I.U.B.:3.4.24.3
CollagenaseRawMaterialCertification
Crudecollagenasepreparationscontainseveralisoformsoftwodifferentcollagenases,asulfhydrylprotease,clostripain,atrypsin-likeenzyme,andanaminopeptidase.Thiscombinationofcollagenolyticandproteolyticactivitiesiseffectiveatbreakingdownintercellularmatrices,theessentialpartoftissuedissociation.OnecomponentofthecomplexisahydrolyticenzymewhichdegradesthehelicalregionsinnativecollagenpreferentiallyattheY-GlybondinthesequencePro-Y-Gly-Pro,whereYismostfrequentlyaneutralaminoacid.ThiscleavageyieldsproductssusceptIBLetofurtherpeptidasedigestion.Crudecollagenaseisinhibitedbymetalchelatingagentssuchascysteine,EDTAoro-phenanthrolinebutnotDFP.Itisalsoinhibitedbyα2-macroglobulin,alargeplasmaglycoprotein.Ca2+isrequiredforenzymeactivity.Particularenzymaticprofilesofeachcollagenasehavebeencorrelatedwiththetissuesfromwhichthecellsforstudywereobtained(orwiththeusestowhichthecellsareput)andasaresultofthecorrelationsseveraltypesofcrudecollagenaseshavebeenestablishedbyWorthington:Types1,2,3,and4.
•Type1crudecollagenasehastheoriginalbalanceofcollagenase,caseinase,clostripainandtrypticactivities.
•Type2containshigherrelativelevelsofproteaseactivity,particularlyclostripain.
•Type3containslowestlevelsofsecondaryproteases.
•Type4isdesignedtobeespeciallylowintrypticactivitytolimitdamagetomembraneproteinsandreceptors.
•Purifiedcollagenase,Code:CLSPA,containsminimalsecondaryproteolyticactivitiesalongwithhighcollagenaseactivity.
NEW!AnimalOriginFreeTypesAFA,AFBandAFC,STZ1,andSTZ2collagenasesarederivedfromculturesgrowninmediumcompletelydevoidofanimalbasedcomponentsanddesignedforbioprocessingapplicationswhereintroductionofpotentialanimalderivedpathogensmustbeprevented.LevelsofsecondaryproteasesaresimilartoTypes1and2collagenase. • CLSAFA istheoriginalAFAgradedesignedtohavecollagenaseandsecondaryproteasessimilartoTypes1and2collagenase.• CLSAFB containshighercollagenaseandcaseinaseactivitiesthanCLSAFA. • CLSAFC hasespeciallylowtrypticactivitysimilartoType4collagenase. • STZ1&STZ2,0.22μfilteredSTEMxyme®AOFCollagenase/NeutralProtease(Dispase®)blendsforprimaryandstemcellisolation. |
Worthingtonalsooffers0.22µmfilteredpreparationsofeachtypeinpre-packagedformfordirectreconstitutionanduseincellisolationandcultureprocedures.Correlationsbetweenenzymetypeandeffectivenesswithdifferenttissueshavebeengood,butnotperfect,dueinparttothevariableparametersofuse.Neverthelessmostresearchersconsiderthetissue-typingofcrudecollagenaselotstobeavaluableservice.AdetaileddescriptionoftheWorthingtoncollagenaseandcontaminantassayscanbefoundintheWorthingtonEnzymeManual.Inaddition,tissuespecificreferencesanddetailedisolationconditionscanbefoundintheWorthingtonTissueDissociationGuide.
ThecollagenaseassayisamodificationofMandlwhereincollagenaseisincubatedforfivehourswithnativecollagenandtheextentofcollagenbreakdownisdeterminedusingtheMooreandStein,JBC,176,367,(1948)colorimetricninhydrinmethod.Aminoacidsreleasedareexpressedasmicromolesleucinepermilligramcollagenase.
Uses:Crudecollagenasesarewidelyusedinenzymaticprimarycellisolationandtissuedissociationprocedures.MostresearchersemployeithercrudecollagenasepreparationssuchasTypes1,2,3,and4orchromatographicallypurifiedcollagenase(Code:CLSPA);thelatterusuallycombinedwithsecondaryenzymessuchaselastase,hyaluronidase,etc.Forbestresults,theprecisemixtureofproteolyticactivitiesmustbetailoredtothetissuetobedissociated.Correlationsbetweentypeandeffectivenesswithdifferenttissueshavebeengood,butnotperfect,andmaybedependentpartlyonparametersofuseandobjectivesaswellaslot-to-lotvariations.FormoreinformationseetheCollagenaseSamplingPrograminformation.WorthingtonalsopublishesaTissueDissociationGuidewhichprovidesadditionalinformationregardingtheenzymesusedfortheseapplicationsandspecificreferencesfornumerouscellandtissuetypes.
UnitDefinition:OneUnitreleasesonemicromoleofL-leucineequivalentsfromcollagenin5hoursat37°C,pH7.5
RelatedProducts:
Collagen(CL)
DeoxyribonucleaseI(DP/D/DCLS/D2/DPFF/DPRF)
Elastase(ES/ESL/ESFF)
Hyaluronidase(HSE/HSEP)
NeutralProtease(Dispase,NPRO)
Papain(PAP/PAPL/PAP2)ProteinaseK(PROK)
Trypsin(TL/TRL/TRL3/TRLS/TRTPCK)
TrypsinInhibitors(LBI/OI/SI/SIC)
CellIsolationOptimizingSystem(CIT)
HepatocyteIsolationSystem(HIS)
NeonatalCardiomyocyteIsolationSystem(NCIS)
PapainDissociiationSystem(PDS/PDS2)
