Formulation50%glycerol/water(v/v)Storage-20°CPurity>95%bySDS-PAGEActivityDeterminationFactorXIclottingassayShelfLife(properlystored)12monthsProteolyticActivationofFactorXITheactivationoffactorXIbyfactorXIIaisillustrated.AlthoughfactorXIcanbindindependentlytonegativelychargedsurfaces,proteolysisbyfactorXIIacanonlyoccurinthepresenceofsurface-boundhighmolecularweightkininogen(HMWKa).TheboundHMWKaissusceptIBLetoproteolysisbythenewlygeneratedfactorXIawhichresultsinalossofitsBIOLOGicalactivity(HMWKi).FactorXIacatalyzestheproteolyticconversionoffactorIXtofactorIXaeitherinsolutionphaseorwhenboundtoanegativelychargedsurface.SampleGelInformation:GelNovex4-12%Bis-TrisLoadHumanFactorXIa,1µgperlaneBufferMOPSStandardSeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa)Overview:ProteolysisofXIbyfactorXIIainthepresenceofhighmolecularweightkininogen(HMWK),yieldstheenzymefactorXIa(1-3).FactorXIaisaserineproteasewhichparticipatesintheintrinsicpathwayofcoagulationbycatalyzingtheconversionoffactorIXtofactorIXa.BecausefactorXIisahomodimer,theenzyme,factorXIa,iscomposedoftwoidenticalheavychains(Mr=50,000),andtwoidenticallightchains(Mr=30,000),allofwhichareheldtogetherbydisulfidebonds.ThislatterpropertymakesfactorXIauniqueamongtheserineproteases,sinceitcontainstwoactivesitespermolecule(1-3).FactorXIa,likeitsprecursorfactorXI,remainsincomplexwithHMWK(4).Incomplexform,factorXIa/HMWKiscapableofactivatingfactorXIItofactorXIIaandprekallikreintokallikrein.Additionally,inthecomplexedform,factorXIaislessaffectedbyproteaseinhibitors(5).ThemajorplasmainhibitoroffactorXIaisa1-antitrypsinandthentoamuchlesserextent,antithrombin-III(5,6).Whileinthecomplexedform,factorXIamaycatalyzetheproteolysisofHMWK,yieldingabiologicallyinactiveformofHMWK(HMWKi),thusallowingdissociationoftheFactorXIa/HMWKicomplex.EitherfreefactorXIaorthefactorXIa/HMWKcomplexcatalyzestheproteolyticconversionoffactorIXtofactorIXa.FactorXIaispreparedbyactivatingpurifiedfactorXIwithfactorXIIa.Followingactivation,themixtureispassedoveracolumnofimmobilizedanti-factorXIItoremovethefactorXIIa,andfurtherpurifiedonheparin-Sepharose.ThefactorXIaissuppliedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20°C.PurityisassessedbySDSPAGEanalysis.ActivityisdeterminedinaclottingassayusingfactorXIdeficientplasma.Properties:LocalizationPlasma;inassociationwithhighmolecularweightkininogenModeofactionAserineproteasethatconvertsfactorIXtofactorIXaMolecularweight160,000(1,2)ExtinctioncoefficientE1%1cm,280nm=13.4(human)(2)-(inferredfromthezymogen,factorXI)Structuretwoapparentlyidenticalheavychains(Mr~50,000)andtwoapparentlyidenticallightchains(Mr~30,000)heldtogetherbydisulfidebonds.Eachlightchaincontainsacatalyticdomain(1,3).Percentcarbohydrate5%(human)(1)