全 文 ：AffinityInteractionBetweenLysozymeand
HyprocrelinA:ElectronTransferand
FluorescenceQuench
SongKaixi1 , ZhouJiahong1 , WuXiaohong3 , ZhouLin1 , GuXiaotian1 ,
WeiShaohua1 , FengYuying1 , WangXuesong2
(1.Analysis＆TestingCenter, JiangsuEngineeringResearchCenterforBio-medicalFunctionMaterials,
NanjingNormalUniversity, Nanjing210097, China)
(2.TechnicalInstituteofPhysicsandChemistry, ChineseAcademyofSciences, Beijing100101, China)
(3.DepartmentofEngineeringandTechnologyJiangsuInstituteofEconomicTradeTechnology, Nanjing210007, China)
Abstract:BasedontheknownbindinginteractionbetweenlysozymeandhypocrellinA, aphotosensitizingdrugusedin
photodynamictherapy, andthefluorescencequenchingmechanismoflysozymebyhypocrellinAwasstudied.Theresults
indicatedthatthephotoinducedelectrontransferbetweenthetryptophanandtyrosineresiduesoflysozymeandhypocrel-
linAplaysanimportantroleinthisfluorescencequenchingprocess.
Keywords:hypocrellinA, lysozyme, bindinginteraction, photoinducedelectrontransfer
CLCnumberO641 DocumentcodeA ArticleID1001-4616(2008)02-0059-06
竹红菌甲素和溶菌酶之间的电子转移与荧光猝灭作用
宋开玺 1 ,周家宏 1 ,吴晓红 3 ,周 林 1 ,顾晓天 1 ,魏少华 1 ,冯玉英 1 ,王雪松2
(1.南京师范大学分析测试中心 ,江苏省生物功能材料重点实验室 ,江苏 南京 210097)
(2.中国科学院理化技术研究所 ,北京 100101)
(3.江苏经贸职业技术学院工程技术系 ,江苏 南京 210007)
[摘要 ] 在光动力疗法中 , 竹红菌甲素作为敏剂与溶菌酶发生了相互作用.本文对竹红菌甲素同溶菌酶作用的
荧光猝灭机理进行了研究.结果表明 , 竹红菌甲素与溶菌酶结构中的酪氨酸和色氨酸之间的光诱导电子转移机
制 , 在荧光猝灭过程中起了重要作用.
[关键词 ] 竹红菌甲素 , 溶菌酶 ,结合作用 , 光诱导电子转移
Receiveddate:2007-09-06.
Foundationitem:SupportedbytheNationalNaturalScienceFoundationofChina(20603018)andtheNaturalScienceFoundationofJiangsuEdu-
cationDepartment(04KJB150068).
Correspondingauthor:FengYuying, professor, majoredinfunctionalmaterialandinstrumentanalysis.E-mail:yyfeng3@ 163.com
Photodynamictherapy(PDT)isatreatmentthatinvolvesinjectionofphotosensitizingandtumor-localizing
dyesfolowedbyexposureofthetumorregiontohighfluenceratesoflight, usualyfromalaser[ 1] .4, 9-di-
hydroxyperylene-3, 10-quinone(HA, Fig.1), extractedfromHypocrelabambuase, hasbeenusedasaphoto-
therapeuticagenttocurevariousskindiseases, andtakenoralyasafolkmedicineforseveralcenturiesinChi-
na[ 2] .Recently, studiesshowthatthisnaturalperylenequinonoidcompoundalsohasantitumorandantiviralac-
tivities, includinghumanimmunodeficiencyvirus[ 3] .Itpossesesseveraladvantagesoverthepresentlyusedhe-
matoporphyrinderivatives(HPD), i.e.readypreparation, easypurificationrelativetoHPD, smalaggregation
tendency, strongredlightabsorptivityandhighquantumyieldsofsingletoxygen[ 4] .Inthephotodynamicthera-
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peuticprocedure, HAoritsanalogueswilbeinjectedintravenouslyintothepatients, thenincubationstepis
neededbeforethetreatmentwithlight.Previousstudiesshowthatduringthisincubationstep, HAwilbindwith
variousserumproteinssuchasalbumin, globulins, andlipoproteinstoformprotein-HAcomplex.Finalythe
bindingwilbegradualyreleasedineithervascularstromaorintercelularloci, suchaslysosomes, golgi, endo-
plasmicreticulum, andcelularmembranes[ 5] .ThisbindingafinityofHAtoproteinsinfluenceontransport, up-
take, andthedistributionofHAinvivo, aswelasthephotophysicalandphotochemicalcharactersofHA.It s
possibledeterminingthephotodynamicpropertiesofHASuchasHAS, C-PC[ 6] .Thesestudiesindicatedthat
HAcaninteractwiththeseproteins, andquenchthefluorescenceoftheseproteins.
Inthispaper, lysozymewaschosenasthetargetofHAduetoitswel-knownstructureandlowermolecular
weight.WefoundthatHAcanefectivelybindwithlysozymeandsignificantlyvarytheconformationoflysozyme,
inaddition, thefluorescenceoflysozymewasquenched.Basedontheseresults, thefluorescencequenching
mechanismwassystematicalydiscussed.
1 MaterialsandMethods
1.1 Materials
LysozymewastheproductofSigmaChemicalCompay(St.Lonis, Mo, USA)anddissolvedin0.04 mol/L
pH7.4 PBScontaining0.10 mol/LNaClandthefinalconcentrationofLysozymewas1.6×10-5 mol/L.HA
wasobtainedaccordingtotheproceduredescribedinliterature, andthepuritywasconfirmedas> 97% by
HPLC.Dimethylsulfoxide(DMSO)wasdriedbydistilationoverKOHpriortouse.2mmol/LHAaqueoussolu-
tionwaspreparedbyadding10mlDMSOsolutionsofHAto50mLdoubledistiledwater.
1.2 Spectroscopicmeasurements
UV-visibleabsorptionspectroscopywasperformedwithLambda17 UV/Visspectrophotometer(Perkin-El-
mer, USA), andabsorptionspectrumfrom210to310nmwasrecorded.Althefluorescencemeasurementswere
donewithaLS50 Bspectrofluorimeter(Perkin-Elmer, USA), andtheexcitationwasat286nm.Unlessother-
wisestated, incabatedfor40 minutes, andalexperimentswerecarriedoutatroomtemperature25℃.
1.3 Electronparamagneticresonance(EPR)measurements
EPRspectrawereobtainedusingaBrukerESP-300Espectrometeroperatingatroomtemperature, andthe
operatingconditionswereasfolows:microwavebridge:X-bandwith100Hzfieldmodulation;sweepwidth:
100Gmodulationamplitude:1.0G;modulationfrequency, 100kHz;receivergain:1×105;microwavepower:
5mW.SampleswereinjectedintothespecialymadequartzcapilariesforEPRanalyses, andpurgedwithar-
gon, airoroxygenfor40minutesinthedarkorderly, accordingtotheexperimentalrequirementsandiluminated
directlyinthecavityoftheEPRspectrometerwithaNd:YAGlaser(355nm, 5-6nsofpulsewidth, repetition
frequency:10Hz, 10mJ/pulse).ThekineticsofspinadductwerestudiedbyrecordingpeakheightsofEPR
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spectraevery30s.
2 ResultsandDiscussion
2.1 BindinginteractionbetweenlysozymeandHA
TheexperimentsoftheUV-visibleabsorptionspectroscopyandthefluorescencespectroscopywerecarried
outtostudytheinteractionbetweenlysozymeandHA.
Intheregionof210— 310nm, lysozymeandHAhavetwocharacteristicabsorptionbandswithabsorption
maximaat219nmand280nm, respectively(Fig.2).AfteradditionofdiferentconcentrationofHAtoaseries
oflysozymeaqueoussolution, notablechangesintheabsorptionspectrumoflysozymewereobserved.Theab-
sorptionpeaksat210nmshiftedtolongerwavelength, andtheintensitiesdecreasedgreatly, whiletheotherab-
sorptionpeaksat280nmshowednoobviouschanges.WhentheconcentrationofHAreached40μM, theab-
sorptionpeakat219nmshiftedto233 nm, andtheintensitydecreasedby72%(Fig.2).Accordingtothepre-
viousreports[ 7] , theabsorptionbandat280nmcanbeatributedtotheamidicacidresiduesinlysozyme, and
theabsorptionbandat219nmdependsstronglyontheconformationthatlysozymeadopts, e.g.thehelixoflyso-
zyme.Therefore, theseresultssuggestthatHAcanbindontolysozyme, andthisbindinginteractioncausesthe
conformationoflysozymetochangeremarkably.
Furthermore, thecontroledexperimentsshowthatDMSOpresentintheabove-mentionedsolutionsdidnot
afecttheabsorptionspectrumoflysozyme, whichisconsistentwelwiththereference[ 8] .
ThefluorescencespectraoflysozymewithvaryingconcentrationsofHAareshowninFig.3.Itisclearthat
HAcanquenchthefluorescenceoflysozymeefectively.Fig.4givestheStern-Volmerplotsforthefluorescence
quenchingoflysozymebyHAat25℃ and42℃, respectively.Boththestraightlineshavegoodlinearcorrela-
tionsof0.998 (25℃)and0.999 (42℃)(Fig.4).onthebaseofStern-Volmerequation[ 9] (equation1):
F0 /F=1 +Kqτ0 [ Q] . (1)
Inequation1, F0 andFarethefluorescenceintensitiesofthefluorophoreintheabsenceorpresenceofthe
quencherQ, τ0 isthesingletexcitedstatelifetimeofthefluorophoreintheabsenceofthequencher.Forbiopoly-
mers, τ0 of10-8 scanbeusedincalculation.Sothebimolecularquenchingconstants(Kq)arecalculatedtobe
6.43×1012 mol-1L· s-1(25℃)and5.75 ×1012 mol-1L· s-1(42℃), respectively.ThetwoKqarenearly
threeordersofmagnitudehigherthanthedifusionconstantofwater(7×109 mol-1Ls-1 at25℃ estimatedfrom
itsviscosity[ 10] ), indicatingthatthefluorescencequenchingmayresultfromthestaticinteractionduetothe
bindingofHAontolysozyme.Thestaticquenchingmechanismisinlinewiththeobservedsmalerquenching
constantatelevatedtemperature(42℃ vs25℃).Generaly, bimolecularquenchingconstantwilincreasewith
theincreaseofthetemperaturewhenquenchingisdynamic, i.e., relyingonthedifusioncolisionbetweenex-
citedfluorophoreandquencher.Incontrast, elevatedtemperaturefavorsthedissociationofthecomplexformed
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non-covalently, e.g.bywayofhydrogenbond, electrostaticatraction, orhydrophobicforce, thus, aleviates
thefluorescencequenchingoriginatedfromthecomplexformationbetweenfluorophoreandquencher.Thecon-
clusionisconsistentwiththeresultsofUV-visibleabsorptionspectroscopy.
2.2 Fluorescencequenchingmechanism
Fluorescenceemissionsofproteinsmainlyoriginatefromtryptophanresiduesandtyrosineresidues, andtheir
emissionbandsoverlapcloselyinthenormalfluorescencespectrum, thus, toknowthefluorescencequenching
objectofHAinthisfluorescencequenchingprocess, thesynchronousfluorescenceexperimentswerecarriedout.
Insynchronousfluorescencespectrum, whenthewavelengthinterval(Δλ)betweentheexcitationandemission
wavelengths(λexandλem)issettobe20nm, theobtainedemissionpeakcanbeascribedtotyrosineresidues.
Incontrast, whenthevalueofΔλis80nm, theobtainedemisionpeakcanbeatributedtotryptophanresi-
dues[ 11] .
Fig.5 showsthesynchronousfluorescencespectraoflysozymeinthepresenceofvariedconcentrationsofHA
withΔλ=20nm(A)or80nm(B).IntheabsenceofHA, theemissionmaximumoftyrosineresidueslocates
at314nm, whilethatoftryptophanresiduesisat349nm.UponadditionofHA, boththeemissionbandsde-
creasedmarkedlyinintensity, andthepeakpositionsredshiftedslightly.Itiswelknownthattheemissionmax-
imaoftyrosineresiduesortryptophanresiduesinproteinsdependgreatlyonthemicroenvironmentssurrounding
them.Theredshiftoftheemissionmaximasuggestsachangeofmicroenvironmentfromhydrophobictohydro-
philic, astheresultoftheconformationvariationofproteins.Inourexperimentalconditions, theemissionpeak
oftryptophanresiduesshiftedbyabout6nmuponadditionofHA, from349nmto355nm, veryclosetotheval-
ueinthetryptophanaqueoussolution, indicatingthattheinteractionsbetweenHAandlysozymemadethetrypto-
phanresiduesexposedalmostcompletelytotheaqueousmedium.Thesefindingsareingoodagreementwiththe
remarkablechangesoftheshort-wavelengthabsorptionbandoflysozymeafterbindingbyHA(Fig.2), asthere-
sultofconformationchangeoflysozyme.
BoththenormalandsynchronousfluorescencespectrashowthatthebindingofHAquenchedthefluores-
cenceemissionoflysozymeeficientlythroughformingthecomplexbetweenlysozymeandHA, butthefluores-
cencequenchingmayeitherviaenergytransfermechanismorelectrontransfermechanismorbothofthem, so
furtherexperimentsarenecessary..
Toexaminetheposibilityofenergytransfer, wemeasuredthefluorescencespectraofHAuponadditionof
lysozyme, inwhichtheexcitationwavelengthwassetat286 nm.Itwasfoundthatthefluorescenceintensityof
HAdecreasedwiththeincreaseoftheconcentrationoflysozyme(Fig.6).Thisindicatesthattheenergytransfer
fromtheexcitedlysozymetoHA, ifany, didnotplayanimportantroleintheinteractionsbetweenHAandlyso-
zyme.
Incontrast, thephotoinducedelectrontransferbetweenHAandlysozymeisevident.Fig.7showstheEPR
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spectraoftheiradiatedHAsolutionswithorwithoutthepresenceoflysozyme.Iradiationofthedeaeratedaque-
oussolutionofHA(20μM)byaNd:YAGlaserat355nmfor1mingaveanEPRsignal(spectrumb, Fig.7),
whichcanbereasonablyatributedtothesemiquinoneanionradicalofHA(HA-.)bycomparisonwithourpre-
viousstudy[ 12] .ThesemiquinoneanionradicalofHAisbelievedtobegeneratedbytheelectrontransferbetween
anexcitedHAwithagroundstateHA(equation2).
HA＊ +HA※ HA-.+HA+.. (2)
WhenlysozymewasaddedintothedeoxygenatedaqueoussolutionofHA, theintensityoftheHA-.signal
wasenhancedsignificantly(spectruma, Fig.7), Thisimplieslysozymemayserveaselectrondonortofavorthe
formationofHA-..
Theoxidationpotentialsoftryptophanandtyrosineare0.88Vand0.93VvsNHE, respectively[ 13] , and
thereductionpotentialofHAis-0.37VvsNHE[ 14] .Thus, basedontheirabsorptionedges(313nmfortrypto-
phan, 290nmfortyrosine, and640nmforHA)[ 15] , theoxidationpotentialsoftheexcitedtryptophanandtyro-
sineareestimatedtobe-3.08Vand-3.35VvsNHErespectively, andthereductionpotentialoftheexcited
HAis1.57VvsNHE.AccordingtoRehm-Welerequation[ 16] ,
ΔG=Eox(donor)-Ered(acceptor)-E0, 0(excitedstateenergy). (3)
thefreeenergychangeofthephotoinducedelectrontransfer
procescanbeestimated.Whenthephotoinducedelectron
transferoccurredbetweentheexcitedHA andlysozyme,
G(Trp)is-15.9kcal/mol, G(Tyr)is-14.8kcal/mol, re-
spectively.Ontheotherhand, whenthephotoinducedelec-
trontransferoccuredbetweentheexcitedlysozymeandHA,
G(Trp)is-62.5kcal/mol, G(Tyr)is-68.7kcal/mol, re-
spectively.Fromthesevalues, itcanbeknownthatthephoto-
induecdelectrontransferfromtryptophanortyrosinetoHAwil
beathermodynamicfavorableprocessnomaterwhetherHAor
lysozymeisexcited(Fig.8).Inaddition, theparticipationoftryptophanresiduesandtyrosineresiduesinthe
photoinducedelectrontransferbetweenlysozymeandHAagreeverywelwiththefactthattheirfluorescencee-
missionswerequenchedefectivelybyHA(Fig.5).
Accordingtotheaboveanalysis, thephotoinducedelectrontransferbetweenlysozymeandHAcanbeilus-
tratedasfolowingprocess.
1Trp hv 1Trp＊ ,
1Trp＊ +HA※[ TrpH.+HA.- ] ※TrpH.++HA.-,
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SongKaixi, etal:AfinityInteractionBetweenLysozymeandHyprocrelinA:ElectronTransferandFluorescenceQuench
1Trp＊※3Trp＊ ,
3Trp＊ +HA※Trp.++HA.-.
TheHA.- isformedduetothephotoinducedelectrontransferfromtryptophanresiduesofproteintoHAei-
therinsingletorintripletstate.TheTrp.+aregeneratedpredominantlythroughelectrontransferratherthanen-
ergytransfer.
3 Conclusions
Insummary, theexperimentalresultsindicatethatHAcanefectivelybindonlysozyme, whichisdriven
mainlybyhydrophobicinteractions, andcausethefluorescenceoflysozymetobequenched.Inaddition, Based
ontheabovediscussions, itisveryclearthatthefluorescencequenchingmechanismisthephotoinduecdelectron
transferfromtryptophanortyrosineresiduesoflysozymetoHAnomaterwhetherHAorlysozymeisexcited.
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