N-β-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H 2O2) 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 (O2•-) 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.
Bibliographical noteFunding Information:
We thank Dr Okuyama (Biotech Healthcare Partners, Inc.), Dr Chihara, Dr Asami and Dr Sakaguchi (RIKEN) for their helpful discussions and comments. This work was supported in part by a grant for ‘Chemical Biology Research Program’ from RIKEN.
- Electron spin resonance
- Hydrogen peroxide
- O-semiquinone radical anion
- Superoxide dismutase