The most direct assay to measure substrate adenylation is the colorimetric hydroxylaminetrapping iron complex method. Hydroxylamine is used as an acceptor molecule, displacing AMP to form a hydroxamic acid with the acyl substrate that can be detected spectroscopically upon addition of a solution containing Fe 3+ . This assay is not dependent on PPi release from the A domain, but drawbacks include discontinuity, low sensitivity, instability of the complex, and the observation that not all hydroxamate-iron complexes are detectable spectroscopically. Another direct detection method is the radiolabeled formation of a PCP-substrate species. This reaction requires an activated PCP domain and the addition of a radiolabeled substrate (usually 3 H, C, or S), which can then be measured after precipitation of the protein using trichloroacetic acid (TCA). Due to the necessity of separating the enzyme from the remaining aqueous labeled substrate, this assay is discontinuous, and its utilization of radioactive materials makes it hazardous and inconvenient.