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 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.