SelectiveantagoNISTofthealpha(1A)-adrenoceptor
AdTx1(rho-Da1a–ρ-Da1a)isa65amino-acidpeptideoriginallyisolatedfromthevenomofthegreenmamba,anAfricansnake(Dendroaspisangusticep).AdTx1isstABIlizedbyfourdisulfidebridgesandbelongstothefamilyofthethree-finger-foldpeptide.AdTx1hassubnanomolaraffinity(K(i)=0.35nM)andhighspecificityforthehumanalpha(1A)-adrenoceptorsubtypeandis1000timesmorepotentonthissubtypethanonotheradrenoceptorsubtypes.AdTx1isapotentrelaxantofsmoothmuscles.
Description:
AAsequence:LTC3VTSKSIFGITTEDC17PDGQNLC24FKRRHYVVPKIYDSTRGC42AATC46PIPENYDSIHC57C58KTDKC63NE
Disulfidebonds:Cys3-Cys24,Cys17-Cys42,Cys46-Cys57,Cys58-Cys63
Length(aa):65
Formula:C310H481N87O100S8
MolecularWeight(average): 7283.29Da
Appearance:<hitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: notavailable
Source:synthetic
Purityrate: >98%
Reference:
IsolationandpharmacologicalcharacterizationofAdTx1,anaturalpeptidedisplayingspecificinsurmountableantagonismofthea1A-adrenoceptor
Abstract:
Venomsarearichsourceofligandsforionchannels,butverylittleisknownabouttheircapacitytomodulateG-proteincoupledreceptor(GPCR)activity.WedevelopedastrategytoidentifynoveltoxinstargetingGPCRs.Westudiedtheinteractionsofmambavenomfractionswithalpha(1)-adrenoceptorsinbindingexperimentswith(3)H-prazosin.Theactivepeptide(AdTx1)wassequencedbyEdmandegradationandmassspectrometryfragmentation.Itssynthetichomologuewaspharmacologicallycharacterizedbybindingexperimentsusingclonedreceptorsandbyfunctionalexperimentsonrabbitisolatedprostaticsmoothmuscle.AdTx1,a65amino-acidpeptidestabilizedbyfourdisulphidebridges,belongstothethree-finger-foldpeptidefamily.Ithassubnanomolaraffinity(K(i)=0.35nM)andhighspecificityforthehumanalpha(1A)-adrenoceptorsubtype.Weshowedhighselectivityandaffinity(K(d)=0.6nM)ofrADIo-labelledAdTx1indirectbindingexperimentsandrevealedaslowassociationconstant(k(on)=6x10(6).M(-1).min(-1))withanunusuallystablealpha(1A)-adrenoceptor/AdTx1complex(t(1/2diss)=3.6h).AdTx1displayedpotentinsurmountableantagonismofphenylephrine’sactionsinvitro(rabbitisolatedprostaticmuscle)atconcentrationsof10to100nM.AdTx1isthemostspecificandselectivepeptideinhibitorforthealpha(1A)-adrenoceptoridentifiedtodate.Itdisplaysinsurmountableantagonism,actingasapotentrelaxantofsmoothmuscle.Itspeptidicnaturecanbeexploitedtodevelopnewtools,asaradio-labelled-AdTx1orafluoro-labelled-AdTx1.IdentificationofAdTx1thusoffersnewperspectivesfordevelopingnewdrugsfortreatingbenignprostatichyperplasia.
Gprotein-coupledreceptors,anunexploitedanimaltoxintargets:Explorationofgreenmambavenomfornoveldrugcandidatesactiveagainstadrenoceptors
Abstract:
AtatimewhenpharmaceuticalcompaniesarehavingtroublefindingnewlowMWdrugsandwhenBIOLOGicsarebecomingmorecommon,animalvenomscouldconstituteanunderexploitedsourceofnoveldrugcandidates.WelookedforidentifyingnovelanimaltoxinsactiveagainstGprotein-coupledreceptors(GPCR),themostfrequentlyexploitedclassoftreatmenttargets,withtheaimtodevelopnovelresearchtoolsanddrugcandidates.Screeningofgreenmamba(Dendroaspisangusticeps)venomagainstadrenoceptorsidentifiedtwonovelvenompeptides.ρ-Da1ashownanaffinityof0.35 nMfortheα1a-ARwhileρ-Da1bdisplayedaffinitiesbetween14and73 nMforthethreeα2-ARs.Thesetwovenompeptideshavesequencessimilartothoseofmuscarinictoxinsandbelongtothethree-finger-foldproteinfamily.α1a-ARistheprimarytargetforthetreatmentofprostatehypertrophy.Invitroandinvivotestsdemonstratedthatρ-Da1areducedprostaticmuscletoneasefficientlyastamsulosin(anantagonistpresentlyused),butwithfewercardiovascularsideeffects.α2-ARsaretheprototypeofGPCRsnotcurrentlyusedastreatmenttargetsduetoalackofspecificligands.Blockageofthesereceptorsincreasesintestinalmotility,whichmaybecompromisedbyaBDominalsurgeryandreducesorthosterichypotension.Invitroandinvivotestsdemonstratedthatρ-Da1bantagonizesα2-ARsinsmoothmusclesandincreasedheartrateandbloodcatecholamineconcentrations.TheseresultshighlightpossIBLeexploitationofρ-Da1aandρ-Da1binimportantpathologies.
Crystallizationofrecombinantgreenmambaρ-Da1atoxinduringalyophilizationprocedureanditsstructuredetermination
MaïgaA.,etal.(2013)Crystallizationofrecombinantgreenmambaρ-Da1atoxinduringalyophilizationprocedureanditsstructuredetermination.ActaCrystallogrSectFStructBiolCrystCommun.PMID:23722859
Abstract:
ρ-Da1atoxinfromeasterngreenmamba(Dendroaspisangusticeps)venomisapolypeptideof65aminoacidswithastrongaffinityfortheG-protein-coupledα(1A)-adrenoceptor.Thisneurotoxinhasbeencrystallizedfromresolubilizedlyophilizedpowder,butthebestcrystalsgrewspontaneouslyduringlyophilization.ThecrystalsbelongedtothetrigonalspacegroupP3(1)21,withunit-cellparametersa=b=37.37,c=66.05Å,anddiffractedto1.95Åresolution.Thestructuresolvedbymolecularreplacementshowedstrongsimilaritiestogreenmambamuscarinictoxins.
Effectsofρ-Da1aapeptidicα(1)(A)-adrenoceptorantagonistinhumanisolatedprostaticadenomaandanaesthetizedrats
Abstract
ρ-Da1a,a65amino-acidpeptide,hassubnanomolaraffinityandhighselectivityforthehumanα(1)(A)-adrenoceptorsubtype.Thepurposeofthisstudywastocharacterizethepharmacologicaleffectsofρ-Da1aonprostaticfunction,bothinvivoandinvitro.ρ-Da1awastestedasanantagonistofadrenaline-inducedeffectsonCOScellstransfectedwiththehumanα(1)(A)-adrenoceptoraswellasonhumanisolatedprostaticadenomaobtainedfrompatientssufferingfrombenignprostatichyperplasia.Moreover,wecomparedtheeffectsofρ-Da1aandtamsulosinonphenylephrine(PHE)-inducedincreasesinintra-urethral(IUP)andarterialpressures(AP)inanaesthetizedrats,followingi.v.orp.o.administration.OnCOScellsexpressinghumanα(1)(A)-adrenoceptorsandonhumanprostaticstrips,ρ-Da1ainhibitedadrenaline-andnoradrenaline-inducedeffects.Inanaesthetizedrats,ρ-Da1aandtamsulosinadministeredi.v.30 minbeforePHEsignificantlyantagonizedtheeffectsofPHEonIUP.ThepK(B)valuesfortamsulosinandρ-Da1aforthiseffectweresimilar.WithregardstoAP,ρ-Da1aonlyreducedtheeffectofPHEonAPatthelowestdosetested(10 μg·kg(-1)),whereastamsulosinsignificantlyreducedPHEeffectsatdosesbetween10and150 μg·kg(-1).ρ-Da1aexhibitedarelevanteffectonIUPandasmalleffectonAP.Incontrast,tamsulosinantagonizedtheeffectsofPHEonbothIUPandAP.Weconcludethatρ-Da1aismoreuroselectivethantamsulosin.ρ-Da1aisthemostselectivepeptidicantagonistforα(1A)-adenoceptorsidentifiedtodateandcouldbeanewtreatmentforvariousurologicaldiseases.
Orthostericbindingofρ-Da1a,anaturalpeptideofsnakevenominteractingselectivelywiththeα1A-adrenoceptor
Abstract:
ρ-Da1aisathree-fingerfoldtoxinfromgreenmambavenomthatishighlyselectivefortheα1A-adrenoceptor.Thistoxinhasatypicalpharmacologicalproperties,includingincompleteinhibitionof(3)H-prazosinor(125)I-HEATbindingandinsurmountableantagonistaction.Weaimedtoclarifyitsmodeofactionattheα1A-adrenoceptor.Theaffinity(pKi9.26)andselectivityofρ-Da1afortheα1A-adrenoceptorwereconfirmedbycomparingbindingtohumanadrenoceptorsexpressedineukaryoticcells.Equilibriumandkineticbindingexperimentswereusedtodemonstratethatρ-Da1a,prazosinandHEATcompeteattheα1A-adrenoceptor.ρ-Da1adidnotaffectthedissociationkineticsof(3)H-prazosinor(125)I-HEAT,andtheIC50ofρ-Da1a,determinedbycompetitionexperiments,increasedlinearlywiththeconcentrationofradioligandsused,whiletheresidualbindingbyρ-Da1aremainedstable.Theeffectofρ-Da1aonagonist-stimulatedCa(2+)releasewasinsurmountableinthepresenceofphenethylamine-orimidazoline-typeagonists.Tenmutationsintheorthostericbindingpocketoftheα1A-adrenoceptorwereevaluatedforalterationsinρ-Da1aaffinity.TheD106(3.32)AandtheS188(5.42)A/S192(5.46)Areceptormutationsreducedtoxinaffinitymoderately(6and7.6times,respectively),whiletheF86(2.64)A,F288(6.51)AandF312(7.39)Amutationsdiminisheditdramaticallyby18-to93-fold.Inaddition,residueF86(2.64)wasidentifiedasakeyinteractionpointfor(125)I-HEAT,asthevariantF86(2.64)Ainduceda23-foldreductioninHEATaffinity.UnliketheM1muscarinicacetylcholinereceptortoxinMT7,ρ-Da1ainteractswiththehumanα1A-adrenoceptororthostericpocketandsharesreceptorinteractionpointswithantagonist(F86(2.64),F288(6.51)andF312(7.39))andagonist(F288(6.51)andF312(7.39))ligands.Itsselectivityfortheα1A-adrenoceptormayresult,atleastpartly,fromitsinteractionwiththeresidueF86(2.64),whichappearstobeimportantalsoforHEATbinding.
Polypharmacologyprofilesandphylogeneticanalysisofthree-fingertoxinsfrommambavenom:caseofaminergictoxins
Compositionofmamba’svenomisquiteatypicalandcharacterizedbythepresenceofalargediversityofthree-fingerfoldtoxins(3FTx)interactingwithvariousenzymes,receptorsandionchannels.Inparticular,3FTxfrommambasdisplaytheuniquepropertytointeractwithclassAGPCRs,sometimeswithahighaffinityandselectivity.Ascreeningoffiveofthesetoxins(MT1,MT3,MT7,ρ-Da1aandρ-Da1b)on29differentsubtypesofbioaminergicreceptors,usingcompetitionbindingexperiments,highlightsthediversityoftheirpharmacologicalprofiles.Thesetoxinsmaydisplayeitherabsoluteselectivityforonereceptorsubtypeorapolypharmacologicalpropertyforvariousbioaminergicreceptors.Nevertheless,adrenoceptoristhemainreceptorfamilytargetedbythesetoxins.FurThermore,anewreceptortargetwasidentifiedfor3FTxandtoxinsingeneral,theρ-Da1binteractingcompetitivelywiththehumandopamineD3receptorinthemicromolarrange.ThisresultexpandsthediversityofGPCRstargetedbytoxinsandmoregenerallyhighlightsthemultipotentinteractingpropertyof3FTx.Phylogenicanalyzesofthesetoxinsshowthatmuscarinic,adrenergicanddopaminergictoxinsmaybepooledinonefamilycalledaminergictoxins,thisfamilycomingprobablyfromaspecificradiationofligandspresentinmambavenoms.
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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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