Tertiapin hasbeenisolatedfromthevenomoftheHoneybee Apismellifera. Tertiapin-Q isanoxidation-resistantmutantofthewild-typetertiapinwhereMethionine13hasbeenreplacedbyaGlutamine. Tertiapin-Q blockstheinwardlyrectifying Kir1.1(ROMK1) and Kir3.1/3.4(GIRK1/GIRK4alsoknownasIKACh) potassiumchannelswithKdvaluesofaround2nMand8nMrespectively.Tertiapin-Q alsoinhibitscalcium-activatedlargeconductance BKpotassiumchannels (KCa1.1)inaconcentrationandvoltage-dependentmanner(IC50 ~5nM),inadditiontoinhibiting Kir3.1/3.2(GIRK1/GIRK2) heteromultimerpotassiumchannelswithaKdcloseto270nM. Tertiapin-Q canpreventdose-dependentacetylcholine(ACh)-inducedatrioventricularblocksinmammalianhearts,asKCNJ3/KCNJ5channels(alsonamedI(KACh)),areactivatedbyAChfoundinmammalianmyocytes.
Description:
AAsequence: Ala-Leu-Cys3-Asn-Cys5-Asn-Arg-Ile-Ile-Ile-Pro-His-Gln-Cys14-Trp-Lys-Lys-Cys18-Gly-Lys-Lys-NH2
Disulfidebonds: Cys3-Cys14 andCys5-Cys18
Length(aa): 21
Formula: C106H175N35O24S4
MolecularWeight: 2452Da
Appearance:Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: [252198-49-5]Source: Synthetic
Purityrate: >97%
Reference:
CharacterizationofKir1.1channelswiththeuseofarADIolabeledderivativeoftertiapin,Biochemistry
Inwardrectifierpotassium channels (Kir)playcriticalrolesincellphysiology.Despiterepresentingthesimplesttetramericpotassiumchannelstructures,thepharmacologyofthischannelfamilyremainslargelyundeveloped.Inthisrespect, tertiapin (TPN),a21aminoacidpeptideisolatedfrombeevenom,hasbeenreportedtoinhibit Kir1.1 andKir3.1/3.4 channels withhighaffinitybybindingtotheM1-M2linkerregionofthese channels.Thefeaturesofthepeptide-channelinteractionhavebeenexploredelectrophysiologically,&thesestudieshaveidentifiedwaysbywhichtoalterthecompositionofthepeptidewithoutaffectingitsBIOLOGicalactivity.Inthepresentstudy,theTPN derivative,TPN-Y1/K12/Q13,hasbeensynthesized&radiolabeled tohighspecificactivitywith(125)I.TPN-Y1/K12/Q13&mono-iodo-TPN-Y1/K12/Q13([(127)I]TPN-Y1/K12/Q13)inhibitwithhighaffinityratbutnothuman Kir1.1 channels stablyexpressedinHEK293cells.[(125)I]TPN-Y1/K12/Q13bindsinasaturable,time-dependent,&reversIBLemannertoHEK293cellsexpressingrat Kir1.1,aswellastomembranesderivedfromthesecells,&thepharmacologyofthebindingreactionisconsistentwithpeptidebindingto Kir1.1 channels.Studiesusingchimeric channels indicatethatthedifferencesinTPNsensitivitybetweenrat&human Kir1.1 channels areduetothepresenceoftwononconservedresidueswithintheM1-M2linkerregion.Whentheseresultsaretakentogether,theydemonstratethat[(125)I]TPN-Y1/K12/Q13representsthefirsthighspecificactivityradioligandforstudyingrat Kir1.1 channels&suggestitsutilityforidentifyingotherKirchannelmodulators.
Felix,J.P.,etal.(2006)CharacterizationofKir1.1channelswiththeuseofaradiolabeledderivativeoftertiapin,Biochemistry. PMID: 16906771
Tertiapin-QblocksrecombinantandnativelargeconductanceK+channelsinause-dependentmanner
Tertiapin,ashortpeptidefromhoneybeevenom,hasbeenreportedtospecificallyblocktheinwardlyrectifying K(+)(Kir) channels,includingGprotein-coupledinwardlyrectifyingpotassiumchannel(GIRK)1+GIRK4heteromultimers&ROMK1homomultimers.Inthepresentstudy,theeffectsofastable&functionallysimilarderivativeoftertiapin, tertiapin-Q,wereexaminedon recombinant humanvoltage-dependentCa(2+)-activated largeconductance K(+)channel(BKorMaxiK;alpha-subunitorhSlo1homomultimers)&mouseinwardlyrectifyingGIRK1+GIRK2(i.e.,Kir3.1&Kir3.2)heteromultimeric K(+) channels expressedinXenopusoocytes&inculturednewbornmousedorsalrootganglion(DRG)neurons.Intwo-electrodevoltage-clampedoocytes, tertiapin-Q (1-100nM)inhibitedBK-type K(+) channels inause-&concentration-dependent manner.Wealsoconfirmedtheinhibitionof recombinant GIRK1+GIRK2heteromultimersby tertiapin-Q,whichhadnoeffectonendogenousdepolarization-&hyperpolarization-activatedcurrentssensitivetoextracellulardivalentcations(Ca(2+),Mg(2+),Zn(2+),&Ba(2+))indefolliculatedoocytes.Involtage-clampedDRGneurons, tertiapin-Q voltage-&use-dependentlyinhibitedoutwardlyrectifying K(+)currents,butCs(+)-blockedhyperpolarization-activatedinwardcurrentsincludingI(H)wereinsensitiveto tertiapin-Q,baclofen,barium,&zinc,suggestingabsenceoffunctionalGIRK channels inthenewborn.Undercurrent-clampconditions, tertiapin-Q blockedtheactionpotentialafterhyperpolarization(AHP)&increasedactionpotentialdurationinDRGneurons.Takentogether,theseresultsdemonstratethattheblockingactionsof tertiapin-Q arenotspecifictoKir channels&thattheblockadeofrecombinant BK channels&native neuronalAHPcurrentsis use-dependent.InhibitionofspecifictypesofKir&voltage-dependentCa(2+)-activated K(+) channels by tertiapin-Q atnanomolarrangeviadifferentmechanismsmayhaveimplicationsinpainphysiology&therapy.
Kanjhan,R.,C etal.(2005)Tertiapin-QblocksrecombinantandnativelargeconductanceK+channelsinause-dependentmanner, JPharmacolExpTher. PMID: 15947038
Titrationoftertiapin-QinhibitionofROMK1channelsbyextracellularprotons
Tertiapin-Q (TPN(Q)),ahoneybeetoxinderivative,inhibitsinward-rectifierK(+) channels bybindingtotheirexternalvestibule.InthepresentstudywefoundthatTPN(Q) inhibition ofthe channels isprofoundlyaffectedby extracellular pH.ThispHdependencemainlyreflects titration ofhistidineresidue12inTPN(Q)by extracellular protons,sinceitlargelyvanisheswhenthehistidineresidueisreplacedwithalanine.Notsurprisingly,thisalaninederivativeofTPN(Q)bindstothechannelwithmuchloweraffinity.QuantitativeThermodynamiccycleanalysisshowsthatdeprotonationofthehistidineresiduereducestheTPN(Q)-ROMK1 bindingenergyby1.6kcal/mol.ToeliminatepHsensitivitybutretainhighaffinity,wederivatizedTPN(Q)byreplacinghistidine12withlysine.Thisderivative-denotedtertiapin-KQ(TPN(KQ))-notonlyispracticallyinsensitiveto extracellular pHbutalsobindstothechannelwithevenhigheraffinitythanTPN(Q)at extracellular pH7.6.
Ramu,Y., etal.(2001)Titrationoftertiapin-QinhibitionofROMK1channelsbyextracellularprotons, Biochemistry. PMID: 11297426
TertiapinpotentlyandselectivelyblocksmuscarinicK(+)channelsinrabbitcardiacmyocytes
Tertiapin isa21-residuepeptideisolatedfromhoneybeevenoms.Arecentstudyindicatedthat tertiapin isapotentblockerofcertaintypesofinwardlyrectifying K(+)(Kir) channels ().Weexaminedtheeffectof tertiapin onionchannelcurrentsin rabbit cardiac myocytes usingthepatch-clamptechnique.Inthewhole-cellconfiguration, tertiapin fullyinhibitedacetylcholine(1microM)-induced muscarinic K(+)(K(ACh))channelcurrentsinatrialmyocytes withthehalf-maximuminhibitoryconcentrationofapproximately8nMthroughapproximately1:1stoichiometry.Thepotencyof tertiapin ininhibiting K(ACh) channels wasnotsignificantlydifferentat-40&-100mV. Tertiapin alsoinhibitedthe K(ACh)channelpreactivatedbyintracellularguanosine5′-O-(3-thiotriphosphate),anonhydrolyzableGTPanalog.AconstitutivelyactiveKirchannel,theI(K1)channel,wasatleast100timeslesssensitiveto tertiapin.AnotherKirchannelin cardiac myocytes,theATP-sensitive K(+)channel,wasvirtuallyinsensitiveto tertiapin (1microM).Thevoltage-dependent K(+)&theL-typeCa(2+) channels werenotaffectedby tertiapin (1microM).Atthesingle-channellevel, tertiapin inhibitedtheK(ACh)channelfromtheoutsideofthemembranebyreducingtheNP(o)(Nisthenumberoffunctional channels,&theP(o)istheopenprobABIlityofeachchannel)withoutaffectingthesingle-channelconductanceorfastkinetics.Therefore, tertiapin potently&selectively blocks the K(ACh)channelin cardiac myocytes inareceptor-&voltage-independentmanner. Tertiapin isanovelpharmacologicaltooltoidentifythefunctionalroleofthe K(ACh)channelintheparasympatheticregulationoftheheartbeat.
Kitamura,H., etal.(2000)TertiapinpotentlyandselectivelyblocksmuscarinicK(+)channelsinrabbitcardiacmyocytes, JPharmacolExpTher. PMID: 10734170
Mechanismsofinward-rectifierK+channelinhibitionbytertiapin-Q,Biochemistry
Tertiapin-Q(TPN(Q))isaderivativeofhoneybeetoxintertiapin(TPN)whosemethionineresidueisreplacedwithaglutamineresidue.TPN(Q)inhibitstheROMK1&GIRK1/4inward-rectifierK(+)channelswithaffinitiesverysimilartoTPN.However,unlikenativeTPN,TPN(Q)isnonoxidizablebyair.ThestabilityofTPN(Q)allowsustoinvestigatehowitinteractswiththetargetedchannels.WefoundthattheinteractionbetweenTPN(Q)&theROMK1channelisabimolecularreaction,i.e.,oneTPN(Q)moleculebindstoonechannel.TheinteractionsurfaceinTPN(Q)isprimarilyformedbyitsalphahelixratherthanthebetasheetswithwhichscorpiontoxinsformtheirinteractionsurface.Themutagenesisstudiesonboththechannel&TPN(Q)togetherstronglysuggestthattoblocktheK(+)poreTPN(Q)plugsitsalphahelixintothevestibuleoftheK(+)pore,whileleavingtheextendedstructuralportionstickingoutofthevestibuleintotheextracellularmedia.
Jin,W., etal.(1999)Mechanismsofinward-rectifierK+channelinhibitionbytertiapin-Q, Biochemistry. PMID: 10572004
Synthesisofastableformoftertiapin:ahigh-affinityinhibitorforinward-rectifierK+channels
Tertiapin (TPN),asmallproteinderivedfromhoneybeevenom,inhibitstheGIRK1/4&ROMK1 channels withnanomolaraffinities.Methionineresidue13inTPNinteractswithresidueF148inthechannel,locatedjustoutsideofthenarrowregionoftheROMK1pore.ThemethionineresidueinTPNcanbeoxidizedbyair,whichsignificantlyhindersTPNbindingtothe channels.ToovercomethereductioninTPNaffinityduetooxidationofM13,wereplacedM13inTPNwithfourteendifferentresidues.Outofthefourteenderivatives,onlytheoneinwhichM13wasreplacedbyglutamine,TPNQ,bindstothechannelwithaKivalueverysimilartothatofnativeTPN.SinceTPNQis stable&functionallyresemblesnativeTPN,itwillbeaveryusefulmolecularprobeforstudyingthe inward-rectifier K+ channels.
Jin,W.,andLu,Z.(1999)Synthesisofastableformoftertiapin:ahigh-affinityinhibitorforinward-rectifierK+channels, Biochemistry. PMID: 10572003
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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!
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。
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!
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?
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