Pyrophosphate Fluorometric (Blue) Assay Kit, EnzChek® from Molecular Probes (Invitrogen)

The EnzChek Pyrophosphate Assay Kit includes the enzyme inorganic pyrophosphatase, which catalyzes conversion of PPi present into two equivalents of Pi. The Pi is then consumed by the MESG/PNP reaction and detected by an increase in absorbance at 360 nm. Additional sensitivity is gained by the amplification of one molecule of PPi to two molecules of Pi. The assay can detect as little as 1 nanomole of PPi in 1 mL reactions (Figure 2) and can be performed over a pH range of 6.5 to 8.5.2 The MESG/PNP reaction has been used for continuous spectrophotometric determination of aminoacyl-tRNA synthetase activity.3 In addition, we have tested the ability of this assay to detect the PPi liberated by the activities of acetyl-CoA synthetase (Figure 3) and luciferase (Figure 4). In these experiments, we have detected as few as 5 10-5 U of acetyl-CoA synthetase activity and 10-2 U of luciferase activity.