Recombinant Human Arylsulfatase A/ARSA Protein, CF

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

Recombinant Human Arylsulfatase A/ARSA Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the substrate 4-Nitrocatechol Sulfate (PNCS). The specific activity is >25 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Arylsulfatase A/ARSA protein
  Arg19-Ala507, with a C-terminal 10-His tag
• Accession #: AAH14210
• N-terminal Sequence: Arg19
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ARSA
• Purity: >95%, 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: 53 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: 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: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM NaOAc, 0.5 M NaCl, pH 4.5
  • Recombinant Human Arylsulfatase A/ARSA (rhARSA) (Catalog # 2485-SU)
  • Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N-7251), 100 mM stock in diH₂O
  • Sodium Hydroxide (NaOH), 2.0 M stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhARSA to 20 µg/mL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. Mix 75 µL of 20 µg/mL of rhARSA and 75 µL of 2 mM of Substrate. As a Substrate Blank combine 75 µL Assay Buffer with 75 µL Substrate only. Prepare in triplicate.
  4. Incubate at 37 °C for 1 hour.
  5. Stop reaction by adding 150 µL of 0.2 M NaOH to reaction vials.
  6. Load in a clear 96-well plate 200 µL from each reaction vial.
  7. Read 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:
  • rhARSA: 1 µg
  • PNCS: 0.5 mM

Notes

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

Alternate Names for Recombinant Human Arylsulfatase A/ARSA Protein, CF

• ARSA
• Arylsulfatase A
• ASA
• Cerebroside 3-sulfatase
• Cerebroside-sulfatase
• EC 3.1.6
• EC 3.1.6.8
• MLD

Background

As a member of the sulfatase family, ARSA is required for the lysosomal degradation of cerebroside-3-sulfate, a sphingolipid sulfate ester and a major constituent of the myelin sheet (1). The ARSA deficiency results in metachromatic leukodystrophy (MLD), a lysosomal storage disease in the central and peripheral nervous systems with severe and progressive neurological symptoms (2). The deduced amino acid sequence of human ARSA consists of a signal peptide (residues 1-18) and a mature chain (residues 19-507) (3). Recombinant human ARSA corresponds to the mature chain and has sulfatase activity described above.

1. Lukatela, G. et al. (1998) Biochemistry 37:3654.
2. Parenti, G. et al. (1997) Curr. Opin. Genet. & Dev. 7:386.
3. Stein, C. et al. (1989) J. Biol. Chem. 264:1252.

Publications for Arylsulfatase A/ARSA (2485-SU)(1)

Publication using 2485-SU

• MC Babcock, CR Mikulka, B Wang, S Chandriani, S Chandra, Y Xu, K Webster, Y Feng, HR Nelvagal, A Giaramita, BK Yip, M Lo, X Jiang, Q Chao, JC Woloszynek, Y Shen, S Bhagwat, MS Sands, BE Crawford Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor Scientific Reports, 2021-07-14;11(1):14486. 2021-07-14 [PMID: 34262084] (Bioassay, Human)

Bioinformatics

• Gene Symbol: ARSA
• Uniprot:
  • AAH14210()