• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• 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 >1.0 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Thr36-Pro552 with an N-terminal signal sequence and a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Thr36

• Predicted Molecular Mass

  60 kDa

• SDS-PAGE

  77 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM NaOAc, pH 5.0

• Recombinant Mouse Iduronate 2?Sulfatase/IDS (rmIDS) (Catalog # 2486-SU)

• Substrate: 4-Nitrocatechol Sulfate (4-PNCS) (Sigma, Catalog # N-7251)

• Sodium Hydroxide (NaOH) (Sigma, Catalog # S-0899)

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

• Plate Reader (Model: Spectra-Max Plus by Molecular Devices) or equivalent

1. Dilute rmIDS to 20 μg/mL in Assay Buffer.

2. Dilute Substrate to 2 mM in Assay Buffer.

3. Combine 150 μL of 20 μg/mL rmIDS and 150 μL of 2 mM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate only.

4. Incubate at 37 °C for 24 hours.

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

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

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:

• rmIDS: 1.0 μg

• Substrate: 1 mM

Background: Iduronate 2-Sulfatase/IDS

As a member of the sulfatase family, Iduronate 2-Sulfatase encoded by the IDS gene is required for the lysosomal degradation of the glycosaminoglycans (GAG) heparan sulfate and dermatan sulfate (1, 2). It hydrolyzes the 2-sulfate group of the L-iduronate 2-sulfate units of the GAG. The IDS deficiency results in mucopolysaccharidosis II (MPS II or Hunter syndrome), an X-linked inborn error leading to lysosomal accumulation of the GAG and their excretion in urine. MPS II has a wide spectrum of clinical manifestations ranging from mild to severe. The mouse IDS has sulfatase activity described in the Activity Assay Protocol. In addition, recombinant human IDS is also available (Catalog # ).

• References:

1. Parenti, G. et al. (1997) Curr. Opin. Genet. & Dev. 7:386.

2. Neufeld, E.F. and J. Muenzer (2001) in The Metabolic and Molecular Basis of Inherited Disease, Scriver, C.R. et al. (eds.) p. 3421, New York, McGraw-Hill.

• Entrez Gene IDs:

  3423 (Human); 15931 (Mouse)

• Alternate Names:

  Alpha-L-iduronate sulfate sulfatase; EC 3.1.6.13; IDS; iduronate 2-sulfatase 14 kDa chain; iduronate 2-sulfatase 42 kDa chain; iduronate 2-sulfatase; idursulfase; MPS2; S; SIDS