科大高分子四聚合物在生物高分子分離中的應(yīng)用1課件

２．聚合物在蛋白質(zhì)分離中的應(yīng)用蛋白質(zhì)是由氨基酸通過酰胺鍵連接而成的高分子，兩性，存在等電點(diǎn)，因其空間結(jié)構(gòu)的復(fù)雜性，有親水或疏水之分。蛋白質(zhì)在低于等電點(diǎn)時(shí)會(huì)帶上正電荷，在高于等電點(diǎn)時(shí)帶負(fù)電荷．若緩沖溶液的pH值低于蛋白質(zhì)等電點(diǎn)，蛋白質(zhì)則吸附H＋而帶正電荷，由于靜電相互作用，蛋白質(zhì)與管壁發(fā)生吸附。（a）堿性蛋白質(zhì)（pI＝8）所帶電荷量隨pH的變化曲線；（b）在pH＝4時(shí)，堿性蛋白質(zhì)（pI＝8）與毛細(xì)管壁發(fā)生吸附１.極端pH值法

pH高于蛋白質(zhì)的pI時(shí)，毛細(xì)管內(nèi)壁對(duì)蛋白質(zhì)產(chǎn)生靜電排斥抑制吸附，但是，蛋白質(zhì)存在的自然狀態(tài)為pH4－10，高pH如11.0時(shí)，容易使蛋白質(zhì)變性或者水解。pH小于1.5（熔融硅或石英的等電點(diǎn)）時(shí)，蛋白質(zhì)的質(zhì)子化導(dǎo)致其質(zhì)荷比差別較小，分辨率難以提高。對(duì)等電點(diǎn)相近的蛋白質(zhì)不易實(shí)現(xiàn)分離。一般認(rèn)為，使蛋白質(zhì)混合物分離的首選pH值應(yīng)和蛋白質(zhì)混合物的pKa值基本一致。McManigillD.AnalChem,1986，58：166～170.(2)物理吸附的毛細(xì)管涂層目前使用的聚合物：中性的聚合物－已經(jīng)廣泛用于DNA和其它一些生物大分子的分析，但是由于蛋白質(zhì)的吸附作用，使得對(duì)蛋白質(zhì)的分析仍有一定難度。親水性的聚合物－如甲基纖維素，聚糖等不能很好的吸附在管壁上，穩(wěn)定性差。帶疏水基團(tuán)的聚合物－聚乙烯基吡咯烷酮，聚N,N-二甲基丙烯酰胺可以形成穩(wěn)定的涂層，但是由于疏水基團(tuán)與蛋白質(zhì)之間的相互作用使得分離效率下降。MadabhushiRS.Electrophoresis，1998，19：224～230.VerzolaB,GelfiC,RighettiPG.JChromatogrA，2000，874：293～303.羥乙基纖維素的改性

Formationofthecat-HEC

Schematicdiagramofadsorptionofcat-HEContosilicasurface.(1)Cat-HECNo.HEC(g)Glycidyltrimethylamminiumchloride(g)Viscosity(mp.s)Nitrogencontent(%)Cat-HEC-110.02.53800.8Cat-HEC-210.05.03751.3Cat-HEC-310.07.53701.7PreparationandpropertiesofCat-HECElectroosmoticflowasafunctionofpH.Comparisonbetweenabarefused-silicacapillary,HECandcat-HECcoatedcapillary

Electropherogramsofamixtureofstandardproteins.Separationswerecarriedoutusingcat-HEC-2coatedcapillary1=Lysozyme;2=CytochromeC;3=RibonucleaseA.ElectropherogramsofamixtureofstandardproteinsatpH4.6.ProteinBarecapillaryN(plate/m)bHECN(plate/m)RSD(%)Cat-HEC-2N(plate/m)RSD(%)CytochromeC93001040003.521860000.86Lysozyme--1620002.452650000.85RibonucleaseA173001800003.562820000.96Electrophoresis,

2008,29,1460-1466.ElectropherogramsofamixtureofstandardproteinsindifferentpH.Separationswerecarriedoutusingcat-HEC-2coatedcapillary1=Lysozyme;2=CytochromeC;3=RibonucleaseA.Migration

timereproducibility(n=3)andpeakefficiencyofproteinsseparatedinpolymer-coatedcapillariesatpH4.6(２)HEC-g-P4VPFormationoftheHEC-g-P4VPElectroosmoticflowasafunctionofpH.Comparisonbetweenabarefused-silicacapillary,HECandHEC-g-P4VPcoatedcapillaryElectropherogramsofamixtureofstandardproteins.Separationswerecarriedoutusingcat-HEC-g-P4VPcoatedcapillary.1=Lysozyme;2=CytochromeC;3=RibonucleaseA.２．結(jié)構(gòu)規(guī)整的聚合物PEO-b-P4VPNOOBrOOOONN+BrsamplesMn

1HNMRa)(×10-4)Mn，GPCb)(×10-4)Mw/Mn(GPC)PEO113-b-P4VP450.971.41.21PEO113-b-P4VP901.442.01.29PEO113-b-P4VP1131.692.41.26PEO113-b-P4VP2943.595.21.35a.MnofPEO-b-P4VPestimatedby1HNMR；b.MnofPEO-b-P4VPdeterminedbyGPC.FormationofthePEO-b-P4VP

1HNMRandGPCdataofPEO-b-P4VPcopolymers

EffectofmolecularweightofP4VPblockontheseparationofbasicproteinsatpH4.6.1,lysozyme;2,cytochromec;3,ribonuclease.(A)Typical

TEMimagesof(a)PEO113-b-P4VP45,(b)PEO113-b-P4VP90,(c)PEO113-b-P4VP113,and(d)PEO113-b-P4VP294;(B)Schematicillustrationofcopolymersofdifferentmorphologiescapillarycoatingprocedure(a)PEO113-b-P4VP45,(b)PEO113-b-P4VP90,(c)PEO113-b-P4VP113,and(d)PEO113-b-P4VP294

AnalysisofsalivasamplesbyPEO113-b-P4VP294coatedcapillary.Salivasamplesweredilutedto2-foldusingdeionizedwater.Samples(A)withoutand(B)withspikingof0.2mg/mLlysozymewereinjected.Separationconditions:40mMphosphate-citratebuffer,pH5.1;500V/cm;40cmcapillary(30cmtothedetector);temperature25℃.EffectofbufferpHontheseparationofbasicproteins.separationsweretakeninPEO113-b-P4VP294coatedcapillary

Electropherogramsofplasmasampleinbarecapillary(A)andaPEO113-b-P4VP294coatedcapillary(B).Separationbuffer:19mMNaOH-Na2B4O7atpH9.7.HSA,humanserumalbumin;α1-AT,α1-antitrypsin;α2-M,α2-macroglobulin;β-lp,β-lipoprotein;Tf,transferrin;IgA,immunoglobulinA;IgG,immunoglobulinG.Electrophoresis2008,29,2812-2819

3.多功能分離介質(zhì)的合成及應(yīng)用研究(1)HEC-g-PDMA研究背景能進(jìn)行DNA、蛋白質(zhì)、氨基酸、同分異構(gòu)體等的分離SeparationofΦ×174/HaeIIIdigestbyCEwithHEC-g-PDMAat(a)1.0%w/v;(b)1.5%w/v;(c)2.0%w/vEffectofpHvaluesonseparationsofbasicproteinsusingHEC-g-PDMAcoatedcapillary.1,lysozyme;2,cytochromec;3,ribonucleaseA.Electrophoresis,

2008,29,4351–4354.

(2)quasi-interpenetratingnetworkofLPAandPDMA

Propertiesofquasi-IPNcontainingLPAwithdifferentmolecularmassesPolymerMvofLPA(MDa)IntrinsicviscosityofLPA(mL/g)MolarratioofAM/DMAIntrinsicviscosityofIPN(mL/g)quasi-IPN-10.2510618.6:188quasi-IPN-20.251065.1:1145quasi-IPN-30.471686.8:1167quasi-IPN-40.471682.6:1266quasi-IPN-50.922786.5:1260quasi-IPN-64.609288.4:1840

EOFasafunctionofpH.Electropherogramsofamixtureofstandardproteins.

SeparationofΦ×174/HaeIIIdigestbyCEwithquasi-IPN-3at(a)2%w/v;(b)3%w/v;(c)4%w/v;(d)4%w/v.Conditions:Separationelectricfieldstrengthof(a),(b),and(c)is–6kV,separationelectricfieldstrengthof(d)is–8kV.Separationofbasicproteinsusingquasi