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 °C as supplied.
  • 3 months, -70 °C 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()