© 2007 The Japanese Biochemical Society.
A Long-lived o-Semiquinone Radical Anion is Formed from N-ß-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an Insect-derived Antibacterial Substance
1Natori Laboratory, and; 2Cellular Biochemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198; 3Redox Regulation Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences (NIRS), Inage-ku, Chiba 263-8555; 4Department of Material and Life Sciences, Graduate School of Engineering, Osaka University, SORST, Japan Science and Technology Agency (JST), Suita, Osaka 565-0871; 5Department of Anatomy, School of Medicine, Showa University, Shinagawa-ku, Tokyo 142-8555; 6Department of Medical Pharmacology, The University of Tokushima, Institute of Health Biosciences, Tokushima 770-8505; and 7The New Industry Creation Hatchery Center (NICHe), Tohoku University, Aoba-ku, Sendai, Miyagi 980-8579, Japan
*To whom correspondence should be addressed. Tel: +81 48 467 9515, Fax: +81 48 462 4670, E-mail: fnobuko{at}riken.jp
Received April 4, 2007; Accepted April 16, 2007
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Abstract |
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N-ß-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H2O2) that exerts antitumour activity. We have investigated the precise mechanism of H2O2 production from 5-S-GAD by autoxidation aiming to understand its action toward tumour cells. Using the electron spin resonance (ESR) technique, we detected a strong signal due to radical formation from 5-S-GAD. Surprisingly, the ESR signal of the radical derived from 5-S-GAD appeared after incubation for 30 min at 37°C in the buffer at pH 7.4; the signal was persistently detected for 10 h in the absence of catalytic metal ions. The computer simulation of the observed ESR spectrum together with the theoretical calculation of the spin density of the radical species indicates that an o-semiquinone radical anion was formed from 5-S-GAD. We demonstrated that H2O2 is produced via the formation of superoxide anion (
) by the electron-transfer reduction of molecular oxygen by the 5-S-GAD anion, which is in equilibrium with 5-S-GAD in the aqueous solution. The radical formation and the subsequent H2O2 production were inhibited by superoxide dismutase (SOD), when the antitumour activity of 5-S-GAD was inhibited by SOD. Thus, the formation of the o-semiquinone radical anion would be necessary for the antitumour activity of 5-S-GAD as an intermediate in the production of cytotoxic H2O2.
Key Words: electron spin resonance, hydrogen peroxide, o-semiquinone radical anion, superoxide dismutase, 5-S-GAD
Abbreviations:
5-S-GAD, N-ß-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine; H2O2, hydrogen peroxide; 
, superoxide anion; SOD, superoxide dismutase; GSH, reduced glutathione
These authors have equally contributed to this work.