Neuraminidase,N-acetylneuraminateglycohydrolase,Exo-alpha-sialidase

α(2-3,6)SialidaseCpcleavesallnon-reducingterminalnon-branched&alpha(2-3)-andα(2-6)sialicacidresiduesfromcomplexcarbohydratesandglycoproteins.Thereisnodetectableactivityonα(2-8)orα(2-9)linkagesoronbranchedα(2-3)orα(2-6)linkages.Therelativecleavageratesfordifferentlinkagesare:α(2-3)>α(2-6).

α(2-3,6)SialidaseCpwillnotcleavebranchedsialicacids(linkedtoaninternalresidue).Useα(2-3,6,8,9)Sialidase(E-S001)forα(2-8)orbranchedsialicacids.Tocleaveonlynon-reducingterminalα(2-3)unbranchedsialicacidresidues,useα(2-3)Sialidase(E-S007).

α(2-3,6)SialidaseCpisisolatedfromacloneofClostridiumperfringens.Theenzymehasbeenextensivelycharacterizedusingoligosaccharidestandards.

SourcerecombinantfromClostridiumperfringensinE.Coli.

EC3.2.1.18

Contents
SialidaseCpin20mMTris-HCl,25mMNaCl,pH7.5

Includedits20µLand60µLpacksizes:
5xReactionBuffer250mMsodiumphosphate,pH6.0

SpecificActivity>250U/mg
Activity15U/ml

Molecularweight41,000daltons

pHrange50mMsodiumphosphate(pH6.0)providestheoptimalbufferforenzymeactivitywith3’-siayllactose,astandardsubstrate.IfglycosidasetreatmentisperformedatsuboptimalpHbecauseofglycoproteinsolubilityoractivityrequirements,expectsomediminutioninenzymeactivity.

Suggestedusage
1.Addupto100μgofglycoproteinor1nmolofoligosaccharidetotube.
2.Addwaterto14μL
3.Add4μL5XReactionBuffer.
4.Add2μLα(2-3,6)Sialidase.
5.Incubateat37°Cfor1hour.

DesialylationmaybemonitoredbySDS-PAGEifthesizedifferentialbetweennativeanddesialylatedproteinissufficientfordetection.

SpecifictityCleavesallnonreducingterminalnon-branchedalpha-(2-3)andalpha-(2-6)-sialicacidresiduesfromcomplexcarbohydratesandglycoproteins.

Relativecleavageratesfordifferentlinkagesare:(2-3)>(2-6).

SpecificActivityAssayDefinedastheamountofenzymerequiredtoproduce1µmoleofmethylumbelliferonein1minuteat37˚C,pH5.0fromMU-NANA[2′-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminicacid].

StorageStoreenzymeat4˚C.

SialidaseCPReferences

Corfield,A.P.,H.Higa,J.C.PaulsonandR.Schauer.Thespecificityofviralandbacterialsialidasesforalpha(2-3)andalpha(2-6)-linkedsialicacidsinglycoproteins.BiochemBiophysActa744:121-126(1983).

Dwek,R.A.,C.J.Edge,D.J.Harvey,M.R.WormaldandR.B.Parekh.Analysisofglycoprotein-associatedoligosaccharides.AnnRevBiochem62:65-100(1993).

Kobata,A.Useofendo-andexoglycosidasesforstructuralstudiesofglycoconjugates.AnalBiochem100:1-14(1979).

Prime,S.,J.Dearnley,A.M.Venton,R.B.ParekhandC.J.Edge.Oligosaccharidesequencingbasedonexo-andendoglycosidasedigestionandliquidchromatographicanalysisoftheproducts.JChromatogrA720:263-274(1996).

Uchida,Y.,Y.TsukadaandT.Sugimori.EnzymaticpropertiesofneuraminidasesfromArthrobacterureafaciens.JBiochem(Tokyo)86:573-585(1979).

Roggentin,P,B.Rothe,FLottspeichandR.Schauer.CloningandsequencingofaClostridiumperfringenssialidasegene.FEBSLett238:31-34(Sept1988).

RoggentinP.,R.G.KleineidamandR.Schauer.DiversityinthepropertiesoftwosialidaseisoenzymesproducedbyClostridiumperfringensspp.BiolChemHoppe-Seyler376:569-575(1995)