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.Cy5-HuwentoxinIVisafluorescentlytaggedversionofHuwentoxinIV.
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
FormulaofHwTx-IV: C174H277N51O52S6
MolecularWeightofHwTx-IV: 4107.20Da
Appearance:darklyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber:
Source: Synthetic
Purityrate: >95%
Cy5:λex646nm,λem662nm
Stoichiometry:1:1
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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我在做一细菌不同酸碱度生长状况时,发现这些奇怪现象:pH=3的培养基灭菌(TSB液体培养基)灭菌后pH上升到到9.2!而原来pH=9.0的降到8.7(基本没多少变化),请问各位大侠,这是什么原因?
一般做不同酸碱度生长实验时,该如何才能防止pH在湿热灭菌后基本不变化?
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！
因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
拼音名：Chunhuashui
英文名：PurifiedWater
【性状】本品为无色的澄清液体；无臭，无味。
【检查】酸碱度取本品10ml，加甲基红指示液2滴，不得显红色；另取10ml，加溴麝香草酚蓝指示液5滴，不得显蓝色。氯化物、流酸盐与钙盐取本品，分置三支试管中，每管各50ml。第一管中加硝酸5滴与硝酸银试液1ml，第二管中加氯化钡试液2ml，第三管中加草酸铵试液2ml，均不得发生浑浊。
硝酸盐取本品5ml置试管中，于冰浴中冷却，加10%氯化钾溶液0.4ml与0.1%二苯胺硫酸溶液0.1ml，摇匀，缓缓滴加硫酸5ml，摇匀，将试管子50℃水浴中放置15分钟，溶液产生的蓝色与标准硝酸盐溶液［取硝酸钾0.163g，加水溶解并稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，再精密量取10ml，加水稀释成100ml，摇匀，即得（每1ml相当于1pgNO3）0.3ml，加无硝酸盐的水4.7ml，用同一方法处理后的颜色比较，不得更深（0.000006%）。
亚硝酸盐取本品10ml，置纳氏管中，加对氨基苯磺酰胺的稀盐酸溶液（1→100）lml与盐酸菜乙H肢溶液（0．l＋100）1ml，产生的粉红色，与标准亚硝酸盐溶液〔取亚硝酸钠0.750g（按干燥品计算），加水溶解，稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，摇匀，再精密量取1ml，加水稀释成50ml，摇匀，即得（每1ml相当于1μgNO2）]0.2ml，加无亚硝酸盐的水9.8ml，用同一方法处理后的颜色比较，不得更深（0.000002%）。
氨取本品50ml，加碱性碘化汞钾试液2ml，放置15分钟；如显色，与氯化铵溶液（取氯化铵31.5mg，加无氨水适量使溶解并稀释成1000ml）1.5ml，加元氨水48ml与碱性碘化汞钾试液2ml制成的对照液比较，不得更深（0.00003%）。
二氧化碳取本品25ml，置50ml具塞量筒中，加氢氧化钙试液25ml，密塞振摇，放置，小时内不得发生浑浊。
易氧化物取本品100ml，加稀硫酸10ml，煮沸后，加高锰酸钾滴定液（0.02mol/L）0.10ml，再煮沸10分钟，粉红色不得完全消失。
不挥发物取本品100ml，置105℃恒重的蒸发皿中，在水浴上蒸干，并在105℃干燥至恒重，遗留残渣不得过1mg。
重金属取本品50ml，加水18.5ml，蒸发至20ml，放冷，加醋酸盐缓冲液（pH3.5）2ml与水适量使成25ml，加硫代乙酰胺试液2ml，摇匀，放置2分钟，与标准铅溶液1.5ml加水18.5ml用同一方法处理后的颜色比较，不得更深（0.00003%）。
微生物限度取本品，采用薄膜过滤法处理后，依法检查（附录ⅪJ），细菌、霉菌和酵母菌总数每1ml不得过100个。
【贮藏】密闭保存。
【化学成分】本品为蒸馏法、离子交换法、反渗透法或其他适宜的方法制得的供药用的水，不含任何附加剂。
【分子式与分子量】H2O18.02
【药理作用】溶剂、稀释剂
这里药典纯化水标准中并无PH值项目，请问对纯化水有PH值的要求吗，范围应在多少？请说明出处？
在纯化水检测中，检验酸碱度合格，但是发现PH在8左右。如果按以上标准检验合格，是否要考虑PH值？请知道的解答，谢谢！
1、弱酸和它的盐（如：HAc---NaAc）的水溶液组成；
2、弱碱和它的盐（如：NH3·H2O---NH4Cl）的水溶液组成；
3、多元弱酸的酸式盐及其对应的次级盐（如：NaH2PO4---Na2HPO4）的水溶液组成。
酸碱缓冲溶液的选型一般应根据具体情况进行选择。缓冲酸性可选用碱性缓冲液，缓冲酸性可采用碱性缓冲液。常用作缓冲溶液的酸类由弱酸及其共轭酸盐组合成的溶液具有缓冲作用。生化实验室常用的缓冲系主要有磷酸、柠檬酸、碳酸、醋酸、巴比妥酸、Tris（三羟甲基氨基甲烷）等系统，生化实验或研究工作中要慎重地选择缓冲体系，因为有时影响实验结果的因素并不是缓冲液的pH值，而是缓冲液中的某种离子。如硼酸盐、柠檬酸盐、磷酸盐和三羟甲基甲烷等缓冲剂都可能产生不需要的化学反应。
【酸碱缓冲溶液】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。这种溶液称为酸碱缓冲溶液。
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。
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