
(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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pH(1)=pKa+lg[c(CH₃COONa)/c(CH₃COOH)]=pKa=4.74
通HCl后,溶液是c(CH₃COOH)=0.2mol/L、c(NaCl)=0.1mol/L的混合溶液,溶液pH按照弱酸溶液pH的求法求.
c(H⁺)=√[Ka*c(CH₃COOH)]=√(10^-4.74*0.2)=0.00191(mol/L)(采用了近似公式)
pH(2)=-lg{c(H⁺)}=2.72
两个pH求得,那么pH的变化量也就可得了.pH的变化量=|pH(2)-pH(1)|=|2.72-4.74|=2.02
1)PH缓冲溶液作用原理和pH值
当往某些溶液中加入一定量的酸和碱时,有阻碍溶液pH变化的作用,称为缓冲作用,这样的溶液叫做缓冲溶液.弱酸及其盐的混合溶液(如HAc与NaAc),弱碱及其盐的混合溶液(如NH3·H2O与NH4Cl)等都是缓冲溶液.
由弱酸HA及其盐NaA所组成的缓冲溶液对酸的缓冲作用,是由于溶液中存在足够量的碱A-的缘故.当向这种溶液中加入一定量的强酸时,H离子基本上被A-离子消耗:
所以溶液的pH值几乎不变;当加入一定量强碱时,溶液中存在的弱酸HA消耗OH-离子而阻碍pH的变化.
2)PH缓冲溶液的缓冲能力
在缓冲溶液中加入少量强酸或强碱,其溶液pH值变化不大,但若加入酸,碱的量多时,缓冲溶液就失去了它的缓冲作用.这说明它的缓冲能力是有一定限度的.
缓冲溶液的缓冲能力与组成缓冲溶液的组分浓度有关.0.1mol·L-1HAc和0.1mol·L-1NaAc组成的缓冲溶液,比0.01mol·L-1HAc和0.01mol·L-1NaAc的缓冲溶液缓冲能力大.关于这一点通过计算便可证实.但缓冲溶液组分的浓度不能太大,否则,不能忽视离子间的作用.
组成缓冲溶液的两组分的比值不为1∶1时,缓冲作用减小,缓冲能力降低,当c(盐)/c(酸)为1∶1时△pH最小,缓冲能力大.不论对于酸或碱都有较大的缓冲作用.缓冲溶液的pH值可用下式计算:
此时缓冲能力大.缓冲组分的比值离1∶1愈远,缓冲能力愈小,甚至不能起缓冲作用.对于任何缓冲体系,存在有效缓冲范围,这个范围大致在pKaφ(或pKbφ)两侧各一个pH单位之内.
弱酸及其盐(弱酸及其共轭碱)体系pH=pKaφ±1
弱碱及其盐(弱碱及其共轭酸)体系pOH=pKbφ±1
例如HAc的pKaφ为4.76,所以用HAc和NaAc适宜于配制pH为3.76~5.76的缓冲溶液,在这个范围内有较大的缓冲作用.配制pH=4.76的缓冲溶液时缓冲能力最大,此时(c(HAc)/c(NaAc)=1.
3)PH缓冲溶液的配制和应用
为了配制一定pH的缓冲溶液,首先选定一个弱酸,它的pKaφ尽可能接近所需配制的缓冲溶液的pH值,然后计算酸与碱的浓度比,根据此浓度比便可配制所需缓冲溶液.
以上主要以弱酸及其盐组成的缓冲溶液为例说明它的作用原理、pH计算和配制方法.对于弱碱及其盐组成的缓冲溶液可采用相同的方法.
PH缓冲溶液在物质分离和成分分析等方面应用广泛,如鉴定Mg2离子时,可用下面的反应:
白色磷酸铵镁沉淀溶于酸,故反应需在碱性溶液中进行,但碱性太强,可能生成白色Mg(OH)2沉淀,所以反应的pH值需控制在一定范围内,因此利用NH3·H2O和NH4Cl组成的缓冲溶液,保持溶液的pH值条件下,进行上述反应.
这就是说不用酸碱预处理吗?
Whatman的网站上没有DE52最大耐受压力,请问又经验的战友应该是多少?
Whatman的网站上:
DE32DryMicrogranularDEAECellulose
SimilarperformancecharacteristicsafterprecyclingasDE52.
DE52PreswollenMicrogranularDEAECellulose
ProbablythemostwidelyusedDEAEcelluloseintheworld;usedforbiopolymerswithlowtohighnegativecharges;exhibitsexcellentresolutionwithgoodflowrates.
附件是一本图书(MethodsinMolecularMedicine,)的章节,上面说:
WhatmanDEAE52comesalreadypreswollenandonlyneedstobetransferred
totherunningbuffer50mMTE8.
lAntibodiesUsingIonExchangeChromatography.pdf(87.06k)
是否可以理解为纯化水得PH范围为6.3-7.6?能否直接用pH计测量?谢谢!


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