingthestructureofBSAlooser,sothattheinternalhydrophobicgroupssuchastryptophan(Trp)andotheraromaticresiduesweregraduallye*posed.Whenweobservedthemwithuorescencespectra,wefounduorescencequenchingwithincreasingCu2+dose.Al3+isshownaslittlesignicantinuenceontheBSA,butBSAwasfoundtoaggregatewiththedoseofAl3+bymeansofRLSbecauseofthehydrolysisandionstrengtheffectofAl3+.Theresultsalsoprovednormalsalinecouldkeepliveshealthyandgood-workingasabiologicalhumour,however,heavymetalsmadeharmfuleffectstothebodywhentheye*ceededtheminimaleffectlevel(MEL),suchasCu2+choseninourwork.

2022ElsevierB.V.Allrightsreserved.

Articlehistory:

Received2September2022

Receivedinrevisedform13October2022Accepted15November2022Keywords:Metalions

Bovineserumalbumin(BSA)SpectratechniquesTo*icinteraction

1.Introduction

Asisknowntoall,Proteindenaturationreferstothedestructionofthemoleculestructurebyseveralenvironmentalfactorsinclud-ingphysicalandchemicalones,suchastemperature,dehydration,ultravioletradiationandallkindsofto*icchemicals[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)asthetargettoevaluatetheto*iceffectofmetaliononhealthbecauseofitssimilarstructuretohumanserumalbumin(HSA)[11,12]anditslowprice.

Theto*iceffectsofmetalionstoproteinhavebeenstudiedbyto*icologistssince1990s[13,14].Moreandmoreresearchersstart

tostudytheseeffectsinmolecularlevelatpresent,especiallyana-lyzebindingsitesandparametersbetweenproteinandmetalions.ChuqiaoTuetal.[15]investigatedtheinteractionbetweenCd2+andHSAbymeansofbalanceddialysis,andhefoundthecom-ple*compoundconformationsimilartoatetrahedronafterbindingsitesbetweenCd2+andHSAwereanalyzed.Sadle[16]propoundedapatternbetweenserumalbuminandCd2+,Zn2+withsomeionscoe*istinginthesolutionsbynuclearmagneticresonance.Cam-marotetal.[17]learnedtheinhibitionofmatri*metalloproteinase(mmps)tobodyto*icitybynickelandchromium.Butnoschol-arshavestudiedtheto*icitiesbetweenionsandproteinfromthestandpointofionelectriccharge.Soourgroupsetupthise*peri-menttoinvestigatethisissuethoroughly.WeselectedthreeionswithdifferentelectricchargesNa+,Cu2+,Al3+,andtookBSAasthetargetmoleculetoanalyzethesystemswithspectroscopictech-niques.

2.Materialsandmethods2.1.Apparatus

AHitachiF-4600uorescencespectrophotometerwasusedtomeasuretheintensityofuorescence,synchronousuores-cenceandRLS.TheUV–visabsorptionspectrumwasobtainedbyaShimadzuUV–2450spectrophotometer.AJasco-810circulardichroismspectrometerwasusedtomeasurethechangesofthesecondarystructureoftheproteinandthepHs-3CpHmeter(Peng-shun,Shanghai,People’sRepublicofChina)wasusedtomeasurethepHinallthee*periments.

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

E-mailaddress:rutaoliu@(R.Liu).1386-1425/$–seefrontmatter2022ElsevierB.V.Allrightsreserved.doi:10.1016/j.saa.2022.11.021

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H.Zhangetal./SpectrochimicaActaPartA78(2022)523–527

2.2.Reagents

BSA(electrophoreticreagentgrade)purchasedfromBeijingChemicalReagentCorporationwaspreparedat105mol/Landpre-servedat0–4C.

A0.2mol/LofNaH2PO4-Na2HPO4bufferwaspreparedfromNaH2PO4andNa2HPO4,whichadjustedtotheappropriatepH.

A1.0103mol/LofNaClwaspreparedbydissolving0.0146gNaCl(TsingtaoSifangChemicalReagentFactory)in100mLofultrapurewater.

A1.0103mol/LofCuCl2waspreparedbydissolving0.0427gCuCl22H2O(TianjinKermelChemicalReagentResearchInstitute)in100mLofultrapurewater.

A1.0103mol/LofAlCl3waspreparedbydissolving0.0602gAlCl36H2O(TianjinDamaoChemicalReagentFactory)in100mLofultrapurewater.3.Methods

Alluorescencespectra,RLS,andsynchronousuorescencespectrawererecordedonF-4600uorescencespectrophotometer(HitachiJapan)ina1-cmcell.

Firstly,uorescencespectraweremeasuredwiththeto*iceffectsofNa+,Cu2+,Al3+onBSAwithdifferentconcentrationgradientsofNa+,Cu2+,Al3+.Thee*citationwavelengthwas278nm.Thescanscopewasfrom290nmto500nm.Thee*ci-tationandemissionslitwidthsweresetat5nm.Scanspeedwas1200nm/min.PMT(PhotoMultiplierTube)voltagewas700V.

Secondly,RLSweremeasuredduringtheNa+–BSA,Cu2+–BSA,andAl3+–BSAsystems,aswellasNa+,Cu2+andAl3+alone.Theconditionswereasfollowse*=em,e*=200–600nm.PMTweredifferentamongNa+–BSA,Cu2+–BSA,andAl3+–BSAsystems,PMT(Na+–BSA,Al3+–BSA)=700V,whilePMT(Cu2+–BSA)=600Vbecausethissystemwasbeyondthedetectionlimitwhenwetookthise*perimentwithPMT700V.

SynchronousuorescencespectraofBSAinthepresenceofNa+,Cu2+,Al3+weremeasured(e*=15nm,e*=250–320nmande*=60nm,e*=250–320nm,respectively).Thee*citationandemissionslitswereboth5nminwidth.Scanspeedwas1200nm/min.PMTvoltagewas*edat700V.

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+didnotquenchedtheuorescenceofBSAbutincreaseit,whichindicatedthatNa+madethestructureofBSAtighterandhydrophobicityenhanced,andprovedthatphysio-logicalsalinewasprotectivetowardsourbodies.ThisresultisconsistentwithChen’sresearch[18].InCu2+–BSAsystem,wefounduorescencequenchingwiththedoseofCu2+increased,whichindicatedCu2+reactedwithseveralresiduesofBSAthatcouldemituorescence,suchastryptophan(Trp),tyrosine(Tyr)

y

tisnetniecnecseroulFConcentration(10-6mol/L)

Fig.1.FluorescenceintensityofBSAwithdifferentconcentrationsofionsNa+,Cu2+,Al3+.Conditions:c(BSA)=1.0106mol/L,c(Na+,Cu2+,Al

3+)/(1.0106mol/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)=1.0106mol/L,c(Na+,Cu2+,Al3+)/(1.0106mol/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+e*ceeded4.0106mol/L.WehavetestedtheRLSofAl3+aloneinordertorecognizetherealeffectprinci-plebetweenAl3+andBSA.TheresultswereshowninTable1.1.Table1.1showedpeaksignalsofAl3+–BSAwerestrongerthanthoseofAl3+alonewhenc(Al3+)≤2.0105mol/L.WhiletheresultswouldgobycontrarieswithincreasingAl3+dose,andthesubtrac-

H.Zhangetal./SpectrochimicaActaPartA78(2022)523–527

525

Table1.1

ThecomparisonofRLSbetweenAl3+–BSAandAl3+alone.Systems01102040

BSA+Al3+

4910

4788474050055131Al3+0242633514046168RLS

4910

2362

1389

959

2037

Ps:CmeansconcentrationofAl3+.Signale*ceedsthedetectionlimitwhenC5.0105mol/L.

RLSstandsforthesubtractionbetweenthepeaksignalsofBSA+Al3+andAl3+alone.

Table1.2

ThecomparisonofRLSbetweenNa+–BSAandNa+alone.SystemsC(106mol/L)110100BSA+Na+

464746864854Na+416529212931RLS

482

1765

1923

tion(RLS)wasbecomingsmaller.TheresultindicatedthatBSAwouldnotreactwithAl3+inlowconcentrationofAl3+,andthepeaksignalsofRLSremainedstable;Whenc(Al3+)≥2.0105mol/L,peaksignalsofthesystemraisedwithAl3+increased,butweakerthanthoseofAl3+alone,soBSAreactedwithAl3+initshighcon-centration,andformedalargeraggregationsimultaneously.Atthesametime,hydrolysisofAl3+-binedwiththeresultofuorescenceemissionspectrainAl3+–BSAsystem,wecancometoaconclusionthattheto*iceffectofAl3+onBSAisweakenoughtoinuencethestructureofBSAvisi-bly,onlychangingthesolutionenvironmentofBSAbyhydrolysisofAl3+.

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

TheresultwhichwecangetfromuorescenceemissionspectraandRLSgivesusanilluminationthationelectricchargeisnotthemainfactoraffectingthestructureofBSA.4.3.Synchronousuorescencespectra

Synchronousuorescencespectroscopycangiveinformationaboutthemolecularenvironmentinthevicinityofachro-mophoresuchastryptophanandtyrosine.Theshiftintheemissionma*imum(em)reectsthechangesofpolarityaroundthechro-mophoremolecule[19].WetestedCu2+–BSAwithsynchronousuorescencespectrainordertondoutthequenchingmechanisminwhichCu2+playedarolewiththeuorescenceofBSAresidues,Trp,TyrandPheinparticular.Whenbetweene*citationandemissionwavelengthissetat15nm,thesynchronousuorescencegivescharacteristicinformationofTyr.Andwhenis*edat60nm,aspectrumcharacteristicofTrpisobtained[20,21].TheresultsdetectedbyF-4600uorescencespectrophotometerwereillustratedinFig.3.

Table1.3

ThecomparisonofRLSbetweenCu2+–BSAandCu2+alone.SystemsC(106mol/L)110100BSA+Cu2+

155817419066Cu2+77111094935RLS

787

632

4131

y

tisnetniecnecseroulFy

tisnetniecnecseroulFFig.3.SynchronousFluorescenceSpectraofBSAwithdifferentconcen-trationsofcopperchloride,(A)=15nm,(B)=60nm.Conditions:c(BSA)=1.0106mol/L,c(Cu2+)/(1.0106mol/L):0,1,5,20,50,100respectively;pH7.4.

WecangureoutfromFig.3thatbothTrpandTyrwerequenchedsimultaneously,andtheintensityofTrpdecreasedsharperthanthatofTyr,whichindicatedbindingsitesbetweenCu2+andBSAwasclosertoTrp[22].WiththeconcentrationofCu2+increasedgradually,aslightredshiftofTrpandTyrpeakcouldbeobserved,whichprovedmolecularconformationofBSAchangedintheto*iceffectofCu2+,thehydrophobicityoftheTrpandTyrdecreased,andbothoftwochromophoresburiedinthenon-polarhydrophobiccavitiesweremovedtoamorehydrophilicenvironment[23,24].

Theinteractionbetweenheavymetalsandbiomoleculesisavastresearchareathatmanyresearchershavestudiedinthiseld.LiWangetal.[25]hasinvestigatedthatNi2+hadanobviousto*iceffectonbovinehemoglobin(BHb).Zn2+,Cd2+,Mn2+,Co2+andCr2+[26–29]havealreadybeenstudiedontheto*icityofserumalbumin.Alloftheseresultsabovewarnusthatheavymetalmakesamajorhazardtobiomolecules.4.4.UV–visabsorptionspectra

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

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H.Zhangetal./SpectrochimicaActaPartA78(2022)523–527

2.5

A

2.0

1.5

s

bA1.0

0.5

0.0

202220240260280300320

Wavelength(nm)

2.5

B

2.0

1.5

s

bA1.0

0.5

0.0

202220240260280300320

Wavelength(nm)

s

bAWavelength(nm)

Fig.4.UV–Vis.AdsorptionspectraofBSAwithdifferentconcentrationsofsodiumchloride,copperchlorideandaluminiumchlorideConditions:c(BSA)=1.0107mol/L,c(Na+,Al3+)/(1.0106mol/L):0,1,5,10,50,100respec-tively;c(Cu2+)/(1.0106mol/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,whichindicatedthattheto*iceffectofCu2+onBSAenhancedthee*posureofaromaticresidues,andmadethehydrophobicityoftheTrpandTyrdecreased,peptidechainse*tendedsimultaneously[30].

)

gedm(θWavelength(nm)

Fig.5.CirculardichroismspectraofBSAwithdifferentconcentrationsofcopperchlorideConditions:c(BSA)=2.0107mol/L,c(Cu2+)/(1.0106mol/L)=0,1,10,100respectively;pH7.4.

Table2

Thecomparisonofthe-heli*ofBSAintheabsenceandpresenceofCu2+.System

-Heli*contentBSA

64.1%BSA+Cu2+(1.0106mol/L)61.2%BSA+Cu2+(5.0105mol/L)58.1%BSA+Cu2+(1.0104mol/L)

53.6%

4.5.Circulardichroismspectra

TheinformationhasbeenrevealedthattheCDspectraofBSAe*hibitedtwonegativebandsat208and222nm,whichischar-acteristicofthe-heli*ofproteins[31,32].Fig.5showedtheCDspectraofBSAintheabsenceandpresenceofCu2+,andwecangureoutthatthe-heli*ofBSAdecreasedwithincreasingCu2+dose.The-heli*ofBSAintheabsenceandpresenceofCu2+werecalculatedfromEqs.(1)and(2)[33–35].MRE=

CD(mdeg)(1)P-Heli*(%)=

MRE2084000

330004000

100

(2)

whereCPisthemolarconcentrationoftheprotein,nisthenum-berofaminoacidresiduesandlisthepathlength.MRE208istheobservedMRE(meanresidualellipticity)at208nm,4000istheMREofthe-formandrandomcoilconformationcrossat208nm,and33000istheMREvalueofapure-heli*at208nm.Accord-ingtothetwoequationsabove,quantitativeanalysisresultsfortheamountof-heli*inthesecondarystructureofBSAwereobtained(Table2).TheCDspectraofBSAinpresenceofNa+andAl3+areshowedinFig.6AandB,respectively.The-heli*con-tentofNa+–BSAandAl3+–BSAwascalculatedinthesamemethod(Tables3and4).Bothofthesetwoionsdidnotshowgreatinu-enceon-heli*contentsofBSA,whichrevealedthatbothNa+andAl3+didnotaffectthesecondarystructureofBSA.

Table3

Thecomparisonofthe-heli*ofBSAintheabsenceandpresenceofNa+.System

-Heli*contentBSA

64.1%BSA+Al3+(1.0106mol/L)63.4%BSA+Al3+(1.0104mol/L)

65.1%

H.Zhangetal./SpectrochimicaActaPartA78(2022)523–527

527

403020

)

g10edm(θ0

-10-20-30190202210220230240250260270

Wavelength(nm)

40B

3020

)

g10edm(θ0-10-20

-30

190202210220230240250260270

Wavelength(nm)

Fig.6.CirculardichroismspectraofBSAwithdifferentconcentrationsofsodiumchlorideandaluminiumchloride.Conditions:c(BSA)=2.0107mol/L,c(Na+,Al3+)/(1.0106mol/L):0,1,100respectively;pH7.4.

Table4

Thecomparisonofthe-heli*ofBSAintheabsenceandpresenceofAl3+.System

-Heli*contentBSA

64.1%BSA+Na+(1.0106mol/L)61.4%BSA+Na+(1.0104mol/L)

59.2%

Wecangureoutfromtheresultsabovethatthe-heli*ofBSAdecreasedclearlyinthepresenceofCu2+,inotherwords,theconformationofBSAwasdestroyedharmfullyinto*icityofCu2+.However,bothNa+andAl3+didlittleharmtothesecondarystruc-tureofBSA.5.Conclusions

Inthispaper,theeffectofmetalionsNa+,Cu2+,Al3+onBSAstruc-turehasbeeninvestigatedbymultiplespectroscopictechniquesincludinguorescence,RLS,synchronousuorescence,UV–visabsorptionandCDinvitroconditions.WecangettheconclusionthatthereislittlecorrelationbetweenionelectricchargesandthestructureofBSA.Specically,Na+didlittleinuencewiththestruc-tureofBSA,butcouldbeprotectivetowardsit.AndCu2+couldbindwithBSAintoalargerparticle,anddenatureitirreversibly,whichtaughtusthatCu2+cancausespeciclossoffunctionof

proteintotheproductionofharmfulto*iceffectonourphysicalbodies.Therefore,heavymetalpollutionintheenvironmentanddietisamajorhazardtohumanhealth,anditisofgreatimpor-tancetoimproveandprotecttheto*icityevaluationmechanism.Theresultsofuorescence,synchronousuorescence,ultravioletabsorptionandCDspectra,Al3+didnoharmtothestructureofBSA,butRLSofAl3+–BSAindicatedthatAl3+couldchangethesolutionenvironmentofBSAbyhydrolysisofAl3+inhighconcentrations.Acknowledgments

ThisworkwasaccomplishedundernancialsupportofNatu-ralScienceFoundationofChina(20875055),theCultivationFundoftheKeyScienticandTechnicalInnovationProject,MinistryofEducationofChina(708058),andFoundationforE*cellentYoungScientistsandKeyScience-TechnologyProjectinShandongProvince(2022GG10006012).Appendi*A.Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,atdoi:10.1016/j.saa.2022.11.021.References

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