Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave alpha -N-acetylgalactosaminyl from 4-Nitrophenyl N-acetyl-alpha -D-galactosaminide. The specific activity is >5,000 pmol/min/ug, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
Source
E. coli-derived Lys2-Lys619 with an N-terminal Met and 6-His tag Accession # AAM55479
Accession #
N-terminal Sequence
AnalysisMet
Predicted Molecular Mass
75 kDa
SDS-PAGE
65 kDa, reducing conditions
5705-GH |
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Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. | ||
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Assay Procedure
Materials
Assay Buffer: 100 mM MES, pH 6.5
Recombinant C. perfringens alpha -N-acetylgalactosaminidase (rC. perfringens alpha -N-galactosaminidase) (Catalog # 5705-GH)
Substrate: 4-Nitrophenyl N-acetyl-alpha-D-galactosaminide (Sigma-Aldrich, Catalog # N4264)
0.2 M NaOH, prepare in deionized water
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute rC. perfringens alpha -N-galactosaminidase to 1 ng/µL in Assay Buffer.
Dilute Substrate to 2 mM in Assay Buffer.
Combine 50 µL of alpha -N-galactosaminidase and 50 µL of Substrate in wells of a 96-well plate. Substitute alpha -N-galactosaminidase with Assay Buffer for Substrate Blank.
Incubate at room temperature for 5 minutes.
Stop the reaction by adding 100 µL of 0.2 M NaOH, which also develops the color.
Read at 402 nm in endpoint mode.
Calculate specific activity:
Specific Activity (pmoles/min/µg) = | Adjusted Abs* (OD) x well volume (L) x 1012 pmol/M |
| Inc. time (min) x epsilon ** (M-1cm-1) x path corr.***(cm) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 17700 M-1cm-1
***Using the path correction 0.600 cm
Per Well:
rC. perfringens alpha -N-galactosaminidase: 0.05 µg
Substrate: 0.5 mM
Background: alpha-N-acetylgalactosaminidase/NAGA
The ABO blood group system is the most important blood type system and alpha -N-acetylgalactoside is key to the ABO blood group antigen (1). alpha -N-acetylgalactosidase from Clostridium perfringens is a useful tool for removing alpha linked N-acetylgalactosamine from blood type A antigen to produce H antigen and blood type O (2, 3). Blood type O is universally compatible in the ABO system and is widely used in blood transfusion (2). The enzyme was highly selective for terminal N‑acetylgalactosamine residues (3). No other exoglycosidase activities, particularly neuraminidase, was detected. Recombinant alpha -N-acetylgalactosidase from Clostridium perfringens can potentially be used in enzymatic conversion of human blood type A red blood cells to universally transfusable type O red blood cells.
References:
Watkins, W.M. (1980) Adv. Hum. Genet. 10:1.
Liu, Q.P. et al. (2007) Nature Biotechnol. 25:454.
Calcutt, M. J. et al. (2002) FEMS Microbiol. Lett. 214:77.
Hsieh, H.Y. et al. (2003) Protein Expr. Purif. 32:309.
Entrez Gene IDs:
4668 (Human); 17939 (Mouse); 315165 (Rat)
Alternate Names:
alpha-N- (alpha-galactosidase B); alphaNacetylgalactosaminidase; alpha-N-acetylgalactosaminidase; EC 3.2.1; EC 3.2.1.49; GALB; N-acetylgalactosaminidase, alpha-; NAGA
