EndoH,endo-beta-N-acetylglucosaminidaseH,EndoglycosidaseH

EndoHcleavesAsparagine-linkedhybridorhighmannoseoligosaccharides,butnotcomplexoligosaccharides.ItcleavesbetweenthetwoN-acetylglucosamineresiduesinthediacetylchitobiosecoreoftheoligosaccharide,generatingatruncatedsugarmoleculewithoneN-acetylglucosamineresidueremainingontheasparagine.Incontrast,PNGaseFremovestheoligosaccharideintact.Detergentandheatdenaturationmayincreasetherateofcleavageforsomeglycoproteins.

SourcerecombinantfromStreptomycesplicatusinE.Coli

EC3.2.1.96

SpecificActivityOneunitofEndoHactivityisdefinedastheamountofenzymerequiredtocatalyzethereleaseofN-linkedoligosaccharidesfrom1µmoleofdenaturedRibonucleaseB.CleavageismonitoredbySDS-PAGE(cleavedRibonucleaseBmigratesfaster).

Contents
60µlaliquotofenzyme(300mU)in20mMTris-HCl,25mMNaCl,1mMEDTA(pH7.5).
Includedwith20µLand60µLpacksizes:
200µl5xReactionBuffer5.5(250mMsodiumphosphate,pH5.5)
DenaturationSolution–2%SDS,1MBeta-mercaptoethanol

SpecificActivity>40U/mg

Activity>5U/ml

Molecularweight29,000daltons

pHrange5-6,optimum5.5

Suggestedusage
1.Addupto200µgofglycoproteintoEppendorftube.Adjustto37.5µlfinalvolumewithdeionizedwater.
2.Add10µl5XEndoHBufferand2.5µlofDenaturationSolution(SDS/-ME).Heatat100˚Cfor5minutes.
3.Cool,andthenadd2.0µlofEndoHtothereaction.Incubate3hoursat37˚C.

StorageStoreenzymeat4˚C.

StABIlityStableatleast12monthswhenstoredproperly.Severaldaysexposuretoambienttempertureswillnotreduceactivity.

PurityEndoglycosidaseHistestedforcontaminatingproteaseasfollows;10μgofdenaturedBSAisincubatedfor24hoursat37oCwith2μLofenzyme.SDS-PAGEanalysisofthetreatedBSAshowsnoevidenceofdegradation.

Theproductionhoststrainhasbeenextensivelytestedanddoesnotproduceanydetectableglycosidases.

EndoHReferences

RobbinsP.W.,D.F.WirthandC.J.Hering.ExpressionoftheStreptomycesenzymeendoglycosidaseHinEscherichiacoli.BiolChem256:10640-10644(1981).

RobbinsP.W.,R.B.Trimble,D.F.Wirth,C.Hering,F.Maley,G.F.Maley,R.Das,B.W.Gibson,N.RoyalandK.Biemann.PrimarystructureoftheStreptomycesenzymeendo-beta-N-acetylglucosaminidaseH.JBiolChem259:7577-7583(1984).

TrimbleR.B.,A.L.Tarentino,G.EAumickandF.Maley.Endo-beta-N-acetylglucosaminidaseLfromStreptomycesplicatus.MethodsEnzymol83:603-610(1982).

TrimbleR.B.andF.Maley.OptimizinghydrolysisofN-linkedhigh-mannoseoligosaccharidesbyendo-beta-N-acetylglucosaminidaseH.AnalBiochem141:515-522(1984).

TrimbleR.B.,R.J.TrumblyandF.Maley.Endo-beta-N-acetylglucosaminidaseHfromStreptomycesplicatus.MethodsEnzymol138:763-770(1987).

TrumblyR.J.,P.W.Robbins,M.Belfort,F.D.Ziegler,F.MaleyandR.B.Trimble.AmplifiedexpressionofStreptomycesendo-beta-N-acetylglucosaminidaseHinEscherichiacoliandcharacterizationoftheenzymeproduct.JBiolChem260:5683-5690(1985).

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