PNPP

p-nitrophenyl phosphate (pNPP) is a chromogenic substrate widely used to assay phosphatase enzymes. It is typically supplied as the disodium salt (pNPP-Na2) and is especially common in assays for alkaline phosphatase.

During the enzymatic reaction, a phosphatase removes the phosphate group to yield p-nitrophenol (pNP) and inorganic phosphate. The pNP product displays a pH-dependent color: under alkaline conditions it forms a yellow p-nitrophenolate ion that absorbs at approximately 405 nm, enabling spectrophotometric measurement of enzyme activity.

Assay procedure generally involves mixing substrate with the sample in an appropriate buffer, incubating, stopping the reaction with an alkaline stopping solution (for example sodium carbonate or bicarbonate), and measuring absorbance at 405 nm. The rate of color development is proportional to phosphatase activity. Calibration with p-nitrophenol standards allows conversion of absorbance to activity units.

Preparation and storage: pNPP is sparingly soluble in water and is typically dissolved in an organic solvent such as dimethyl sulfoxide or dimethylformamide to make a stock solution, which is then diluted into assay buffer. The dry powder is stored at low temperature and protected from light; working solutions should be prepared fresh or stored for short periods according to manufacturer recommendations.

Safety and handling: pNPP is an irritant; handle with appropriate personal protective equipment and consult the safety data sheet for details.