New England Biolabs, P0737L, Bacteroides Heparinase III

t is active on both heparin and heparan sulfate. Related Categories Heparinases Applications Analysis of Heparin/HS Specification Unit Definition One unit is defined as the amount of enzyme that will liberate 0.25 nmol unsaturated oligosaccharides from heparan sulfate per minute at 30°C and pH 7.0 in a total reaction volume of 100 μl. Reaction Conditions 1X Bacteroides Heparinase Reaction Buffer Incubate at 30°C Storage Buffer 20 mM Tris-HCl 100 mM NaCl 5 mM CaCl2 1 mM EDTA pH 7.5 @ 25°C Heat Inactivation 100°C for 1 minute Molecular Weight Apparent: 75 kDa Unit Assay Conditions Two fold dilutions of Bacteroides Heparinase III are incubated with 1 mg/ml porcine mucosal heparan sulfate in 1X Bacteroides Heparinase Reaction Buffer, in a 100 μl reaction. The reaction mix is incubated at 30°C. Liberation of unsaturated oligosaccharides is detected by real-time UV spectroscopy at 232 nm. FAQ Q: What are Bacteroides Heparinase Enzymes? A: Heparinases, also called Heparin Lyase enzymes, are enzymes that cleave the glycosidic linkage between hexosamines and uronic acids and are known to cleave heparin and HS chains selectively, via an elimination mechanism. Heparinase enzymes create a double bond on the non-reducing end of the uronic acid that absorbs at 232nm and can be used for the detection of oligosaccharide and disaccharide products. Q: What is the difference between Bacteroides Heparinase I, II and III? A: Heparinase I cleaves highly sulfated heparin/HS chains, heparinase III cleaves less sulfated HS chains, while heparinase II cleaves domains of both high and low sulfation on both heparin and HS. Heparinase I, II and III used in combination can produce a near-complete depolymerization of heparin/HS polysaccharide chains to disaccharides. Q: Can Bacteroides Heparinase I, II, and III be used together in one digest? A: Yes, all three Heparinase enzymes are supplied with the same Heparinase Reaction Buffer (1X formulation is 20mM TrisHCl pH 7, 100 mM NaCl, 1.5 mM CaCl2), and have optimal incubation temperatures of 30°C. Q: What is the optimal pH range for Bacteroides Heparinase III? A: Bacteroides Heparinase III is most active between pH 7.0-8.0 Q: Is Bacteroides Heparinase III activated by Calcium? A: Yes, Bacteroides Heparinase III has greatest activity between the range of 1.5 -5.0mM CaCl2. The storage buffer and reaction buffer both contain calcium for this reason. Q: What is the formulation of the Heparinase Reaction Buffer? A: The 1X Heparinase Reaction Buffer contains 20 mM TrisHCl pH 7, 100 mM NaCl, 1.5 mM CaCl2.