Alkaline phosphatase (ALP) catalyze the hydrolysis of 4-nitrophenylphosphate (4-NPP) with the formation of free 4-nitrophenol and inorganic phosphate, acting the alkaline buffer as a phosphate-group acceptor.
The reaction is monitored kinetically at 405 nm by the rate of formation of 4-nitrophenol, proportional to the activity of ALP present in the sample.