Properties and Usage
One unit of O-Glycosidase is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of Neuraminidase digested, non-denatured fetuin in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).
Non-denaturing Unit Definition of O-Glycosidase:
Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of Neuraminidase digested fetuin with 1X GlycoBuffer 2. The reaction is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by Morgan and Elson Assay (4). Note: Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.
Unit Definition of Neuraminidase:
1X Glycoprotein Denaturing Buffer
One unit of Neuraminidase is defined as the amount of enzyme required to cleave > 95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 µl.
40 mM DTT
1% NP-40 in MilliQ-H2O
1X GlycoBuffer 2
Incubate at 37°C
1X GlycoBuffer 2:
50 mM sodium phosphate
pH 7.5 @ 25°C