Applications of Fluorescence Correlation Spectroscopy Measurement of Size-Mass Relationship of Native and Denatured Schizophyllan.pdf

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1、XiaojingLengKonstantinStarchevApplicationsofFluorescenceJacquesBuf¯eCABE(AnalyticalandCorrelationSpectroscopy:BiophysicalEnvironmentalChemistry),MeasurementofSize±MassDepartmentofInorganic,RelationshipofNativeandAnalyticalandAppliedChemistry,Denature

2、dSchizophyllanUniversityofGeneva,ScienceII,30QuaiErnestAnsermet,1211Geneva4,SwitzerlandReceived20June2000;accepted8March2001Publishedonline00Month2001Abstract:Diffusiondynamicsofapolysaccharide,schizophyllanhasbeenstudiedby¯uorescencecorrelationspect

3、roscopy(FCS).Severaldifferentsizesofnondenaturedanddenaturedschizophyl-lanhavebeenlabeledwithrhodamine6Ginboratebuffer.ThelengthofthenondenaturedschizophyllanwascalculatedfromFCSdatabyusingtheBroersma'srelationshipforrod-likemacromolecules.Theobtaine

4、dlengthwasclosetothatobtainedbyatomicforcemicroscopy(AFM)measurements.Denaturedschizophyllanpossessesarandomcoilconformation.ItshydrodynamicradiusRhwasmeasuredbyFCS.TherelationshipbetweenRhandthemolecularmassMhasbeen0.59studiedandthescalingrelationsh

5、ipRh;Mhasbeenobtained,whichisinagreementwiththerandomcoilmodelwithexcludedvolumeeffect.ThepersistencelengthqdenatofthedenaturedschizophyllanwasdeterminedbyHearst'srelationship,tobeequalto5.1660.75(nm).TheworkdemonstratestheutilityofFCSmethodfordynami

6、csinvestigationsofbiopolymersespeciallyin28dilutedregime(concentrationlowerthan10Mcouldbemeasured)whereothertechniquescouldnotbeused.©2001JohnWiley&Sons,Inc.Biopolymers59:290±299,2001Keywords:¯uorescencecorrelationspectroscopy;schizophyllan;rhodamine

7、6G;diffusion;nat-uralpolysaccharide;polydispersity;denaturation;randomcoilconformation;triple-helicalconformation4±16INTRODUCTIONandsincethenithasbeensubstantiallydeveloped.ThismethodprovidesthecharacteristicdiffusiontimeThebasicconceptsof¯uorescence

8、correlationspec-andtheaveragenumberof¯uorescentmoleculesin1±3troscopy(FCS)methodwas®rstreportedin1974theconfocalvolumeby®ttingtheautocorrelationCorrespondenceto:XiaojingLeng;email:xiao.leng@cabe.unige.chContractgrantsponsor:SwissNationalFoundationCon