| Formulation | DMSO |
| MolecularWeight | 846 |
| Storage | -20°C |
| Purity | HPLCandTLCanalysis |
| Compound | FFPR-CK.1HCl |
| Assay | |
| ShelfLife(properlystored) | 12months |
| ChemicalFormula | C42H42O9N6Cl2 |
ThestructureofbiotinylatedFPRchloromethylketones(BFPRCK)(TOP)andbiotinylatedEGRchloromethylketone(BEGRCK)(BOTTOM)areshown.TheSPACERrepresentacarbon-spacerusedtooptimizethereactivityofthebiotin-groupaftertheprobehasbeenreactedwiththeactivesiteofaserineprotease.
Overview:
Tri-peptidechloromethylketoneshavebeenutilizedextensivelytoirreversIBLyinhibitvariousserineproteases(1-5).AmongthemostcommonchloromethylketonesareFPRCK(Phe-Pro-Arg-chloromethylketone;commonlyreferredtoasPPACK),whichisarapidthrombininhibitorandEGRCK(Glu-Gly-Arg-chloromethylketone;commonlyreferredtoasGGACK),whichisarapidfactorXainhibitor(1).BothFPRCKandEGRCKareusedextensivelyduringproteinisolationprocedurestoinhibitserineproteaseactivityandpreventfurtherconversionofzymogenstoactiveenzymes.Recently,themodificationofthesetri-peptidechloromethylketoneswithreportinggroups,suchasfluorescentprobes(6-8,14),rADIoactivelabels(9)orthioreactive-labels(10),hasprovidedauniqueapproachtothestudyofvariousserineproteases.Theseprobesareusefulbecausetheyallowameansofreportingmolecularchangesinanenzyme,andnotitszymogen,whilealsoinhibitingtheenzymaticactivity.
TheuseofbiotinasareportinggrouphasbeenusedextensivelywithantibodiesinELISAbasedassaysandinwesternblotting.Thebiotin,inconjunctionwithavidin,createsahighlysensitivemethodfordetectingantibodies,andtherefore,antigens.Bymodifyingthetripeptide-chloromethylketoneswithabiotingroup,thesensitivityoftheavidin/biotinsystemcanbeextendedtostudyserineproteaseswithouttheneedforspecificantibodiestotheactiveenzymes.
Biotinylatedtripeptidechloromethylketonescanbeusedinavarietyofways(11-13).First,thecompoundscanbereactedwithunwantedserineproteasesinasampleorpreparation,andcanthenberemovedalongwiththeproteaseusingavidin-Sepharose(11).Second,thebiotinylated-serineproteasecanbevisualizedonablotwithouttheuseofspecificantibodies(11).Third,thebiotinylatedserineproteasecanbequantitatedinanactive-sitespecificimmunoassay(12,13,15),suchasthetPA-CASSIA(seeAssayKits).ThespacerutilizedonthesecompoundshasbeenoptimizedtoallowgoodreactivityofthebiotinylatedFPRCKandthebiotinylatedEGRCKintheabovementionedprocedures.
Inadditiontobiotinylatedchloromethylketones,fluoresceinlabelledcompoundsarealsoavailable.ThefluoresceinlabelledcompoundsareusefulinbothWesternblotandfluorescentimagingapplications.
BiotinylatedandfluoresceinlabelledFPRCKandEGRCKarepreparedbythemethodofWilliamsetal.(11).
Properties:
| SpecialProperties | Tri-peptidechloromethylketones(CMK)areverypotentandirreversibleinhibitorsofserineproteases.BFPRCKisespeciallyusefulforinhibitionofthrombinandtPA,whileBEGRCKisusefulforinhibitionoffactorXa.ThebiotinmoietyprovidestheABIlitytousethepeptide-CMKsasspecificprobesfordetectionand/orcaptureofserineproteasesviatheavidin/biotininteraction. |
|---|---|
| SpecialNotes | FPRCKandEGRCKaresuppliedlyophilized,andshouldbestoredat4oC.BiotinylatedCMKsaresuppliedin10mMHClandshouldbestoredfrozedat-20oCorcolder.FluorosceinCMKsaresuppliedinDMSO,andshouldalsobestoredat-20oCorcolder. |
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本人目前需从电鳗放电器官中提取乙酰胆碱受体蛋白，提纯需要的步骤和电鳗器官都可以提供，有没有提供外包提纯服务的公司或者实验室。
