Para-Nitrophenylphosphate: Difference between revisions
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{{DISPLAYTITLE:''para''-Nitrophenylphosphate}} |
{{DISPLAYTITLE:''para''-Nitrophenylphosphate}} |
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| verifiedrevid = 393645519 |
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| ImageAltL1 = Skeletal formula of para-nitrophenylphosphate |
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| ImageFileR1 = Para-Nitrophenylphosphate-3D-spacefill.png |
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| ImageSizeR1 = 130 |
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| ImageAltR1 = Space-filling model of the para-nitrophenylphosphate molecule |
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| CASNo_Ref = {{cascite|correct|CAS}} |
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| UNII_Ref = {{fdacite|correct|FDA}} |
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| UNII = 59NF9TE3BA |
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| ChEBI_Ref = {{ebicite|changed|EBI}} |
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| ChEBI = 17440 |
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| ChEMBL_Ref = {{ebicite|changed|EBI}} |
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| ChEMBL = 24231 |
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| ChemSpiderID_Ref = {{chemspidercite|changed|chemspider}} |
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| ChemSpiderID = 369 |
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| InChI = 1/C6H6NO6P/c8-7(9)5-1-3-6(4-2-5)13-14(10,11)12/h1-4H,(H2,10,11,12) |
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| InChIKey = XZKIHKMTEMTJQX-UHFFFAOYAU |
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| StdInChI_Ref = {{stdinchicite|changed|chemspider}} |
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| StdInChI = 1S/C6H6NO6P/c8-7(9)5-1-3-6(4-2-5)13-14(10,11)12/h1-4H,(H2,10,11,12) |
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| StdInChIKey_Ref = {{stdinchicite|changed|chemspider}} |
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| StdInChIKey = XZKIHKMTEMTJQX-UHFFFAOYSA-N |
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|Section2= |
|Section2={{Chembox Properties |
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| Formula=C<sub>6</sub>H<sub>6</sub>NO<sub>6</sub>P |
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| MolarMass=219.09 |
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|Section3= |
|Section3={{Chembox Hazards |
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|Section4={{Chembox Related |
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| OtherCompounds=[[Paraoxon]] |
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'''''para''-Nitrophenylphosphate''' ('''pNPP''') is a non-proteinaceous [[chromogenic substrate]] for [[Alkaline phosphatase|alkaline and acid phosphatases]] used in [[ELISA]] and conventional spectrophotometric assays.<ref>{{Cite journal|last=Lorenz|first=Ulrike|date=2017-05-07|title=Protein Tyrosine Phosphatase Assays|journal=Current Protocols in Immunology |volume=91 |issue=1 |pages=11.7.1–12|doi=10.1002/0471142735.im1107s93|pmc=3097125|pmid=21462163|isbn=978-0471142737}}</ref> Phosphatases catalyze the hydrolysis of pNPP liberating inorganic phosphate and the conjugate base of [[4-nitrophenol|''para''-nitrophenol]] (pNP). The resulting phenolate is yellow, with a maximal absorption at 405 nm.<ref>MacKintosh, C. (1993). In D.G. Hardie (Ed.). ''Protein Phosphorylation: A Practical Approach''. 221. New York: IRL Press.</ref> This property can be used to determine the activity of various phosphatases including alkaline phosphatase (AP) and protein tyrosine phosphatase (PTP).<ref>{{Cite journal|last1=Matsushima|first1=Ayako|last2=Inoue|first2=Yorinao|last3=Shibata|first3=Kazuo|date=1975|title=Derivative absorption spectrophotometry of native proteins|url=http://dx.doi.org/10.1016/0003-2697(75)90520-5|journal=Analytical Biochemistry|volume=65|issue=1–2|pages=362–368|doi=10.1016/0003-2697(75)90520-5|pmid=1169006|issn=0003-2697}}</ref> |
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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.<ref>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 .</ref> 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.<ref>{{Cite journal |last=Mercan |first=Fatih |last2=Bennett |first2=Anton M. |date=July 2010 |title=Analysis of Protein Tyrosine Phosphatases and Substrates |url=https://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb1816s91 |journal=Current Protocols in Molecular Biology |language=en |volume=91 |issue=1 |doi=10.1002/0471142727.mb1816s91 |issn=1934-3639|pmc=4351734 }}</ref> |
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'''''para''-Nitrophenylphosphate''' (pNPP) is a [[chromogenic substrate]] for [[acid phosphatase|acid]] and [[alkaline phosphatase]] in [[ELISA]] assays. Under their influence the decay to yellow [[4-nitrophenol|''para''-nitrophenol]] is catalysed. This product can be measured with a 405 nm [[spectrophotometer]]. |
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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.<ref>{{Cite journal |last=Li |first=Tong |last2=Zhong |first2=Wen |last3=Jing |first3=Chuanyong |last4=Li |first4=Xuguang |last5=Zhang |first5=Tong |last6=Jiang |first6=Chuanjia |last7=Chen |first7=Wei |date=2020-07-21 |title=Enhanced Hydrolysis of p -Nitrophenyl Phosphate by Iron (Hydr)oxide Nanoparticles: Roles of Exposed Facets |url=https://pubs.acs.org/doi/10.1021/acs.est.9b07473 |journal=Environmental Science & Technology |language=en |volume=54 |issue=14 |pages=8658–8667 |doi=10.1021/acs.est.9b07473 |issn=0013-936X}}</ref> |
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:<math>PNPP+H_2O \; \overrightarrow{_{AP}} \; PNP + P_i</math> |
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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.<ref>{{cite web|url=https://www.neb.com/products/p0757-p-nitrophenyl-phosphate-pnpp|title=p-Nitrophenyl Phosphate (PNPP) - NEB|first=New England|last=Biolabs|website=www.NEB.com|access-date=29 October 2017}}</ref> |
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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.<ref>{{Cite journal |last=Terefe |first=N.S. |last2=Arimi |first2=J.M. |last3=VanLoey |first3=A. |last4=Hendrickx |first4=M. |date=2004-10-01 |title=Kinetics of the Alkaline Phosphatase Catalyzed Hydrolysis of Disodium p-Nitrophenyl Phosphate: Effects of Carbohydrate Additives, Low Temperature, and Freezing |url=http://doi.wiley.com/10.1021/bp0498894 |journal=Biotechnology Progress |language=en |volume=20 |issue=5 |pages=1467–1478 |doi=10.1021/bp0498894 |issn=8756-7938}}</ref> |
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== References == |
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{{Reflist}} |
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==External links== |
==External links== |
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*[http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=N22002|ALDRICH&N5=Product%20No.|BRAND_KEY&F=SPEC pNPP disodium salt hexahydrate (SigmaAldrich.com)] |
* [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=N22002|ALDRICH&N5=Product%20No.|BRAND_KEY&F=SPEC pNPP disodium salt hexahydrate (SigmaAldrich.com)] |
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*[http://www.nuncbrand.com/NAG/DP0010.htm Nunc Brand Products] |
* [http://www.nuncbrand.com/NAG/DP0010.htm Nunc Brand Products] |
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{{DEFAULTSORT:Nitrophenylphosphate, para-}} |
{{DEFAULTSORT:Nitrophenylphosphate, para-}} |
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[[Category:Organophosphates]] |
[[Category:Organophosphates]] |
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[[Category: |
[[Category:Nitrobenzene derivatives]] |
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[[Category:Phenol esters]] |
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{{Organic-compound-stub}} |