α-conotoxinGID isaconopeptideoriginallyisolatedfromthevenomofthe Conusgeographus.Itiscomposedof19aminoacidsandisfoldedbytwodisulphidebondsconnectingCys1-Cys3 andCys2-Cys4.Basedonthenumberofaminoacidsbetweenthesecondandthethirdcysteineresidues(loopI)andthethirdandfourthcysteineresidues(loopII), α-conotoxinGID belongstotheα4/7-conotoxinfamily. α-conotoxinGID blocksselectively neuronalnicotinicacetylcholinereceptorswithIC50 valuesof3nM (α3β2nicotinicreceptors),5nM (α7) and150nM (α4β2). α-conotoxinGID isatleast1000-foldlesspotentontotheα1β1γδ,α3β4,andα4β4nicotinicreceptors. α-conotoxinGID isauniqueα4/7-conotoxinbecauseofitsABIlitytoblockbothα7andα3β2isoforms,contrarytoconotoxinPnIAorPnIBthataremoreselective.
Description:
AAsequence: Ile-Arg-Asp-Gla-Cys5-Cys6-Ser-Asn-Pro-Ala-Cys11-Arg-Val-Asn-Asn-Hyp-His-Val-Cys19-OH
Disulfidebonds:Cys5-Cys11 andCys6-Cys19
Length(aa): 19
Formula:C84H135N31O30S4
MolecularWeight:2185.45g/mol
Modifications:Gla4 aminoacid(gamma-carboxyglutamicacid)andHyp16 aminoacid(Hydroxyproline)
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:
Source:Synthetic
Purityrate:>95%
Reference:
Designandsynthesisofα-conotoxinGIDanaloguesasselectiveα4β2nicotinicacetylcholinereceptorantagoNISTs
Theα4β2nicotinicacetylcholinereceptor(nAChR)isanimportanttargetforcurrentlyapprovedsmokingcessationtherapeutics.However,thedevelopmentofhighlyselectiveα4β2nAChRantagonistsremainsasignificantchallenge.α-ConotoxinGIDisanantagonistofα4β2nAChRs,thoughitissignificantlymorepotenttowardtheα3β2andα7subtypes.Withthegoalofobtainingfurtherinsightsintoα-conotoxinGID/nAChRinteractionsthatcouldleadtothedesignofGIDanalogueswithimprovedaffinityforα4β2nAChRs,webuiltahomologymodeloftheGID/α4β2complexusinganX-rayco-crystalstructureofanα-conotoxin/acetylcholinebindingprotein(AChBP)complex.SeveraladditionalinteractionsthatcouldpotentiallyenhancetheaffinityofGIDforα4β2nAChRswereobservedinourmodel,whichledtothedesignandsynthesisof22GIDanalogues.Sevenanaloguesdisplayedinhibitoryactivitytowardα4β2nAChRsthatwascomparabletoGID.Significantly,bothGID[A10S]andGID[V13I]demonstratedmoderatelyimprovedselectivitytowardα4β2overα3β2whencomparedwithGID,whileGID[V18N]exhibitednomeasurableinhibitoryactivityfortheα3β2subtype,yetretainedinhibitoryactivityforα4β2.Inthisregard,GID[V18N]isthemostα4β2nAChRselectiveα-conotoxinanalogueidentifiedtodate.©2013WileyPeriodicals,Inc.Biopolymers(PeptSci)102:78-87,2014.
BanerjeeJ., etal. (2014) Designandsynthesisofα-conotoxinGIDanaloguesasselectiveα4β2nicotinicacetylcholinereceptorantagonists. Biopolymers. PMID: 24122487
InhibitionofNeuronalNicotinicAcetylcholineReceptorSubtypesbyα-ConotoxinGIDandAnalogues
Alpha-Conotoxinsaresmalldisulfide-richpeptidesfromthevenomoftheConusspeciesthattargetthenicotinicacetylcholinereceptor(nAChR).Theyarevaluablepharmacologicaltoolsandalsohavepotentialtherapeuticapplicationsparticularlyforthetreatmentofchronicpain.alpha-ConotoxinGIDisisolatedfromthevenomofConusgeographusandhasanunusualN-terminaltailsequencethathasbeenshowntobeimportantforbindingtothealpha4beta2subtypeofthenAChR.Todate,onlyfourconotoxinsthatinhibitthealpha4beta2subtypehavebeencharacterized,buttheyareofconsiderableinterestasitisthemostabundantnAChRsubtypeinthemammalianbrainandhasbeenimplicatedinarangeofdiseases.Inthisstudy,analysisofalanine-scanandtruncationmutantsofGIDrevealsthataconservedprolineinalpha-conotoxinsisimportantforactivityatthealpha7,alpha3beta2,andalpha4beta2subtypes.Althoughtheprolineresiduewasthemostcriticalresidueforactivityatthealpha3beta2subtype,Asp(3),Arg(12),andAsn(14)arealsocriticalatthealpha7subtype.Interestingly,veryfewofthemutationstestedretainedactivityatthealpha4beta2subtypeindicatingatightlydefinedbindingsite.Thislackoftolerancetosequencevariationmayexplainthelackofselectiveligandsdiscoveredforthealpha4beta2subtypetodate.Overall,ourfindingscontributetotheunderstandingofthestructure-activityrelationshipsofalpha-conotoxinsandmaybebeneficialfortheongoingattemptstoexploitmodulatorsoftheneuronalnAChRsastherapeuticagents.
MillardE., etal. (2009) InhibitionofNeuronalNicotinic AcetylcholineReceptorSubtypesby α-ConotoxinGIDandAnalogues.JBC. PMID: 19098004
Isolation,Structure,andActivityofGID,aNovel4/7-ConotoxinwithanExtendedN-terminalSequence
Usingassay-directedfractionationofConusgeographuscrudevenom,weisolatedalpha-conotoxinGID,whichactsselectivelyatneuronalnicotinicacetylcholinereceptors(nAChRs).Unlikeotherneuronallyselectivealpha-conotoxins,alpha-GIDhasafouraminoacidN-terminaltail,gamma-carboxyglutamate(Gla),andhydroxyproline(O)residues,andlacksanamidatedCterminus.GIDinhibitsalpha7andalpha3beta2nAChRswithIC(50)valuesof5and3nm,respectivelyandisatleast1000-foldlesspotentatthealpha1beta1gammadelta,alpha3beta4,andalpha4beta4combinations.GIDalsopotentlyinhibitsthealpha4beta2subtype(IC(50)of150nm).DeletionoftheN-terminalsequence(GIDDelta1-4)significantlydecreasedactivityatthealpha4beta2nAChRbuthardlyaffectedpotencyatalpha3beta2andalpha7nAChRs,despiteenhancingtheoff-ratesatthesereceptors.Incontrast,Arg(12)contributedtoalpha4beta2andalpha7activitybutnottoalpha3beta2activity.Thethree-dimensionalstructureofGIDiswelldefinedoverresidues4-19withasimilarmotiftootheralpha-conotoxins.However,despiteitsinfluenceonactivity,thetailappearstobedisorderedinsolution.ComparisonofGIDwithotheralpha4/7-conotoxinswhichpossessanNN(P/O)motifinloopII,revealedacorrelationbetweenincreasinglengthofthealiphaticside-chaininposition10(equivalentto13inGID)andgreateralpha7versusalpha3beta2selectivity.
NickeA., etal. (2003) Isolation,Structure,andActivityofGID,aNovel4/7-ConotoxinwithanExtendedN-terminalSequence. JBC. PMID: 12419800
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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。
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。
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.
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!
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)
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!
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