µ-conotoxinGIIIBisa22aminoacidconopeptideoriginallyisolatedfromthevenomofthepiscivorousmarinesnailConusgeographus.µ-conotoxinGIIIBadoptsacompactstructureconsistingofadistorted310-helixandasmallß-hairpin.µ-conotoxinGIIIBisstABIlizedbythreedisulphidebridgesandishighlyenrichedinlysineandarginineresidues,formingpotentialsitesofinteractionwithNachannels.Anunusualfeatureisthepresenceofthreehydroxyprolineresidues.µ-conotoxinGIIIBisausefulprobetodiscriminatebetweenneuronalandmusclesodiumchannelsasitexhibitsatleasta1000-foldspecificityformuscleversusnervesodiumchannels.µ-ConotoxinGIIIBselectivelyblocksNav1.4voltage-dependentsodiumchannels,whicharepredominantlyexpressedinmuscle,withanaffinitycloseto20nM.µ-ConotoxinGIIIBappearstophysicallyoccludethechannelporebybindingonsiteIoftheNa+channel.
Description:
AAsequence:Arg-Asp-Cys3-Cys4-Thr-Hyp-Hyp-Arg-Lys-Cys10-Lys-Asp-Arg-Arg-Cys15-Lys-Hyp-Met-Lys-Cys20-Cys21-Ala-NH2
Disulfidebonds:Cys3-Cys15,Cys4-Cys20andCys10-Cys21
Length(aa):22
Formula:C101H175N39O30S7
MolecularWeight:2640.26Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:
Source:Synthetic
Purityrate:>97%
Reference:
MolecularBasisofIsoform-specificμ-ConotoxinBlockofCardiac,SkeletalMuscle,andBrainNa+Channels
mu-Conotoxins(mu-CTXs)blockskeletalmuscleNa(+)channelswithanaffinity1-2ordersofmagnitudehigherthancardiacandbrainNa(+)channels.Althoughanumberofconservedporeresiduesarerecognizedascriticaldeterminantsofmu-CTXblock,themolecularbasisofisoform-specifictoxinsensitivityremainsunresolved.SequencecomparisonofthedomainII(DII)S5-S6loopsofratskeletalmuscle(mu1,Na(v)1.4),humanheart(hh1,Na(v)1.5),andratbrain(rb1,Na(v)1.1)Na(+)channelsrevealssubstantialdivergenceintheirN-terminalS5-PlinkerseventhoughtheP-S6andC-terminalPsegmentsarealmostidentical.WeusedNa(v)1.4asthebackboneandsystematicallyconvertedtheseDIIS5-PisoformvariantstothecorrespondingresiduesinNa(v)1.1andNa(v)1.5.TheNa(v)1.4–>Na(v)1.5variantsubstitutionsV724R,C725S,A728S,D730S,andC731S(Na(v)1.4numbering)reducedblockofNa(v)1.4by4-,86-,12-,185-,and55-foldrespectively,renderingtheskeletalmuscleisoformmore“cardiac-like.”Conversely,anNa(v)1.5–>Na(v)1.4chimericconstructinwhichtheNa(v)1.4DIIS5-PlinkerreplacestheanalogoussegmentinNa(v)1.5showedenhancedmu-CTXblock.However,thesevariantdeterminantsareconservedbetweenNa(v)1.1andNa(v)1.4andthuscannotexplaintheirdifferentsensitivitiestomu-CTX.ComparisonoftheirsequencesrevealstwovariantsatNa(v)1.4positions729and732:SerandAsninNa(v)1.4comparedwithThrandLysinNa(v)1.1,respectively.ThedoublemutationS729T/N732KrenderedNa(v)1.4more“brain-like”(30-folddownwardarrowinblock),andtheconversemutationT925S/K928NinNa(v)1.1reproducedthehighaffinityblockingphenotypeofNa(v)1.4.WeconcludethattheDIIS5-Plinker,althoughlyingoutsidetheconventionalion-conductingpore,playsaprominentroleinmu-CTXbinding,thusshapingisoform-specifictoxinsensitivity.
RonaldA.Li, etal.(2003)MolecularBasisofIsoform-specificμ-ConotoxinBlockofCardiac,SkeletalMuscle,andBrainNa+ Channels. JBC. PMID: 12471026
Conusgeographustoxinsthatdiscriminatebetweenneuronalandmusclesodiumchannels
Wedescribethepropertiesofafamilyof22-aminoacidpeptides,themu-conotoxins,whichareusefulprobesforinvestigatingvoltage-dependentsodiumchannelsofexcitabletissues.Themu-conotoxinsarepresentinthevenomofthepiscivorousmarinesnail,ConusgeographusL.Wehavepurifiedsevenhomologsofthemu-conotoxinsetanddeterminedtheiraminoacidsequences,asfollows,whereHyp=trans-4-hydroxyproline.GIIIAR.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2[Pro6]GIIIAR.D.C.C.T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2[Pro7]GIIIAR.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2GIIIBR.D.C.C.T.Hyp.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2[Pro6]GIIIBR.D.C.C.T.P.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2[Pro7]GIIIBR.D.C.C.T.Hyp.P.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2GIIICR.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2.Usingthemajorpeptide(GIIIA)inelectrophysiologicalstudiesonnerve-musclepreparationsandinsinglechannelstudiesusingplanarlipidbilayers,wehaveestablishedthatthetoxinblocksmusclesodiumchannels,whilehavingnodiscernIBLeeffectonnerveorbrainsodiumchannels.InbilayerstheblockingkineticsofGIIIAwerederivedbystatisticalanalysisofdiscretetransitionsbetweenblockedandunblockedstatesofbatrachotoxin-activatedsodiumchannelsfromratmuscle.Thekineticsconformtoasingle-site,reversiblebindingequilibriumwithavoltage-dependentbindingconstant.ThemeasuredvalueoftheequilibriumKDforGIIIAis100nMatOmV,decreasinge-fold/34mVofhyperpolarization.Thisvoltagedependenceofblockingissimilartothatoftetrodotoxinandsaxitoxinasmeasuredbythesametechnique.Thetissuespecificityandkineticcharacteristicssuggestthatthemu-conotoxinsmayserveasusefulligandstodistinguishsodiumchannelsubtypesindifferenttissues.
CruzLJ, etal. (1985)Conusgeographustoxinsthatdiscriminatebetweenneuronalandmusclesodiumchannels. JBC. PMID: 2410412
NovelStructuralDeterminantsofm-Conotoxin(GIIIB)BlockinRatSkeletalMuscle(m1)Na+Channels
mu-Conotoxin(mu-CTX)specificallyoccludestheporeofvoltage-dependentNa(+)channels.IntheratskeletalmuscleNa(+)channel(mu1),weexaminedthecontributionofchargedresiduesbetweenthePloopsandS6inallfourdomainstomu-CTXblock.ConversionofthenegativelychargeddomainII(DII)residuesAsp-762andGlu-765tocysteineincreasedtheIC(50)formu-CTXblockbyapproximately100-fold(wild-type=22.3+/-7.0nm;D762C=2558+/-250nm;E765C=2020+/-379nm).Restorationorreversalofchargebyexternalmodificationofthecysteine-substitutedchannelswithmethanethiosulfonatereagents(methanethiosulfonateethylsulfonate(MTSES)andmethanethiosulfonateethylammonium(MTSEA))didnotaffectmu-CTXblock(D762C:IC(50,MTSEA+)=2165.1+/-250nm;IC(50,MTSES-)=2753.5+/-456.9nm;E765C:IC(50,MTSEA+)=2200.1+/-550.3nm;IC(50,MTSES-)=3248.1+/-2011.9nm)comparedwiththeirunmodifiedcounterparts.Incontrast,thecharge-conservingmutationsD762E(IC(50)=21.9+/-4.3nm)andE765D(IC(50)=22.0+/-7.0nm)preservedwild-typeblockingbehavior,whereasthechargereversalmutantsD762K(IC(50)=4139.9+/-687.9nm)andE765K(IC(50)=4202.7+/-1088.0nm)destabilizedmu-CTXblockevenfurther,suggestingaprominentelectrostaticcomponentoftheinteractionsbetweentheseDIIresiduesandmu-CTX.Kineticanalysisofmu-CTXblockrevealsthatthechangesintoxinsensitivityarelargelyduetoacceleratedtoxindissociation(k(off))rateswithlittlechangesinassociation(k(on))rates.Weconcludethattheacidicresiduesatpositions762and765arekeydeterminantsofmu-CTXblock,primarilybyvirtueoftheirnegativecharge.TheinabilityofthebulkyMTSESorMTSEAsidechaintomodifymu-CTXsensitivityplacesstericconstraintsonthesitesoftoxininteraction.
RonaldA.Li, etal. (2000)NovelStructuralDeterminantsofm-Conotoxin(GIIIB)BlockinRatSkeletalMuscle(m1)Na+ Channels.JBC.PMID: 10859326
HyperpolarizedshiftsinthevoltagedependenceoffastinactivationofNav1.4andNav1.5inaratmodelofcriticalillnessmyopathy
Criticalillnessmyopathyisadisorderinwhichskeletalmusclebecomeselectricallyinexcitable.Wepreviouslydemonstratedthatashiftinthevoltagedependenceoffastinactivationofsodiumcurrentscontributestoinexcitabilityofaffectedfibresinananimalmodelofcriticalillnessmyopathyinwhichdenervatedratskeletalmuscleistreatedwithcorticosteroids(steroid-denervated;SD).InthecurrentstudyweexaminedwhetherexpressionofNav1.5contributestothealteredvoltagedependenceofsodiumchannelinactivationinSDmuscle.WeusedTTXandmu-conotoxinGIIIBtoselectivelyblockNav1.4inSDmuscleandfoundthatthelevelofNav1.5didnotcorrelatecloselywiththeshiftinfastinactivation.Surprisingly,wefoundthatthevoltagedependenceofinactivationofNav1.4wassimilartothatofNav1.5inskeletalmuscleinvivo.Inseverelyaffectedfibres,inactivationofbothNav1.4andNav1.5wasshiftedtowardshyperpolarizedpotentials.Weexaminedtheroleofdenervationandsteroidtreatmentintheshiftofthevoltagedependenceofinactivationandfoundthatbothdenervationandsteroidtreatmentcontributetotheshiftininactivation.OurresultssuggestthatmodulationofthevoltagedependenceofinactivationofbothNav1.4andNav1.5invivocontributestolossofelectricalexcitabilityinSDmuscle.
GregoryN.FilatovandMarkM.Rich(2004)HyperpolarizedshiftsinthevoltagedependenceoffastinactivationofNav1.4andNav1.5inaratmodelofcriticalillnessmyopathy. J.Physiol. PMID: 15254148
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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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