北大生物化学完整课件Chapter3 2

AminoAcids,PeptidesandProteins

氨基酸、肽及蛋白质Nelson,D.L.,andCox,M.M.(2005)LehningerPrinciplesofBiochemistry,4thedition.ProteinsequencesandEvolution！IntroductiontoBioinformatics:Sequencealignment;Homologs;Paralogs;orthologs;Blosum(blockssubstitutionmatrix);Signaturesequences;

Currentphylogenytreeoflife(byCarlWoese)

CharacteristictitrationcurvesofaminoacidsTheprincipalcomponentsaspectrophotometerLevelsofstructureinproteinsProteinsAndProstheticgroups(辅基)AnalyticalversusPreparative;Sourcesofproteins:Blood(serum);Tissuecellsandmicrobialcells;Extraction,fractionation,separationandpurification.

WorkingwithProteinsMethodsforseparatingproteinstakeadvantageofthephysicalpropertiessuchascharge,size,andsolubility,whichvaryfromoneproteintothenext.Becausemanyproteinsbindtootherbiomolecules,proteinscanalsobeseparatedonthebasisoftheirbindingproperties.

ProteinscanbeseparatedandpurifiedCentrifugation;Electrophoresis;LiquidChromatography;Edmandegradation;Massspectrometry;

OtherPhysicalmeans:X-ray,NMR,ElectronMicroscopy;lightscatteringmethods;differentspectrophotometries;Thermomeasurements;etc…ManywaystoworkwithProteinsbasedontheirphysicalandchemicalproperties

Svedbergstudiedproteinswiththemethodsofultra-centrifugation(超速离心),anddefinedsedimentationcoefficient(s).e.g.Hemoglobin:The(odor)Svedberg

UppsalaUniversityBorn1884Ph.D.1908Professor1912Nobelprice1926蛋白质可以通过各种生物化学技术纯化利用蛋白质的溶解度、净电荷、大小以及与配体结合特异性上的微小差异。有透析、凝胶过滤、离子交换层析、亲和层析、电泳（垂直板电泳、等电聚焦电泳、双向电泳）等分离纯化方法。透析Saltingoutanddialysis(透析)Columnchromatography

Ion-exchangechromatographySize-exclusionchromatographyHydrophobicinteractionchromatographyIsoelectricfocusingchromatography

Affinitychromatography:Antibodies,His-tags,Protein-A,Protein-G,GST-,MBP-fusionproteinsetc…,Ion-exchangeChromatography离子交换

分离氨基酸常用的是带有耐酸性非常强的磺酸根SO3－Na＋（以盐的形式出现）的强阳离子交换树脂。首先将这种树脂填充到柱子中，然后注入含有样品的流动相，样品中含有阳离子成分X＋，通过静电吸引，与树脂中的带电基团相互作用，结果X＋与Na＋交换，即发生阳离子交换后，形成SO3－X＋。

Size-exclusionChromatography分子筛Thismethodseparatesproteinsaccordingtosize.Thecolumncontainsacross-linkedpolymerwithporesofselectedsize.Largerproteinsmigratefasterthansmallerones,becausetheyaretoolargetoentertheporesinthebeadsandhencetakeamoredirectroutethroughthecolumn.Thesmallerproteinsentertheporesandareslowedbythemorelabyrinthianpaththeytakethroughthecolumn.

AffinityChromatography亲和层析Affinitychromatographyseparatesproteinsbytheirbindingspecificities.Theproteinsretainedonthecolumnarethosethatbindspecificallytoaligandcross-linkedtothebeads.(Inbiochemistry,theterm"ligand"isusedtorefertoagroupormoleculethatisbound.)Afternonspecificproteinsarewashedthroughthecolumn,theboundproteinofparticularinterestiselutedbyasolutioncontainingfreeligand.

Isoelectricfocusing

Isoelectricfocusingisaprocedureusedtodeterminetheisoelectricpoint(pI)ofaprotein.ApHgradientisestablishedbyallowingamixtureoflowmolecularweightorganicacidsandbasestodistributethemselvesinanelectricfieldgeneratedacrossthegel.Whenaproteinmixtureisapplied,eachproteinmigratesuntilitreachesthepHthatmatchesitspI.Proteinswithdifferentisoelectricpointsarethusdistributeddifferentlythroughoutthegel.AmershamBiosciencesAKTApurifierQuantificationofprotein,anEnzyme:ActivityversusspecificactivityProteinscanbecharacterizedbyelectrophoresisInadditiontochromatography,anotherimportantsetofmethodsisavailablefortheseparationofproteins,basedonthemigrationofchargedproteinsinanelectricfield,aprocesscalledelectrophoresis.Electrophoresisisespeciallyusefulasananalyticalmethod.Itsadvantageisthatproteinscanbevisualizedaswellasseparated,permittingaresearchertoestimatequicklythenumberofproteinsinamixtureorthedegreeofpurityofaparticularproteinpreparation.Also,electrophoresisallowsdeterminationofcrucialpropertiesofaproteinsuchasitsisoelectricpointandapproximatemolecularweight.ArneWilhelmKaurinTiseliusUppsalaUnuversityBorn1902Ph.D.1930Prof.1938Nobelprice1948Tiseliusdevelopedthemethodsofelectrophoresis

sepratingandpurifyingproteins.

20vg10vg5vg2vgM1vg500ng200ng100ng50ng20ngBSACoomassiebluestainingSensitivityofsilverstain:~3ngonBSA300ng30ng3ng1ngBSAMark1vl5vl66.2KDSDS(SDS-PolyAcrylamideGelElectrophoresis)Isoelectricfocusing

Isoelectricfocusingisaprocedureusedtodeterminetheisoelectricpoint(pI)ofaprotein(Fig.6-6).ApHgradientisestablishedbyallowingamixtureoflowmolecularweightorganicacidsandbases(ampholytes;seep.118)todistributethemselvesinanelectricfieldgeneratedacrossthegel.Whenaproteinmixtureisapplied,eachproteinmigratesuntilitreachesthepHthatmatchesitspI.Proteinswithdifferentisoelectricpointsarethusdistributeddifferentlythroughoutthegel.FrederickSangerCam