Direct quantification of chemogenetic H2O2production in live human cells

Abstract

AbstractReactive Oxygen Species (ROS) in the form of H2O2can act both as physiological signaling molecules as well as damaging agents, depending on its concentration and localization. The downstream biological effects of H2O2were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2production by for instance mitochondrial respiration. The enzyme D-Amino Acid Oxidase (DAAO) catalyzes H2O2formation 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 H2O2. However, a method to directly quantify the amount of H2O2produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2production. The oxygen consumption rate of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, allowing to estimate whether the ensuing level of H2O2production is within the range of physiological mitochondrial ROS production. We show that the assay can also be used to select clones that express differently localized DAAO with the same absolute level of H2O2production to be able to discriminate the effects of H2O2production 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.