Analytical Sciences

Colorimetric Determination of Pyrophosphate Anion and Its Application to Adenylation Enzyme Assay

Hajime KATANO, Hiro WATANABE, Masahiro TAKAKUWA, Chitose MARUYAMA, and Yoshimitsu HAMANO

Faculty of Biotechnology, Fukui Prefectural University, Eiheiji, Fukui 910-1195, Japan

A colorimetric pyrophosphate assay based on the formation and reduction of the 18-molybdopyrophosphate ([(P₂O₇)Mo₁₈O₅₄]⁴⁻) anion in an acetonitrile–water mixed solvent was modified and improved. The [(P₂O₇)Mo₁₈O₅₄]⁴⁻ anion is precipitated from the acetonitrile–water solution containing MoO₄²⁻ and HCl, and is re-dissolved in neat acetonitrile or propylene carbonate. This separation process decreases the interference by ATP, and prevents a yellow coloration of the reducing agent, ascorbic acid, due to excess Mo(VI) species. In the organic solvent, the [(P₂O₇)Mo₁₈O₅₄]⁴⁻ anion is reduced to a more intense blue molybdopyrophosphate species. The application of the colorimetry to the assay of adenylation enzymes is also described in this note.

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