Presentation アスコルビン酸のラジカル消去速度に及ぼす生体関連金属イオンの効果

中西, 郁夫  ,  大久保, 敬  ,  福住, 俊一  ,  小澤, 俊彦  ,  松本, 謙一郎

Ascorbic acid (AscH(2)) is a representative water-soluble antioxidant that scavenges reactive oxygen species (ROS) as well as related radicals and believed to prevent diseases induced by oxidative stress. However, the detailed radical-scavenging mechanism of AscH(2) has yet to be fully clarified. On the other hand, metal ions play essential roles not only as an active center of metalloenzymes, but also as an electron-transfer catalyst in biological redox systems. In this study, we investigated the effect of magnesium ion (Mg(2+)) as a bio-related metal ion on the radical-scavenging rates of AscH(2) in aqueous and non-aqueous media using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as a reactivity model of ROS. Upon mixing of AscH(2) with the DPPH radical in deaerated methanol (MeOH) on a stopped-flow spectrophotometer (UNISOKU RSP-1000-02NM), the absorption band at 516 nm due to the DPPH radical disappeared immediately. The decay of the absorbance at 516 nm obeyed pseudo-first-order kinetics, when the concentration of AscH(2) ([AscH(2)]) was maintained at more than a 10-fold excess of the DPPH radical concentration. The pseudo-first-order rate constants (k(obs)) increased with increasing [AscH(2)] and reached a constant value. When MeOH was replaced by a phosphate buffer solution (0.05 M, pH 7.4), where the DPPH radical was solubilized by β-cyclodextrin (β-CD) in water (Nakanishi I., et al., Chem. Common., 51, 8311 (2015)), the k(obs) values increased linearly with increasing [AscH(2)]. Because the pK(a) value of AscH(2) is known to be 4.1, AscH(2) exists in its anionic form, AscH(-), in phosphate buffer (0.05 M, pH 7.4). From the slope of the linear plot, the second-order rate constant (k) for the DPPH radical-scavenging reaction of AscH(-) was determined to be 7.9 × 10(3) M(-1) s(-1) in phosphate buffer (0.05 M, pH 7.4) at 298 K. Upon addition of 0.1 M Mg(ClO(4))(2) to the AscH(2)-the DPPH radical system in MeOH, the kobs values were significantly decreased. On the other hand, the k value (7.3 × 10(3) M(-1) s(-1)) for the reaction between AscH(-) and the β-CD-solubilized DPPH radical in the presence of 0.01 M MgCl(2) in phosphate buffer (0.05 M, pH 7.4) was virtually the same as that in its absence. Thus, the effect of Mg(2+) on the DPPH radical-scavenging rates was different depending on the solvents. In MeOH, the deprotonation of AscH(2) may be the rate-determining step, leading to the saturation dependence of the k(obs) values on [AscH(2)]. Furthermore, the complex formation between AscH(2) and Mg(2+) may retard the DPPH radical-scavenging reaction in MeOH.

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