Oxygen-consumption based quantification of chemogenetic HO production in live human cells.

Reactive Oxygen Species (ROS) in the form of HO can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of HO were often studied making use of exogenously added HO, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular HO production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes HO formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular HO. However, a method to directly quantify the amount of HO produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of HO. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during HO production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, to estimate whether the ensuing level of HO production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTERT cells, addition of 5 mM d-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of HO. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of HO production to be able to discriminate the effects of HO production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.