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. 2013 Dec:65:1310-1314.
doi: 10.1016/j.freeradbiomed.2013.09.017. Epub 2013 Sep 27.

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

Jacek Zielonka  1 , J David Lambeth  2 , Balaraman Kalyanaraman  3
Affiliations

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

Jacek Zielonka et al. Free Radic Biol Med. 2013 Dec.

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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Figures

Figure 1
Figure 1
Superoxide radical anion and HRP-dependent kinetics of L-012 and HE consumption. (a) Chemical structures of L-012 and HE probes. (b) The concentration of L-012 and HE was monitored by HPLC. Reaction mixtures contained L-012 (50 μM) or HE (50 μM) in phosphate buffer (pH 7.4, 50 mM) containing dtpa (100 μM), HX (200 μM) and XO (1 mU/ml). Where indicated, HRP (1 mU/ml) was present. Samples were placed in an HPLC autosampler thermostated at 25°C and repeatedly injected over a 4 h incubation time.
Figure 2
Figure 2
Comparison of the kinetics of luminescence formed from L-012. (a) Incubations contained L-012 (50 μM) in phosphate buffer (pH 7.4, 50 mM) containing HX (200 μM), dtpa (100μM), and XO ± HRP at concentrations indicated. (b) Incubations contained L-012 (50 μM) in the presence of different concentrations of HRP and H2O2 (at the indicated concentrations) in phosphate buffer (pH 7.4, 50 mM) containing dtpa (100 μM). Reactions were carried out in a 96-well plate thermostated at 37°C and the luminescence monitored with time for 2 h. The data points represent the mean values of three replicates and the error bars represent standard deviation. (c) Luminescence spectrum recorded during oxidation of L-012 in H2O2/HRP system. (d) Changes in luminescence intensity during consecutive addition of reactants as follows: L-012 (100 μM), H2O2 (50 μM) and HRP (0.1 U/ml).
Figure 3
Figure 3
The dynamics of generation of luminescence from L-012 probe. (a–d) L-012 (100 μM) was incubated under aerobic conditions in phosphate buffer (50 mM, pH 7.4) containing dtpa (100 μM) and HX (250 μM) at 37°C. Where indicated, XO (1 mU/ml), HRP (0.1 U/ml), SOD (50 μg/ml) or CAT (3 kU/ml) were added during reaction monitoring.
Figure 4
Figure 4
The dynamics of generation of luminescence from L-012 probe during deoxygenation and reoxygenation. (a–d) L-012 (100 μM) was incubated in aqueous solution of phosphate buffer (50 mM, pH 7.4) containing dtpa (100 μM) and H2O2 (50 μM) at 37°C. Where indicated, HRP (0.1 U/ml), SOD (50 μg/ml) or CAT (3 kU/ml) were added during reaction monitoring. In (d), sample was deoxygenated by passing argon gas through the solution before and during the reaction. In (c) and (d), Ar and O2 denote time points at which purging of argon gas and oxygen gas was started.
Figure 5
Figure 5
A modified mechanism proposed for luminescence formation during oxidation of luminol-based probes.
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