Cell-penetratingpeptideandionchannelsblocker
Crotamine isa basicpeptide presentinthe venom ofthe SouthAmerican rattlesnakeCrotalusdurissusterrificus.MultipleBIOLOGicalfunctionshavebeenattributedtoCrotamine.Itisanaturalcell-penetratingpeptidewithselectivebiologicalactiontowardsactivelyproliferatingcelltypes.Moreover,ithasbeenreportedthatcrotamineisablockerofKv1.3(IC50around300nM)aswellasKv1.1andKv1.2.Ithasanalgesicpropertiesandmyonecroticeffects.Inaddition,crotaminebelongstothebeta-defensinpeptidesandassuchdemonstratesantibacterialpropertiesbyinteractingwithlipidmembranes.
Description:
AAsequence: YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG-OH
Disulfidebridges: Cys4-Cys36;Cys11-Cys30;Cys18-Cys37
Length(aa):41
Formula:C214H326N64O54S7
MolecularWeight:4883.82Da
Appearance:whitelyophilizedsolid
Solubility:waterorsalinebuffer
CASnumber:
Source:synthetic
Purityrate:>95%
Reference:
CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus
DeLuccaFL.,etal.(1974)CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus(Ophidia,Reptilia)aftermanualextractionofthevenom.Studiesontemplateactivityandbasecomposition.BiochemJ.PMID:4463939
Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus
Crotamine,a4.88kDaneurotoxicprotein,hasbeenpurifiedtoapparenthomogeneityfromCrotalusdurissusvenombygelfiltrationonSephadexG-75.Wheninjected(i.p.ors.c.)inadultmaleSwissmice(20-25g),itinducedatime-dosedependentanalgesiceffectwhichwasinhibitedbynaloxone,thussuggestinganopioidactionmechanism.Whencomparedwithmorphine(4mg/kg),crotamine,eveninextremelylowdoses(133.4microg/kg,i.p.,about0.4%ofaLD50isapproximately30-foldmorepotentthanmorphine(w/w)asananalgesic.Onamolarbasisitismorethan500-foldmorepotentthanmorphine.Itisalsomuchmorepotentthanthelowermolecularweightcrudefractionsofthesamevenom.Theantinociceptiveeffectsofcrotamineandmorphinewereassayedbythehotplatetestandbytheaceticacid-inducedwrithingmethod.Therefore,bothcentralandperipheralmechanismsshouldbeinvolved.Histopathologicalanalysisofthebrain,liver,skeletalmuscles,stomach,lungs,spleen,heart,kidneysandsmallintestineofthecrotamineinjectedmicedidnotshowanyvisiblelesioninanyoftheseorgansbylightmicroscopy.Sincecrotamineaccountedfor22%(w/w)ofthedesiccatedvenom,itwasidentifiedasitsmajorantinociceptivelowmolecularweightpeptidecomponent.
MancinAC.,etal.(1998) Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus(SouthAmericanrattlesnake)venom:abiochemicalandpharmacologicalstudy.Toxicon.PMID:9839677
Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus
Hereinwereportthatcrotamine,asmalllysine-andcysteine-richproteinfromthevenomoftheSouthAmericanrattlesnake,canrapidlypenetrateintodifferentcelltypesandmouseblastocystsinvitro.Invivocrotaminestronglylabelscellsfrommousebonemarrowandspleenandfromperitonealliquid,asshownbyfluorescentconfocallaser-scanningmicroscopy.Nuclearlocalizationofcrotaminewasobservedinbothfixedandunfixedcells.Inthecytoplasm,crotaminespecificallyassociateswithcentrosomesandthusallowsustofollowtheprocessofcentrioleduplicationandseparation.Inthenucleus,itbindstothechromosomesatS/G2phase,whencentriolesstartdividing.Moreover,crotamineappearsasaMarkerofactivelyproliferatingcells,asshownby5-BrdUcell-proliferationassay.CrotamineinthemicromolarrangeprovednontoxictoanyofthecellculturestestedanddidnotaffectthepluripotencyofEScellsorthedevelopmentofmouseembryos.
KerkisA.,etal.(2004)Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus.FASEBJ.PMID:15231729
BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake
Moleculesisolatedfromanimals,insects,plantsormicroorganismscanprovideprototypesfordesignofbiopharmaceuticalproducts.Somevenomtoxinsandtheirderivativesareusedinmedicine,whileothersprovidetemplatesfordevelopmentofnewdrugs.Themildtoxin,crotamine,asmallbasiclow-molecular-weightpolypeptidepurifiedfromthevenomofaSouthAmericanrattlesnake,Crotalusdurissusterrificus.Crotaminewasdiscoveredmorethan50yearsagoandonlyinthepastsixyearshasitsexceptionalbiologicalversatilitybeendemonstrated.Particularly,itscell-penetratingABIlity,whichallowscrotaminetocrosscellmembranesandtoaccumulateinthenucleus;itsuseforintracellularvesicletrackingandasacellcyclemarkeranditscapabilityfordeliveringDNAintoreplicatingmammaliancells.Bothantimicrobialactionandpotentialselectiveantitumoractivityofcrotaminehavealsobeenfound.Multidisciplinaryapproachesandpathwaysofdiscoveryplacedcrotamineinararecategoryofversatilebiomolecules,inwhichconcentration,moleculartargetpreference,structuralancestryandspecificitytowardbiologicalmembranesplayanintegralrole.Crotamineisadruggablepeptidewithhighpotentialforuseasanimagingagentfordetectingdividingcells,forintracellulardeliveryofhydrophilicbiomolecules,andasanalternativechemotherapeuticcompoundagainstaggressivetypesofcancer.
KerkisI.,etal.(2010)BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake.ExpertOpinInvestigDrugs.PMID:21062230
Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake
Animalvenomscompriseanaturallyselectedcocktailofbioactivepeptides/proteinsandothermolecules,eachofwhichplayingadefinedrolethankstothehighlyspecificinteractionswithdiversemoleculartargetsfoundintheprey.Researchfocusedonisolation,structural,andfunctionalcharacterizationsofnovelnaturalbiologics(bioactivepeptides/proteinsfromnaturalsources)hasalongwaytogothroughfromthebasicsciencetoclinicalapplications.Herein,weoverviewthestructuralandfunctionalcharacteristicsofthemyoneurotoxincrotamine,firstlyisolatedfromtheSouthAmericanrattlesnakevenom.Crotamineisthefirstvenompeptideclassifiedasanaturalcellpenetratingandantimicrobialpeptide(CPPandAMP)withamorepronouncedantifungalactivity.IncontrasttootherknownnaturalCPPsandAMPs,crotaminedemonstratesawidespectrumofbiologicalactivitieswithpotentialbiotechnologicalandtherapeuticvalues.Morerecentstudieshavedemonstratedtheselectiveinvitroanticanceractivityofcrotamine.Invivo,usingamurinemelanomamodel,itwasshownthatcrotaminedelaystumorimplantation,inhibitstumorcellsproliferation,andalsoincreasesthesurvivalofmiceengraftedwithsubcutaneousmelanoma.Thestructuralandfunctionalpropertiesandalsothepossiblebiotechnologicalapplicationsofminimizedmoleculesderivedfromcrotaminearealsodiscussed.
KerkisI.,etal.(2014)Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake.BiomedResInt.PMID:24551848
Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.
Ourgoalwastodemonstratetheinvivotumorspecificaccumulationofcrotamine,anaturalpeptidefromthevenomoftheSouthAmericanrattlesnakeCrotalusdurissusterrificus,whichhasbeencharacterizedbyourgroupasacellpenetratingpeptidewithahighspecificityforactivelyproliferatingcellsandwithaconcentration-dependentcytotoxiceffect.Crotaminecytotoxicityhasbeenshowntobedependentonthedisruptionoflysosomesandsubsequentactivationofintracellularproteases.Inthiswork,weshowthatthecytotoxiceffectofcrotaminealsoinvolvesrapidintracellularcalciumreleaseandlossofmitochondrialmembranepotentialasobservedinrealtimebyconfocalmicroscopy.Theintracellularcalciumoverloadinducedbycrotaminewasalmostcompletelyblockedbythapsigargin.Microfluorimetryassaysconfirmedtheimportanceofinternalorganelles,suchaslysosomesandtheendoplasmicreticulum,ascontributorsfortheintracellularcalciumincrease,aswellastheextracellularmedium.Finally,wedemonstrateherethatcrotamineinjectedintraperitoneallycanefficientlytargetremotesubcutaneoustumorsengraftedinnudemice,asdemonstratedbyanoninvasiveopticalimagingprocedurethatpermitsinvivoreal-timemonitoringofcrotamineuptakeintotumortissue.Takentogether,ourdataindicatethatthecytotoxicpeptidecrotaminecanbeusedpotentiallyforadualpurpose:totargetanddetectgrowingtumortissuesandtoselectivelytriggertumorcelldeath.
NascimentoFD(2012)Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.MolPharm.PMID:22142367
Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.
Crotamineisabasic,42-residuepolypeptidederivedfromsnakevenomthathasbeenshowntopossesscell-penetratingproperties.CrotamineformsnanoparticleswithavarietyofDNAandRNAmolecules,andcrotamine-plasmidDNAnanoparticlesareselectivelydeliveredintoactivelyproliferatingcellsincultureorinmice.Assuch,thesenanoparticlescouldformthebasisforanucleicaciddrug-deliverysystem.Herewedescribethepreparation,purification,andbiochemicalandbiophysicalanalysisofvenom-derived,recombinant,chemicallysynthesized,andfluorescent-labeledcrotamine;theformationandcharacterizationofcrotamine-DNAand-RNAnanoparticles;andthedeliveryofthesenanoparticlesintocellsandanimals.
HayashiMA.,etal.(2012)Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.MethodsMolBiol.PMID:22791447
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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.
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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