• Purity

  >95%, 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 hydrolyze the substrate 4-Nitrocatechol Sulfate (PNCS). The specific activity is >0.4 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Arg23-Leu502, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Arg23

• Predicted Molecular Mass

  56 kDa

• SDS-PAGE

  60 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM MES, pH 5.5

• Recombinant Mouse Sulfamidase/SGSH (mSGSH) (Catalog # 2969-SU)

• Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N-7251), 100 mM stock in deionized water

• Sodium Hydroxide (NaOH) (Sigma, Catalog # 221465), 2 M stock in deionized water

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rmSGSH to 40 μg/mL in Assay Buffer.

2. Dilute Substrate to 20 mM in Assay Buffer.

3. Mix 150 μL of 40 μg/mL of rmSGSH and 150 μL of 20 mM of Substrate. Include a Substrate Blank with 150 μL of Assay Buffer and 150 μL of Substrate.

4. Incubate at 37 °C for 72 hours.

5. Stop reaction by adding 300 μL of 0.2 M NaOH to the reaction vials.

6. Load in a plate 200 μL in duplicate from each reaction vial.

7. Read at 510 nm (absorbance) in endpoint mode.

8. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard P-Nitrocatechol (PNC) (Sigma, Catalog # N15553).

Per Well:

• rmSGSH: 2 μg

• Substrate: 5 mM

Background: Sulfamidase/SGSH

Also known as N-sulfoglucosamine sulfohydrolase and heparan N-sulfatase, Sulfamidase/SGSH is an important member of the sulfatase family involved in the degradation of heparan sulfate (1). The SGSH deficiency results in mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome), an autosomal recessive lysosomal storage disease characterized by neurological dysfunction but relatively mild somatic manifestations (2). The deduced amino acid sequence of mouse SGSH consists of a signal peptide (residues 1-22) and a mature chain (residues 23-502) (1). Recombinant Mouse Sulfamidase/SGSH corresponds to the mature chain and has sulfatase activity as described in the Activity section.

• References:

1. Diez-Roux, G. and Ballabio, A. (2005) Annu. Rev. Genomics Hum. Genet. 6:355.

2. Gliddon, B.L. et al. (2004) Mol. Genet. Metab. 83:239.

• Entrez Gene IDs:

  6448 (Human); 27029 (Mouse)

• Alternate Names:

  heparan sulfate sulfatase; HSS; HSSN-sulphoglucosamine sulphohydrolase; MPS3A; MPS3Asulphamidase; N-sulfoglucosamine sulfohydrolase; SFMD; SFMDEC 3.10.1.1; SGSH; Sulfamidase; Sulfoglucosamine sulfamidase; Sulphamidase