Recombinant Human SPR His-tag Protein, CF

Recombinant Human SPR His-tag Protein (Catalog # 10209-SP) is measured by its ability to catalyze the reduction of phenanthrenequinone.

2 μg/lane of Recombinant Human SPR His-tag was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at 28 kDa under reducing conditions.

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

Recombinant Human SPR His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to catalyze the reduction of phenanthrenequinone. The specific activity is >700 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human SPR protein
  Met1-Lys261
  with a C-terminal 6-His tag
• Accession #: P35270
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 29 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: 28 kDa under 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, NaCl and Glycerol.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Potassium Phosphate, 150 mM NaCl, pH 6.0
  • Recombinant Human SPR His-tag (rhSPR) (Catalog # 10209-SP)
  • Substrate: 9,10-Phenanthrenequinone (PQ) (Sigma, Catalog # 156507), 5 mM stock in N,N-Dimethylformamide (DMF)
  • beta -Nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt ( beta -NADPH) (Sigma, Catalog# N7505), 10 mM stock in deionized water
  • 96-well clear plate (Catalog # DY990)
  • Fluorescent Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhSPR to 10 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 200 µM PQ and 800 µM beta -NADPH in Assay Buffer.
  3. Load 50 µL of 10 µg/mL rhSPR into a plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  4. Read at an absorbance of 340 nm, respectively, in kinetic mode of 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol x -1) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Using the extinction coefficient 6270 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD

  Per Well:
  • rhSPR: 0.5 µg
  • PQ: 100 µM
  • beta -NADPH: 400 µM

Notes

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

Alternate Names for Recombinant Human SPR His-tag Protein, CF

• EC 1.1.1.153
• Gm10328
• SDR38C1
• sepiapterin reductase (7,8-dihydrobiopterin:NADP+ oxidoreductase)
• sepiapterin reductase
• short chain dehydrogenase/reductase family 38C, member 1
• SPR

Background

Sepiapterin reductase (SPR) catalyzes the biosynthesis of tetra-hydrobiopterin (BH4), an important cofactor in aromatic amino acid metabolism and key regulator of nitric oxide biosynthesis which relates the BH4 cofactor to many pathophysiological processes (1, 2). SPR has additionally been shown to be efficient at mediating chemical redox cycling of quinones and herbicides through a mechanism distinct from sepiapterin reduction (3). SPR is an NADPH-dependent, cytoplasmic enzyme classified as a member of the short-chain dehydrogenase/reductase (SDR) family based on its structural catalytic domain and NADPH binding motifs (4). SPR forms an active homodimer where each monomer contains a key c-terminal asparagine required for activity (3) in addition to the conserved catalytic triad that serves to stabilize protein structure while maintaining cofactor and substrate proximity in a catalytic site composed of several hydrophobic amino acids (4-6). SPR contains the conserved NADPH binding motif at the N-terminus (4-6). SPR has broad tissue expression (7). Identified human mutations in SPR result in compromised production of biopterin cofactor and are associated with neurological deficits (8, 9) linked to dystonia (8, 10). Knockout in mice likewise results in reduced neurotransmitters and movement disorders (11). Loss of BH4 can also reduce nitric oxide production leading to alterations in cardiac function and inflammation (2). SPR has been pharmacologically targeted to reduce pathologically elevated BH4 for pain management (12, 13) and to regulate T-cell function in pathological diseases and tumor growth (14).

1. Werner, E.R. et al. (2011) Biochem. J 348:397.
2. Bendall, J. K. et al. (2014) Antiox. Redox. Signal 20:3040.
3. Yang, S. et al. (2013) J. Biol. Chem. 288:19222.
4. Jornvall, H. et al. (1995) Biochemistry 34:6003.
5. Auerbach, G. et al. (1997) EMBO J. 16:7219.
6. Fujimoto, K. et al. (2001) Chem. Biol. Interact. 130:825.
7. Katoh, S. (1971) Arch. Biochem. Biophys. 146:202.
8. Bonafe, L. et al. (2001) Am. J. Hum. Genet 69:269.
9. Farrugia, R. et al. (2007) Mol. Genet. Metab. 90:277.
10. Abeling, N.G. et al. (2006) Mol. Genet. Metab. 89:116.
11. Yang, S. et al. (2006) Am. J. Hum. Genet. 78:575.
12. Latremoliere, A. et al. (2015) Neuron. 86:1393.
13. Fujita, M. et al. (2019) Arthritis Rheumatol. [Epub ahead of print]
14. Cronin, S.J.F. et al. (2018) Nature. 563:7732.

Bioinformatics

• Uniprot:
  • P35270()