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

  • Free Radic Biol Med. 2013 Dec;65:1310-1314. doi: 10.1016/j.freeradbiomed.2013.09.017.
Jacek Zielonka  1 ,  J David Lambeth  2 ,  Balaraman Kalyanaraman  3
Affiliations
  • 1. Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
  • 2. Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA.
  • 3. Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: [email protected].
PMID: 24080119 DOI: 10.1016/j.freeradbiomed.2013.09.017
Abstract

L-012, a luminol-based chemiluminescent (CL) probe, is widely used in vitro and in vivo to detect NADPH Oxidase (Nox)-derived superoxide (O2(*-)) 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. O2(*-) alone does not react with L-012 to emit luminescence. Self-generated O2(*-) 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 O2(*-) and NOX inhibitors.

Keywords
8-amino-5-chloro-7-phenylpyrido[3,4–d]pyridazine-1,4(2H,3H)dione; CAT; CL; Free radicals; HE; HRP; HX; L-012; Luminescence; Luminol; NADPH oxidase; NADPH oxidases; Nox; O(2)(−); ROS; Redox cycling; Redox probe; SOD; Superoxide radical anion; XO; catalase; chemiluminescence; diethylenetriaminepentaacetate; dtpa; horseradish peroxidase; hydroethidine; hypoxanthine; reactive oxygen species; superoxide dismutase; superoxide radical anion; xanthine oxidase.
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