Endoglycosidase H (Endo-b-N-acetylglucosaminidase H)

Suitable for studies of glycopeptide and glycoprotein with �High Mannose� sugar chains. Enzyme Commission No: EC 3.2.1.96 Trivial Name: Endo-b-N-acetylglucosaminidase H Systematic Name: Mannosyl-glycoprotein-1, 4-N-acetamidodeoxy-b-D-glycohydrolase Stabilizer: < 1mg/0.1u BSA (bovine serum albumin Activity: (same/more than)0.1units Protein: (same/more than)6ug/vial Specific Activity: ~30 units/mg protein Contaminants: No detectable amount of various exoglycosidases or proteases Quality Control: Endoglycosidase H (0.01 unit) was incubated with appropriate p-nitrophenylglycosides (0.1mg) in 0.2ml of citrate-phosphate buffer (pH 5.0) at 37 degrees C for 12 hours. No activites of a- and b-galactosidase or a-L-fucosidase were observed. Absence of contaminating a-mannosidase was also confirmed by using oligosaccharides from ovalbumin and yeast mannan. Endoglycosidase H (0.01 unit) was incubated with 10mg casein in 1ml of 0.1M Tris-CHl buffer at 37 degrees C for 20 hours. 1ml of 10% TCA was added. Release of acid soluble material was below the limit of detection (OD275 less than 0.05). To inhibit potential protease activity during extended Endo-H digestions, 1mM PMSF and/or 10mM pepstatin A may be added to incubations. PMSF is stable as a 2mM stock solution in absolute methanol. Optimum pH: 5.0 Km Value: 0.44mM toward [14C]-acetyl-Asn-GlcNAc2Man6 Unit Definition: One unit is that amount of enzyme required to hydrolyze 1mmole (Man)5- (GlcNAc)2Asn- [14C]-acetyl, pH 5.0 and 37 degrees C per minute at the saturated substrate concentration using Muramatsu�s method. Storage and Stability: Lyophilized powder may be stored at -20 degrees C. Stable for 12 months at -20 degrees C. Reconstitute with sterile ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Reconstituted product is stable at pH 5.0�9.0 at 37 degrees C for 20 hours. Stable for 6 months at -20 degrees C. Repeated freezing and thawing may decrease the enzyme acitivity.