Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical.
The fluorogenic oxidation of hydroethidine (HE) to ethidium (E+) has been used as a measure of O2-. Evaluation of this method confirms that O2-, but not O2 or H2O2, rapidly oxidizes HE to E+. However the ratio of E+ produced per O2- introduced decreased as the flux of O2- was increased. This suggested that HE can catalyze the dismutation of O2- and this was affirmed. HE was oxidized to a red product, distinct from E+ by ferricytochrome c and a similar oxidation may occur within Escherichia coli. HE inhibited the growth and killed a SOD-null strain to a greater extent than the SOD-replete parental strain and these effects were much diminished under anaerobic conditions. This indicated that E+ was responsible for the toxicity of HE and indeed E+ was seen to be toxic under both aerobic and anaerobic conditions. In view of the data presented HE can be recommended as a qualitative but not as a quantitative measure of O2(-1).
Benov
L
L
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Sztejnberg
L
L
Fridovich
I
I
D000166:Acridines; D000838:Anions; D003574:Cytochrome c Group / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D004996:Ethidium / Q000737:chemistry / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D010617:Phenanthridines / Q000737:chemistry* / Q000378:metabolism; D013482:Superoxide Dismutase / Q000032:analysis; D013481:Superoxides / Q000032:analysis* / Q000737:chemistry
D000166:Acridines; D000838:Anions; D003574:Cytochrome c Group; D010617:Phenanthridines; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; C058428:hydroethidine; D005949:Glucose Oxidase; D013482:Superoxide Dismutase; D004996:Ethidium