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 (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.
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Jacek
J
Zielonka
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA.
J David
JD
Lambeth
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: balarama@mcw.edu.
Balaraman
B
Kalyanaraman
D002374:Catalase / Q000737:chemistry; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000737:chemistry; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000031:analogs & derivatives* / Q000737:chemistry; D019255:NADPH Oxidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D009195:Peroxidase / Q000737:chemistry; D013482:Superoxide Dismutase / Q000737:chemistry*; D013481:Superoxides / Q000032:analysis* / Q000737:chemistry
D005609:Free Radicals; D013481:Superoxides; C081614:L 012; D008165:Luminol; D006861:Hydrogen Peroxide; D002374:Catalase; D009195:Peroxidase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D010100:Oxygen
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