Recombinant Human Sulfamidase/SGSH Protein, CF

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

Recombinant Human Sulfamidase/SGSH Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the substrate 4-Nitrocatechol Sulfate (PNCS). The specific activity is >0.3 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Sulfamidase/SGSH protein
  Arg23-Leu502 with a C-terminal 6-His tag
• Accession #: P51688
• N-terminal Sequence: Arg23
• Protein/Peptide Type: Recombinant Enzymes
• Gene: SGSH
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Theoretical MW: 55 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: 55-63 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -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 visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, pH 5.5
  • Recombinant Human Sulfamidase/SGSH (rhSGSH) (Catalog # 8380-SU)
  • Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N7251), 100 mM stock in deionized water
  • Sodium Hydroxide (NaOH) (Sigma, Catalog # 221465), 2 M stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhSGSH to 40 µg/mL in Assay Buffer.
  2. Dilute PNCS to 20 mM in Assay Buffer.
  3. Combine 150 µL of 40 µg/mL of rhSGSH and 150 µL of 20 mM of PNCS in microtubes. Include a Substrate Blank with 150 µL of Assay Buffer and 150 µL of 20 mM of PNCS.
  4. Incubate at 37 °C for 72 hours.
  5. Stop reactions and Substrate Blank by adding 300 µL of 0.2 M NaOH to each tube.
  6. Load 200µL from each stopped reaction and Substrate Blank vial into a plate.
  7. Read plate at 510 nm (absorbance) in endpoint mode.
  8. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Abs* (OD) x Conversion Factor** (pmol/OD)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard 4-Nitrocatechol (PNC) (Sigma, Catalog # N15553).

  Per Well:
• rhSGSH: 2 µg
• Substrate: 5 mM

Notes

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

Alternate Names for Recombinant Human Sulfamidase/SGSH Protein, CF

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

Background

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 (HS) (1). Different from the HS specific endosulfatases that remove sulfate from internal GlcNAc residues (2), SGSH removes sulfate group from the non-reducing end glucosamine residues on HS. 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 (3). Human SGSH shows 88.6% sequence identity with that of mouse sequence.

1. DiezRoux, G. and Ballabio, A. et al. (2005) Annu. Rev. Genomics Hum. Genet. 6:355.
2. Morimoto-Tomita, M. et al. (2002) J. Biol. Chem. 277:49175.
3. Blanch, L. et al. (1997) Hum. Mol. Genet. 6:787.

Bioinformatics

• Gene Symbol: SGSH
• Uniprot:
  • P51688()