• 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    
  Analysis

  Met

• Predicted Molecular Mass

  75 kDa

• SDS-PAGE

  65 kDa, reducing conditions

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

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:

     *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:

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.

• 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