Recombinant C. perfringens a-N-acetylgalactosaminidase, CF

• Reactivity: Cp
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant C. perfringens a-N-acetylgalactosaminidase, CF Summary

• Details of Functionality: 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.
• Source: E. coli-derived c. perfringens alpha-N-acetylgalactosaminidase/NAGA protein Lys2-Lys619 with an N-terminal Met and 6-His tag Accession # AAM55479
• Accession #: AAM55479
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 75 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 65 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  6 months from date of receipt, -70 degreesC as supplied. 3 months, -70 degreesC under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • 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
  1. Dilute rC. perfringens alpha -N-galactosaminidase to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. 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.
  4. Incubate at room temperature for 5 minutes.
  5. Stop the reaction by adding 100 µL of 0.2 M NaOH, which also develops the color.
  6. Read at 402 nm in endpoint mode.
  7. Calculate specific activity:

  Specific Activity (pmoles/min/µg) = (Adjusted Abs* (OD) x well volume (L) x 10^12 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant C. perfringens a-N-acetylgalactosaminidase, CF

• alpha-N- (alpha-galactosidase B)
• alphaNacetylgalactosaminidase
• alpha-N-acetylgalactosaminidase
• EC 3.2.1
• EC 3.2.1.49
• GALB
• N-acetylgalactosaminidase, alpha-
• NAGA

Background

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.

1. Watkins, W.M. (1980) Adv. Hum. Genet. 10:1.
2. Liu, Q.P. et al. (2007) Nature Biotechnol. 25:454.
3. Calcutt, M. J. et al. (2002) FEMS Microbiol. Lett. 214:77.
4. Hsieh, H.Y. et al. (2003) Protein Expr. Purif. 32:309.

Bioinformatics

• Uniprot:
  • AAM55479()