A Nonradioactive DEHAL Assay for Testing Substrates, Inhibitors, and Monitoring Endogenous Activity

Iodotyrosine deiodinase (DEHAL1) is a crucial enzyme in iodine homeostasis. Unbound mono- and diiodotyrosines are indispensable byproducts of thyroid hormone biosynthesis. Their iodine needs to be recovered to avoid iodine deficiency, as observed in genetic defects in DEHAL1. Despite its importance, the enzyme is rarely studied. The deiodination process can be monitored by radioactive tracers or via techniques involving mass spectrometry. However, isotope-labeled molecules are expensive, not always commercially available, and their use is legally restricted, whereas mass spectrometry requires sophisticated, costly, and sensitive instrumentation. To circumvent these difficulties, we adapted the nonradioactive iodothyronine deiodinase assay to determine DEHAL1 activity by a colorimetric readout, based on the Sandell-Kolthoff reaction. DEHAL1 was recombinantly expressed and used to optimize the assay in microtiter format. We applied the setup to scenarios of alternative substrate screening or search for comp...