北大生物化學(xué)完整課件Chapter3 2

AminoAcids,PeptidesandProteins

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

Currentphylogenytreeoflife(byCarlWoese)

CharacteristictitrationcurvesofaminoacidsTheprincipalcomponentsaspectrophotometerLevelsofstructureinproteinsProteinsAndProstheticgroups(輔基)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(超速離心),anddefinedsedimentationcoefficient(s).e.g.Hemoglobin:The(odor)Svedberg

UppsalaUniversityBorn1884Ph.D.1908Professor1912Nobelprice1926蛋白質(zhì)可以通過(guò)各種生物化學(xué)技術(shù)純化利用蛋白質(zhì)的溶解度、凈電荷、大小以及與配體結(jié)合特異性上的微小差異。有透析、凝膠過(guò)濾、離子交換層析、親和層析、電泳（垂直板電泳、等電聚焦電泳、雙向電泳）等分離純化方法。透析Saltingoutanddialysis(透析)Columnchromatography

Ion-exchangechromatographySize-exclusionchromatographyHydrophobicinteractionchromatographyIsoelectricfocusingchromatography

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

分離氨基酸常用的是帶有耐酸性非常強(qiáng)的磺酸根SO3－Na＋（以鹽的形式出現(xiàn)）的強(qiáng)陽(yáng)離子交換樹(shù)脂。首先將這種樹(shù)脂填充到柱子中，然后注入含有樣品的流動(dòng)相，樣品中含有陽(yáng)離子成分X＋，通過(guò)靜電吸引，與樹(shù)脂中的帶電基團(tuán)相互作用，結(jié)果X＋與Na＋交換，即發(fā)生陽(yáng)離子交換后，形成SO3－X＋。

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

AffinityChromatography親和層析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