海藻糖对重组人铜锌SOD酶的保护作用

SOD(,,200237)rhCuZn2SOD,,5%10%(w/v)ProtectiveEffectofTrehaloseonRecombinantHumanCopperandZincSu2peroxideDismutase(rhCuZn2SOD)GEYu,LIULing,GAOJunjieetal(StateKeyLaboratoryofBioreactorEngineering,CollegeofBioengineering,EastChinaUniversityofScienceandTechnology,Shanghai200237)AbstractObjectiveToexploretheinfluenceofvarioussaccharidesontheresidualactivityofre2combinanthumancopperandzincsuperoxidedismutase(rhCuZn2SOD)duringtreatmentbyvariousprocesses.MethodsMixrhCuZn2SODandvariousdisaccarideswell,andobservetheactivitiesoftheenzymebeforeandaftertreatmentbyvariousprocesses,suchascoldstorage,repeatfreezingandthawing,vacuumdrying,lyophilization.ResultsComparedwithothersaccharides,trehaloseshowedgoodprotectiveeffectagainstfreez2inganddehydration.Theprotectiveeffectwasrelatedtoprocessatacertainextent.Theoptimalconcentrationoftrehalosewas5%210%(w/v).ConclusionThestudylaidafoundationofdesigningtheoptimalprotectivesystemofbiologicalactivity.KeywordsTrehaloseProtectiveeffectSuperoxidedismutaseEnzymeactivity(Superoxidedismutase,SOD),O2rhCuZn2SOD,,rhCuZn2SOD,,,,,,rhCuZn2SOD,,11,,,98%rhCuZn2SOD,,,21,10mmol/LpH7.4PBS,,,,pH7.4:242003161ChinJBiologicals2003,Vo1.16.No.131SOD[1]25,1ml50%141Lowry[2],514,61-25,25,3,71,,3048h81,24h,2%914(Tris2HClPBSNa2HPO42),,1013760,372,6011SODrhCuZn2SOD,,,,25d,,75%,121SOD,,380%,2,,1SODFig1.ActivityofSODstoredinliquidform2Fig2.InfluenceofsaccharideonactivityofrhCuZn2SODduringrepeatfreezingandthawing31SOD,,,,141SOD,100%,SOD,2342003161ChinJBiologicals2003,Vo1.16.No.11Tab1.InfluenceofvaccumdryingonactivityofrhCuZn2SODandeffectofprotectiveagentsProtectiveagentBlankTrehaloseSucroseLactoseMaltoseGlucoseActivityremainrate(%)75.185.3981.3576.9483.9880.962Tab2.InfluenceoflyophilizationonactivityofrhCuZn2SODandeffectofprotectiveagentsProtectiveagentBlankTrehaloseSucroseLactoseMaltoseGlucoseActivityremainrate(%)79.7399.8887.0680.8687.0675.9951SOD,,,5%10%,33Tab3.InfluenceofbuffersonactivityofrhCuZu2SODBufferTris2HClPBSBoricacid2sodiumborateNa2HPO42citricacid10mmol/Lactivitymaintainrate80.7680.9410094.220mmol/Lactivitymaintainrate87.5788.3498.521003SODFig3.InfluenceoftrehaloseconcentrationonSODactivity61hCu2ZnSODSOD,4,Na2HPO42,171SOD,rhCuZnSOD372,70%,60160%,45,437Fig4.ActivityofrhCuZn2SODstoredat37560Fig5.ActivityofrhCuZn2SODstoredat60442003161ChinJBiologicals2003,Vo1.16.No.1,,,,,,[3],,[4],,,Tadanori[5],,2,,,,,ATR2FTIRSOD,,2,,,[6][7],,,,,,,,,,rhCuZn2SOD,,,[8][1],,..,1988,19(5):217.[2]LowryOH,RoscbroughNJ,FarrA,etal.Proteinmeasurementwiththefolinphenolreagent.JBiolChem,1954,193,265-275.[3]Sola2PennaM,Meyer2FernandesJR.Stabilizationagainstthermalinacti2vationpromotedbysugarsonenzymestructureandfunction:whyisthre2halosemoreeffectivethanothersugars.ArchBiochemBiophy,1998,360(1):10-14.[4],,,..,2001,30(1):4446.[5]TadanoriS,TakehikoG,YoshiyasuA.Growthrateandmorphologyoficecrystalsgrowinginasolutionoftrehaloseandwater.JCrystalGrowth,2002,240(1-2):218-229.[6]CroweJH.Anhydrobiosis:Anunsolvedproblem.AmeNaturalist,1971,105:563-573.[7]LevineH,SladeL.Anotherviewoftrehalosefordryingandstabilizingbi2ologicalmolecules.Biopharm,1992,5:36-40.[8]SunWQ,DavidsonP.Proteininactivationinamorphoussucroseandtre2halosematrices:effectsofphaseseparationandcrystallization.BiochemBiophyActa,1998,1425:235-244.(:2002-04-22,:2002-05-21)542003161ChinJBiologicals2003,Vo1.16.No.1