Kuthan H, Ullrich V, Estabrook R W
Biochem J. 1982 Jun 1;203(3):551-8. doi: 10.1042/bj2030551.
The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.
本文报道了一种部分琥珀酰化细胞色素c的制备方法及其性质，该细胞色素c适用于检测肝微粒体中的超氧阴离子自由基。通过将马心细胞色素c的45%的伯氨基进行琥珀酰化，与天然细胞色素c相比，其对溶解的NADPH-细胞色素P-450还原酶的活性降低了99%。细胞色素b5和细胞色素c氧化酶分别还原琥珀酰化高铁细胞色素c和氧化琥珀酰化亚铁细胞色素c的能力也有类似程度的下降。然而，在pH 7.7时，O2-还原部分琥珀酰化高铁细胞色素c的双分子速率常数估计仅为O2-与天然高铁细胞色素c反应速率常数的十分之一。基于此，对由补充NADPH的肝微粒体产生的O2-进行定量成为可能。在细胞色素c衍生物的各种有限浓度下测定的琥珀酰化高铁细胞色素c还原的初始速率可以外推得到均相体系中O2-产生的真实速率。文中还讨论了在生物膜（如微粒体）中产生的O2-定量测定中遇到的问题。
