Enzymatic assay of inorganic phosphate with use of sucrose phosphorylase and phosphoglucomutase.

We developed a new enzymatic method for the assay of inorganic phosphate (Pi) by using sucrose phosphorylase (SP; EC 2.4.1.7) and phosphoglucomutase (PGM; EC 5.4.2.2). Pi is transferred to sucrose by SP, producing alpha-D-glucose 1-phosphate (G1P) and alpha-D-fructose. G1P is transphosphorylated by PGM in the presence of alpha-D-glucose 1,6-bisphosphate to form alpha-D-glucose 6-phosphate, which is oxidized by NAD+ and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to form 6-phosphogluconate (6PG) and NADH. Finally, the oxidation of 6PG by NAD+, catalyzed by 6-phosphogluconic dehydrogenase (EC 1.1.1.44), yields D-ribulose 5-phosphate and NADH. Thus two molecules of NADH are formed for each molecule of Pi, and the reaction is monitored at 340 nm. The Km values of SP for Pi and sucrose were 4.44 and 5.31 mmol/L, respectively. The best buffer was 1,4-piperazinediethanesulfonic acid (PIPES) at 50 mmol/L and pH 6-7. Implementing this method with a Cobas-Bio centrifugal analyzer allowed us to measure Pi accurately and precisely.