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Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Published on: August 18, 2012
Oxygen radicals photo-induced by ferric nitrilotriacetate complex
Koichiro Tsuchiya1, Kaori Akai, Akira Tokumura
1Department of Clinical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78-1 Sho-machi, Tokushima 770-8505, Japan.
Abstract:
This study examined the photo-induced generation of reactive oxygen species (ROS) by the carcinogenic iron(III)-NTA complex. Iron(III)-NTA complex (1:1) has three conformations (type (a) in acidic conditions of pH 1-6, type (n) in neutral conditions of pH 3-9, and type (b) in basic conditions of pH 7-10) with two pK(a) values (pK(a1) approximately 4, pK(a2) approximately 8). The iron(III)-NTA complex was reduced to iron(II) under cool-white fluorescent light without the presence of any reducing agent, and the reduction rates of the three conformations of iron(III)-NTA were in the order type (a)>type (n)>type (b) as reported previously (Akai K. et al., Free Radic. Res. 38, 951-962, 2004). ROS generation was investigated by electron paramagnetic resonance (EPR) spectroscopy with a spin-trapping technique. Apparent EPR signals attributed to PBN/*(13)CH(3) and PBN/*OCH(3) spin adducts were observed after incubation of the iron(III)-NTA complex was mixed with alpha-phenyl-tert-butylnitrone (PBN) and (13)C-DMSO in an aerobic condition. The addition of catalase effectively attenuated the PBN adducts, but superoxide dismutase enhanced them. Taken together, these results indicate that the iron(III)-NTA complex is spontaneously reduced to the iron(II)-NTA complex by light under acidic to neutral pH, and in turn transfers an electron to molecular oxygen to form ROS.
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