On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: a reevaluation

L-012, a luminol-based chemiluminescent (CL) probe, is widely used in vitro and in vivo to detect NADPH oxidase (Nox)-derived superoxide (O-2(.-)) and identify Nox inhibitors. Yet understanding of the free radical chemistry of the L-012 probe is still lacking. We report that peroxidase and H2O2 induce superoxide dismutase (SOD)-sensitive, L-012-derived CL in the presence of oxygen. O-2(.-) alone does not react with L-012 to emit luminescence. Self-generated 02 during oxidation of L-012 and luminol analogs artifactually induce CL inhibitable by SOD. These aspects make assays based on luminol analogs less than ideal for specific detection and identification of O-2(.-) and NOX inhibitors. (C) 2013 Published by Elsevier Inc.