Possibly the largest number of adenylation assays involves indirectly measuring activity via the release of PPi from the enzyme. There is some discussion surrounding the applicability of PPi release assays to various A domains, as in some cases, the pyrophosphate remains tightly bound to the enzyme active site. The first of these is a mainstay in the NRPS field, having been used most frequently for analysis of diverse systems: the ATP-[ 32 P]PPi exchange assay. Dependent on the reverse hydrolysis of ATP in the first half-reaction, excess radiolabeled PPi is added to the reaction, and the resulting 32 P-ATP is adsorbed on activated charcoal before washing and liquid scintillation counting. In early days, this assay required large volumes of hazardous and expensive radioactive materials, and it was time-consuming and technically demanding. More recently, a high-throughput, 96-well plate optimized procedure has become more feasible. This PPi exchange assay is discontinuous, but kinetic constants derived from it are a good approximation of physiologically relevant reaction rates. Other colorimetric, discontinuous strategies for measuring PPi release utilize the malachite green Pi detection assay or direct precipitation of PPi as an 18-molybdopyrophosphate anion. The malachite green method involves the addition of a pyrophosphatase enzyme to the reaction mixture, which rapidly converts any released PPi to Pi.