Kooy N W, Royall J A, Ischiropoulos H
Department of Pediatrics, University of Iowa, Iowa City 52242, USA.
Free Radic Res. 1997 Sep;27(3):245-54. doi: 10.3109/10715769709065763.
The simultaneous production of nitric oxide and superoxide anion leads to the formation of peroxynitrite, a potent oxidant which may be an important mediator of cellular injury. Oxidation of dichlorofluorescin to the fluorescent dichlorofluorescein has been used as a marker for cellular oxidant production. The mechanisms of peroxynitrite-mediated oxidation of dichlorofluorescin to dichlorofluorescein were investigated. Chemically synthesized peroxynitrite (50-500 nM) induced the oxidation of dichlorofluorescin to dichlorofluorescein in a linear fashion. In addition, the simultaneous generation of nitric oxide and superoxide anion induced the oxidation of dichlorofluorescin to dichlorofluorescein, while nitric oxide (1-10 microM) alone under aerobic conditions did not. Peroxynitrite-mediated oxidation of dichlorofluorescin was not inhibited by the hydroxyl radical scavengers mannitol (100 mM) or dimethylsulfoxide (100 mM). Moreover, peroxynitrite-mediated oxidation of dichlorofluorescin was not dependent upon metal ion-catalyzed reactions. Furthermore, dichlorofluorescein formation was diminished at alkaline pH. These findings suggest that peroxynitrite-mediated dichlorofluorescein formation results directly from the protonation of peroxynitrite to form the conjugate peroxynitrous acid. L-cysteine was an efficient inhibitor (KI approximately 25 microM) of dichlorofluorescin oxidation through competitive oxidation of free sulfhydryls. Urate was a less efficient with a maximum inhibition of only 49%. These results demonstrate that dichlorofluorescin is efficiently oxidized by peroxynitrite. Therefore, under conditions where nitric oxide and superoxide are produced simultaneously, oxidation of dichlorofluorescin may be mediated by the formation of peroxynitrite.
一氧化氮和超氧阴离子的同时产生会导致过氧亚硝酸盐的形成，过氧亚硝酸盐是一种强效氧化剂，可能是细胞损伤的重要介质。二氯荧光素氧化为荧光性二氯荧光黄已被用作细胞氧化剂产生的标志物。研究了过氧亚硝酸盐介导的二氯荧光素氧化为二氯荧光黄的机制。化学合成的过氧亚硝酸盐（50 - 500 nM）以线性方式诱导二氯荧光素氧化为二氯荧光黄。此外，一氧化氮和超氧阴离子的同时产生诱导二氯荧光素氧化为二氯荧光黄，而在有氧条件下单独的一氧化氮（1 - 10 μM）则不会。过氧亚硝酸盐介导的二氯荧光素氧化不受羟基自由基清除剂甘露醇（100 mM）或二甲基亚砜（100 mM）的抑制。此外，过氧亚硝酸盐介导的二氯荧光素氧化不依赖于金属离子催化的反应。此外，在碱性pH条件下二氯荧光黄的形成减少。这些发现表明，过氧亚硝酸盐介导的二氯荧光黄形成直接源于过氧亚硝酸盐质子化形成共轭过亚硝酸。L - 半胱氨酸是二氯荧光素氧化的有效抑制剂（抑制常数约为25 μM），通过竞争性氧化游离巯基发挥作用。尿酸是一种效率较低的抑制剂，最大抑制率仅为49%。这些结果表明二氯荧光素可被过氧亚硝酸盐有效氧化。因此，在一氧化氮和超氧同时产生的条件下，二氯荧光素的氧化可能是由过氧亚硝酸盐的形成介导的。
