Greenstock C L, Miller R W
Biochim Biophys Acta. 1975 Jul 8;396(1):11-6. doi: 10.1016/0005-2728(75)90184-x.
The rate of reaction between superoxide anion (O2) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O2. A kinetic spectrophotometric method utilizing competition between p-benzoquinone and tiron for O2 was employed. In this system, the known rate of reduction of p-benzoquinone was compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5x10-8 M-minus1-s-minus1. Ascorbate reduced O2 to hydrogen peroxide with a rate constant of 10-8 M-minus1-s-minus1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O2 compared to the rate of O2 formation is most enzymatic systems. Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O2 since O2 was consumed in the reaction and tiron did not reduce the P700 cation radical or other components of Photosystem I under anaerobic conditions.
利用脉冲辐解产生的超氧阴离子（O₂⁻）测定了其与1,2 - 二羟基苯 - 3,5 - 二磺酸（钛铁试剂）之间的反应速率。采用了一种动力学分光光度法，该方法利用对苯醌和钛铁试剂对O₂⁻的竞争作用。在这个体系中，将已知的对苯醌还原速率与钛铁试剂氧化为半醌的速率进行比较。根据钛铁试剂氧化速率的浓度依赖性，确定该反应的绝对二级速率常数为5×10⁻⁸ M⁻¹·s⁻¹。用相同方法测定，抗坏血酸将O₂⁻还原为过氧化氢的速率常数为10⁻⁸ M⁻¹·s⁻¹。由于与大多数酶促体系中O₂⁻的生成速率相比，儿茶酚被O₂氧化的速率很快，所以钛铁试剂半醌可作为生物体系中超氧阴离子形成的指示自由基。利用钛铁试剂氧化来跟踪肿胀叶绿体中超氧阴离子的形成。叶绿体通过光化学作用还原分子氧，该分子氧又被钛铁试剂进一步还原为过氧化氢。钛铁试剂氧化特别需要O₂，因为在反应中消耗了O₂，并且在厌氧条件下钛铁试剂不会还原P700阳离子自由基或光系统I的其他组分。
