Titration of human placental alkaline phosphatase with radioactive orthophosphate

Human placental alkaline phosphatase incorporates radioactive phosphate specifically and covalently at acid pH. By titration of solutions of the purified enzyme with radioactive orthophosphate, the enzyme was shown to incorporate up to 2 phosphate groups per molecule. No evidence was found to suggest that the two sites had different affinities for phosphate. Similar titrations can be used to determine the molarity of solutions of non-placental alkaline phosphatases of unknown purity, if these also are assumed to possess 2 binding sites per molecule.