Superoxide radical detection in cells, tissues, organisms (animals, plants, insects, microorganisms) and soils.
A simple protocol is presented for the assessment of superoxide radical in organisms (animal/plant tissues, microorganisms, cell cultures, biological/culture fluids) and soils, through the quantification of 2-hydroxyethidium (2-OH-E+), its specific reaction product with hydroethidine (HE). It is an alternative to the quantification of 2-OH-E+ by HPLC (restricted to cell cultures), offering the advantage of the in vivo assessment of superoxide radical in a wide range of experimental systems. The protocol includes alkaline-acetone extraction of the sample, purification by microcolumn cation exchange and hydrophobic chromatographies, and fluorescence detection of the isolated 2-OH-E+/HE-oxidation products mixture before and after consumption of 2-OH-E+ by a horseradish peroxidase/hydrogen peroxide system. The protocol is sensitive at <1 pmol 2-OH-E+ per mg protein (extended to the femto level when using large samples) in biological systems, and in soils at 9 pmol superoxide radical per gram of soil. The protocol includes a cytochrome c-based subprotocol for superoxide radical detection in soils at 770 pmol g(-1) soil. For processing ten samples and depending on the experimental material used (soil or biological), the approximate procedure time would be 2-7 h.
Department of Biology, Section of Genetics, Cell Biology and Development, University of Patras, Patras 26100, Greece. c.georgiou@upatras.gr
Christos D
CD
Georgiou
Ioannis
I
Papapostolou
Konstantinos
K
Grintzalis
D000818:Animals; D015415:Biomarkers; D002478:Cells, Cultured; D002845:Chromatography; D004331:Drosophila melanogaster / Q000737:chemistry*; D004996:Ethidium / Q000031:analogs & derivatives / Q000737:chemistry; D005658:Fungi / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D015394:Molecular Structure; D049878:Mytilus / Q000737:chemistry; D010617:Phenanthridines / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D012987:Soil / Q000032:analysis*; D018724:Spinacia oleracea / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry*
C506392:2-hydroxyethidium; D015415:Biomarkers; D010617:Phenanthridines; D012987:Soil; D013481:Superoxides; C058428:hydroethidine; D004996:Ethidium