ShK(StichodactylahelianthusNeurotoxin)hasbeenisolatedfromthevenomoftheCarribeanseaanemoneStoichactishelianthus.ShKinhibitsvoltage-dependentpotassiumchannels.ItblocksKv1.3(KCNA3)potentlyandalsoKv1.1(KCNA1),Kv1.4(KCNA4)andKv1.6(KCNA6)respectivelywithaKdof11pM,16pM,312pMand165pM.Interestingly,itwasalsodemonstratedthatShKpotentlyinhibitsthehKv3.2bchannelwithanIC50valueofapproximately0.6nM.
Description:
AAsequence:Arg-Ser-Cys3-Ile-Asp-Thr-Ile-Pro-Lys-Ser-Arg-Cys12-Thr-Ala-Phe-Gln-Cys17-Lys-His-Ser-Met-Lys-Tyr-Arg-Leu-Ser-Phe-Cys28-Arg-Lys-Thr-Cys32-Gly-Thr-Cys35-OH
Disulfidebonds:Cys3-Cys35,Cys12-Cys28andCys17-Cys32
Length(aa):35
Formula:C169H274N54O48S7
MolecularWeight:4054.85Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:165168-50-3
Source:Synthetic
Purityrate:>97%
Reference:
DurablepharmacologicalresponsesfromthepeptideShK-186,aspecificKv1.3channelinhibitorthatsuppressesTcellmediatorsofautoimmunedisease
TheKv1.3channelisarecognizedtargetforpharmaceuticaldevelopmenttotreatautoimmunediseasesandorganrejection.ShK-186,aspecificpeptideinhibitorofKv1.3,hasshownpromiseinanimalmodelsofmultiplesclerosisandrheumatoidarthritis.Here,wedescribethepharmacokinetic-pharmacodynamicrelationshipforShK-186inratsandmonkeys.ThepharmacokineticprofileofShK-186wasevaluatedwithavalidatedhigh-performanceliquidchromatography-tandemmassspectrometrymethodtomeasurethepeptide’sconcentrationinplasma.Theseresultswerecomparedwithsingle-photonemissioncomputedtomography/computedtomographydatacollectedwithan¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid-conjugateofShK-186toassesswhole-bloodpharmacokineticparametersaswellasthepeptide’sabsorption,distribution,andexcretion.AnalysisofthesedatasupportamodelwhereinShK-186isabsorbedslowlyfromtheinjectionsite,resultinginbloodconcentrationsabovetheKv1.3channel-blockingIC₅₀valueforupto7daysinmonkeys.PharmacodynamicstudiesonhumanperipheralbloodmononuclearcellsshowedthatbriefexposuretoShK-186resultedinsustainedsuppressionofcytokineresponsesandmaycontributetoprolongeddrugeffects.Indelayed-typehypersensitivity,chronicrelapsing-remittingexperimentalautoimmuneencephalomyelitis,andpristane-inducedarthritisratmodels,asingledoseofShK-186every2to5dayswasaseffectiveasdailyadmiNISTration.ShK-186’sslowdistributionfromtheinjectionsiteanditslongresidencetimeontheKv1.3channelcontributetotheprolongedtherapeuticeffectofShK-186inanimalmodelsofautoimmunedisease.
TarchaEJ.,etal.(2012)DurablepharmacologicalresponsesfromthepeptideShK-186,aspecificKv1.3channelinhibitorthatsuppressesTcellmediatorsofautoimmunedisease.JPharmacolExpTher.PMID: 22637724
ThebeneficialeffectofblockingKv1.3inthepsoriasiformSCIDmousemodel
TheKv1.3channelisimportantintheactivationandfunctionofeffectormemoryTcells.Recently,specificblockersoftheKv1.3channelhavebeendevelopedasapotentialtherapeuticoptionfordiverseautoimmunediseases.Inpsoriaticlesions,mostlymphocytesarememoryeffectorTcells.TheaimofthepresentstudywastodetecttheexpressionofKv1.3channelsinthesecellsinpsoriaticlesionsaswellasinhumanpsoriasiformskingraftsusingtheseverecombinedimmunodeficient(SCID)mousemodel.HistologicalandimmunohistochemicalstainingforKv1.3expressionandvariousinflammatoryMarkerswasperformedinsectionsobtainedfromsixpsoriaticpatientsand18beige-SCIDmicewithpsoriasiformhumanskingrafts.SixgraftedmiceweretreatedwithStichodactylahelianthusneurotoxin(ShK),aknownKv1.3blocker.TheresultsshowedanincreasednumberofKv1.3+cellsinthepsoriaticskinaswellasinthepsoriasiformskingraftsascomparedwithnormalskinandnormalskingrafts.InjectionsofShKshowedamarkedtherapeuticeffectinthreeofsixpsoriasiformskingrafts.AsignificantlydecreasednumberofKv1.3+cellswasobservedintheresponderscomparedwiththecontrolgrafts.Thispilotstudy,althoughperformedinasmallnumberofmice,revealsthepossIBLebeneficialeffectofKv1.3blockersinpsoriasispatients.
GilharA.,etal.(2011)ThebeneficialeffectofblockingKv1.3inthepsoriasiformSCIDmousemodel.JInvestDermatol.PMID: 20739949
Modelingthebindingofthreetoxinstothevoltage-gatedpotassiumchannel(Kv1.3)
Theconductionpropertiesofthevoltage-gatedpotassiumchannelKv1.3anditsmodesofinteractionwithseveralpolypeptidevenomsareexaminedusingBrowniandynamicssimulationsandmoleculardynamicscalculations.Employinganopen-statehomologymodelofKv1.3,wefirstdeterminecurrent-voltageandcurrent-concentrationcurvesandascertainthatsimulatedresultsaccordwithexperimentalmeasurements.Wetheninvestigate,usingamoleculardockingmethodandmoleculardynamicssimulations,thecomplexesformedbetweentheKv1.3channelandseveralKv-specificpolypeptidetoxinsthatareknowntointerferewiththeconductingmechanismsofseveralclassesofvoltage-gatedK(+)channels.ThedepthsofpotentialofmeanforceencounteredbycharyBDotoxin,α-KTx3.7(alsoknownasOSK1)andShKare,respectively,-19,-27,and-25kT.Thedissociationconstantscalculatedfromtheprofilesofpotentialofmeanforcecorrespondcloselytotheexperimentallydeterminedvalues.Wepinpointtheresiduesinthetoxinsandthechannelthatarecriticalfortheformationofthestablevenom-channelcomplexes.
ChenR.,etal.(2011)Modelingthebindingofthreetoxinstothevoltage-gatedpotassiumchannel(Kv1.3).BiophysJ.PMID: 22261053
BlockadeofT-lymphocyteKCa3.1andKv1.3channelsasnovelimmunosuppressionstrategytopreventkidneyallograftrejection
Currently,thereisanunmetclinicalneedfornovelimmunosuppressiveagentsforlong-termpreventionofkidneytransplantrejectionasalternativestothenephrotoxiccalcineurininhibitorcyclosporine(CsA).RecentstudieshaveshownthatK(+)channelshaveacrucialroleinT-lymphocyteactivity.WeinvestigatedwhethercombinedblockadeoftheT-cellK(+)channelsK(Ca)3.1andK(v)1.3,bothofwhichregulatecalciumsignalingduringlymphocyteactivation,iseffectiveinpreventionofrejectionofkidneyallograftsfromFisherratstoLewisrats.AllrecipientswereinitiallytreatedwithCsA(5mg/kgd)for7days.Inratswithintactallograftfunction,treatmentwascontinuedfor10dayswitheitherCsA(5mg/kgd),oracombinationofTRAM-34(K(Ca)3.1inhibitor;120mg/kgd)plusStichodactylahelianthustoxin(ShK,K(v)1.3inhibitor;80microg/kg3timesdaily),orvehiclealone.Kidneysectionswerestainedwithperiodicacid-Schifforhematoxylin-eosinandhistochemicallyformarkersofmacrophages(CD68),T-lymphocytes(CD43),orcytotoxicT-cells(CD8).OurresultsshowedthattreatmentwithTRAM-34andShKreducedtotalinterstitialmononuclearcellinfiltration(-42%)andthenumberofCD43+T-cells(-32%),cytotoxicCD8+T-cells(-32%),andCD68+macrophages(-26%)inallograftswhencomparedtovehicletreatmentalone.EfficacyofTRAM-34/ShKtreatmentwascomparablewiththatofCsA.Inaddition,novisibleorgandamageorotherdiscernibleadverseeffectswereobservedwiththistreatment.Thus,selectiveblockadeofT-lymphocyteK(Ca)3.1andK(v)1.3channelsmayrepresentanovelalternativetherapyforpreventionofkidneyallograftrejection.
GrgicI.,etal.(2009)BlockadeofT-lymphocyteKCa3.1andKv1.3channelsasnovelimmunosuppressionstrategytopreventkidneyallograftrejection.TransplantProc.PMID: 19715983
MolecularmechanismoftheseaanemonetoxinShKrecognizingtheKv1.3channelexploredbydockingandmoleculardynamicsimulations
ComputationalmethodsareemployedtosimulatetheinteractionoftheseaanemonetoxinShKincomplexwiththevoltage-gatedpotassiumchannelKv1.3frommice.Alloftheavailable20structuresofShKintheProteinDataBankwereconsideredforimprovingtheperformanceoftherigidproteindockingofZDOCK.ThetrADItionalandnovelbindingmodeswereobtainedamongalargenumberofpredictedcomplexesbyusingclusteringanalysis,screeningwithexpertknowledge,energyminimization,andmoleculardynamicsimulations.Thequalityandvalidityoftheresultingcomplexeswerefurtherevaluatedtoidentifyafavorablecomplexstructureby500psmoleculardynamicsimulationsandthechangeofbindingfreeenergieswithacomputationalalaninescanningtechnique.ThenovelandreasonableShK-Kv1.3complexstructurewasfoundtobedifferentfromthetraditionalmodelbyusingtheLys22residuetoblockthechannelpore.FromtheresultingstructureoftheShK-Kv1.3complex,ShKmainlyassociatesthechanneloutervestibulewithitssecondhelicalsegment.StructuralanalysisfirstrevealedthattheLys22residuesidechainoftheShKpeptidejusthangsbetweenCandDchainsoftheKv1.3channelinsteadofphysicallyblockingthechannelpore.TheobviouslossoftheShKSer20AlaandTyr23AlamutantbindingABIlitytotheKv1.3channeliscausedbytheconformationalchange.ThefivehydrogenbondsbetweenArg24inShKandH404(A)andD402(D)inKv1.3makeArg24themostcrucialforitsbindingtotheKv1.3channel.BesidesthedetailedinteractionbetweenShKandKv1.3attheatomlevel,thesignificantconformationalchangeinducedbytheinteractionbetweentheShKpeptideandtheKv1.3channel,accompaniedbythegradualdecreaseofbindingfreeenergies,stronglyimpliesthatthebindingoftheShKpeptidetowardtheKv1.3channelisadynamicprocessofconformationalrearrangementandenergystabilization.AllofthesecanacceleratethedevelopmentofShKstructure-basedimmunosuppressants.
JinL,WuY.(2007)MolecularmechanismoftheseaanemonetoxinShKrecognizingtheKv1.3channelexploredbydockingandmoleculardynamicsimulations.JChemInfModel. PMID: 17718553
K+channelblockers:noveltoolstoinhibitTcellactivationleadingtospecificimmunosuppression
DuringthelasttwodecadessincetheidentificationandcharacterizationofTcellpotassiumchannelsgreatadvanceshavebeenmadeintheunderstandingoftheroleofthesechannelsinTcellfunctions,especiallyinantigen-inducedactivation.TheirlimitedtissuedistributionandtherecentdiscoverythatdifferentTcellsubtypescarryingoutdistinctimmunefunctionsshowspecificexpressionlevelsofthesechannelshavemadeTcellpotassiumchannelsattractivetargetsforimmunomodulatorydrugs.Manytoxinsofvariousanimalspeciesandastructurallydiversearrayofsmallmoleculesinhibitingthesechannelswithvaryingaffinityandselectivitywerefoundandtheirsuccessfuluseinimmunosuppressioninvivowasalsodemonstrated.Betterunderstandingofthetopologicaldifferencesbetweenpotassiumchannelpores,detailedknowledgeoftoxinandsmall-moleculestructuresandtheidentificationofthebindingsitesofblockingcompoundsmakeitpossibletoimprovetheselectivityandaffinityoftheleadcompoundsbyintroducingmodificationsbasedonstructuralinformation.Inthisreviewthebasicpropertiesandphysiologicalrolesofthevoltage-gatedKv1.3andtheCa2+-activatedIKCa1potassiumchannelsarediscussedalongwithanoverviewofcompoundsinhibitingthesechannelsandapproachesaimingatproducingmoreefficientmodulatorsofimmunefunctionsforthetreatmentofdiseaseslikesclerosismultiplexandtypeIdiabetes.
PanyiG,etal.(2006)K+channelblockers:noveltoolstoinhibitTcellactivationleadingtospecificimmunosuppression.CurrPharmDes. PMID: 16787250
Stichodactylahelianthuspeptide,apharmacologicaltoolforstudyingKv3.2channels
Voltage-gatedpotassium(Kv)channelsregulatemanyphysiologicalfunctionsandrepresentimportanttherapeutictargetsinthetreatmentofseveralclinicaldisorders.Althoughsomeofthesechannelshavebeenwell-characterized,thestudyofothers,suchasKv3channels,hasbeenhinderedbecauseoflimitedpharmacologicaltools.ThecurrentstudywasinitiatedtoidentifypotentblockersoftheKv3.2channel.Chinesehamsterovary(CHO)-K1cellsstablyexpressinghumanKv3.2b(CHO-K1.hKv3.2b)wereestablishedandcharacterized.Stichodactylahelianthuspeptide(ShK),isolatedfromS.helianthusvenomandaknownhigh-affinityblockerofKv1.1andKv1.3channels,wasfoundtopotentlyinhibit86Rb+effluxfromCHO-K1.hKv3.2b(IC50approximately0.6nM).InelectrophysiologicalrecordingsofKv3.2bchannelsexpressedinXenopuslaevisoocytesorinplanarpatch-clampstudies,ShKinhibitedhKv3.2bchannelswithIC50valuesofapproximately0.3and6nM,respectively.DespitethepresenceofKv3.2proteininhumanpancreaticbetacells,ShKhasnoeffectontheKvcurrentofthesecells,suggestingthatitisunlikelythathomotetramericKv3.2channelscontributesignificantlytothedelayedrectifiercurrentofinsulin-secretingcells.InmousecorticalGABAergicfast-spikinginterneurons,however,applicationofShKproducedeffectsconsistentwiththeblockadeofKv3channels(i.e.,anincreaseinactionpotentialhalf-width,adecreaseintheamplitudeoftheactionpotentialafterhyperpolarization,andadecreaseinmaximalfiringfrequencyinresponsetodepolarizingcurrentinjections).Takentogether,theseresultsindicatethatShKisapotentinhibitorofKv3.2channelsandmayserveasausefulpharmacologicalprobeforstudyingthesechannelsinnativepreparations.
YanL.,etal.(2005)Stichodactylahelianthuspeptide,apharmacologicaltoolforstudyingKv3.2channels.MolPharmacol.PMID: 15709110
TargetingeffectormemoryTcellswithaselectivepeptideinhibitorofKv1.3channelsfortherapyofautoimmunediseases
Thevoltage-gatedKv1.3K(+)channelisanoveltargetforimmunomodulationofautoreactiveeffectormemoryT(T(EM))cellsthatplayamajorroleinthepathogenesisofautoimmunediseases.WedescribethecharacterizationofthenovelpeptideShK(L5)thatcontainsl-phosphotyrosinelinkedviaanine-atomhydrophiliclinkertotheNterminusoftheShKpeptidefromtheseaanemoneStichodactylahelianthus.ShK(L5)isahighlyspecificKv1.3blockerthatexhibits100-foldselectivityforKv1.3(K(d)=69pM)overKv1.1andgreaterthan250-foldselectivityoverallotherchannelstested.ShK(L5)suppressestheproliferationofhumanandratT(EM)cellsandinhibitsinterleukin-2productionatpicomolarconcentrations.NaiveandcentralmemoryhumanTcellsareinitially60-foldlesssensitivethanT(EM)cellstoShK(L5)andthenbecomeresistanttothepeptideduringactivationbyup-regulatingthecalcium-activatedK(Ca)3.1channel.ShK(L5)doesnotexhibitinvitrocytotoxicityonmammaliancelllinesandisnegativeintheAmestest.Itisstableinplasmaandwhenadministeredoncedailybysubcutaneousinjection(10mug/kg)attains“steadystate”bloodlevelsofapproximately300pM.ThisregimendoesnotcausecardiactoxicityassessedbycontinuousEKGmonitoringanddoesnotalterclinicalchemistryandhematologicalparametersafter2-weektherapy.ShK(L5)preventsandtreatsexperimentalautoimmuneencephalomyelitisandsuppressesdelayedtypehypersensitivityinrats.ShK(L5)mightproveusefulfortherapyofautoimmunedisorders.
BeetonC.,etal.(2005)TargetingeffectormemoryTcellswithaselectivepeptideinhibitorofKv1.3channelsfortherapyofautoimmunediseases.MolPharmacol. PMID: 15665253
PotassiumchannelblockadebytheseaanemonetoxinShKforthetreatmentofmultiplesclerosisandotherautoimmunediseases
Expressionofthetwolymphocytepotassiumchannels,thevoltage-gatedchannelKv1.3andthecalciumactivatedchannelIKCa1,changesduringdifferentiationofhumanTcells.WhileIKCa1isthefunctionallydominantchannelinnaiveand“early”memoryTcells,Kv1.3iscrucialfortheactivationofterminallydifferentiatedeffectormemory(TEM)Tcells.BecauseoftheinvolvementofTEMcellsinautoimmuneprocesses,Kv1.3isregardedasapromisingtargetforthetreatmentofT-cellmediatedautoimmunediseasessuchasmultiplesclerosisandthepreventionofchronictransplantrejection.ShK,a35-residuepolypeptidetoxinfromtheseaanemone,Stichodactylahelianthus,blocksKv1.3atlowpicomolarconcentrations.ShKadoptsacentralhelix-kink-helixfold,andalanine-scanningandothermutagenesisstudieshavedefineditschannel-bindingsurface.ModelshavebeendevelopedofhowthistoxineffectsK+-channelblockadeandhowitsdockingconfigurationmightdifferinShK-Dap22,whichcontainsasinglesidechainsubstitutionthatconfersspecificityforKv1.3blockade.ShK,ShK-Dap22andtheKv1.3blockingscorpiontoxinkaliotoxinhavebeenshowntopreventandtreatexperimentalautoimmuneencephalomyelitisinrats,amodelformultiplesclerosis.AfluoresceinatedanalogofShK,ShK-F6CA,hasbeendeveloped,whichallowsthedetectionofactivatedTEMcellsinhumanandanimalbloodsamplesbyflowcytometryandthevisualizationofKv1.3channeldistributioninlivingcells.ShKanditsanalogsarecurrentlyundergoingfurtherevaluationasleadsinthedevelopmentofnewbiopharmaceuticalsforthetreatmentofmultiplesclerosisandotherT-cellmediatedautoimmunedisorders.
NortonRS.,etal.(2004)PotassiumchannelblockadebytheseaanemonetoxinShKforthetreatmentofmultiplesclerosisandotherautoimmunediseases.CurrMedChem.PMID: 15578998
SolutionstructureofShKtoxin,anovelpotassiumchannelinhibitorfromaseaanemone
AnessentialbindingsurfaceforShKtoxininteractionwithratbrainpotassiumchannels.
An“Alascan”analysisofShKtoxin,a35-residuebasicpeptidepossessingthreedisulfidebonds,identifiessevensidechainswhichinfluencebindingtobraindelayedrectifierpotassiumchannels.Additionalanalogsweresynthesizedandtestedtofurtherdeciphertherolesoftheseresidues,particularlyTyr23.Theinhibitoryeffectsoftheseanalogson125I-labeleddendrotoxinbindingtoratbrainmembranesshowedthatreplacementofTyr23withAladrasticallyloweredtheaffinityofthetoxinfortheKv1.2channels.AlasubstitutionofPhe27reducedpotencymorethan15-fold.MonosubstitutedAlaanalogsforIle7,Ser20,orLys30eachdisplayed5-foldreductionsinpotency.Thus,aromaticityatposition23isimportantforeffectivedelayedrectifierbrainKchannelbinding.Incontrast,thearomaticresidueatposition27wasnotcritical,sincecyclohexylalaninesubstitutionincreasedaffinity.ThesolutionstructureofShKtoxinclustersIle7,Arg11,Ser20,Lys22,Tyr23,andPhe27incloseproximity,formingthepotassiumchannelbindingsurfaceofthetoxin.WeproposeanessentialbindingsurfaceonthetoxininwhichLys22andTyr23aremajorcontributors,throughionicandaromatic(hydrophobic)interactions,withthepotassiumchannel.
PenningtonMW.,etal.(1996)AnessentialbindingsurfaceforShKtoxininteractionwithratbrainpotassiumchannels.Biochemistry. PMID: 8987971
CharacterizationofapotassiumchanneltoxinfromtheCaribbeanSeaanemoneStichodactylahelianthus
Apeptidetoxin,ShK,thatblocksvoltage-dependentpotassiumchannelswasisolatedfromthewholebodyextractoftheCaribbeanseaanemoneStichodactylahelianthus.ItcompeteswithdendrotoxinIandalpha-dendrotoxinforbindingtosynaptosomalmembranesofratbrain,facilitiesacetylcholinereleaseatanavianneuromuscularjunctionandsuppressesK+currentsinratdorsalrootganglionneuronesinculture.ItsaminoacidsequenceisR1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35.ThereisnohomologywithotherK+channel-blockingpeptides,exceptforBgKfromtheseaanemoneBunodosomagranulifera.ShKandBgKappeartobeinadifferentstructuralclassfromothertoxinsaffectingK+channels.
Castaneda,O.,etal.(1995)CharacterizationofapotassiumchanneltoxinfromtheCaribbeanSeaanemoneStichodactylahelianthus,Toxicon. PMID: 7660365
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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。
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~
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！
拼音名：Chunhuashui
英文名：PurifiedWater
【性状】本品为无色的澄清液体；无臭，无味。
【检查】酸碱度取本品10ml，加甲基红指示液2滴，不得显红色；另取10ml，加溴麝香草酚蓝指示液5滴，不得显蓝色。氯化物、流酸盐与钙盐取本品，分置三支试管中，每管各50ml。第一管中加硝酸5滴与硝酸银试液1ml，第二管中加氯化钡试液2ml，第三管中加草酸铵试液2ml，均不得发生浑浊。
硝酸盐取本品5ml置试管中，于冰浴中冷却，加10%氯化钾溶液0.4ml与0.1%二苯胺硫酸溶液0.1ml，摇匀，缓缓滴加硫酸5ml，摇匀，将试管子50℃水浴中放置15分钟，溶液产生的蓝色与标准硝酸盐溶液［取硝酸钾0.163g，加水溶解并稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，再精密量取10ml，加水稀释成100ml，摇匀，即得（每1ml相当于1pgNO3）0.3ml，加无硝酸盐的水4.7ml，用同一方法处理后的颜色比较，不得更深（0.000006%）。
亚硝酸盐取本品10ml，置纳氏管中，加对氨基苯磺酰胺的稀盐酸溶液（1→100）lml与盐酸菜乙H肢溶液（0．l＋100）1ml，产生的粉红色，与标准亚硝酸盐溶液〔取亚硝酸钠0.750g（按干燥品计算），加水溶解，稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，摇匀，再精密量取1ml，加水稀释成50ml，摇匀，即得（每1ml相当于1μgNO2）]0.2ml，加无亚硝酸盐的水9.8ml，用同一方法处理后的颜色比较，不得更深（0.000002%）。
氨取本品50ml，加碱性碘化汞钾试液2ml，放置15分钟；如显色，与氯化铵溶液（取氯化铵31.5mg，加无氨水适量使溶解并稀释成1000ml）1.5ml，加元氨水48ml与碱性碘化汞钾试液2ml制成的对照液比较，不得更深（0.00003%）。
二氧化碳取本品25ml，置50ml具塞量筒中，加氢氧化钙试液25ml，密塞振摇，放置，小时内不得发生浑浊。
易氧化物取本品100ml，加稀硫酸10ml，煮沸后，加高锰酸钾滴定液（0.02mol/L）0.10ml，再煮沸10分钟，粉红色不得完全消失。
不挥发物取本品100ml，置105℃恒重的蒸发皿中，在水浴上蒸干，并在105℃干燥至恒重，遗留残渣不得过1mg。
重金属取本品50ml，加水18.5ml，蒸发至20ml，放冷，加醋酸盐缓冲液（pH3.5）2ml与水适量使成25ml，加硫代乙酰胺试液2ml，摇匀，放置2分钟，与标准铅溶液1.5ml加水18.5ml用同一方法处理后的颜色比较，不得更深（0.00003%）。
微生物限度取本品，采用薄膜过滤法处理后，依法检查（附录ⅪJ），细菌、霉菌和酵母菌总数每1ml不得过100个。
【贮藏】密闭保存。
【化学成分】本品为蒸馏法、离子交换法、反渗透法或其他适宜的方法制得的供药用的水，不含任何附加剂。
【分子式与分子量】H2O18.02
【药理作用】溶剂、稀释剂
这里药典纯化水标准中并无PH值项目，请问对纯化水有PH值的要求吗，范围应在多少？请说明出处？
在纯化水检测中，检验酸碱度合格，但是发现PH在8左右。如果按以上标准检验合格，是否要考虑PH值？请知道的解答，谢谢！
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