Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems

Yunbo Li, Hong Zhu, Periannan Kuppusamy, Valerie Roubaud, Jay L. Zweier, Michael A. Trush

Research output: Contribution to journalArticle

Abstract

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O2/(·-)) production by biological systems. However, its validity as a O2/(·-)detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no O2/(·-). Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O2/(·-) has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O2/(·-) production by 5-(diethoxyphosphoryl)-5-methyl- 1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O2/(·-) in various enzymatic and cellula systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 μM in all of the O2- generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O2/(·-) production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/xanthine and phagocytic NADPH oxidase systems. In contrast to the above O2/(·-)generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O2/(·-). Thus, LDCL still appears to be a valid probe for detecting O2/(·-) production by enzymatic and cellular sources.

Original languageEnglish (US)
Pages (from-to)2015-2023
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number4
DOIs
StatePublished - Jan 23 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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