Nonradioactive detection of recombinant DEHAL1 activity. The photometric readout dOD at 415nm represents iodide release quantified by the Sandell-Kolthoff reaction. A, Iodide-release was concentration dependently related to the amount of added protein from DEHAL1-expressing HEK293 cells. A direct comparison indicates that DTN constitutes the superior reductant in comparison with NADPH with respect to the maximal velocity of iodide release (1 h of incubation, n = 4, mean ± SD). B, The K_m value for MIT was determined in an experiment with various concentrations of MIT as substrate and incubation over a short time frame (1 h of incubation, 50-μg protein/reaction). C, Recombinant enzyme (50 μg/reaction, time course, n = 3, mean ± SD) was incubated with 20μM MIT with or without addition of DBT (f.c. 1mM). DBT inhibited the DEHAL1-dependent iodide release over the whole period of incubation time. The noninhibited reaction displayed an almost linear DEHAL1-dependent release of iodide from the substrate. D, Titrations of NADPH and DBT indicate that NADPH can serve as cosubstrate of the enzyme, whereas DBT inhibits DEHAL1-mediated iodide release (50 μg/reaction, 2 h of incubation, n = 1). E, Robustness of the assay was tested by comparing a high number of reactions with and without enzyme, as well as reactions with enzyme and surplus addition of DBT (f.c. 1mM). The DEHAL1-generated signal was distinguishable from BG with high significance. DBT in the given concentration was able to generate a BG signal without any significant difference from the reactions without enzyme (100-μg protein/reaction, 2 h of incubation, n = 32, mean ± SD, ANOVA with Bonferroni’s post hoc test). F, Repeated freeze-thaw cycles (without additional storage time) did not decrease enzymatic activity in murine kidney homogenate (100-μg protein/reaction, 3 h of incubation, n = 3).