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]
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]
Referencesedit
^Lorenz, Ulrike (2017-05-07). "Protein Tyrosine Phosphatase Assays". Current Protocols in Immunology. 91 (1): 11.7.1–12. doi:10.1002/0471142735.im1107s93. ISBN978-0471142737. PMC3097125. PMID 21462163.
^MacKintosh, C. (1993). In D.G. Hardie (Ed.). Protein Phosphorylation: A Practical Approach. 221. New York: IRL Press.
^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. PMC4351734.
^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.
^Biolabs, New England. "p-Nitrophenyl Phosphate (PNPP) - NEB". www.NEB.com. Retrieved 29 October 2017.
^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.
External linksedit
pNPP disodium salt hexahydrate (SigmaAldrich.com)
Nunc Brand Products
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para, nitrophenylphosphate, pnpp, proteinaceous, chromogenic, substrate, alkaline, acid, phosphatases, used, elisa, conventional, spectrophotometric, assays, phosphatases, catalyze, hydrolysis, pnpp, liberating, inorganic, phosphate, conjugate, base, para, nit. 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 NamesPreferred IUPAC name 4 Nitrophenyl dihydrogen phosphateOther names pNPPIdentifiersCAS Number 330 13 2 Y3D model JSmol Interactive imageChEBI CHEBI 17440 NChEMBL ChEMBL24231 NChemSpider 369 NECHA InfoCard 100 005 777PubChem CID 378UNII 59NF9TE3BA YCompTox Dashboard EPA DTXSID60861876InChI InChI 1S C6H6NO6P c8 7 9 5 1 3 6 4 2 5 13 14 10 11 12 h1 4H H2 10 11 12 NKey XZKIHKMTEMTJQX UHFFFAOYSA N NInChI 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 UHFFFAOYAUSMILES C1 CC CC C1 N O O OP O O OPropertiesChemical formula C6H6NO6PMolar mass 219 09Related compoundsRelated compounds ParaoxonExcept where otherwise noted data are given for materials in their standard state at 25 C 77 F 100 kPa N verify what is Y 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 edit 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 MacKintosh C 1993 In D G Hardie Ed Protein Phosphorylation A Practical Approach 221 New York IRL Press 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 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 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 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 amp Technology 54 14 8658 8667 doi 10 1021 acs est 9b07473 ISSN 0013 936X Biolabs New England p Nitrophenyl Phosphate PNPP NEB www NEB com Retrieved 29 October 2017 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 External links editpNPP disodium salt hexahydrate SigmaAldrich com Nunc Brand Products nbsp This article about an organic compound is a stub You can help Wikipedia by expanding it vte Retrieved from https en wikipedia org w index php title Para Nitrophenylphosphate amp oldid 1201497401, wikipedia, wiki, book, books, library,
