Question

Phosphoryl chloride is a hazardous compound to work with. It is an extremely strong acid. Additionally, a chemist can generate a phosphate triester, and phosphate triesters are known as AChEI inhibitors. How does a chemist ensure that, during the synthesis of pNPP, a phosphate triester is not inadvertently synthesized?

Answer 1

-p Nitrophenyl Phosphate (PNPP) is a non-proteinaceous, non-specific substrate used to assay protein, alkaline and acid phosphatases.

• Can be used for quick analysis of the protein phosphatase activity under any conditions
• Advantageous to a radioactive assay because the substrate concentration can be much higher than the Km
• Activity is measured using a continuous or single-point spectrophotometric assay based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol, a chromogenic product that absorbs maximally at 405 mm.
• Nitrophenyl Phosphate (PNPP) is a non-proteinaceous, non-specific substrate used to assay protein, alkaline and acid phosphatases. The PNPP phosphatase activity is measured using a continuous or single-point spectrophotometric assay based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol, a chromogenic product with absorbance at 405 nm (1). The assay can be used for the quick analysis of the protein phosphatase activity under any non-standard conditions.
  
  The advantage of the PNPP phosphatase activity assay is that unlike radioactive assays the substrate concentration can be much higher than the Km. The initial velocity can be recorded in the continuous assay, but the assay volume is larger than in a radioactive assay (about 1 ml to fill a 1 ml spetrophotometer cuvette) (1,2). The reaction volume in a single-point assay can be very small because the reaction is stopped with the amount of NaOH enough to fill the cuvette (1,3,4).