HuwentoxinIV (HwTx-IV) isaneurotoxinthatwasoriginallyisolatedfromHaplopelmaschmidti(Chinesebirdspider).Thislethalneurotoxinactsselectivelyon tetrodotoxin-sensitive(TTX-S)voltage-gatedsodiumchannels,withanIC50 of30nMinratDRGneurons.Itpreferentiallyinhibitsneuronal voltage-gatedsodiumchannelsubtypehNav1.7 (SCN9A,IC50 is26nM), rNav1.2 (SCN2A,IC50 is150nM),and rNav1.3 (SCN3A,IC50 is338nM),comparedwithmusclesubtypesrNav1.4(SCN4A)andhNav1.5(SCN5A)(IC50 is>10µM).HuwentoxinIV inhibitstheactivationofsodiumchannelsbytrappingthevoltagesensorofdomainIIofthesite4intheinward,closedconfiguration.
Description:
AAsequence: Glu-Cys2-Leu-Glu-Ile-Phe-Lys-Ala-Cys9-Asn-Pro-Ser-Asn-Asp-Gln-Cys16-Cys17-Lys-Ser-Ser-Lys-Leu-Val-Cys24-Ser-Arg-Lys-Thr-Arg-Trp-Cys31-Lys-Tyr-Gln-Ile-NH2
Disulfidebonds: Cys2-Cys17,Cys9-Cys24 andCys16-Cys31
Length(aa): 35
Formula: C174H277N51O52S6
MolecularWeight: 4107.20Da
Appearance:Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber:
Source: Synthetic
Purityrate: >97%
Reference:
Analysisofthestructuralandmolecularbasisofvoltage-sensitivesodiumchannelinhibitionbythespidertoxin,Huwentoxin-IV(μ-TRTX-Hh2a)
Voltage-gatedsodiumchannels(VGSCs)areessentialtothenormalfunctionofthevertebratenervoussystem.AberrantfunctionofVGSCsunderliesavarietyofdisorders,includingepilepsy,arrhythmia,andpain.Alargenumberofanimaltoxinstargettheseionchannelsandmayhavesignificanttherapeuticpotential.Mostofthesetoxins,however,havenotbeencharacterizedindetail.Here,bycombiningpatchclampelectrophysiologyandrADIoligandbindingstudieswithpeptidemutagenesis,NMRstructuredetermination,andmolecularmodeling,wehaverevealedkeymoleculardeterminantsoftheinteractionbetweenthetarantulatoxinhuwentoxin-IVandtwoVGSCisoforms,Nav1.7andNav1.2.Ninehuwentoxin-IVresidues(F6A,P11A,D14A,L22A,S25A,W30A,K32A,Y33A,andI35A)wereimportantforblockofNav1.7andNav1.2.Importantly,moleculardynamicssimulationsandNMRstudiesindicatedthatfoldingwasnormalforseveralkeymutants,suggestingthattheseaminoacidsprobablymakespecificinteractionswithsodiumchannelresidues.Additionally,weidentifiedseveralaminoacids(F6A,K18A,R26A,andK27A)thatareinvolvedinisoform-specificVGSCinteractions.Ourstructuralandfunctionaldatawereusedtomodelthedockingofhuwentoxin-IVintothedomainIIvoltagesensorofNav1.7.ThemodelpredictsthatahydrophobicpatchcomposedofTrp-30andPhe-6,alongwiththebasicLys-32residue,docksintoagrooveformedbytheNav1.7S1-S2andS3-S4loops.Theseresultsprovidenewinsightintothestructuralandmolecularbasisofsodiumchannelblockbyhuwentoxin-IVandmayprovideabasisfortherationaldesignoftoxin-basedpeptideswithimprovedVGSCpotencyand/orselectivity.
MinassianNA.,etal.(2013)Analysisofthestructuralandmolecularbasisofvoltage-sensitivesodiumchannelinhibitionbythespidertoxin,Huwentoxin-IV(μ-TRTX-Hh2a). JBC. PMID: 23760503
Commonmoleculardeterminantsoftarantulahuwentoxin-IVinhibitionofNa+channelvoltagesensorsindomainsIIandIV
ThevoltagesensorsofdomainsIIandIVofsodiumchannelsareimportantdeterminantsofactivationandinactivation,respectively.AnimaltoxinsthatalterelectrophysiologicalexcitABIlityofmusclesandneuronsoftenmodifysodiumchannelactivationbyselectivelyinteractingwithdomainIIandinactivationbyselectivelyinteractingwithdomainIV.Thissuggeststhattheremaybesubstantialdifferencesbetweenthetoxin-bindingsitesinthesetwoimportantdomains.Hereweexploretheabilityofthetarantulahuwentoxin-IV(HWTX-IV)toinhibittheactivityofthedomainIIandIVvoltagesensors.HWTX-IVisspecificfordomainII,andweidentifyfiveresiduesintheS1-S2(Glu-753)andS3-S4(Glu-811,Leu-814,Asp-816,andGlu-818)regionsofdomainIIthatarecrucialforinhibitionofactivationbyHWTX-IV.ThesedataindicatethatasingleresidueintheS3-S4linker(Glu-818inhNav1.7)iscrucialforallowingHWTX-IVtointeractwiththeotherkeyresiduesandtrapthevoltagesensorintheclosedconfiguration.MutagenesisanalysisindicatesthatthefivecorrespondingresiduesindomainIVareallcriticalforendowingHWTX-IVwiththeabilitytoinhibitfastinactivation.Ourdatasuggestthatthetoxin-bindingmotifindomainIIisconservedindomainIV.Increasingourunderstandingofthemoleculardeterminantsoftoxininteractionswithvoltage-gatedsodiumchannelsmaypermitdevelopmentofenhancedisoform-specificvoltage-gatingmodifiers.
Xiao,Y., etal.(2011)Commonmoleculardeterminantsoftarantulahuwentoxin-IVinhibitionofNa+channelvoltagesensorsindomainsIIandIV, JBC. PMID: 21659528
ThetarantulatoxinsProTx-IIandhuwentoxin-IVdifferentiallyinteractwithhumanNav1.7voltagesensorstoinhibitchannelactivationandinactivation
Thevoltage-gatedsodiumchannelNa(v)1.7playsacrucialroleinpain,anddrugsthatinhibithNa(v)1.7mayhavetremendoustherapeuticpotential.ProTx-IIandhuwentoxin-IV(HWTX-IV),cystineknotpeptidesfromtarantulavenoms,preferentiallyblockhNa(v)1.7.Understandingtheinteractionsofthesetoxinswithsodiumchannelscouldaidthedevelopmentofnovelpaintherapeutics.WhereasbothProTx-IIandHWTX-IVhavebeenproposedtopreferentiallyblockhNa(v)1.7activationbytrappingthedomainIIvoltage-sensorintherestingconfiguration,weshowthatspecificresiduesinthevoltage-sensorpaddleofdomainIIplaysubstantiallydifferentrolesindeterminingtheaffinitiesofthesetoxinstohNa(v)1.7.ThemutationE818CincreasesProTx-II‘sandHWTX-IV‘sIC(50)forblockofhNa(v)1.7currentsby4-and400-fold,respectively.Incontrast,themutationF813GdecreasesProTx-IIaffinityby9-foldbuthasnoeffectonHWTX-IVaffinity.ItisnoteworthythatwealsoshowthatProTx-II,butnotHWTX-IV,preferentiallyinteractswithhNa(v)1.7toimpedefastinactivationbytrappingthedomainIVvoltage-sensorintherestingconfiguration.MutationsE1589QandT1590KindomainIVeachdecreasedProTx-II’sIC(50)forimpairmentoffastinactivationby~6-fold.IncontrastmutationsD1586AandF1592Aindomain-IVincreasedProTx-II’sIC(50)forimpairmentoffastinactivationby~4-fold.OurresultsshowthatwhereasProTx-IIandHWTX-IVbindingdeterminantsondomain-IImayoverlap,domainIIplaysamuchmorecrucialroleforHWTX-IV,andcontrarytowhathasbeenproposedtobeaguidingprincipleofsodiumchannelpharmacology,moleculesdonothavetoexclusivelytargetthedomainIVvoltage-sensortoinfluencesodiumchannelinactivation.
Xiao,Y., etal.(2010)ThetarantulatoxinsProTx-IIandhuwentoxin-IVdifferentiallyinteractwithhumanNav1.7voltagesensorstoinhibitchannelactivationandinactivation, MolPharmacol. PMID: 20855463
Mechanismofactionoftwoinsecttoxinshuwentoxin-IIIandhainantoxin-VIonvoltage-gatedsodiumchannels
SelenocosmiahuwenaandSelenocosmiahainanaaretwotarantulaspeciesfoundinsouthernChina.Theirvenomscontainabundantpeptidetoxins.Twonewneurotoxicpeptides,huwentoxin-III(HWTX-III)andhainantoxin-VI(HNTX-VI),wereobtainedfromthevenomusingion-exchangechromatographyandreverse-phasehighperformanceliquidchromatography(RP-HPLC).ThemechanismofactionofHWTX-IIIandHNTX-VIoninsectneuronalvoltage-gatedsodiumchannels(VGSCs)wasstudiedviawhole-cellpatchclamptechniques.Inafashionsimilartodelta-atracotoxins,HNTX-VIcaninduceaslowdownofcurrentinactivationoftheVGSCandreductioninthepeakofNa+currentincockroachdorsalunpairedmedian(DUM)neurons.Meanwhile,10micromol/LHNTX-IVcausedapositiveshiftofsteady-stateinactivationofsodiumchannel.HWTX-IIIinhibitedVGSCsonDUMneurons(concentrationoftoxinathalf-maximalinhibition(IC(50))approximately1.106micromol/L)inawaymuchsimilartotetrodotoxin(TTX).HWTX-IIIhadnoeffectonthekineticsofactivationandinactivation.Theshiftinthesteady-stateinactivationcurvewasdistinctfromotherdepressantspidertoxins.ThediverseeffectandthemechanismofactionofthetwoinsecttoxinsillustratethediverseBIOLOGicalactivitiesofspidertoxinsandprovideafreshtheoreticalfoundationtodesignanddevelopnovelinsecticides.
WangRL, etal.(2010)Mechanismofactionoftwoinsecttoxinshuwentoxin-IIIandhainantoxin-VIonvoltage-gatedsodiumchannels. PMID: 20506577
Tarantulahuwentoxin-IVinhibitsneuronalsodiumchannelsbybindingtoreceptorsite4andtrappingthedomainiivoltagesensorintheclosedconfiguration
Peptidetoxinswithhighaffinity,divergentpharmacologicalfunctions,andisoform-specificselectivityarepowerfultoolsforinvestigatingthestructure-functionrelationshipsofvoltage-gatedsodiumchannels(VGSCs).Althoughanumberofinterestinginhibitorshavebeenreportedfromtarantulavenoms,littleisknownaboutthemechanismfortheirinteractionwithVGSCs.Weshowthathuwentoxin-IV(HWTX-IV),a35-residuepeptidefromtarantulaOrnithoctonushuwenavenom,preferentiallyinhibitsneuronalVGSCsubtypesrNav1.2,rNav1.3,andhNav1.7comparedwithmusclesubtypesrNav1.4andhNav1.5.OfthefiveVGSCsexamined,hNav1.7wasmostsensitivetoHWTX-IV(IC(50)approximately26nM).Followingapplicationof1micromHWTX-IV,hNav1.7currentscouldonlybeelicitedwithextremedepolarizations(>+100mV).RecoveryofhNav1.7channelsfromHWTX-IVinhibitioncouldbeinducedbyextremedepolarizationsormoderatedepolarizationslastingseveralminutes.Site-directedmutagenesisanalysisindicatedthatthetoxindockedatneurotoxinreceptorsite4locatedattheextracellularS3-S4linkerofdomainII.MutationsE818QandD816NinhNav1.7decreasedtoxinaffinityforhNav1.7byapproximately300-fold,whereasthereversemutationsinrNav1.4(N655D/Q657E)andthecorrespondingmutationsinhNav1.5(R812D/S814E)greatlyincreasedthesensitivityofthemuscleVGSCstoHWTX-IV.Ourdataidentifyanovelmechanismforsodiumchannelinhibitionbytarantulatoxinsinvolvingbindingtoneurotoxinreceptorsite4.Incontrasttoscorpionbeta-toxinsthattraptheIIS4voltagesensorinanoutwardconfiguration,weproposethatHWTX-IVtrapsthevoltagesensorofdomainIIintheinward,closedconfiguration.
Xiao,Y., etal.(2008)Tarantulahuwentoxin-IVinhibitsneuronalsodiumchannelsbybindingtoreceptorsite4andtrappingthedomainiivoltagesensorintheclosedconfiguration, JBC. PMID: 18628201
Functionandsolutionstructureofhuwentoxin-IV,apotentneuronaltetrodotoxin(TTX)-sensitivesodiumchannelantagoNISTfromChinesebirdspiderSelenocosmiahuwena
WehaveisolatedahighlypotentneurotoxinfromthevenomoftheChinesebirdspider,Selenocosmiahuwena.This4.1-kDatoxin,whichhasbeennamedhuwentoxin-IV,contains35residueswiththreedisulfidebridges:Cys-2-Cys-17,Cys-9-Cys-24,andCys-16-Cys-31,assignedbyachemicalstrategyincludingpartialreductionofthetoxinandsequenceanalysisofthemodifiedintermediates.Itspecificallyinhibitstheneuronaltetrodotoxin-sensitive(TTX-S)voltage-gatedsodiumchannelwiththeIC(50)valueof30nminadultratdorsalrootganglionneurons,whilehavingnosignificanteffectonthetetrodotoxin-resistant(TTX-R)voltage-gatedsodiumchannel.ThistoxinseemstobeasiteItoxinaffectingthesodiumchannelthroughamechanismquitesimilartothatofTTX:itsuppressesthepeaksodiumcurrentwithoutalteringtheactivationorinactivationkinetics.Thethree-dimensionalstructureofhuwentoxin-IVhasbeendeterminedbytwo-dimensional(1)HNMRcombinedwithdistantgeometryandsimulatedannealingcalculationbyusing527nuclearOverhausereffectconstraintsand14dihedralconstraints.Theresultingstructureiscomposedofadouble-strandedantiparallelbeta-sheet(Leu-22-Ser-25andTrp-30-Tyr-33)andfourturns(Glu-4-Lys-7,Pro-11-Asp-14,Lys-18-Lys-21andArg-26-Arg-29)andbelongstotheinhibitorcystineknotstructuralfamily.Aftercomparisonwithothertoxinspurifiedfromthesamespecies,weareconvincedthatthepositivelychargedresiduesofloopIV(residues25-29),especiallyresidueArg-26,mustbecrucialtoitsbindingtotheneuronaltetrodotoxin-sensitivevoltage-gatedsodiumchannel.
Peng,K., etal.(2002)Functionandsolutionstructureofhuwentoxin-IV,apotentneuronaltetrodotoxin(TTX)-sensitivesodiumchannelantagonistfromChinesebirdspiderSelenocosmiahuwena, JBiolChem.PMID: 12228241
Smartox"scitation:Anaturalpointmutationchangesbothtargetselectivityandmechanismofactionofseaanemonetoxins
APETx3,anovelpeptideisolatedfromtheseaanemoneAnthopleuraelegantissima,isanaturallyoccurringmutantfromAPETx1,onlydifferingbyaThrtoProsubstitutionatposition3.APETx1isbelievedtobeaselectivemodulatorofhumanether-á-go-gorelatedgene(hERG)potassiumchannelswithaK(d)of34nM.Inthisstudy,APETx1,2,and3havebeensubjectedtoanelectrophysiologicalscreeningonawiderangeof24ionchannelsexpressedinXenopuslaevisoocytes:10clonedvoltage-gatedsodiumchannels(Na(V)1.2-Na(V)1.8,theinsectchannelsDmNa(V)1,BgNa(V)1-1a,andthearachnidchannelVdNa(V)1)and14clonedvoltage-gatedpotassiumchannels(K(V)1.1-K(V)1.6,K(V)2.1,K(V)3.1,K(V)4.2,K(V)4.3,K(V)7.2,K(V)7.4,hERG,andtheinsectchannelShakerIR).Surprisingly,theThr3ProsubstitutionresultsinacompleteabolishmentofAPETx3modulationonhERGchannelsandprovidesthistoxintheabilitytobecomeapotent(EC(50)276nM)modulatorofvoltage-gatedsodiumchannels(Na(V)s)becauseitslowsdowntheinactivationofmammalianandinsectNa(V)channels.OurstudyalsoshowsthatthehomologoustoxinsAPETx1andAPETx2displaypromiscuouspropertiessincetheyarealsocapableofrecognizingNa(V)channelswithIC(50)valuesof31nMand114nM,respectively,causinganinhibitionofthesodiumconductancewithoutaffectingtheinactivation.Ourresultsprovidenewinsightsinkeyresiduesthatallowtheseseaanemonetoxinstorecognizedistinctionchannelswithsimilarpotencybutwithdifferentmodulatoryeffects.FurThermore,wedescribeforthefirsttimethetargetpromiscuityofafamilyofseaanemonetoxinsthusfarbelievedtobehighlyselective.
PeigneurS, etal. (2012)Anaturalpointmutationchangesbothtargetselectivityandmechanismofactionofseaanemonetoxins. FASEBJ. PMID: 22972919
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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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