ProtoxinI(ProTx-I;β-theraphotoxin-Tp1a) isatoxinthatwasoriginallyisolatedfromthevenomofThrixopelmapruriens(Peruviangreenvelvettarantula).ThistoxinreversIBLyinhibitsthetetrodotoxin(TTX)-resistantchannel Nav1.8(IC50 =27nM)and Nav1.2,Nav1.5andNav1.7 withIC50 valuesbetween50and100nM.FurThermore, ProTx-I shiftsthevoltagedependenceactivityof T-typeCav3.1channels (IC50=50nM)withoutaffectingthevoltagedependenceofinactivation. ProTx-I isavaluabletooltodiscriminatebetweenCav3.1andCav3.2
Description:
AAsequence:Glu-Cys2-Arg-Tyr-Trp-Leu-Gly-Gly-Cys9-Ser-Ala-Gly-Gln-Thr-Cys15-Cys16-Lys-His-Leu-Val-Cys21-Ser-Arg-Arg-His-Gly-Trp-Cys28-Val-Trp-Asp-Gly-Thr-Phe-Ser-OH
Disulfidebridges:Cys2-Cys16,Cys9-Cys21,Cys15-Cys28
Length(aa):35
Formula:C171H245N53O47S6
MolecularWeight:3987.50Da
Appearance:Whitelyophilizedsolid
Solubility:waterorsalinebuffer
CASnumber:Notavailable
Source:Synthetic
Purityrate:>95%
Reference:
Twotarantulapeptidesinhibitactivationofmultiplesodiumchannels
Twopeptides,ProTx-IandProTx-II,fromthevenomofthetarantulaThrixopelmapruriens,havebeenisolatedandcharacterized.ThesepeptideswerepurifiedonthebasisoftheirABIlitytoreversiblyinhibitthetetrodotoxin-resistantNachannel,Na(V)1.8,andareshowntobelongtotheinhibitorycystineknot(ICK)familyofpeptidetoxinsinteractingwithvoltage-gatedionchannels.Thefamilyhasseveralhallmarks:cystinebridgeconnectivity,mechanismofchannelinhibition,andpromiscuityacrosschannelswithinandacrosschannelfamilies.ThecystinebridgeconnectivityofProTx-IIisverysimilartothatofothermembersofthisfamily,i.e.,C(2)toC(16),C(9)toC(21),andC(15)toC(25).Thesepeptidesarethefirsthigh-affinityligandsfortetrodotoxin-resistantperipheralnerveNa(V)channels,butalsoinhibitotherNa(V)channels(IC(50)’s<100nM).ProTx-IandProTx-IIshiftthevoltagedependenceofactivationofNa(V)1.5tomorepositivevoltages,similartoothergating-modifierICKfamilymembers.ProTx-IalsoshiftsthevoltagedependenceofactivationofCa(V)3.1(alpha(1G),T-type,IC(50)=50nM)withoutaffectingthevoltagedependenceofinactivation.Toenablefurtherstructuralandfunctionalstudies,syntheticProTx-IIwasmade;itadoptsthesamestructureandhasthesamefunctionalpropertiesasthenativepeptide.SyntheticProTx-Iwasalsomadeandexhibitsthesamepotencyasthenativepeptide.SyntheticProTx-I,butnotProTx-II,alsoinhibitsK(V)2.1channelswith10-foldlesspotencythanitspotencyonNa(V)channels.ThesepeptidesrepresentnoveltoolsforexploringthegatingmechanismsofseveralNa(V)andCa(V)channels.
MiddeltonR.E, etal. (2002)Twotarantulapeptidesinhibitactivationofmultiplesodiumchannels.Biochemestry.PMID: 12475222
ProTx-IandProTx-II:gatingmodifiersofvoltage-gatedsodiumchannels
ThetarantulavenompeptidesProTx-IandProTx-IIinhibitvoltage-gatedsodiumchannelsbyshiftingtheirvoltagedependenceofactivationtoamorepositivepotential,thusactingbyamechanismsimilartothatofpotassiumchannelgatingmodifierssuchashanatoxinandVSTX1.ProTx-IandProTx-IIinhibitallsodiumchannel(Nav1)subtypestestedwithsimilarpotencyandrepresentthefirstpotentpeptidylinhibitorsofTTX-resistantsodiumchannels.Likegatingmodifiersofpotassiumchannels,ProTx-IandProTx-IIconformtotheinhibitorycystineknotmotif,andProTx-IIwasdemonstratedtobindtosodiumchannelsintheclosedstate.Bothtoxinshavebeensynthesizedchemically,andProTx-II,producedbyrecombinantmeans,hasbeenusedtomaptheinteractionsurfaceofthepeptidewiththeNav1.5channel.Incomparison,beta-scorpiontoxinsactivatesodiumchannelsbyshiftingthevoltagedependenceofactivationtomorenegativepotentials,andtogetherthesepeptidesrepresentvaluabletoolsforexploringthegatingmechanismofsodiumchannels.
PriestB.T., etal.(2007)ProTx-IandProTx-II:gatingmodifiersofvoltage-gatedsodiumchannels. Toxicon.PMID: 17087985
TarantulatoxinProTx-IdifferentiatesbetweenhumanT-typevoltage-gatedCa2+ChannelsCav3.1andCav3.2
ProTx-Ipeptide,avenomtoxinofthetarantulaThrixopelmapruriens,hasbeenreportedtointeractwithvoltage-gatedionchannels.ProTx-IreducedBa(2+)currentsthroughrecombinanthumanT-typevoltage-gatedCa(2+)channels,Ca(v)3.1(hCa(v)3.1),withroughly160-foldmorepotencythanthroughhCa(v)3.2channels.Chimericchannelproteins(hCa(v)3.1/S3S4andhCa(v)3.2/S3S4)wereproducedbyexchangingfourteenaminoacidsinthehCa(v)3.1domainIVS3-S4linkerregionandthecorrespondingregionofhCa(v)3.2betweeneachother.TheProTx-IsensitivitywasmarkedlyreducedinthehCa(v)3.1/S3S4chimeraascomparedtotheoriginalhCa(v)3.1channel,whilethehCa(v)3.2/S3S4chimeraexhibitedgreaterProTx-IsensitivitythantheoriginalhCa(v)3.2channel.TheseresultssuggestthatthedomainIVS3-S4linkerinthehCa(v)3.1channelmaycontainresiduesinvolvedintheinteractionofProTx-IwithT-typeCa(2+)channels.
OhkuboT, etal. (2010)TarantulatoxinProTx-IdifferentiatesbetweenhumanT-typevoltage-gatedCa2+ ChannelsCav3.1andCav3.2. JPharmacolSci. PMID: 20351484
ATarantula-VenomPeptideAntagonizestheTRPA1NociceptorIonChannelbyBindingtotheS1-S4GatingDomain
BACKGROUND:
Thevenomsofpredatorshavebeenanexcellentsourceofdiversehighlyspecificpeptidestargetingionchannels.HerewedescribethefirstknownpeptideantagoNISTofthenociceptorionchanneltransientreceptorpotentialankyrin1(TRPA1).
RESULTS:
WeconstructedarecombinantCDNAlibraryencoding∼100diverseGPI-anchoredpeptidetoxins(t-toxins)derivedfromspidervenomsandscreenedthislibrarybycoexpressioninXenopusoocyteswithTRPA1.Thisscreenresultedinidentificationofprotoxin-I(ProTx-I),a35-residuepeptidefromthevenomofthePeruviangreen-velvettarantula,Thrixopelmapruriens,asthefirstknownhigh-affinitypeptideTRPA1antagonist.ProTx-Iwaspreviouslyidentifiedasanantagonistofvoltage-gatedsodium(NaV)channels.Weconstructedat-toxinlibraryofProTx-Ialanine-scanningmutantsandscreenedthislibraryagainstNaV1.2andTRPA1.ThisrevealeddistinctpartiallyoverlappingsurfacesofProTx-Ibywhichitbindstothesetwoionchannels.Importantly,thismutagenesisyieldedtwonovelProTx-IvariantsthatareonlyactiveagainsteitherTRPA1orNaV1.2.Bytestingitsactivityagainstchimericchannels,weidentifiedtheextracellularloopsoftheTRPA1S1-S4gatingdomainastheProTx-Ibindingsite.
CONCLUSIONS:
Thesestudiesestablishourapproach,whichweterm“toxineering,”asagenerallyapplicablemethodforisolationofnovelionchannelmodifiersanddesignofionchannelmodifierswithalteredspecificity.TheyalsosuggestthatProTx-IwillbeavaluablepharmacologicalreagentforaddressingbiophysicalmechanismsofTRPA1gatingandthephysiologyofTRPA1functioninnociceptors,aswellasforpotentialclinicalapplicationinthecontextofpainandinflammation.
GuiJ,etal.(2014)ATarantula-VenomPeptideAntagonizestheTRPA1NociceptorIonChannelbyBindingtotheS1-S4GatingDomain. CurrBiol. PMID: 24530065
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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!
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!
由弱酸及其盐、弱碱及其盐组成的混合溶液,能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响,从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
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~
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.
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