identification of secondary structure：二级结构的识别

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1、StructuralAnalysisofProteinStructureCircularDicroismFluorescenceX-rayNMRMethodsforSecondaryStructuralAnalysisAnumberofexperimentaltechniquescanselectivelyexaminecertaingeneralaspectsofmacromolecularstructurewithrelativelylittleinvestmentoftimeandsample.Reasonableestimatesofproteinsecondarys

2、tructurecontentandstructurechangecanbedeterminedempiricallythroughtheuseofCirculardichroism(CD)spectroscopyFluorescencespectroscopyNuclearMagneticResonance(NMR)spectroscopyFT-infraredspectroscopyCircularDichroismCirculardichroism(CD)spectroscopyisaformoflightabsorptionspectroscopythatmeasur

3、esthedifferenceinabsorbanceofright-andleft-circularlypolarizedlight(ratherthanthecommonlyusedabsorbanceofisotropiclight)byasubstance.ItismeasuredwithaCDspectropolarimeter.TheinstrumentneedstobeabletomeasureaccuratelyinthefarUVatwavelengthsdownto190-170nm(170-260nm).Thedifferenceinleftandrig

4、hthandedabsorbanceA(l)-A(r)isverysmall(usuallyintherangeof0.0001)correspondingtoanellipticityofafew1/100thofadegree.RotationofPlane-polarizedLightbyanOpticallyActiveSamplePockelscellproducesabeamthatisalternatelyswitchedbetweenLandR.Thebeamthenpassesthroughthesampletoaphotomultiplier.Thedet

5、ectedsignalcanthenbeprocessedasΔAvsλ.InstrumentationThemostcommoninstrumentsaroundarethecurrentlyproducedJASCO,JobinYvon,OLIS,andAVIVmodels.WehavetheJasco710and810modelswithtemperaturecontrollers.Theaircooled150WXenonlampdoesnotnecessitatewatercooling.Youstillneedtopurgewithamplenitrogentog

6、ettolowerwavelengths(below190nm).TypicalInitialConcentrationsProteinConcentration:0.5mg/ml(Theproteinconcentrationneedstobeadjustedtoproducethebestdata).CellPathLength:0.5-1.0mm.Ifabsorptionposesaproblem,cellswithshorterpath(0.1mm)andacorrespondinglyincreasedproteinconcentrationandlongersca

7、ntimecanbeemployed.Stabilizers(Metalions,etc.):minimumBufferConcentration:5mMoraslowaspossible,whilemaintainingproteinstability.AtypicalbufferusedinCDexperimentsis10mMphosphate,althoughlowconcentrationsofTris,perchlorateorborateisalsoacceptable.Asagenera