Practice question

Para-nitrophenyl phosphate serves as artificial substrate for alkaline phosphatase, which catalyzes hydrolysis of phosphate ester at alkaline pH optimum around 9.5. Cleavage liberates para-nitrophenol, which under basic conditions exists as para-nitrophenolate anion exhibiting intense yellow color with absorbance near 405 nm. Reaction is widely used for quantitative ELISA because product remains soluble and obeys Beer-Lambert law. Horseradish peroxidase with TMB generates blue intermediate. Luciferase-luciferin pair emits light rather than yellow. Alkaline phosphatase with NBT produces purple precipitate. Hence pNPP uniquely yields yellow signal in phosphatase systems.