Kunitomo R, Miyauchi Y, Inoue M
Department of Biochemistry, Kumamoto University Medical School, Japan.
J Biol Chem. 1992 May 5;267(13):8732-8.
Since cytochrome c and acetylated cytochrome c disappear from the circulation with a half-life of 4 min, these proteins cannot be used for in vivo detection of superoxide radicals and related metabolites. To determine superoxide and other radicals in vivo, a cytochrome c derivative (SMAC) was synthesized by linking 1 mol of poly(styrene-co-maleic acid) butyl ester (SM) to cytochrome c, followed by acetylation of its lysyl amino groups. SMAC retained 8 and 80% of cytochrome c activity to react with ascorbyl and superoxide radicals, respectively. However, SMAC did not serve as a substrate for cytochrome c reductase and cytochrome c oxidase. When injected intravenously to the rat, SMAC circulated bound to albumin with a half-life of 130 min. SMAC was rapidly reduced in the circulation of intact animals. Treatment of animals with paraquat markedly enhanced the reduction of the circulating SMAC. We have synthesized an SM-conjugated superoxide dismutase (SOD) derivative (SM-SOD) that circulates bound to albumin with a half-life of 6 h. Kinetic analysis revealed that SM-SOD effectively inhibited the superoxide-dependent reduction of SMAC either in the presence or absence of 0.5 mM albumin. However, the reduction of the circulating SMAC was not inhibited by SM-SOD both in normal and paraquat-treated animals. Plasma samples from both animal groups also reduced cytochrome c and SMAC by an SOD-insensitive mechanism. However, after treatment with ascorbate oxidase, both plasma samples lost their activity to reduce cytochrome c and SMAC. These and other results suggest that ascorbyl radical might principally be responsible for the reduction of circulating SMAC and that plasma levels of ascorbyl radical might increase in paraquat-treated animals.
由于细胞色素c和乙酰化细胞色素c以4分钟的半衰期从循环系统中消失，因此这些蛋白质不能用于体内超氧自由基及相关代谢产物的检测。为了测定体内的超氧自由基和其他自由基，通过将1摩尔的聚（苯乙烯 - 马来酸）丁酯（SM）与细胞色素c连接，随后对其赖氨酰氨基进行乙酰化，合成了一种细胞色素c衍生物（SMAC）。SMAC分别保留了细胞色素c与抗坏血酸自由基和超氧自由基反应活性的8%和80%。然而，SMAC不是细胞色素c还原酶和细胞色素c氧化酶的底物。静脉注射到大鼠体内后，SMAC与白蛋白结合在循环系统中，半衰期为130分钟。在完整动物的循环系统中，SMAC迅速被还原。用百草枯处理动物显著增强了循环中SMAC的还原。我们合成了一种与SM共轭的超氧化物歧化酶（SOD）衍生物（SM-SOD），它与白蛋白结合在循环系统中，半衰期为6小时。动力学分析表明，无论有无0.5 mM白蛋白存在，SM-SOD都能有效抑制超氧自由基依赖的SMAC还原。然而，在正常动物和百草枯处理的动物中，循环中SMAC的还原均未被SM-SOD抑制。两组动物的血浆样本也通过一种SOD不敏感的机制还原细胞色素c和SMAC。然而，用抗坏血酸氧化酶处理后，两组血浆样本都失去了还原细胞色素c和SMAC的活性。这些以及其他结果表明，抗坏血酸自由基可能是循环中SMAC还原的主要原因，并且在百草枯处理的动物中抗坏血酸自由基的血浆水平可能会升高。
