A long-lived o-semiquinone radical anion is formed from N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial substance.
N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H(2)O(2)) that exerts antitumour activity. We have investigated the precise mechanism of H(2)O(2) 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 degrees 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2).
Akiyama
Nobuko
N
Natori Laboratory, and Cellular Biochemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. f.nobuko@riken.jp
Nakanishi
Ikuo
I
Ohkubo
Kei
K
Satoh
Kazue
K
Tsuchiya
Koichiro
K
Nishikawa
Takeshi
T
Fukuzumi
Shunichi
S
Ikota
Nobuo
N
Ozawa
Toshihiko
T
Tsujimoto
Masafumi
M
Natori
Shunji
S
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D016227:Benzoquinones / Q000737:chemistry* / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D000975:Antioxidants; D016227:Benzoquinones; D005609:Free Radicals; C100496:N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine; D013481:Superoxides; C025232:semiquinone radicals; D004295:Dihydroxyphenylalanine; D006861:Hydrogen Peroxide; D005978:Glutathione