Detection of mitochondria-derived reactive oxygen species production by the chemilumigenic probes lucigenin and luminol

Yunbo Li, Hong Zhu, Michael A. Trush

Research output: Contribution to journalArticle

Abstract

Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide (O2-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2-. and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2-. production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)1-12
Number of pages12
JournalBiochimica et Biophysica Acta - General Subjects
Volume1428
Issue number1
DOIs
StatePublished - Jun 28 1999

Fingerprint

Luminol
Mitochondria
Chemiluminescence
Luminescence
Reactive Oxygen Species
Macrophages
Horseradish Peroxidase
10,10'-dimethyl-9,9'-biacridinium
Porphyrins
Alveolar Macrophages
Substrates
Electron Transport
Superoxides
Catalase
Superoxide Dismutase
Rats
Monocytes
Respiration
Membranes

Keywords

  • Chemiluminescence
  • Lucigenin
  • Luminol
  • Mitochondria
  • Reactive oxygen species
  • Superoxide

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Detection of mitochondria-derived reactive oxygen species production by the chemilumigenic probes lucigenin and luminol. / Li, Yunbo; Zhu, Hong; Trush, Michael A.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1428, No. 1, 28.06.1999, p. 1-12.

Research output: Contribution to journalArticle

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abstract = "Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide (O2-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2-. and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2-. production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria. Copyright (C) 1999 Elsevier Science B.V.",
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AU - Li, Yunbo

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N2 - Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide (O2-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2-. and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2-. production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria. Copyright (C) 1999 Elsevier Science B.V.

AB - Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide (O2-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2-. and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2-. production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria. Copyright (C) 1999 Elsevier Science B.V.

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