• 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 cleave a fluorogenic substrate, 4-Methylumbelliferyl  alpha -L-iduronide.  The specific activity is >7,500 pmol/min/ug, as measured under the described conditions.    
  See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Glu17-Ser634, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Glu17

• Predicted Molecular Mass

  70 kDa

• SDS-PAGE

  83-95 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM NaOAc, 150 mM NaCl, 0.02% Brij-35 (w/v), pH 3.5

• Developing Buffer: 0.1 M Tris, pH 9.0

• Recombinant Mouse alpha -L-Iduronidase/IDUA (rmIDUA) (Catalog # 9348-GH)

• Substrate: 4-methylumberlliferyl-alpha -L-Iduronide (Glycosynth, Catalog # 44076), 20 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

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

1. Dilute rmIDUA to 0.2 μg/mL in Assay Buffer. Minimize the number of dilution steps to obtain the best activity results.

2. Dilute Substrate to 200 μM in Assay Buffer.

3. Combine equal volumes of 0.2 μg/mL rmIDUA and 200 μM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate.

4. Incubate for 10 minutes at room temperature.

5. Dilute mixtures to 0.005 μg/mL rmIDUA in Developing Buffer.

6. In a plate load 100 μL of diluted mixtures.

7. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in endpoint mode.

8. Calculate specific activity:

*Adjusted for Substrate Blank.
**Derived using calibration standard 4-methylumbelliferone (Sigma, Catalog # M1381).

Per Well:

• rmIDUA: 0.0005 μg

• Substrate: 5 μM

Background: alpha-L-Iduronidase/IDUA

alpha -L-Iduronidase encoded by the IDUA gene is an important enzyme required for the lysosomal degradation of glycosaminoglycans (GAGS). It hydrolyzes the  
non-reducing terminal alpha -L-iduronic acid residues in GAGS including dermatan sulfate and heparan sulfate.  Mature mouse IDUA shares 80% aa identity with human IDUA. Mutations in IDUA that result in enzymatic deficiency lead to the autosomal recessive disease mucopolysaccharidosis type I (MPS I) (1). MPS I can be classified as three clinical subtypes; Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome with decreasing severity, respectively. MPS I causes progressive cellular, tissue and organ damage, and several clinical studies using enzyme replacement therapy show positive results (2, 3). Recently, the IDUA gene has been linked to osteoporosis (4, 5).

• References:

1. Scott, H.S. et al. (1995) Hum. Mutat. 6:288.

2. Wraith, J.E. (2005) Expert Opin. Pharmacother. 6:489.

3. Jameson, E. (2016) Cochrane Database Syst. Rev. 4: CD009354.

4. Kodric, K. et al. (2016) Wien Klin Wochenschr. 128:480.

5. Niu, T. et al. (2016) J. Bone Miner. Res. 31:358.

• Entrez Gene IDs:

  3425 (Human); 15932 (Mouse); 360904 (Rat)

• Alternate Names:

  alphaLIduronidase; alpha-L-Iduronidase; IDA; IDUA