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

M. Tedokon, K. Suzuki, Y. Kayamori, S. Fujita, Y. Katayama

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26 Citations (Scopus)

Abstract

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 α-D-glucose 1-phosphate (G1P) and α-D-fructose. G1P is transphosphorylated by PGM in the presence of α-D-glucose 1,6-bisphosphate to form α-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 K(m) 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.

Original languageEnglish
Pages (from-to)512-515
Number of pages4
JournalClinical Chemistry
Volume38
Issue number4
DOIs
Publication statusPublished - 1992

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

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