(Dap22)-ShK(ShKDap22)peptideisasyntheticderivativeofthewell-knownShKtoxin#08SHK001(Stichodactylahelianthusneurotoxin)isolatedfromthevenomoftheCarribeanseaanemoneStoichactishelianthus.Wild-typeShKblockspotentlyKv1.3(KCNA3),Kv1.1(KCNA1),Kv1.4(KCNA4),andKv1.6(KCNA6)respectivelywithaKdof11pM,16pM,312pMand165pM.In(Dap22)-ShK,lysine22hasbeenreplacedbyadiaminopropionicacid(Dap)residuethatgreatlyimprovestheselectivityofthepeptideforthevoltage-gatedpotassiumchannelKv1.3(IC50around23pM)againstKv1.1(1.8nM),Kv1.4(37nM)andKv1.6(10nM)channels.Thehighselectivityof(Dap22)-ShkisachievedthankstothestrongbindingbetweentheDapandHis404/Asp386residuesofKv1.3channel.(Dap22)-ShKinhibitsTcellproliferationinducedbyanti-CD3atsubnanomolarconcentrations.
Description:
AAsequence:Arg-Ser-Cys3-Ile-Asp-Thr-Ile-Pro-Lys-Ser-Arg-Cys12-Thr-Ala-Phe-Gln-Cys17-Lys-His-Ser-Met-Dap-Tyr-Arg-Leu-Ser-Phe-Cys28-Arg-Lys-Thr-Cys32-Gly-Thr-Cys35-OH
Disulfidebonds:Cys3-Cys35,Cys12-Cys28andCys17-Cys32
Length(aa):35
Formula:C166H268N54O48S7
MolecularWeight:4012.8Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:220384-25-8
Source:Synthetic
Purityrate:>97%
Reference:
HypoxiamodulatesearlyeventsinTcellreceptor-mediatedactivationinhumanTlymphocytesviaKv1.3channels
Tlymphocytesareexposedtohypoxiaduringtheirdevelopmentandwhentheymigratetohypoxicpathologicalsites.AlthoughithasbeenshownthathypoxiainhibitsKv1.3channelsandproliferationinhumanTcells,themechanismsbywhichhypoxiaregulatesTcellactivationarenotfullyunderstood.HereinwetestthehypothesisthathypoxicinhibitionofKv1.3channelsinducesmembranedepolarization,thusmodulatingtheincreaseincytoplasmicCa2+thatoccursduringactivation.HypoxiacausesmembranedepolarizationinhumanCD3+Tcells,asmeasuredbyfluorescence-activatedcellsorting(FACS)withthevoltage-sensitivedyeDiBAC4(3).SimilardepolarizationisproducedbytheselectiveKv1.3channelblockersShK-Dap22andmargatoxin.FurThermore,pre-exposuretosuchblockerspreventsanyfurtherdepolarizationbyhypoxia.SincemembranedepolarizationisunfavourabletotheinfluxofCa2+throughtheCRACchannels(necessarytodrivemanyeventsinTcellactivationsuchascytokineproductionandproliferation),theeffectofhypoxiaonTcellreceptor-mediatedincreaseincytoplasmicCa2+wasdeterminedusingfura-2.HypoxiadepressestheincreaseinCa2+inducedbyanti-CD3/CD28antibodiesinapproximately50%oflymphocytes.Intheremainingcells,hypoxiaeitherdidnotelicitanychangeorproducedasmallincreaseincytoplasmicCa2+.Similareffectswereobservedinrestingandpre-activatedCD3+cellsandweremimickedbyShK-Dap22.TheseeffectsappeartobemediatedsolelybyKv1.3channels,aswefindnoinfluenceofhypoxiaonIKCa1andCRACchannels.OurfindingsindicatethathypoxiamodulatesCa2+homeostasisinTcellsviaKv1.3channelinhibitionandmembranedepolarization.
RobbinsJR.,etal.(2005)HypoxiamodulatesearlyeventsinTcellreceptor-mediatedactivationinhumanTlymphocytesviaKv1.3channels.JPhysiol.PMID: 15677684
PotassiumchannelblockadebytheseaanemonetoxinShKforthetreatmentofmultiplesclerosisandotherautoimmunediseases
Expressionofthetwolymphocytepotassiumchannels,thevoltage-gatedchannelKv1.3andthecalciumactivatedchannelIKCa1,changesduringdifferentiationofhumanTcells.WhileIKCa1isthefunctionallydominantchannelinnaiveand“early”memoryTcells,Kv1.3iscrucialfortheactivationofterminallydifferentiatedeffectormemory(TEM)Tcells.BecauseoftheinvolvementofTEMcellsinautoimmuneprocesses,Kv1.3isregardedasapromisingtargetforthetreatmentofT-cellmediatedautoimmunediseasessuchasmultiplesclerosisandthepreventionofchronictransplantrejection.ShK,a35-residuepolypeptidetoxinfromtheseaanemone,Stichodactylahelianthus,blocksKv1.3atlowpicomolarconcentrations.ShKadoptsacentralhelix-kink-helixfold,andalanine-scanningandothermutagenesisstudieshavedefineditschannel-bindingsurface.ModelshavebeendevelopedofhowthistoxineffectsK+-channelblockadeandhowitsdockingconfigurationmightdifferinShK-Dap22,whichcontainsasinglesidechainsubstitutionthatconfersspecificityforKv1.3blockade.ShK,ShK-Dap22andtheKv1.3blockingscorpiontoxinkaliotoxinhavebeenshowntopreventandtreatexperimentalautoimmuneencephalomyelitisinrats,amodelformultiplesclerosis.AfluoresceinatedanalogofShK,ShK-F6CA,hasbeendeveloped,whichallowsthedetectionofactivatedTEMcellsinhumanandanimalbloodsamplesbyflowcytometryandthevisualizationofKv1.3channeldistributioninlivingcells.ShKanditsanalogsarecurrentlyundergoingfurtherevaluationasleadsinthedevelopmentofnewbiopharmaceuticalsforthetreatmentofmultiplesclerosisandotherT-cellmediatedautoimmunedisorders.
NortonRS.,etal.(2004)PotassiumchannelblockadebytheseaanemonetoxinShKforthetreatmentofmultiplesclerosisandotherautoimmunediseases.CurrMedChem. PMID: 15578998
SubstitutionofasingleresidueinStichodactylahelianthuspeptide,ShK-Dap22,revealsanovelpharmacologicalprofile
ShK,apeptideisolatedfromStichodactylahelianthusvenom,blocksthevoltage-gatedpotassiumchannels,K(v)1.1andK(v)1.3,withsimilarhighaffinity.ShK-Dap(22),asyntheticderivativeinwhichadiaminopropionicacidresiduehasbeensubstitutedatpositionLys(22),hasbeenreportedtobeaselectiveK(v)1.3inhibitorandtoblockthischannelwithequivalentpotencyasShK[Kalmanetal.(1998)J.Biol.Chem.273,32697-32707].Inthisstudy,alargebodyofevidenceispresentedwhichindicatesthatthepotenciesofwild-typeShKpeptideforbothK(v)1.3andK(v)1.1channelshavebeenpreviouslyunderestimated.Therefore,theaffinityofShK-Dap(22)forbothchannelsappearstobeca.10(2)-10(4)-foldweakerthanShK.ShK-Dap(22)doesdisplayca.20-foldselectivityforhumanK(v)1.3vsK(v)1.1whenmeasuredbythewhole-cellvoltageclampmethodbutnotinequilibriumbindingassays.ShK-Dap(22)haslowaffinityforK(v)1.2channels,butheteromultimericK(v)1.1-K(v)1.2channelsformareceptorwithca.200-foldhigheraffinityforShK-Dap(22)thanK(v)1.1homomultimers.Infact,K(v)1.1-K(v)1.2channelsbindShK-Dap(22)withonlyca.10-foldlesspotencythanShKandrevealanovelpharmacologynotpredictedfromthehomomultimersofK(v)1.1orK(v)1.2.TheconcentrationsofShK-Dap(22)neededtoinhibithumanTcellactivationwereca.10(3)-foldhigherthanthoseofShK,ingoodcorrelationwiththerelativeaffinitiesofthesepeptidesforinhibitingK(v)1.3channels.Allofthesedata,takentogether,suggestthatShK-Dap(22)willnothavethesameinvivoimmunosuppressantefficacyofotherK(v)1.3blockers,suchasmargatoxinorShK.Moreover,ShK-Dap(22)mayhaveundesiredsideeffectsduetoitsinteractionwithheteromultimericK(v)1.1-K(v)1.2channels,suchasthosepresentinbrainand/orperipheraltissues.
MiddletonRE.,etal.(2003)SubstitutionofasingleresidueinStichodactylahelianthuspeptide,ShK-Dap22,revealsanovelpharmacologicalprofile.Biochemistry. PMID: 14622016
MutatingacriticallysineinShKtoxinaltersitsbindingconfigurationinthepore-vestibuleregionofthevoltage-gatedpotassiumchannel,Kv1.3
Thevoltage-gatedpotassiumchannelinTlymphocytes,Kv1.3,animportanttargetforimmunosuppressants,isblockedbypicomolarconcentrationsofthepolypeptideShKtoxinanditsanalogueShK-Dap22.ShK-Dap22showsincreasedselectivityforKv1.3,andourgoalwastodeterminethemolecularbasisforthisselectivitybyprobingtheinteractionsofShKandShK-Dap22withtheporeandvestibuleofKv1.3.Thefreeenergiesofinteractionsbetweentoxinandchannelresiduesweremeasuredusingmutantcycleanalyses.Thesedata,interpretedasapproximatedistancerestraints,guidedmoleculardynamicssimulationsinwhichthetoxinsweredockedwithamodelofKv1.3basedonthecrystalstructureofthebacterialK(+)-channelKcsA.Despitethesimilartertiarystructuresofthetwoligands,themutantcycledataimplythattheymakedifferentcontactswithKv1.3,andtheycanbedockedwiththechannelinconfigurationsthatareconsistentwiththemutantcycledataforeachtoxinbutquitedistinctfromoneanother.ShKbindstoKv1.3withLys22occupyingthenegativelychargedporeofthechannel,whereastheequivalentresidueinShK-Dap22interactswithresiduesfurtheroutinthevestibule,producingasignificantchangeintoxinorientation.TheincreasedselectivityofShK-Dap22isachievedbystronginteractionsofDap22withHis404andAsp386onKv1.3,withonlyweakinteractionsbetweenthechannelporeandthetoxin.PotentandspecificblockadeofKv1.3apparentlyoccurswithoutinsertionofapositivelychargedresidueintothechannelpore.Moreover,thefindingthatasingleresiduesubstitutionaltersthebindingconfigurationemphasizestheneedtoobtainconsistentdatafrommultiplemutantcycleexperimentsinattemptstodefineproteininteractionsurfacesusingthesedata.
LaniganMD.,etal.(2002)MutatingacriticallysineinShKtoxinaltersitsbindingconfigurationinthepore-vestibuleregionofthevoltage-gatedpotassiumchannel,Kv1.3.Biochemistry. PMID: 12356296
SelectiveblockadeofTlymphocyteK(+)channelsamelioratesexperimentalautoimmuneencephalomyelitis,amodelformultiplesclerosis
Adoptivetransferexperimentalautoimmuneencephalomyelitis(AT-EAE),adiseaseresemblingmultiplesclerosis,isinducedinratsbymyelinbasicprotein(MBP)-activatedCD4(+)Tlymphocytes.Bypatch-clampanalysis,encephalitogenicratTcellsstimulatedrepeatedlyinvitroexpressedauniquechannelphenotype(“chronicallyactivated”)withlargenumbersofKv1.3voltage-gatedchannels(approximately1500percell)andsmallnumbersofIKCa1Ca(2+)-activatedK(+)channels(approximately50-120percell).Incontrast,restingTcellsdisplayed0-10Kv1.3and10-20IKCa1channelspercell(“quiescent”phenotype),whereasTcellsstimulatedonceortwiceexpressedapproximately200Kv1.3andapproximately350IKCa1channelspercell(“acutelyactivated”phenotype).Consistentwiththeirchannelphenotype,[(3)H]thymidineincorporationbyMBP-stimulatedchronicallyactivatedTcellswassuppressedbythepeptideShK,ablockerofKv1.3andIKCa1,andbyananalog(ShK-Dap(22))engineeredtobehighlyspecificforKv1.3,butnotbyaselectiveIKCa1blocker(TRAM-34).ThecombinationofShK-Dap(22)andTRAM-34enhancedthesuppressionofMBP-stimulatedTcellproliferation.Basedontheseinvitroresults,weassessedtheefficacyofK(+)channelblockersinAT-EAE.SpecificandsimultaneousblockadeoftheTcellchannelsbyShKorbyacombinationofShK-Dap(22)plusTRAM-34preventedlethalAT-EAE.BlockadeofKv1.3alonewithShK-Dap(22),butnotofIKCa1withTRAM-34,wasalsoeffective.WhenadmiNISTeredaftertheonsetofsymptoms,ShKorthecombinationofShK-Dap(22)plusTRAM-34greatlyamelioratedtheclinicalcourseofbothmoderateandsevereAT-EAE.WeconcludethatselectivetargetingofKv1.3,aloneorwithIKCa1,mayprovideaneffectivenewmodeoftherapyformultiplesclerosis.
BeetonC.,etal.(2001)SelectiveblockadeofTlymphocyteK(+)channelsamelioratesexperimentalautoimmuneencephalomyelitis,amodelformultiplesclerosis.ProcNatlAcadSciUSA. PMID: 11717451
ShK-Dap22,apotentKv1.3-specificimmunosuppressivepolypeptide
Thevoltage-gatedpotassiumchannelinTlymphocytes,Kv1.3,isanimportantmoleculartargetforimmunosuppressiveagents.Astructurallydefinedpolypeptide,ShK,fromtheseaanemoneStichodactylahelianthusinhibitedKv1.3potentlyandalsoblockedKv1.1,Kv1.4,andKv1.6atsubnanomolarconcentrations.UsingmutantcycleanalysisinconjunctionwithcomplementarymutagenesisofShKandKv1.3,andutilizingthestructureofShK,wedeterminedalikelydockingconfigurationforthispeptideinthechannel.Baseduponthistopologicalinformation,wereplacedthecriticalLys22inShKwiththepositivelycharged,non-naturalaminoaciddiaminopropionicacid(ShK-Dap22)andgeneratedahighlyselectiveandpotentblockeroftheT-lymphocytechannel.ShK-Dap22,atsubnanomolarconcentrations,suppressedanti-CD3inducedhumanT-lymphocyte[3H]thymidineincorporationinvitro.Toxicitywiththismutantpeptidewaslowinarodentmodel,withamedianparalyticdoseofapproximately200mg/kgbodyweightfollowingintravenousadministration.TheoverallstructureofShK-Dap22insolution,asdeterminedfromNMRdata,issimilartothatofnativeShKtoxin,buttherearesomedifferencesintheresiduesinvolvedinpotassiumchannelbinding.Basedontheseresults,weproposethatShK-Dap22orastructuralanaloguemayhaveuseasanimmunosuppressantforthepreventionofgraftrejectionandforthetreatmentofautoimmunediseases.
KalmanK,etal.(1998)ShK-Dap22,apotentKv1.3-specificimmunosuppressivepolypeptide.JBiolChem. PMID: 9830012
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因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
两个CEX方法A和B测定同一单抗，结果碱性峰比例差不多，酸性峰比例相差约7%，相应主峰也差了7%左右。
具体来说，A方法酸性峰高，主峰低，碱性峰稍微低点；B方法酸性峰低，主峰高，碱性峰稍微高点；另外也做了CIEF，结果呢和A方法更接近。
仔细比较起来，AB两个方法的峰性和数量差不多，就不知道为什么会有这么大的差异。两个方法一个用的WCX柱-磷酸缓冲液，一个用SCX柱-MES缓冲液
大家帮我分析下：
1.两个方法哪个方法更准确，是以酸性峰高的为准还是什么？为什么？
2.这显著差异是由方法造成，具体原因是什么？柱子？
3.CIEF的结果和A方法更接近，是不是可以由此证明A方法更好或者CIEF的方法更好（因为CIEF更快更方便）？
欢迎讨论~
纠正下，A方法用的是Tosoh的柱子，B方法用的是SCX柱。TOSOH的柱子是7um的填料，10cm长。SCX是10um的填料。我本人TOSOH的阳离子柱子用的很少，这次信手用用，结果发现差异很大
那我现在就考虑，在以后方法开发过程中，除了通过流动相pH和组成、梯度、柱子选择来获得样品主峰和酸碱性的最大分离，还要关注各峰比例。因为之前比较方法好坏都只看分离度，尤其是主峰和邻近峰的分离度，获得最大分离度，自然可以做到主峰尽可能纯，但从未认真比较过各峰比例。这是一个大疏忽吧！
另外，CIEF和CEX方法原理还是有点差异的，所以分的是不同的异质体，原液放行两个方法肯定是都要做的。问题就是在早期细胞株筛选和工艺开发阶段，哪个方法才是又快又准。CIEF（iCE280)一般15分钟一个样，比CEX快多了。如果CIEF测得主峰要低于CEX结果，是不是真的完全可以取代CEX呢？CEX分离出的峰远比CIEF的多！
欢迎大家继续讨论~
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
拼音名：Chunhuashui
英文名：PurifiedWater
【性状】本品为无色的澄清液体；无臭，无味。
【检查】酸碱度取本品10ml，加甲基红指示液2滴，不得显红色；另取10ml，加溴麝香草酚蓝指示液5滴，不得显蓝色。氯化物、流酸盐与钙盐取本品，分置三支试管中，每管各50ml。第一管中加硝酸5滴与硝酸银试液1ml，第二管中加氯化钡试液2ml，第三管中加草酸铵试液2ml，均不得发生浑浊。
硝酸盐取本品5ml置试管中，于冰浴中冷却，加10%氯化钾溶液0.4ml与0.1%二苯胺硫酸溶液0.1ml，摇匀，缓缓滴加硫酸5ml，摇匀，将试管子50℃水浴中放置15分钟，溶液产生的蓝色与标准硝酸盐溶液［取硝酸钾0.163g，加水溶解并稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，再精密量取10ml，加水稀释成100ml，摇匀，即得（每1ml相当于1pgNO3）0.3ml，加无硝酸盐的水4.7ml，用同一方法处理后的颜色比较，不得更深（0.000006%）。
亚硝酸盐取本品10ml，置纳氏管中，加对氨基苯磺酰胺的稀盐酸溶液（1→100）lml与盐酸菜乙H肢溶液（0．l＋100）1ml，产生的粉红色，与标准亚硝酸盐溶液〔取亚硝酸钠0.750g（按干燥品计算），加水溶解，稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，摇匀，再精密量取1ml，加水稀释成50ml，摇匀，即得（每1ml相当于1μgNO2）]0.2ml，加无亚硝酸盐的水9.8ml，用同一方法处理后的颜色比较，不得更深（0.000002%）。
氨取本品50ml，加碱性碘化汞钾试液2ml，放置15分钟；如显色，与氯化铵溶液（取氯化铵31.5mg，加无氨水适量使溶解并稀释成1000ml）1.5ml，加元氨水48ml与碱性碘化汞钾试液2ml制成的对照液比较，不得更深（0.00003%）。
二氧化碳取本品25ml，置50ml具塞量筒中，加氢氧化钙试液25ml，密塞振摇，放置，小时内不得发生浑浊。
易氧化物取本品100ml，加稀硫酸10ml，煮沸后，加高锰酸钾滴定液（0.02mol/L）0.10ml，再煮沸10分钟，粉红色不得完全消失。
不挥发物取本品100ml，置105℃恒重的蒸发皿中，在水浴上蒸干，并在105℃干燥至恒重，遗留残渣不得过1mg。
重金属取本品50ml，加水18.5ml，蒸发至20ml，放冷，加醋酸盐缓冲液（pH3.5）2ml与水适量使成25ml，加硫代乙酰胺试液2ml，摇匀，放置2分钟，与标准铅溶液1.5ml加水18.5ml用同一方法处理后的颜色比较，不得更深（0.00003%）。
微生物限度取本品，采用薄膜过滤法处理后，依法检查（附录ⅪJ），细菌、霉菌和酵母菌总数每1ml不得过100个。
【贮藏】密闭保存。
【化学成分】本品为蒸馏法、离子交换法、反渗透法或其他适宜的方法制得的供药用的水，不含任何附加剂。
【分子式与分子量】H2O18.02
【药理作用】溶剂、稀释剂
这里药典纯化水标准中并无PH值项目，请问对纯化水有PH值的要求吗，范围应在多少？请说明出处？
在纯化水检测中，检验酸碱度合格，但是发现PH在8左右。如果按以上标准检验合格，是否要考虑PH值？请知道的解答，谢谢！
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