热线13301321103010-56371204QQ:894833566
官网:www.solarbio.cn
联系人:罗敏
CollagenaseⅠ
胶原酶Ⅰ说明书
COLLAGENASE,CRUDE,GeneralUsefromClostridiumhistolyticum
CASNumber:9001-12-1
StorageTemperature:-20℃
ProductNumber:C8140(FromsigmaC0130)
PRODUCTDESCRIPTION
Appearance:Tantobrownpowder.Molecularweight:68,000to125,000.pHoptimum:6.3-8.8.TheenzymeisassayedbySigmaatpH7.4.
Specificity:Collagenaserecognizesthesequence-R-Pro-8-X-Gly-Pro-R-whereXismostoftenaneutralaminoacid.
Crudecollagenasesaremixturesofenzymes(mostlyproteases)secretedbyClostridiumhistolyticum.Thispreparationcontainscollagenase,non-specificproteasesandclostripain.Itcontainsnocarbohydrate.
Collagenaseisactivatedbyfourgramatomcalcium(Ca2+)permoleenzyme.Inhibitorsofcollagenaseincludeethyleneglycol-bis(b-aminoethylether)-N,N,N,N-tetraaceticacid(EGTA);b-mercaptoethanol;glutathione,reduced;thioglycolicacid,sodium;2,2-dipyridyl;8-hydroxyquinoline
Thevarioustypesofcollagenarethenaturalsubstratesforcollagenase.Manysyntheticpeptideshavebeenpreparedtoserveascollagenasesubstrates;theyinclude:N-CBZ-gly-pro-gly-gly-pro-ala(Km=0.71mM);N-CBZ-gly-pro-leu-gly-pro;N-2,4-Dinitrophenyl-pro-gln-gly-ile-ala-gly-gln-D-arg;N-(3-(2-furyl)acryloyl)-leu-gly-pro-ala(FALGPA);4-Phenylazobenzyloxycarbonyl-pro-leu-gly-pro-D-arg.InadditionN-Succinyl-gly-pro-leu-glypro7-amido-4-methylcoumarinislistedasasubstratefor"collagenase-likepeptidase"andN-(2,4-Dinitrophenyl)-proleu-gly-leu-trp-ala-D-argamideislistedasasubstratefor"vertebratecollagenase".
APPLICATIONS:
Thisproductissuitableforthedisaggregationofhumantumor,mousekidney,humanadultandfetalbrain,lungandmanyothertissues,particularlyepithelium.Itisalsoeffectiveinliverandkidneyperfusionstudies,digestionofpancreas,isolationofnonparenchymalratlivercellsandhepatocytepreparation.
AlthoughthetermcollagenaseimpliesthatthereisasingleenzymeproducedbyC.histolyticum,thisisnotthecase.Crudecollagenasesaremixturesofenzyme(mostlyproteases)secretedbyC.histolyticum.Allmaycontain10to18components(byelectrophoresis),only8ofwhichhavebeenidentified.Theproductsdifferbytheamountofthecomponentsabsoluteandrelativetoeachother.
Thecomponentenzymesincrudearetwospecificcollagenases(measuredasFALGAunits/mg),clostripain(measuredasBAEEafterreductionwithDTT)andaneutralprotease(measuredascaseinase).Inthecrudecollagenase,theclostripainismostlyinactive,oxidized.Animportantfeatureforuseintissuedissociationistheratioofcollagenasetoprotease.
Effectivereleaseofcellsfromtissuedependsontheactionofboththetwocollagenaseenzymesandtheneutralprotease,foreitheraloneisnotveryeffective.
ST
ABILITY/STORAGEASSUPPLIED:
Thisproductisstableforatleastoneyearwhenstoredat-20°C.ThereisnolossinFALGPAorproteaseactivityin30daysat37°C,50°Cand-20°C.
SOLUTION/SOLUTIONSTABILITY
Usually,solutionsarepreparedat1-2mg/mlinTESCAbuffer(50mMTES,0.36mMCalciumchloride,pH7.4at37°C.).
Solutionsofcrudecollagenasearestableiffrozenquicklyinaliquots(at10mg/mL)andkeptfrozenat-20°C.
Freeze-thawcycleswilldamagetheenzymesolution.Inaqueousbacterialcollagenaselosesmeasurable
ctivityin3hr.at4°C.AtpH7.0inthepresenceof1mMCa2+thereisnolossofactivityin1hr.at40°C,50%loss
10minat48°Cand100%lossin5min.at60°C.Theoptimumcalciumconcentrationfortissuedissociationis5
M.Theproductretains100%activityover7hourswhenheldonice.