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ElectrochemistryCommunications
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Electro-generatedreactiveoxygenspeciesatAusurfaceasanindicatortoexploreglutathioneredoxchemistryandquantification
GopiKalaiyarasan,AlamVenugopalNarendraKumar,ChinnaiahSivakumar,JamesJoseph
ElectrodicsandElectrocatalysisDivision,CSIR-CentralElectrochemicalResearchInstitute,Karaikudi,630006Tamilnadu,India
articleinfoabstract
Thethioltodisulfideinter-conversionplaysvitalroleintheglutathione(GSH)metabolisminhumanbody.MimickingtheGSHconversiontodisulfidebyelectro-generatedreactiveoxygenspeciesatgoldelectrodeinterfaceandtheuseofelectro-reductionoftheproduceddisulfidesfortheelectro-analysisoflowlevelsofGSHisreported.
©2015ElsevierB.V.Allrightsreserved.
Articlehistory:
Received27February2015
Receivedinrevisedform18March2015Accepted30March2015Availableonline8April2015Keywords:Glutathione
GlutathionedisulfideElectroanalysisOxygenreductionGoldelectrode
1.Introduction
InvestigatingtheGSHmetabolisminhumanatintercellularlevelisoneimportantsubjectresearchersareinterestedinrecenttimes.AnappreciableamountofGSHcanbeboundtothe–SHgroupofproteincysteinylresiduesbyamechanismcalledS-Glutathionylation.TheS-Glutathionylationcanbereversedandthereforecanberegulatedbymeansofenzymaticreactionsinthepresenceofthioltransferases[1].S-Glutathionylationprotectssensitiveproteinthiolsfromirreversibleoxidationduringoxidativestressformationbygeneratedreactiveoxygenspecies(ROS)[1–5].TheratioofGSHanditsoxidizeddisulfideproduct(GSSG)hassignificantroleincreatingcancer[6],Parkinson'sdisease[7],andrelatedproblemsinthebody[8].Totroubleshoottheseproblems,onerequiresstandardmethodformonitoring/quanti-fyingthesebiomoleculesGSH/GSSHinthehumanbodywithhighaccuracy.Although,manyquantifyingmethodsusinghighlysophisti-catedinstrumentations(HPLC)weredeveloped,electrochemicalmodeofquantificationhasfoundmorereverence,becauseofeaseinhandlingandportability.Withtheadventofmicroelectrodesandmicrofabrica-tiontechniques,itispossibletomeasureintercellularanalytesusingelectrochemicaltechniques.Recently,electrochemicaldeterminationofGSHwasextensivelyreviewedbyCompton'sgroup[9].Initially,electrochemicalmethodofGSHdeterminationinvolvesoxidativeconversionofGSHtoGSSGatelectrodessuchasMercury[10]oratotherchemicallymodifiedelectrodes[11,12].AttemptstouseAuelec-trodesfortheelectroanalyticaldeterminationofGSHbyelectro
oxidationhadinherentproblemsduetothestrongconcentrationdependentadsorptionofGSHontoAusurfaceandtheconcomitantfor-mationofgoldoxidewhichoverlapswithGSHelectrooxidationsignal.AttemptsmadeforGSHdeterminationarebasedontheelectrooxida-tionofGSHtoGSSGatachemicallymodifiedinterface.TheformationofGSSGfromGSHNOatneutralpHthroughγ-raysradiolysisgeneratedOH•radicalsfromwaterarereported[13].Onthecontrary,wereportdetailsofourattemptstodetermineGSHbyelectro-reductionofGSSGformedchemicallybyelectro-generatedreactiveoxygenspecies
−•−[14–16](ROS)suchassuperoxides(O•2)hydroxyls(OH)and
−hydroperoxyl(HO•2)atAuelectrode/electrolyteinterfaces.Thisapproachisnewandreportedforthefirsttimeandmimicsthereactionwhichcausesoxidativestress.2.Materialsandmethods2.1.Materials
glutathione(GSH)waspurchasedfromSigmaAldrich,
potassiumchloridewaspurchasedfromMerckandsodiumsulfitewaspurchasedfromSRL,India.Allchemicalswereofanalyticalgradeandusedwithoutfurtherpurification.2.2.Instruments
TheelectrochemicalevaluationwascarriedoutusingAUTOLABpotentiostat(Ecochemie,Netherland)forCyclicVoltammetry(CV)andBAS100BelectrochemicalanalyzerforDifferentialPulse
L-Reduced
E-mailaddresses:jameskavalam@yahoo.com,jamescecri@cecri.res.in(J.Joseph).
http://dx.doi.org/10.1016/j.elecom.2015.03.0211388-2481/©2015ElsevierB.V.Allrightsreserved.
30G.Kalaiyarasanetal./ElectrochemistryCommunications56(2015)29–33
Fig.1.DifferentialPulseVoltammogramfortheadditionofglutathione(a)from100nMto900nMand(b)from1μMto10μMto0.1MKClN2gaspurgedfor30minatgoldelectrode,(c)and(d)arecalibrationcurveswitherrorbarforglutathioneadditionrespectively.Scanrate:50mV/s.
Voltammetry(DPV).Aconventionalthree-electrodecellsetupwasusedinallexperiments.Goldelectrode(Area=0.0232cm2)wasemployedasworkingelectrodeandAg/AgClelectrodewasusedasreferenceelectrode.Thecounterelectrodewasplatinumfoil.
2.2.1.Insituproductanalysis
Insituspectro-electrochemicalstudieswerecarriedoutinPARSTATMCmultichannelpotentiostat(USA)coupledwithaSEC2000spectrom-etersystem,Japan.Thegoldgauzewasusedasworkingelectrodeinaquartzthinlayercell;platinumstickandsilverwirewereusedascounterandreferenceelectroderespectively.2.3.Methods
Theworkingelectrodewaspolishedinapolishingemerypaperusing0.05μmaluminapowderandsonicatedinMilliporewaterfor2mintoremovethealuminaparticleadheringtotheelectrode.Glutathionesolutionwasaddedtothecellcontaining0.1MKClelectrolyteusinganinjectionsyringetopreventtheatmosphericoxygenfromre-dissolvingintheelectrolyte.CyclicVoltammogramintherangefrom0.0Vto1.7VwithrespecttoAg/AgClrevealedthattheoxidationofGSHatAuelectrodeismaskedsignificantlybytheadsorptionofGSHontheelectrodesurface.Asaresult,theuseofGSHoxidationonAuelectrodefortheanalyticaldeterminationiscomplex.Interestinglywhentheworkingelectrodewaselectro-chemicallycycledinthenegativeregionfrom0.0Vto−1.0V,areductionpeakappearedat−0.69V(correspondstoGSSGtoGSH)whichincreaseslinearlywithglutathioneconcentration.Beyond−0.69V,nofurtherelectro-reductionofGSHwasplausible.Atthisstageofanalysis,it'sbelievedthattheaddedGSHischemicallyconvertedtotheoxidizedformnamelyGSSGbytheelectro-reductionproductofmolecularoxygen(i.e.,ROS)at−0.17V.ThisGSSGcanbeelectrochemicallyreducedtoGSHat−0.69V.
Toprovethis,wehavecarriedoutDPVexperimentsatthesamepotentialwindowwithoutthepresenceofoxygeninthemediumtopreventtheH2O2formationbymeansofsodiumsulphite.Theexperi-mentsundertotalabsenceofoxygenshowednoreductionpeakat−0.69VrevealingthatthegenerationofH2O2atAuelectrodeinterfaceisessentialfortheformationofglutathionedisulfide.Thereforetoavoidthechangeintheoxygenconcentrationduringthemeasurement,wehaveconstructedaperfectandcompletelyclosedfour-neckcell.Thethreeelectrodeswereinsertedintothecellandairtightenedusingsuit-ablegroundjoints.Oneneckwasclosedbyrubbercorkandtheglutathi-onewasinjectedthroughrubbercorkusingsyringe.ThefreshlypreparedGSHsolutionwasalsopurgedwithnitrogengasabout30minbeforeadditions.Themagnitudeoftheelectro-reductioncurrentsforGSSGbeforeandafterdeaerationwasfoundtobealmostthesameindicatingthatthechemicalconversionGSHtoGSSGisprob-ablylimitedtoadsorbedinterfacialGSHmolecules.3.Resultsanddiscussion
TheGSHinthemediumadsorbsonAusurfacethroughAu–Slinkage,asafunctionofGSHconcentrationinthesolution.Theobviouscomplexityinusingtheoxidationpeakcurrentfortheanalyticaldeter-minationofGSHisduetoconcomitantformationofAuoxideatnearlysamepotential.Further,theblockingeffectofGSHonAusurfaceleadstosurfacepassivationwhichhasdirectconsequenceinelectrochemicalsignal.However,Aumetalsurfacecanserveasagoodelectrocatalyticmaterialformanyenergyconversionreactionsexceptforoxygenreductionreaction(ORR).IngeneralthecompleteORRshouldfollow4e−pathwaytoformH2Owhereas,inthecaseofAusurfaceitprefersthe2e−reductionpathwayleadingtoH2O2formation(Eq.(1))[17].El-Deabetal.haveprovedthattheORRoncysteineadsorbedAusurfacegoespredominantlythrougha2e−reductionpathwaywhichproduceshydrogenperoxide[18].Inourcase,thereducedglutathioneprobablygetschemicallyoxidizedatpotentialmorenegativeto−0.17Vona
G.Kalaiyarasanetal./ElectrochemistryCommunications56(2015)29–3331
Fig.2.DifferentialPulseVoltammogramof(a)additionofglutathionefrom10μMto200μM,(b)100μMto900μMand(c)1mMto7mMin0.1MKClelectrolytedeaeratedfor30minwithN2gaspurged,(d)and(e)calibrationplotswitherrorbarscorrespondingto(b)and(c)respectively.Scanrate:50mV/s.
Auelectrodein0.1MKClmediumbytheelectrogeneratedROSnamelyH2O2.Similarelectro-reductionbehaviorofGSSGonCobaltphthalocya-ninemodifiedcarbonelectrodewasreported[11].Thetracesofoxygen
remainedevenafterde-aeratingthemediumwithdrynitrogengasfor90min.
O2þ2Hþ2e→H2O2
þ
−
…ð1Þ
Fig.3.InsituUV–VisspectraldataforGSSGformationattwo-timeintervals,(a)at0minand(b)after20min.Appliedpotential:−0.3V.
Inourexperiments,theGSHinteractswithreductionproductsofthemolecularoxygengeneratedelectrochemicallytoinitiallyformGSOH[19,20](seeEq.(2)).SimilarscavengingofelectrochemicallygeneratedoxygenradicalsbyGSHmighthaveresultedintheformationofGSOH.TheformationofGSOHonreactionofhydrogenperoxidewithGSHisconfirmedbyAbedinzadehetal.byUV–Visspectralmeans[19].TheGSOHmayundergooxidationintwodifferentpathwaysdependingupontheconcentrationofGSHattheinterface,(Eqs.(3)&(4))asfollows.i)AtlowerconcentrationofGSH,theGSOHcombineswithH2O2formedbyelectroreductionofavailablemolecularoxygen(representedinEq.(3)).ii)AthighconcentrationofGSH,theGSOHtendstoreactwithGSHandformGSSGchemically(Eq.(4))whichgetsreducedatmore
32G.Kalaiyarasanetal./ElectrochemistryCommunications56(2015)29–33
Table1
DepictingthelistofelectrochemicalmethodsavailableforGSHdetectionintheliteratureanditssensitivity.Electrolyte
PBS
B–RbufferLysisbufferLysisbuffer
10mMTris–HClbufferPlantcellsBoratebufferPBSKCl
Electrode
AuwithMWCNTmodifiedbyGR
PiazselenoleasmediatorusinganAuelectrodeHg/AuamalgamelectrodeHg/Pdamalgamelectrode
2Auelectrodeswith2complimentaryoligonucleoditesHMDE–Co3+ionGCE/PPy–PQQGCE/MWCNTAu
MethodAMPDPVAMPAMPCCDPVAMPCVDPV
Linearrange,(M)9.9×10−5–8.8×10−35.0×10−10–2.2×10−82.5×10−5–1.5×10−42.5×10−5–1.5×10−41.0×10−12–1.0×10−101.0×10−5–1.0×10−4–
4.0×10−4–1.2×10−21.0×10−7–1.0×10−2LoD,(M)6.2×10−68.3×10−118.1×10−69.2×10−60.4×10−121.0×10−51.1×10−53.3×10−61.0×10−7Reference[22][6][23][23][24][25][26][27]
Thiswork
PBS=Phosphatebuffersolution,GR=glutathionereductase,MWCNT=multiwalledcarbonnanotube,AMP=amperometry,B–Rbuffer=Britton–Robinsonbuffer,DPV=DifferentialPulseVoltammetry,CC=chronocoulometry,HMDE=hangingMercurydropelectrode,GCE=glassycarbonelectrode,PPy=polypyrrole,PQQ=pyrroloquinolinequinone,CV=CyclicVoltammetry,Au=goldelectrode.
negativepotentialof−0.69V.TheinvolvementofGSHintheelectrocatalyticreductionofmolecularoxygenisevidentfromthelinearincreaseintheORRcurrentwiththeadditionofGSH.GSHþH2O2→GSOHþH2O
…
ð2Þ2GSOHþH2O2→GSSGþO2þ2H2O…
ð3ÞGSOHþGSH→GSSGþH2O
…
ð4Þ
AtverylowconcentrationofGSH,theintermediateGSOHgeneratedinturnreactswitheitherGSHorH2O2toyieldGSSGatinterface.Inter-estingly,asseenfromFig.1aandb,theoxygenreductioncurrentofthepartiallyde-aeratedmediumat−0.17V,increasesasfunctionofGSHconcentrationatAuelectrode.WehaveruledoutthepossibilityofoxygenenteringintothesystembyusingarubberlidforthecellandtheadditionsofGSHwasperformedusingasyringe.Theincreaseintheoxygenreductioncurrentat−0.17VwithGSHwasobservedtobelinearintheconcentrationrangefrom100nMto10μMasseenfromtheFig.1candd.Thisenhancementofvoltammetricpeakcurrentsat−0.17VwiththeGSHconcentrationupto10μMisobservableonlyinamediumwhichisdeaeratedfor30min.It'sworthmentioningthattheincreaseinpeakcurrentat−0.17V(ORRcurrent)withtheadditionoflowconcentrationsofGSHisnotobservableinnon-deaeratedKClmediumduetosignificantlyhighoxygenreductioncurrents.WhentheGSHconcentrationreaches10μMtheoxygenreductioncurrentstartedgettingsuppressed.Thegradualshiftinthereductionpeaktomorenegativevalueswasalsoobserved(Fig.2a).ThemechanismchangedependingontheconcentrationsofGSHenablesonetouseboththereductionpeaksforthedeterminationofGSHfrom100nMto10mMlevel.
AtconcentrationsofGSHhigherthan10μM,agradualshiftintheoxygenreductionpeaktomorenegativepotentialinadditiontheap-pearanceofanewreductionpeakataround−0.69Vwasseen,whichclearlyindicatesthechemicalconversion(Eq.(4))ofGSHtoGSSGisdominatedathigherGSHconcentration(SeeFig.2a).Butthereductionpeakcurrentat−0.69Vcorrespondingtotheelectro-reductionofGSSGwasfoundtolinearlyincreaseupto10mMconcen-trationofGSHaddition.ThisshiftinthevoltammetricpeakpositionwiththeconcentrationofGSHattheinterfaceclearlyindicatesachangeintheelectro-reductionmechanismathigherGSHconcentration(seeEq.(5)).
GSSGþ2Hþ
þ2e−
→2GSH
…ð5Þ
Controlexperimentscarriedoutoncompleteremovalofoxygenfromthebathbyaddingthecalculatedquantitiesoftheusualscavengernamelysodiumsulfitedidnotshowanyelectro-reductionpeakunderotherwisesimilarexperimentalconditionindicatingclearlythatthegenerationofH2O2isamustfortheformationofGSOHandGSSG.ThisphenomenonmimicstheconversionofGSHtoGSSGbyglutathione
metabolismenzymaticallywhichcausesoxidativestressinhuman[21].TheobservationofelectrochemicalconversionofGSHtoGSSGbytheelectro-generatedROSpromptedustoquantifyglutathioneconcentra-tionbyelectro-reductionofGSSGformedstoichiometricquantitiesattheinterface.Thisapproachofmimickingtheoxidativestresssignalselectrochemicallyandsubsequentrepairbyelectro-reductionisnotreportedintheliteraturetothebestofourknowledge.
Also,thekineticsandmechanismofchemicalconversionofGSHtoGSSGinthepresenceofH2O2wasstudiedindetailbyAbedinzadehetal.[19].Theauthorsclearlyshowedthespectralevidenceforthegen-erationofGSSG.WehavecarriedoutspectroelectrochemicalstudiestoidentifytheproductGSSGformedbyelectro-generatedROSatAusur-faceusingathinlayerspectroelectrochemicalcellunderpotentiostaticconditions.Atpotential−0.3V,(whichisenoughtoproducesufficientquantityofGSSGattheinterface)theabsorptionspectraexhibitedanabsorptionpeakat220nmindicatingtheformationofGSSGattheinterfaceasdepictedinFig.3.Theintensityoftheabsorbancehasshownanincreaseby30%,whenthespectraweretakenafterbiasingtheAuelectrodeat−0.3Vfor20min.Theseexperimentalresultsun-equivocallyprovethattheGSSGhasbeenchemicallygeneratedattheinterfacebychemicaloxidationofGSHbytheelectrogeneratedROS.2GSHþH2O2→GSSGþ2H2O
…
ð6Þ
BasedonthisobservationitislogicaltobelievethattheGSHandelectrochemicallygeneratedROScanleadtocatalyticreductionofoxygentowaterasshowninEq.(6).Inaddition,wehavetabulatedsomeoftheimportantcontributionsintheliteraturewhichdealwithelectrochemicalsensingofGSHusingdifferentelectrodemodifiersinTable1.Fromthecollecteddata,it'sclearthatthereportedmethodolo-gyofquantificationofGSHatAusurfaceisrelativelysuperiorcomparedtothepreviousworkreportedintheliterature.
4.Conclusions
Inconclusion,wehavedemonstratedsuccessfullytheelectrochem-icaldeterminationofGSHbyelectroreductionofGSSGbyROSspeciesgeneratedelectrochemicallydownto100nMconcentrations.ThismethodofquantifyingGSHisbasedonthestoichiometricconversionofGSSGattheinterfaceandgivestotalglutathione(GSH+GSSG).TwopathwaysforconversionofGSHtoGSSGattheinterfacedependingontheconcentrationofGSHinthemediumwereidentified.Hitherto,theelectroanalyticalmethodsdescribedintheliteratureyieldsthecon-centrationofGSHinthemedium.However,bycombiningthismethodwithotherestablishedmethodshasprimeconsequenceindeterminingtheratioofGSH/GSSGinbodyfluids.OurfutureeffortswillbefocusedondeterminingtheGSHinbodyfluidsinthepresenceofothercommoninterferingmoleculesusinggoldcoatedcarbonelectrodes.
G.Kalaiyarasanetal./ElectrochemistryCommunications56(2015)29–3333
Conflictofintereststatement
Theauthorsdeclaretheyhavenoconflictsofinterest.Acknowledgment
AuthorsthankCSIR-Moleculestomaterialstodevices(M2D,CSC-0134)networkprojectforthefinancialsupport.A.V.NarendrakumarthanksCSIR,NewDelhifortheawardofSeniorResearchFellowship.References
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