BDS-1isa43aminoacidpeptidewhichwasoriginallyisolatedfromthevenomoftheseaanemonaAnemoniaViridis.BDS-1wasoriginallydescribedasahighlyselectiveblockeroftherapidlyinactivatingvoltage-gatedpotassiumchannelKv3.4/KCNC4,apotentialtherapeutictargetformajorCNSdisorders(AlzheimerandParkinsondiseases).Thetoxinactsasgatingmodifiers,mainlybyshiftingthevoltage-dependenceofactivation.Channelblockoccurswithhighaffinity(IC50of43nM)andisrapidandreversIBLe.BDS-1alsoblockstheKv3.1andKv3.2channelsalbeitwithaloweraffinity(>200nM).Finally,inamorerecentstudy,itwasdemonstratedthatBDS-1isaselectivegatingactivatoroftheNav1.7channelsubtype,animportanttargetforpainmanagement.Onthehumanisoform,modulationiswitnessedbyadrasticslowingofchannelinactivationwhichoccurswithanIC50of3nM.
Description:
AAsequence:Ala-Ala-Pro-Cys4-Phe-Cys6-Ser-Gly-Lys-Pro-Gly-Arg-Gly-Asp-Leu-Trp-Ile-Leu-Arg-Gly-Thr-Cys22-Pro-Gly-Gly-Tyr-Gly-Tyr-Thr-Ser-Asn-Cys32-Tyr-Lys-Trp-Pro-Asn-Ile-Cys39-Cys40-Tyr-Pro-His-OH
Disulfidebonds: Cys4-Cys39,Cys6-Cys32,Cys22-Cys40
Length(aa):43
Formula: C210H297N57O56S6
Appearance:Whitelyophilizedsolid
MolecularWeight:4708.37Da
CASnumber:
Source:Synthetic
Solubility:Waterorsalinebuffer
Reference:
SeaanemonepeptideswithaspecificblockingactivityagainstthefastinactivatingpotassiumchannelKv3.4
Seaanemonevenomisknowntocontaintoxinsthatareactiveonvoltage-sensitiveNa+channels,aswellasondelayedrectifierK+channelsbelongingtotheKv1family.ThisreportdescribesthepropertiesofanewsetofpeptidesfromAnemoniasulcatathatactasblockersofaspecificmemberoftheKv3potassiumchannelfamily.Thesetoxins,blooddepressingsubstance(BDS)-IandBDS-II,are43aminoacidslonganddifferatonlytwopositions.TheysharenosequencehomologieswithotherK+channeltoxinsfromseaanemones,suchasAsKS,AsKC,ShK,orBgK.InCOS-transfectedcells,theKv3.4currentwasinhibitedinareversiblemannerbyBDS-I,withanIC50valueof47nM.ThisinhibitionisspecificbecauseBDS-IfailedtoblockotherK+channelsintheKv1,Kv2,Kv3,andKv4subfamilies.InwardrectifierK+channelsarealsoinsensitivetoBDS-I.BDS-IandBDS-IIsharethesamebindingsiteonbrainsynapticmembranes,withK0.5valuesof12and19nM,respectively.WeobservedthatBDS-IandBDS-IIhavesomesequencehomologieswithotherseaanemoneNa+channelstoxins,suchasAsI,AsII,andAxI.However,theyhadaweakeffectontetrodotoxin-sensitiveNa+channelsinneuroblastomacellsandnoeffectonNa+channelsincardiacandskeletalmusclecells.BDS-IandBDS-IIarethefirstspecificblockersidentifiedsofarfortherapidlyinactivatingKv3.4channel.
Diochotetal(1998)SeaanemonepeptideswithaspecificblockingactivityagainstthefastinactivatingpotassiumchannelKv3.4.J.Biol.Chem.PMID:9506974.
Up-regulationandincreasedactivityofKV3.4channelsandtheiraccessorysubunitMinK-relatedpeptide2inducedbyamyloidpeptideareinvolvedinapoptoticneuronaldeath
TheaimofthepresentstudywastoinvestigatewhetherK(V)3.4channelsubunitsareinvolvedinneuronaldeathinducedbyneurotoxicbeta-amyloidpeptides(Abeta).Inparticular,totestthishypothesis,threemainquestionswereaddressed:1)whethertheAbetapeptidecanup-regulateboththetranscription/translationandactivityofK(V)3.4channelsubunitanditsaccessorysubunit,MinK-relatedpeptide2(MIRP2);2)whethertheincreaseinK(V)3.4expressionandactivitycanbemediatedbythenuclearfactor-kappaB(NF-kappaB)familyoftranscriptionalfactors;and3)whetherthespecificinhibitionofK(V)3.4channelsubunitrevertstheAbetapeptide-inducedneurodegenerationinhippocampalneuronsandnervegrowthfactor(NGF)-differentiatedPC-12cells.WefoundthatAbeta(1-42)treatmentinducedanincreaseinK(V)3.4andMIRP2transcriptsandproteins,detectedbyreversetranscription-polymerasechainreactionandWesternblotanalysis,respectively,inNGF-differentiatedPC-12cellsandhippocampalneurons.Patch-clampexperimentsperformedinwhole-cellconfigurationrevealedthattheAbetapeptidecausedanincreaseinI(A)currentamplitudecarriedbyK(V)3.4channelsubunits,asrevealedbytheirspecificblockadewithblooddepressingsubstance-I(BDS-I)inbothhippocampalneuronsandNGF-differentiatedPC-12cells.TheinhibitionofNF-kappaBnucleartranslocationwiththecellmembrane-permeablepeptideSN-50preventedtheincreaseinK(V)3.4proteinandtranscriptexpression.Inaddition,theSN-50peptidewasabletoblockAbeta(1-42)-inducedincreaseinK(V)3.4K(+)currentsandtopreventcelldeathcausedbyAbeta(1-42)exposure.Finally,BDS-IproducedasimilarneuroprotectiveeffectbyinhibitingtheincreaseinK(V)3.4expression.Asawhole,ourdataindicatethatK(V)3.4channelscouldbeanoveltargetforAlzheimer’sdiseasepharmacologicaltherapy.
Pannaccioneetal(2007)Up-regulationandincreasedactivityofKV3.4channelsandtheiraccessorysubunitMinK-relatedpeptide2inducedbyamyloidpeptideareinvolvedinapoptoticneuronaldeath.Mol.Pharmacol.PMID:17495071.
Voltage-dependentpotassiumcurrentsduringfastspikesofratcerebellarPurkinjeneurons:inhibitionbyBDS-Itoxin.
MartinaM.,etal.(2007)Voltage-dependentpotassiumcurrentsduringfastspikesofratcerebellarPurkinjeneurons:inhibitionbyBDS-Itoxin.J.Neurophysiol.PMID:17065256
ModulationofKv3subfamilypotassiumcurrentsbytheseaanemonetoxinBDS:significanceforCNSandbiophysicalstudies.
Kv3potassiumchannels,withtheirultra-rapidgatingandhighactivationthreshold,areessentialforhigh-frequencyfiringinmanyCNSneurons.Significantly,theKv3.4subunithasbeenimplicatedinthemajorCNSdisordersParkinson’sandAlzheimer’sdiseases,anditisclaimedthatselectivelytargetingthissubunitwillhavetherapeuticutility.PreviousworksuggestedthatBDStoxins(“blooddepressingsubstance,”fromtheseaanemoneAnemoniasulcata)werespecificblockersforrapidlyinactivatingKv3.4channels,andconsequentlythesetoxinsareincreasinglyusedasdiagnosticagentsforKv3.4subunitsincentralneurons.However,preciselyhowselectivearethesetoxinsforthisimportantCNSprotein?WeshowthatBDSisnotselectiveforKv3.4butmarkedlyinhibitscurrentthroughKv3.1andKv3.2channels.Inhibitioncomesaboutnotby“poreblock”butbystrikingmodificationofKv3gatingkineticsandvoltagedependence.ActivationandinactivationkineticsareslowedbyBDS-IandBDS-II,andV(1/2)foractivationisshiftedtomorepositivevoltages.AlaninesubstitutionmutagenesisaroundtheS3bandS4segmentsofKv3.2revealsthatBDSactsviavoltage-sensingdomains,and,consistentwiththis,ONgatingcurrentsfromnonconductingKv3.2aremarkedlyinhibited.Thealteredkineticsandgatingproperties,combinedwithlackofsubunitselectivitywithKv3subunits,seriouslyaffectstheusefulnessofBDStoxinsinCNSstudies.FurThermore,ourresultsdonoteasilyfitwiththevoltagesensor“paddle”structureproposedrecentlyforKvchannels.OurdatawillbeinformativeforexperimentsdesignedtodissectouttherolesofKv3subunitsinCNSfunctionanddysfunction.
ShukYinM.Yeung,DawnThompson,ZhurenWang,DavidFedida,BrianRobertson.ModulationofKv3subfamilypotassiumcurrentsbytheseaanemonetoxinBDS:significanceforCNSandbiophysicalstudies.TheJournalofNeuroscience25,8735-8745(2005).
ModulationofneuronalsodiumchannelsbytheseaanemonepeptideBDS-I.
Blood-depressingsubstanceI(BDS-I),a43amino-acidpeptidefromseaanemonevenom,isusedasaspecificinhibitorofKv3-familypotassiumchannels.WefoundthatBDS-Iactswithevenhigherpotencytomodulatespecifictypesofvoltage-dependentsodiumchannels.Inratdorsalrootganglion(DRG)neurons,3μMBDS-Istronglyenhancedtetrodotoxin(TTX)-sensitivesodiumcurrentbutweaklyinhibitedTTX-resistantsodiumcurrent.Inratsuperiorcervicalganglion(SCG)neurons,whichexpressonlyTTX-sensitivesodiumcurrent,BDS-Ienhancedcurrentelicitedbysmalldepolarizationsandsloweddecayofcurrentsatallvoltages(EC(50)∼300nM).BDS-IactedwithexceptionallyhighpotencyandefficacyonclonedhumanNav1.7channels,slowinginactivationby6-fold,withanEC(50)ofapproximately3nM.BDS-IalsoslowedinactivationofsodiumcurrentsinN1E-115neuroblastomacells(mainlyfromNav1.3channels),withanEC(50)∼600nM.InhippocampalCA3pyramidalneurons(mouse)andcerebellarPurkinjeneurons(mouseandrat),BDS-Ihadonlysmalleffectsoncurrentdecay(slowinginactivationby20-50%),suggestingrelativelyweaksensitivityofNav1.1andNav1.6channels.ThebiggesteffectofBDS-IincentralneuronswastoenhanceresurgentcurrentinPurkinjeneurons,aneffectreflectedinenhancementofsodiumcurrentduringtherepolarizationphaseofPurkinjeneuronactionpotentials.Overall,theseresultsshowthatBDS-Iactstomodulatesodiumchannelgatinginamannersimilartopreviouslyknownneurotoxinreceptorsite3anemonetoxinsbutwithdifferentisoformsensitivity.Mostnotably,BDS-IactswithveryhighpotencyonhumanNav1.7channels.
PinLiu,SooyeonJo,BruceP.Bean.ModulationofneuronalsodiumchannelsbytheseaanemonepeptideBDS-I.JournalofNeurophysiology107,3155-3167(2012).
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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.
:)
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！
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~
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。
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)
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