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
AnalysisGlu17
Predicted Molecular Mass
70 kDa
SDS-PAGE
83-95 kDa, reducing conditions
9348-GH |
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Formulation Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol. | ||
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. | ||
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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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
Dilute rmIDUA to 0.2 µg/mL in Assay Buffer. Minimize the number of dilution steps to obtain the best activity results.
Dilute Substrate to 200 µM in Assay Buffer.
Combine equal volumes of 0.2 µg/mL rmIDUA and 200 µM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate.
Incubate for 10 minutes at room temperature.
Dilute mixtures to 0.005 µg/mL rmIDUA in Developing Buffer.
In a plate load 100 µL of diluted mixtures.
Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in endpoint mode.
Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) |
| Incubation time (min) x amount of enzyme (µg) |
*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:
Scott, H.S. et al. (1995) Hum. Mutat. 6:288.
Wraith, J.E. (2005) Expert Opin. Pharmacother. 6:489.
Jameson, E. (2016) Cochrane Database Syst. Rev. 4: CD009354.
Kodric, K. et al. (2016) Wien Klin Wochenschr. 128:480.
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
