Colorimetric determination of pyrophosphate anion and its application to adenylation enzyme assay

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 MoO4(2-) 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.