NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules

NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O₂^˙- and H₂O₂. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O₂^˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost.