Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP_i) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PP_i as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PP_i synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PP_i hydrolysis, (b) continuous and fixed-time measurements of PP_i synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.