TY - JOUR
T1 - Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems
AU - Li, Yunbo
AU - Zhu, Hong
AU - Kuppusamy, Periannan
AU - Roubaud, Valerie
AU - Zweier, Jay L.
AU - Trush, Michael A.
PY - 1998/1/23
Y1 - 1998/1/23
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.273.4.2015
DO - 10.1074/jbc.273.4.2015
M3 - Article
C2 - 9442038
AN - SCOPUS:0031941373
SN - 0021-9258
VL - 273
SP - 2015
EP - 2023
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -