Nakamura M
Division of Biophysics, Research Institute of Applied Electricity, Hokkaido University.
J Biochem. 1990 Aug;108(2):245-9. doi: 10.1093/oxfordjournals.jbchem.a123188.
The oxidation mechanism of Trolox C (a vitamin E analogue) by peroxidases was examined by stopped flow and ESR techniques. The results revealed that during the oxidation of Trolox C, peroxidase Compound II was the catalytic intermediate. The rate constants for the reaction of Compound II with Trolox C, which should be the rate-determining step, were estimated to be 2.1 X 10(4) and 7.2 X 10(3) M-1.s-1 for horseradish peroxidase and lactoperoxidase, respectively, at pH 6.0. The formation of the Trolox C radical was followed by ESR. The time course of the signal was similar to that of the optical absorbance changes at 440 nm, assigned as the peak of the Trolox C radical. The signal exhibited a hyperfine structure characteristic of phenoxyl radicals. From an estimation of the radical concentration in the steady state and the velocity of the radical formation, the dismutation constant was calculated to be 5 X 10(5) M-1.s-1. The concentration of the signal in the steady state was reduced by the addition of GSH. The spectrum changed from that of the Trolox C radical to that of the ascorbate radical when the reaction was carried out in the presence of ascorbate.
采用停流技术和电子顺磁共振（ESR）技术研究了过氧化物酶催化的生育三烯酚C（一种维生素E类似物）的氧化机制。结果表明，在生育三烯酚C的氧化过程中，过氧化物酶化合物II是催化中间体。在pH 6.0条件下，化合物II与生育三烯酚C反应的速率常数（该反应应为速率决定步骤）经估算，辣根过氧化物酶和乳过氧化物酶的速率常数分别为2.1×10⁴和7.2×10³ M⁻¹·s⁻¹。通过ESR追踪生育三烯酚C自由基的形成。信号随时间的变化过程与440 nm处的吸光度变化相似，440 nm处被认定为生育三烯酚C自由基的峰值。该信号呈现出苯氧自由基特有的超精细结构。根据稳态下自由基浓度的估算以及自由基形成的速度，计算出歧化常数为5×10⁵ M⁻¹·s⁻¹。加入谷胱甘肽（GSH）后，稳态下信号的浓度降低。当反应在抗坏血酸存在的情况下进行时，光谱从生育三烯酚C自由基的光谱转变为抗坏血酸自由基的光谱。
