Hainantoxin-IV(HNTX-IV)isapeptidethatwasoriginallyisolatedfromthevenomoftheChinesebirdspiderOrnithoctonushainanaLiang(SelenocosmiahainanaLiang).IthasbeenreportedthatthispeptideisapotentantagoNISToftetrodotoxin-sensitive(TTX-S)voltage-gatedsodiumchannels(VGSCs).Hainantoxin-IVbindstoTTX-SwithanIC50valueof34nMinadultratdorsalrootganglion(DRG)neurons.Tetrodotoxin-resistant(TTX-R)voltage-gatedsodiumchannelsarenotaffectedbyHainantoxin-IV.Itprobablyinteractswiththesite1throughamechanismquitesimilartothatofTTXwithoutaffectingtheactivationandinactivationkinetics.
Description:
AAsequence:Glu-Cys2-Leu-Gly-Phe-Gly-Lys-Gly-Cys9-Asn-Pro-Ser-Asn-Asp-Gln-Cys16-Cys17-Lys-Ser-Ser-Asn-Leu-Val-Cys24-Ser-Arg-Lys-His-Arg-Trp-Cys31-Lys-Tyr-Glu-Ile-NH2
(DisulfidebondsbetweenCys2-Cys17,Cys9-Cys24,andCys16-Cys31)
Length(aa):35
Formula:C166H257N53O50S6
MolecularWeight:3987.6Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:Notavailable
Source:Synthetic
Purityrate:>97%
Reference:
Apositivelychargedsurfacepatchisimportantforhainantoxin-IVbindingtovoltage-gatedsodiumchannels
LiuY,etal.(2012)Apositivelychargedsurfacepatchisimportantforhainantoxin-IVbindingtovoltage-gatedsodiumchannels.JPeptSci.PMID:22927181
Structure--activityrelationshipsofhainantoxin-IVandstructuredeterminationofactiveandinactivesodiumchannelblockers.
LiD,etal.(2004)Structure–activityrelationshipsofhainantoxin-IVandstructuredeterminationofactiveandinactivesodiumchannelblockers.JBiolChem.PMID:15201273
Isolationandcharacterizationofhainantoxin-IV,anovelantagonistoftetrodotoxin-sensitivesodiumchannelsfromtheChinesebirdspiderSelenocosmiahainana.
Aneurotoxin,namedhainantoxin-IV,waspurifiedfromthevenomofthespiderSelenocosmiahainana.TheaminoacidsequencewasdeterminedbyEdmandegradation,revealingittobea35-residuepolypeptideamidatedatitsCterminalandincludingthreedisulfidebridges:Cys2-Cys17,Cys9-Cys24,andCys16-Cys31assignedbypartialreductionandsequenceanalysis.Hainantoxin-IVshares80%sequenceidentitywithhuwentoxin-IVfromthespiderS.huwena,apotentantagonistthatactsatsite1ontetrodotoxin-sensitive(TTX-S)sodiumchannels,suggestingthathainantoxin-IVadoptsaninhibitorcystineknotstructuralmotiflikehuwentoin-IV.Underwhole-cellvoltage-clampconditions,thistoxinhasnoeffectontetrodotoxin-resistantvoltage-gatedsodiumchannelsinadultratdorsalrootganglionneurons,whileitblocksTTX-Ssodiumchannelsinamannersimilartohuwentoxin-IV,andtheactionsofbothtoxinsonsodiumcurrentsareverysimilartothatoftetrodotoxin.Thus,theydefineanewfamilyofspidertoxinsaffectingsodiumchannels.
LiuZ,etal.(2003)Isolationandcharacterizationofhainantoxin-IV,anovelantagonistoftetrodotoxin-sensitivesodiumchannelsfromtheChinesebirdspiderSelenocosmiahainana.CellMolLifeSci.PMID:12827284
Inhibitionofneuronaltetrodotoxin-sensitiveNa+channelsbytwospidertoxins:hainantoxin-IIIandhainantoxin-IV.
Hainantoxin-IIIandhainantoxin-IV,isolatedfromthevenomoftheChinesebirdspiderSeleconosmiahainana,areneurotoxicpeptidescomposedof33-35residueswiththreedisulfidebonds.Usingwhole-cellpatch-clamptechnique,weinvestigatedtheiractiononionicchannelsofadultratdorsalrootganglionneurons.ItwasfoundthatthetwotoxinsdidnotaffectCa2+channels(bothhighvoltageactivatedandlowvoltageactivatedtypes)nortetrodotoxin-resistantvoltage-gatedNa+channels(VGSCs).However,hainantoxin-IIIandhainantoxin-IVstronglydepressedtheamplitudeoftetrodotoxin-sensitiveNa+currentswithIC50valuesof1.1and44.6nM,respectively.Bothhainantoxin-III(1nM)andhainantoxin-IV(50nM)causedahyperpolarizingshiftofabout10mVinthevoltagemidpointofsteady-stateNa+channelinactivation,buttheyshoweddifferenceinthereprimekineticsofVGSCs:hainantoxin-IIIsignificantlydecreasedtherecoveryratefrominactivationataprepulsepotentialof-80mVwhilehainantoxin-IVdidnotdo.Itisinterestingtonotethatsimilartohuwentoxin-IV,thetwohainantoxinsdidnotaffecttheactivationandinactivationkineticsofNa+currentsandataconcentrationof1microMtheycompletelyinhibitedtheslowinginactivationcurrentsinducedbyBMK-I(toxinIfromthescorpionButhusmartensiKarsch),ascorpionalpha-liketoxin.Theresultsindicatethathainantoxin-IIIandhainantoxin-IVarenovelspidertoxinsandaffectthemammalneuralNa+channelsthroughamechanismquitedifferentfromotherspidertoxinstargetingtheneuralreceptorsite3,suchasdelta-aractoxinsandmu-agatoxins.
XiaoY,etal.(2003)Inhibitionofneuronaltetrodotoxin-sensitiveNa+channelsbytwospidertoxins:hainantoxin-IIIandhainantoxin-IV.EurJPharmacol.PMID:14512091
Determinationofdisulfidebridgesoftwospidertoxins:Hainantoxin-IIIandHainantoxin-IV
Peptidetoxinsareusuallyhighlybridgedproteinswithmultipairsofintrachaindisulfidebonds.Analysisofdisulfideconnectivityisanimportantfacetofproteinstructuredetermination.Inthispaper,wesuccessfullyassignedthedisulfidelinkageoftwonovelpeptidetoxins,calledHNTX-IIIandHNTX-IV,isolatedfromthevenomofOrnithoctonushainanaspider.BothpeptidesareusefulinhibitorsofTTX-sensitivevoltage-gatedsodiumchannelsandarecomposedofsixcysteineresiduesthatformthreedisulfidebonds,respectively.Firstly,thepeptideswerepartiallyreducedbytris(2-carboxyethyl)-phosphine(TCEP)in0.1Mcitratebuffercontaining6Mguanidine-HClat40°Cfortenminutes.Subsequently,thepartiallyreducedintermediatescontainingfreethiolswereseparatedbyreversed-phasehigh-performanceliquidchromatography(RP-HPLC)andalkylatedbyrapidcarboxamidomethylation.Then,thedisulfidebondsoftheintermediateswereanalyzedbyEdmandegradation.Byusingthestrategyabove,disulfidelinkagesofHNTX-IIIandHNTX-IVweredeterminedasI-IV,II-VandIII-VIpattern.Inaddition,thisstudyalsoshowedthatthismethodmayhaveagreatpotentialfordeterminingthedisulfidebondsofspiderpeptidetoxins.
WangW.,etal.(2009)Determinationofdisulfidebridgesoftwospidertoxins:Hainantoxin-IIIandHainantoxin-IV.JVenomAnimToxinsinclTropDis.
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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~
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!
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!
常用流动相加酸碱后PH的总结,希望大家能够提供一点自己测过的结果,谢谢先
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。
有了源数据之后把源数据按照大小排列,
选中源数据区域-->ALT+A1-->选中图标区右键-->更改图表类型-->散点图
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