Naash M I, Anderson R E
Curr Eye Res. 1984 Nov;3(11):1299-304. doi: 10.3109/02713688409007416.
The properties and kinetic parameters of glutathione peroxidase were measured in retinal homogenates from frogs (R. pipiens) using a spectrophotometric assay in which the oxidation of glutathione is coupled to the oxidation of NADPH+ by exogenous glutathione reductase. The standard assay utilized 5.0 mM glutathione and 0.6 mM cumene hydroperoxide in 50 mM sodium phosphate buffer at pH 7.0. All solutions were bubbled with argon and all reactions were carried out under an atmosphere of argon. The enzyme activity was linear with protein concentration at different concentrations of both substrates. Determination of the pH optimum was complicated by a large increase in non-enzymatic oxidation of glutathione at alkaline pH. The highest ratio of enzymatic to non-enzymatic activity was at pH 7.0. Increasing glutathione concentration showed less effect on the spontaneous reaction than increasing the cumene hydroperoxide concentration. Glutathione peroxidase Km value for glutathione was 3.86 mM and for cumene hydroperoxide was 0.55 mM. Vmax for glutathione at 0.6 mM cumene hydroperoxide was 138 nmoles glutathione oxidized/min/mg protein, while at 5.0 mM glutathione the value for cumene hydroperoxide was 146 nmoles glutathione oxidized/min/mg protein. These studies demonstrate that glutathione peroxidase is active in the retina and establish the optimal experimental conditions for determination of the enzymatic activity.
使用分光光度法测定了牛蛙（R. pipiens）视网膜匀浆中谷胱甘肽过氧化物酶的性质和动力学参数，该方法中谷胱甘肽的氧化与外源谷胱甘肽还原酶将NADPH+氧化相偶联。标准测定在pH 7.0的50 mM磷酸钠缓冲液中使用5.0 mM谷胱甘肽和0.6 mM氢过氧化异丙苯。所有溶液均用氩气鼓泡，所有反应均在氩气气氛下进行。在两种底物的不同浓度下，酶活性与蛋白质浓度呈线性关系。由于在碱性pH下谷胱甘肽的非酶促氧化大幅增加，使得最适pH的测定变得复杂。酶促活性与非酶促活性的最高比值出现在pH 7.0。与增加氢过氧化异丙苯浓度相比，增加谷胱甘肽浓度对自发反应的影响较小。谷胱甘肽过氧化物酶对谷胱甘肽的Km值为3.86 mM，对氢过氧化异丙苯的Km值为0.55 mM。在0.6 mM氢过氧化异丙苯存在下，谷胱甘肽的Vmax为138纳摩尔谷胱甘肽氧化/分钟/毫克蛋白质，而在5.0 mM谷胱甘肽存在下，氢过氧化异丙苯的Vmax值为146纳摩尔谷胱甘肽氧化/分钟/毫克蛋白质。这些研究表明谷胱甘肽过氧化物酶在视网膜中具有活性，并确定了测定酶活性的最佳实验条件。
