para-Nitrophenylphosphate

para-Nitrophenylphosphate (pNPP) is a non-proteinaceous chromogenic substrate for alkaline and acid phosphatases used in ELISA and conventional spectrophotometric assays.[1] Phosphatases catalyze the hydrolysis of pNPP liberating inorganic phosphate and the conjugate base of para-nitrophenol (pNP). The resulting phenolate is yellow, with a maximal absorption at 405 nm.[2] This property can be used to determine the activity of various phosphatases including alkaline phosphatase (AP) and protein tyrosine phosphatase (PTP).[3]

para-Nitrophenylphosphate
Skeletal formula of para-nitrophenylphosphate
Space-filling model of the para-nitrophenylphosphate molecule
Names
Preferred IUPAC name
4-Nitrophenyl dihydrogen phosphate
Other names
pNPP
Identifiers
3D model (JSmol)
ChEBI
ChEMBL
ChemSpider
ECHA InfoCard 100.005.777
UNII
  • InChI=1S/C6H6NO6P/c8-7(9)5-1-3-6(4-2-5)13-14(10,11)12/h1-4H,(H2,10,11,12) ☒N
    Key: XZKIHKMTEMTJQX-UHFFFAOYSA-N ☒N
  • InChI=1/C6H6NO6P/c8-7(9)5-1-3-6(4-2-5)13-14(10,11)12/h1-4H,(H2,10,11,12)
    Key: XZKIHKMTEMTJQX-UHFFFAOYAU
  • C1=CC(=CC=C1[N+](=O)[O-])OP(=O)(O)O
Properties
C6H6NO6P
Molar mass 219.09
Related compounds
Related compounds
 Paraoxon
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is checkY☒N ?)
Infobox references

PNPP is classified as a chromogenic substrate because of its ability to transform from a colorless compound to a colored compound through a biological mechanism, dephosphorylation.[4] PNPP is used because of its low cost and the rate of the reactions can be measured over a wide range of substrate concentrations because the concentration of the substrate is not a limiting factor in the reaction. The limitations of PNPP is that it is a small molecule and perhaps does not entirely represent the conditions and structures that are encountered physiologically.[5]

A PNPP assay involves mixing the sample with a PNPP-containing mixture and permitting the reaction to run its course for a predetermined period of time. After that point, the process is halted through the addition of a stop solution, often made of a potent alkali like sodium hydroxide.[6]

The substance is sensitive to light, and thus should be stored protected from light. This is also important after adding the substrate to the mixture and before reading. −20 °C is the optimal storage temperature.[7]

To get precise and credible findings, the assay parameters must be carefully regulated because the reaction is sensitive to factors such as pH, temperature, and the varying degree of inhibitors or stimulants.[8]

References
  1. Lorenz, Ulrike (2017-05-07). "Protein Tyrosine Phosphatase Assays". Current Protocols in Immunology. 91 (1): 11.7.1–12. doi:10.1002/0471142735.im1107s93. ISBN 978-0471142737. PMC 3097125. PMID 21462163.
  2. MacKintosh, C. (1993). In D.G. Hardie (Ed.). Protein Phosphorylation: A Practical Approach. 221. New York: IRL Press.
  3. Matsushima, Ayako; Inoue, Yorinao; Shibata, Kazuo (1975). "Derivative absorption spectrophotometry of native proteins". Analytical Biochemistry. 65 (1–2): 362–368. doi:10.1016/0003-2697(75)90520-5. ISSN 0003-2697. PMID 1169006.
  4. National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 4686862, p-Nitrophenyl phosphate. Retrieved May 14, 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/p-Nitrophenyl-phosphate .
  5. Mercan, Fatih; Bennett, Anton M. (July 2010). "Analysis of Protein Tyrosine Phosphatases and Substrates". Current Protocols in Molecular Biology. 91 (1). doi:10.1002/0471142727.mb1816s91. ISSN 1934-3639. PMC 4351734.
  6. Li, Tong; Zhong, Wen; Jing, Chuanyong; Li, Xuguang; Zhang, Tong; Jiang, Chuanjia; Chen, Wei (2020-07-21). "Enhanced Hydrolysis of p -Nitrophenyl Phosphate by Iron (Hydr)oxide Nanoparticles: Roles of Exposed Facets". Environmental Science & Technology. 54 (14): 8658–8667. doi:10.1021/acs.est.9b07473. ISSN 0013-936X.
  7. Biolabs, New England. "p-Nitrophenyl Phosphate (PNPP) - NEB". www.NEB.com. Retrieved 29 October 2017.
  8. Terefe, N.S.; Arimi, J.M.; VanLoey, A.; Hendrickx, M. (2004-10-01). "Kinetics of the Alkaline Phosphatase Catalyzed Hydrolysis of Disodium p-Nitrophenyl Phosphate: Effects of Carbohydrate Additives, Low Temperature, and Freezing". Biotechnology Progress. 20 (5): 1467–1478. doi:10.1021/bp0498894. ISSN 8756-7938.


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