Recombinant Human PPA1 Protein, CF

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

Recombinant Human PPA1 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze pyrophosphate. The specific activity is >50,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Inorganic Pyrophosphatase/PPA1 protein
  Met1-Asn289, with a C-terminal 6-His tag
• Accession #: Q15181
• N-terminal Sequence: Ser2
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PPA1
• Purity: >85%, 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: 34-36 kDa

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, NaCl, Glycerol and DTT.
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 25 mM MES, 16 mM MgCl₂, pH 7.0
  • Recombinant Human Inorganic Pyrophosphatase/PPA1 (rhPPA1) (Catalog # 6557-PP)
  • Substrate: Sodium Pyrophosphate (Sigma, Catalog # P8010)
  • ]Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute Substrate to 16 mM in deionized water.
  2. Dilute rhPPA1 to 0.4 µg/mL in Assay Buffer.
  3. Combine 25 µL of 16 mM Substrate with 25 µL of 0.4 µg/mL rhPPA1. Also create a substrate blank by combining 25 µL of 16 mM substrate with 25 µL of Assay Buffer.
  4. Incubate reaction and blank for 10 minutes at room temperature.
  5. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of deionized water for a 100 µM stock.
  6. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in deionized water. The standard curve has a range of 0.078 to 5 nmol per well.
  7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of deionized water.
  8. Dilute the incubated reaction and incubated blank 20-fold by adding 950 μL of deionized water to each tube.
  9. Load 50 µL of the diluted rhPPA1 reaction and substrate blank into the plate.
  10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  11. Add 100 µL deionized water to all wells.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg) x 2*)

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.
  **Note: 2 mol of phosphate are produced for every 1 mol of Pyrophosphate consumed.

  Per Well:
  • rhPPA1: 0.01 µg in reaction, 0.5 ng after 20-fold dilution
  • Substrate: 8 mM in reaction, 0.4 mM after 20-fold dilution

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 PPA1 Protein, CF

• diphosphate phosphohydrolase
• EC 3.6.1.1
• inorganic diphosphatase
• inorganic pyrophosphatase 1
• inorganic pyrophosphatase
• IOPPP
• IOPPPMGC111556
• IPP1
• PP1
• PPA1
• PPase
• PPcytosolic inorganic pyrophosphatase
• pyrophosphatase (inorganic) 1
• pyrophosphatase (inorganic)
• pyrophosphatase 1
• Pyrophosphate phospho-hydrolase
• SID6-8061

Background

Inorganic Pyrophosphatases are enzymes that catalyze the hydrolysis of pyrophosphate to two phosphate ions (1). This reaction is highly exergonic, and is utilized in many biochemical pathways, such as DNA synthesis (2) and bone formation (3), to render reactions effectively irreversible (4, 5). Likewise, inorganic pyrophosphatases can be coupled to unfavorable biochemical reactions in order to drive these reactions to completion, such as in the synthesis of activated sulfur donor 3’‑phosphoadenosine-5’-phosphosulfate (PAPS) (6). Two inorganic pyrophosphatases, i.e. PPA1 and PPA2, are found in humans. PPA1 is highly active on pyrophosphate and is ubiquitously expressed (7).

1. Harold, FM (1966) Bacteriol. Rev. 30:772.
2. Nelson, D.L. and Cox, M.M. (2000) Lehninger Principles of Biochemistry, 3rd ed. pp.937. ISBN 1-57259.
3. Poole, K.E. and Reeve, J. (2005) Curr. Opin. Pharmacol. 5:612.
4. Takahashi, K. et al. (2004) Biochem. Biophys. Res. Commun. 325:203.
5. Terkeltaub, R.A. (2001) Am. J. Physiol., Cell Physiol. 281:C1.
6. Wu, Z.L. et al. (2010) BMC Biotechnology 10:11.
7. Fairchild, T.A. and Patejunas, G. (1999) Biochim. Biophys. Acta 1447:133.

Bioinformatics

• Gene Symbol: PPA1
• Uniprot:
  • Q15181()