Scyllatoxin(Leiurotoxin-1)isaneurotoxinthatwasoriginallyisolatedfromLeiurusquinquestriatushebraeus.ScyllatoxinbindsandblocksSKchannels(smallconductanceCa2+-activatedK+channels)inthebrainandspinalcordandinhibitsthem.
Description:
AAsequence:Ala-Phe-Cys3-Asn-Leu-Arg-Met-Cys8-Gln-Leu-Ser-Cys12-Arg-Ser-Leu-Gly-Leu-Leu-Gly-Lys-Cys21-Ile-Gly-Asp-Lys-Cys26-Glu-Cys28-Val-Lys-His-NH2
Disulfidebondsbetween:Cys3-Cys21,Cys8-Cys26andCys12-Cys28
Length(aa):31
Formula:C142H237N45O39S7
MolecularWeight:3424.1Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:[116235-63-3]Source:Synthetic
Purityrate:>95%
Reference:
EffectofcharyBDotoxinandleiurotoxinIonpotassiumcurrentsinbullfrogsympatheticganglionandhippocampalneurons
TheeffectsofcharybdotoxinandleiurotoxinIwereexaminedonseveralclassesofK+currentsinbullfrogsympatheticganglionandhippocampalCA1pyramidalneurons.Highlypurifiedpreparationsofcharybdotoxinselectivelyblockedalargevoltage-andCa(2+)-dependentK+current(IC)responsIBLeforactionpotentialrepolarization(IC50=6nM)whileleiurotoxinIselectivelyblockedasmallCa(2+)-dependentK+conductance(IAHP)responsiblefortheslowafterhyperpolarizationfollowinganactionpotential(IC50=7.5nM)inbullfrogsympatheticganglionneurons.NeitherofthetoxinshadsignificanteffectsonotherK+currents(M-current[IM],A-current[IA]andthedelayedrectifier[IK])presentinthesecells.LeiurotoxinIataconcentrationof20nMhadnodetectableeffectoncurrentsinhippocampalCA1pyramidalneurons.ThislackofeffectonIAHPincentralneuronssuggeststhatthechannelsunderlyingslowAHPsinthoseneuronsarepharmacologicallydistinctfromanalogouschannelsinperipheralneurons.
GohJW.,etal.(1992)EffectofcharybdotoxinandleiurotoxinIonpotassiumcurrentsinbullfrogsympatheticganglionandhippocampalneurons.BrainRes.PMID: 1280181
Anapamin-andscyllatoxin-insensitiveisoformofthehumanSK3channel
WehaveisolatedanhSK3isoformfromahumanembryonicCDNAlibrarythatwehavenamedhSK3_ex4.Thisisoformcontainsa15aminoacidinsertionwithintheS5toP-loopsegment.TranscriptsencodinghSK3_ex4arecoexpressedatlowerlevelswithhSK3inneuronalaswellasinnon-neuronaltissues.ToinvestigatethepharmacokineticpropertiesofhSK3_ex4,weexpressedtheisoformshSK3andhSK3_ex4intsAcells.BothisoformsweresimilarlyactivatedbycytosolicCa2+(hSK3,EC50=0.91+/-0.4microM;hSK3_ex4,EC50=0.78+/-0.2microM)andby1-ethyl-2-benzimidazolinone(hSK3,EC50=0.17mM;hSK3_ex4,0.19mM).Theywerebothblockedbytetraethylammonium(hSK3,Kd=2.2mM;hSK3_ex4,2.6mM)andshowedsimilarpermeABIlitiesrelativetoK+forCs+(hSK3,0.17+/-0.04,n=3;hSK3_ex4,0.17+/-0.05,n=3)andRb+(hSK3,0.79+/-0.04,n=3;hSK3_ex4,0.8+/-0.07,n=3).Ba2+blockedbothisoforms,andinbothcases,theblockwasstrongestathyperpolarizingmembranepotentials.However,thevoltage-dependenceofhSK3wasstrongerthanthatofhSK3_ex4.ThemostobviousdistinguishingfeatureofthisnewisoformwasthatwhereashSK3wasblockedbyapamin(Kd=0.8nM),scyllatoxin(Kd=2.1nM),andd-tubocurarine(Kd=33.4microM),hSK3_ex4wasnotaffectedbyapaminupto100nM,scyllatoxinupto500nM,andd-tubocurarineupto500microM.Sofar,isoformhSK3_ex4formstheonlysmall-conductancecalcium-activatedpotassium(SK)channels,whichareinsensitivetotheclassicSKblockers.
WittekindtOH.,etal.(2004)Anapamin-andscyllatoxin-insensitiveisoformofthehumanSK3channel.MolPharmacol.PMID: 14978258
LeiurotoxinI,ascorpiontoxinspecificforCa(2+)-activatedK+channels.Structure-activityanalysisusingsyntheticanalogs
Recently,wereportedastructure-activityrelationshipstudyonP05,anovelleiurotoxinI-likescorpiontoxinwhichisselectivefortheapamin-sensitiveCa(2+)-activatedK+channel[Sabatieretal.(1993)Biochemistry32,2763-2770].Arg6,Arg7andC-terminalHis31appearedtobekeyresiduesforP05BIOLOGicalactivity.OwingtothehighsequenceidentitybetweenP05andleiurotoxinI(87%),severalanalogsofleiurotoxinI(Lei-NH2)withpointmutationsatthesepositionsweredesignedandchemicallysynthesizedusinganoptimizedsolid-phasetechnique.Thesynthesizedpeptideswere[L6]Lei-NH2,[R7]Lei-NH2,Lei-OHand[R7]Lei-OH,aswellasfragment[R7,Abu8]N4-S11-NH2.Achimericanalog([M22,K24,R27]Lei-NH2),whichpossessespartoftheiberiotoxinC-terminus,wasalsoconstructed.Circulardichroismanalysesoftheseanalogs,inagreementwiththeirstructuralmodelsobtainedbymoleculardynamics,showedthatthepointmutationsdidnotsignificantlyaffecttheoverallsecondarystructures,ascomparedtonaturalLei-NH2.Allthepeptidesandnaturaltoxinswerecomparedinvitrofortheircapacitytoinhibitbindingof[125I]-apamintoratbrainsynaptosomes,andinvivofortheirspecificneurotoxicityinmice.TheArg6residuewasessentialforhighbiologicalactivityofleiurotoxinI.Further,substitutionofMet7inthenaturaltoxinbyArg7,orC-terminalamidationofHis31,greatlyincreasedaffinityfortheapaminreceptorbutdidnotsignificantlyaffecttoxinneurotoxicity.Remarkably,thechimericanalog[M22,K24,R27]Lei-NH2wasfoundtoretainleiurotoxinI-likeactivity,thusindicatingthatthenegativelychargedresiduesAsp24andGlu27(andIle22)arenotdirectlyinvolvedinthehightoxinbioactivity.However,thechimericmoleculehadnoiberiotoxin-likeeffectonratmuscularmaxi-K+channelsincorporatedinlipidbilayers.
SabatierJM.,etal.(1994)LeiurotoxinI,ascorpiontoxinspecificforCa(2+)-activatedK+channels.Structure-activityanalysisusingsyntheticanalogs.IntJPeptProteinRes. PMID: 8070973
Scyllatoxin,ablockerofCa(2+)-activatedK+channels:structure-functionrelationshipsandbrainlocalizationofthebindingsites
Chemicalmodificationsofscyllatoxin(leiurustoxinI)haveshownthattwoargininesinthesequence,Arg6andArg13,areessentialbothforbindingtotheCa(2+)-activatedK+channelproteinandforthefunctionaleffectofthetoxin.His31isimportantbothforthebindingactivityofthetoxinandfortheinductionofcontractionsontaeniacoli.However,althoughitsiodinationdrasticallydecreasesthetoxinactivity,itdoesnotabolishit.ChemicalmodificationoflysineresiduesorofGlu27doesnotsignificantlyaltertoxinbinding,butitdrasticallydecreasespotencywithrespecttocontractionoftaeniacoli.Thesameobservationhasbeenmadeafterchemicalmodificationofthelysineresidues.ThebraindistributionofscyllatoxinbindingsiteshasbeenanalyzedbyquantitativeautorADIographicanalysis.Itindicatesthatapamin(abeevenomtoxin)bindingsitesarecolocalizedwithscyllatoxinbindingsites.Theresultsareconsonantwiththepresenceofapamin/scyllatoxinbindingsitesassociatedwithCa(2+)-activatedK+channels.High-affinitybindingsitesforapamincanbeassociatedwithvery-high-affinity(lessthan70pM),high-affinity(approximately100-500pM),ormoderate-affinity(greaterthan800pM)bindingsitesforscyllatoxin.
AugusteP.,etal.(1992)Scyllatoxin,ablockerofCa(2+)-activatedK+channels:structure-functionrelationshipsandbrainlocalizationofthebindingsites.Biochemistry. PMID: 1731919
Purificationandcharacterizationofaunique,potentinhibitorofapaminbindingfromLeiurusquinquestriatushebraeusvenom
Aninhibitorofapaminbindinghasbeenpurifiedtohomogeneityinthreechromatographicstepsfromthevenomofthescorpion,Leiurusquinquestriatushebraeus.Theinhibitor,whichwehavenamedleiurotoxinI,representslessthan0.02%ofthevenomprotein.Itisa3.4-kDapeptidewithlittlestructuralhomologytoapaminalthoughithassomehomologytootherscorpiontoxinssuchascharybdotoxin,noxiustoxin,andneurotoxinP2.LeiurotoxinIcompletelyinhibits125I-apaminbindingtoratbrainsynaptosomalmembranes(Ki=75pM).Thus,itis10-20-foldlesspotentthanapamin.LeiurotoxinIisnotastrictlycompetitiveinhibitorofthisbindingreaction.Likeapamin,leiurotoxinIblockstheepinephrine-inducedrelaxationofguineapigteniaecoli(ED50=6.5nM),whilehavingnoeffectontherateorforceofcontractioninguineapigatriaorrabbitportalveinpreparations.Thus,leiurotoxinIofscorpionvenomandapaminofhoneybeevenomdemonstratesimilaractivitiesinavarietyoftissues,yetarestructurallyunrelatedpeptides.Thesetwopeptidesshouldbeusefulinelucidatingtheroleofthesmallconductance,Ca2+-activatedK+channelsindifferenttissues.
ChicchiGG.,etal.(1988)Purificationandcharacterizationofaunique,potentinhibitorofapaminbindingfromLeiurusquinquestriatushebraeusvenom. PMID: 2839478
Iberiotoxin-sensitiveand-insensitiveBKcurrentsinPurkinjeneuronsomata
Purkinjecellshavespecializedintrinsicionicconductancesthatgeneratehigh-frequencyactionpotentials.DisruptionsoftheirCaorCa-activatedK(KCa)currentscorrelatewithalteredfiringpatternsinvitroandimpairedmotorbehaviorinvivo.ToexaminethepropertiesofsomaticKCacurrents,werecordedvoltage-clampedKCacurrentsinPurkinjecellbodiesisolatedfrompostnatalday17-21mousecerebellum.CurrentswereevokedbyendogenousCainfluxwithapproximatelyphysiologicalCabuffering.Purkinjesomataexpressedvoltage-activated,Cd-sensitiveKCacurrentswithiberiotoxin(IBTX)-sensitive(>100nS)andIBTX-insensitive(>75nS)components.IBTX-sensitivecurrentsactivatedandpartiallyinactivatedwithinmilliseconds.Rapid,incompletemacroscopicinactivationwasalsoevidentduring50-or100-Hztrainsof1-msdepolarizations.Incontrast,IBTX-insensitivecurrentsactivatedmoreslowlyanddidnotinactivate.Thesecurrentswereinsensitivetothesmall-andintermediate-conductanceKCachannelblockersapamin,scyllatoxin,UCL1684,bicucullinemethiodide,andTRAM-34,butwerelargelyblockedby1mMtetraethylammonium.Theunderlyingchannelshadsingle-channelconductancesof∼150pS,suggestingthatthecurrentsarecarriedbyIBTX-resistant(β4-containing)large-conductanceKCa(BK)channels.IBTX-insensitivecurrentswereneverthelessincreasedbysmall-conductanceKCachannelagoNISTsEBIO,chlorzoxazone,andCyPPA.Duringtrainsofbriefdepolarizations,IBTX-insensitivecurrentsflowedduringinterstepintervals,andtheaccumulationofinterstepoutwardcurrentwasenhancedbyEBIO.Incurrentclamp,EBIOslowedspiking,especiallyduringdepolarizingcurrentinjections.ThetwocomponentsofBKcurrentinPurkinjesomatalikelycontributedifferentlytospikerepolarizationandfiringrate.Moreover,augmentationofBKcurrentmaypartiallyunderlietheactionofEBIOandchlorzoxazonetoalleviatedisruptedPurkinjecellfiringassociatedwithgeneticataxias.
BentonMD.,etal.(2013)Iberiotoxin-sensitiveand-insensitiveBKcurrentsinPurkinjeneuronsomata.JNeurophysiol. PMID: 23446695
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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。
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~
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！
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！
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