Recombinant Human PNP Protein, CF

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

Recombinant Human PNP Protein, CF Summary

• Details of Functionality: Measured by the phosphorolysis of 7-methyl-6-thioguanosine. The specific activity is >35,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Purine Nucleoside Phosphorylase/PNP protein
  Met1-Ser289, with a C-terminal 6-His tag
• Accession #: P00491
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PNP
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 33 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: 30-35 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -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 Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Potassium Phosphate, pH 7.4
  • Recombinant Human Purine Nucleoside Phosphorylase/PNP (rhPNP) (Catalog # 6486-NP)
  • Substrate: 7-Methyl-6-thioguanosine (MESG) (Berry & Associates, Catalog # PR 3790), 10 mM stock in DMSO
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhPNP to 0.2 ng/μL in Assay Buffer.
  2. Dilute Substrate to 800 μM in Assay Buffer.
  3. Load 50 μL of 0.2 ng/μL rhPNP into the microplate and start the reaction by adding 50 μL of Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate.
  4. Read at an absorbance of 360 nm in kinetic mode for 5 minutes.
  5. Calculate specific activity:

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

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 6220 M^-1cm^-1
  ***Using the path correction 0.320 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhPNP: 0.010 μg
  • Substrate: 400 μM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human PNP Protein, CF

• EC 2.4.2.1
• FLJ94043
• FLJ97288
• Inosine phosphorylase
• MGC117396
• MGC125915
• MGC125916
• NPFLJ97312
• nucleoside phosphorylase
• PNP
• PRO1837
• PUNP
• purine nucleoside phosphorylase
• purine-nucleoside:orthophosphate ribosyltransferase

Background

Purine Nucleoside Phosphorylase (PNP) catalyzes the phophorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. Physiological substrates of PNP include inosine, guanosine, and 2'-deoxyguanosine, but not adenosine (1). PNP is expressed in most tissues, with markedly greater expression in lymphoid tissues. Genetic deficiencies of PNP result in severely compromised T‑lymphocyte function and neurologic dysfunction (2, 3). PNP is used in assays for the measurement of inorganic phosphate (4).

1. Schramm, V.L. (1998) Annu. Rev. Biochem. 67:693.
2. Stoop, W. et al. (1977) N. Eng. J. Med. 296:651.
3. Markert, M.L. (1991) Immunodefic. Rev. 3:45.
4. Webb, M.R. (1992) Proc. Natl. Acad. Sci. USA. 89:4884.

Bioinformatics

• Gene Symbol: PNP
• Uniprot:
  • P00491()