Established phosphate detection assays can then be used, with the addition of molybdate and malachite green providing a color change that can be read at 600 nm. This method is easy to use and the reagents are readily accessible, considering that phosphate detection kits can be purchased. Molybdate-PPi precipitation generates a [(P2O7)Mo18O54] 4-species that can then be further reduced by ascorbic acid to give a more distinguishable color change. 106-108 However, turnover rates of A domains in PPi release assays are thought to be much slower than those from PPi exchange assays due to the lack of an acceptor nucleophile, leading to dependence on PPi leakage from the active site for accurate measurement. Additionally, phosphate is a common contaminant, leading to high background signals. By contrast, a continuous colorimetric readout exists in the form of the MesG assay. Based on older reports of phosphate detection assays and the color-developing conversion of 7-methylthioguanosine (MesG) to 7-methylthioguanine by the enzyme PNP, the coupling of this enzyme activity to both the adenylation reaction and a pyrophosphatase has been used extensively. More recently, the Aldrich group has adapted this assay specifically for A domains by addition of a hydroxylamine acceptor molecule. This hydroxylamine-MesG workflow represents a convenient alternative to radioactive PPi exchange assays with very few drawbacks. The Townsend group also reported recently an improved but discontinuous iron-based colorimetric method employing Chrome Azurol S (CAS), a reagent that chelates iron and has historically been used to detect siderophores. This assay uses the same setup as the original hydroxylamine-trapping iron complex method, but the addition of a CAS-Fe 3+ mixture provides a more sensitive and rapid color change. The authors specifically noted that it was PPi that acted as the predominant iron-sequestering moiety and not the substrate hydroxamate, a property not exhibited by orthophosphate alone. It was recommended that a pyrophosphatase be added to iron-hydroxamate workflows to remove metal binding competition by PPi and improve sensitivity even without the addition of CAS-Fe 3+ . Exploiting reversible reactions from primary metabolic pathways is a common strategy in the development of continuous, NAD + /NADH-coupled assays. There are two such examples that fall in the category of adenylation PPi release, where pyrophosphate formation is connected to NADH generation or consumption via the activity of one or more enzymes. It should also be noted that some coupled assays are able to amplify the detectable adenylation activity by using enzymes that stoichiometrically increase NADH output. The first example couples the activity of three enzymes to the A domain reaction: glucose-1-phosphate uridylyltransferase (UT), phosphoglucomutase (PGM), and glucose-6-phosphate dehydrogenase (G6PD). The second example involves four coupled enzyme reactions, the most of any reported here: PPidependent phosphofructokinase (PPi-PFK), aldolase, triose phosphate isomerase (TPI), and glycerol-3-phosphate dehydrogenase (GDH). It should be noted that the first of these methods measures an increase in NADH, and the second measures a 2-fold decrease. In general, coupled assays are more complicated because they rely on the additional enzymes reacting fast enough that, especially for continuous measurements, their influence on the overall reaction rate (from PPi release to detectable NADH generation or consumption) is almost negligible.