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Journal of Biochemistry Advance Access originally published online on June 29, 2009
Journal of Biochemistry 2009 146(4):571-579; doi:10.1093/jb/mvp101
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Identification of Radicals Formed in the Reaction Mixture of Bovine Kidney Microsomes with NADPH

Kazumasa Kumamoto1,2, Tomihiro Hirai3, Shiroh Kishioka1 and Hideo Iwahashi2,*

1Department of Pharmacology; 2Department of Chemistry, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509; and 3Department of Sport and Health Science, Osaka Sangyo University, 3-1-1 Nakagaito, Daito, Osaka 574-8530, Japan

*To whom correspondence should be addressed. Tel/Fax: +81-73-441-0772, E-mail: chem1{at}wakayama-med.ac.jp

Received May 24, 2009; Accepted June 22, 2009


  Abstract

In order to explore the mechanism of myoglobinuric renal toxicity, detection and identification of free radicals was performed for the reaction mixtures of bovine kidney microsomes. EPR measurements showed prominent signals for the control reaction mixture containing 2.0 mg protein/ml bovine kidney microsomes, 5 mM NADPH, 0.1 M 4-POBN and 29 mM phosphate buffer (pH 7.4). Addition of myoglobin (Mb) to the control reaction mixture resulted in increase of EPR peak height. The result indicates that Mb enhances the radical formation. An HPLC–EPR measurement showed three peaks with retention times of 29.4 min (P1), 32.4 min (P2) and 46.6 min (P3). HPLC–EPR–MS analyses of P1 and P2 gave ions at m/z 282. The results show that 4-POBN/hydroxypentyl radical adducts form in the reaction mixture. An HPLC–EPR–MS analysis of P3 gave ions at m/z 266, indicating that 4-POBN/pentyl radical adduct forms in the reaction mixture.

Key Words: electron spin resonance, kidney, lipid peroxidation, microsomes, myoglobin

Abbreviations: EPR, electron paramagnetic resonance; 4-POBN, {alpha}-(4-pyridyl-1-oxide)-N-tert-butylnitrone; HPLC, high performance liquid chromatography; MS, mass spectrometry; Mb, myoglobin; ROS, reactive oxygen species; NTA, nitrilotriacetic acid; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; DFO, deferoxamine; 13-HPODE, 13-hydroperoxy-9,11-octadecadienoic acid


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