SMCC-结构反应解释

SMCC是一类含有N-羟基琥珀酰亚胺(NHS)活性酯和马来酰亚胺的双功能偶联剂.可以将分别含有巯基和氨基的化合物键接在一起。NHS活性酯与伯胺在PH7-9的环境形成酰胺键。马来酰胺与巯基在PH6.5-7.5的环境下形成稳定的硫醚键。在水溶液中，NHS活性酯的水解是（与氨基的反应）个竞争反应。马来酰胺比NHS稳定，但是在PH大于7.5时，马来酰胺会慢慢水解，失去与巯基反应的特异性。因而，在使用SMCC时通常是在PH7.2-7.5的环境下进行，并且先让NHS发生反应。 SMCC结构里的环己烷环可以降低马来酰胺的水解速率。这使得蛋白质在用SMCC修饰之后可以冻干存放一段时间。很多蛋白质都选用该试剂来进行马来酰亚胺修饰。 用SMCC来制备抗体-酶或者半抗原作载体的蛋白质，经常采用两步合成法。首先，含有氨基的蛋白质与几倍的偶联剂反应，反应结束后通过脱盐柱或者透析的方法除掉没有反应玩的SMCC。然后，再与含有巯基的蛋白质反应。在实际操作中要注意的是，SMCC怕潮湿，存放时要和干燥剂一起存放。并且使用中从冰箱拿出来时要先在室外放置一段时间平衡温度，以免立刻开启，空气中水分遇冷凝结，破坏SMCC结构。INSTRUCTIONSSMCC(succinimidyl4-N-maleimidomethylcyclohexane-1-carboxylate),50mgMolecularWeight:334.32SpacerArm:8.3NetMassAdded:219.09Storage:Uponreceiptstoredesiccatedat4C.Productisshippedatambienttemperature.Sulfo-SMCC(sulfosuccinimidyl4-N-maleimidomethylcyclohexane-1-carboxylate),1gSulfo-SMCC,50mgSulfo-SMCC,No-WeighFormat,82mgmicrotubesMolecularWeight:436.37SpacerArm:8.3NetMassAdded:219.09CAS#:92921-24-9Storage:Uponreceiptstoredesiccatedat-20C.Productisshippedatambienttemperature.IntroductionSMCCanditswater-solubleanalogSulfo-SMCCareheterobifunctionalcrosslinkersthatcontainN-hydroxysuccinimide(NHS)esterandmaleimidegroupsthatallowcovalentconjugationofamine-andsulfhydryl-containingmolecules.NHSestersreactwithprimaryaminesatpH7-9toformamidebonds,whilemaleimidesreactwithsulfhydrylgroupsatpH6.5-7.5toformstablethioetherbonds.Inaqueoussolutions,NHSesterhydrolyticdegradationisacompetingreactionwhoserateincreaseswithpH.ThemaleimidegroupismorestablethantheNHS-estergroupbutwillslowlyhydrolyzeandlosesitsreactionspecificityforsulfhydrylsatpHvalues7.5.Forthesereasons,conjugationswiththesecrosslinkersareusuallyperformedatpH7.2-7.5,withtheNHS-ester(amine-targeted)reactedbeforeorsimultaneouswiththemaleimide(sulfhydryl-targeted)reaction.Thecyclohexaneringinthespacerarmofthesereagentsdecreasestherateofhydrolysisofthemaleimidegroupcomparedtosimilarreagentsthatdonotcontainthisring.1Thisfeatureenablesproteinsthathavebeenmaleimide-activatedwithSMCCorSulfo-SMCCtobelyophilizedandstoredforlaterconjugationtoasulfhydryl-containingmolecule.Manymaleimide-activatedproteinproductsareproducedinthismanner(seeRelatedProducts).SMCCandSulfo-SMCCareoftenusedtoprepareantibody-enzymeandhapten-carrierproteinconjugatesinatwo-stepreactionscheme.First,theamine-containingproteinisreactedwithaseveral-foldmolarexcessofthecrosslinker,followedbyremovalofexcess(nonreacted)reagentbydesaltingordialysis;finally,thesulfhydryl-containingmoleculeisaddedtoreactwiththemaleimidegroupsalreadyattachedtothefirstprotein.Sulfo-SMCCissolubleinwaterandmanyotheraqueousbufferstoapproximately10mM,althoughsolubilitydecreaseswithincreasingsaltconcentration.SMCCisnotdirectlywater-solubleandmustbedissolvedinanorganicsolventsuchasdimethylsulfoxide(DMSO)ordimethylformamide(DMF);subsequentdilutionintoaqueousreactionbufferisgenerallypossible,andmostproteinreactantswillremainsolubleifthefinalconcentrationoforganicsolventislessthan10%.SMCCandSulfo-SMCCImportantProductInformationSMCCandSulfo-SMCCaremoisture-sensitive.Storereagentvialindesiccant.Equilibratevialtoroomtemperaturebeforeopeningtoavoidmoisturecondensationinsidethecontainer.Dissolveneededamountofreagentanduseitimmediatelybeforehydrolysisoccurs.Discardanyunusedreconstitutedreagent.Donotstorereagentinsolution.No-WeighMicrotubeHandling:Immediatelybeforeuse,puncturethemicrotubefoilwithapipettetip,add200lof50mMsodiumphosphatebuffer(pH7.0-7.5)orultrapurewaterandpipetteupanddowntomix.Afteruse,cuttheusedmicrotubefromthemicrotubestripanddiscard.Storetheunusedmicrotubesinthefoilpouchprovided.Note:Donotusephosphate-bufferedsaline(PBS)forinitialdissolutionofSulfo-SMCC;thereagentdoesnotdissolvewellinbuffersexceeding50mMtotalsalts.However,oncedissolved,thesolutioncanbefurtherdilutedinPBSorothernon-aminebuffers.Avoidbufferscontainingprimaryamines(e.g.,Trisorglycine)andsulfhydrylsduringconjugation,becausetheywillcompetewiththeintendedreaction.Ifnecessary,dialyzeordesaltsamplesintoanappropriatebuffersuchasphosphate-bufferedsaline(PBS).Moleculestobereactedwiththemaleimidemoietymusthavefree(reduced)sulfhydryls.ReducepeptidedisulfidebondswithImmobilizedTCEPDisulfideReducingGel(ProductNo.77712).Forproteins,reducedisulfidebondsusing5mMTCEP(1:100dilutionofBond-BreakerTCEPSolution,ProductNo.77720)for30minutesatroomtemperature,followedbytwopassesthroughasuitabledesaltingcolumn(e.g.,ZebaDesaltSpinColumns).Beawarethatproteins(e.g.,antibodies)maybeinactivatedbycompletereductionoftheirdisulfidebonds.Selectivereductionofhinge-regiondisulfidebondsinIgGcanbeaccomplishedwith2-MercaptoethylamineHCl(2-MEA,ProductNo.20408).SulfhydrylscanbeaddedtomoleculesusingN-succinimidylS-acetylthioacetate(SATA,ProductNo.26102)or2-iminothiolaneHCl(TrautsReagent,ProductNo.26101),whichmodifyprimaryamines.ProcedureforTwo-stepProteinCrosslinkingGenerally,a10-to50-foldmolarexcessofcrosslinkerovertheamountofamine-containingproteinresultsinsufficientmaleimideactivationtoenableseveralsulfhydryl-containingproteinstobeconjugatedtoeachamine-containingprotein.Morediluteproteinsolutionsrequiregreaterfoldmolarexcessofreagenttoachievethesameactivationlevel.Empiricaltestingisnecessarytodetermineoptimalactivationlevelsandfinalconjugationratiosfortheintendedapplication.A.MaterialPreparationConjugationBuffer:phosphate-bufferedsaline(PBS=100mMsodiumphosphate,150mMsodiumchloride,pH7.2;e.g.,ProductNo.28372)orotheramine-andsulfhydryl-freebufferatpH6.5-7.5(seeImportantProductInformation)addingEDTAto1-5mMhelpstochelatedivalentmetals,therebyreducingdisulfideformationinthesulfhydryl-containingproteinDesaltingcolumntoseparatemodifiedproteinfromexcesscrosslinkerandreactionbyproducts(e.g.,ZebaDesaltSpinColumns)Amine-containing(Protein-NH2)andsulfhydryl-containingproteins(Protein-SH)tobeconjugatedB.ProtocolNote:Forbestresults,ensurethatProtein-SHis