Formulation	10mMHCl
MolecularWeight	882
Storage	-80°C
Purity	HPLCandTLCanalysis
Compound	BEGR-CK.1HCl
Assay
ShelfLife(properlystored)	12months
ChemicalFormula	C36H62O9N10S1Cl2

StructureofBiotinylatedChloromethylketones
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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