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Haematologic Technologies/FPR-chloromethylketone (PPACK)/FPRCK-01/5 mg, 25 mg, 100 mg
| Formulation | Lyophilized |
| MolecularWeight | 524 |
| Storage | 4°C |
| Purity | HPLCandTLCanalysis |
| Compound | FPR-CK.2HCl |
| Assay | |
| ShelfLife(properlystored) | 12months |
| ChemicalFormula | C21H33O3N6Cl3 |
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. |
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| SpecialNotes | FPRCKandEGRCKaresuppliedlyophilized,andshouldbestoredat4oC.BiotinylatedCMKsaresuppliedin10mMHClandshouldbestoredfrozedat-20oCorcolder.FluorosceinCMKsaresuppliedinDMSO,andshouldalsobestoredat-20oCorcolder. |
