(4-nitrophenyl) dihydrogen phosphate
3D model (Jmol)
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
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para-Nitrophenylphosphate (pNPP) is a chromogenic substrate for acid and alkaline phosphatase in ELISA and conventional spectrophotometric assays. These enzymes catalyse the hydrolysis of pNPP to yellow para-nitrophenol. This product absorbs strongly at 405 nm and can be measured with a visible light spectrophotometer and mainly used in the study of kinetics of enzyme-substrate catalysis reaction catalysed by alkaline phosphatase enzyme in accordance with Lineweaver-Burk formula.Activity assays of alkaline phosphatase with p-nitrophenyl phosphate are terminated by the addition of NaOH. This serves two functions; first, it stops the enzyme-catalysed reaction by changing pH which is unsuitable for the enzyme (the enzyme is denatured at high pH and so unable to function). Second, it deprotonates the p-nitrophenol to give the yellow coloured p-nitrophenylate. The progress of the reaction is monitored by absorption spectrum of p-nitrophenolate (obtained upon deprotonation of p-nitrophenol).
PNPP+H2O → PNP + inorganic phosphate
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