SUPEROXIDE ANION FORMATION DURING PHOTOCHEMICAL EXCITATION OF LUCIGENIN

荧光素(LC++)是一种常用的发光检测试剂 超氧化物。为了检测到超氧化物，必须将光敏素还原为 反应后形成二氧杂环己烷的光泽素阳离子自由基(LCZ+) 有超氧化物。二氧杂环己烷的自发分解 中间体产生化学发光。在现在 我们的研究表明，使用ESR自旋陷阱 5-二乙氧基磷酰基-5-甲基-1-吡咯啉N-氧化物(DEPMPO) 有、无还原条件下光敏素的光激发 NADH试剂产生DEPMPO-超氧化物和羟基自由基加合物。 在存在超氧化物歧化酶的情况下，DEPMPO-超氧化物产生的信号不是 观察到，只有羟基自由基加合物的ESR谱是 观察到的。超氧化物歧化酶和过氧化氢酶的结合抑制了 超氧化物和羟基加合物。超氧化物的产生是 细胞色素-c的减少也证明了这一点 可被超氧化物歧化酶抑制。存在下LC++的厌氧光解 还原剂NADH的作用导致还原光敏素的形成 在可见光范围内具有光谱特征。当着大家的面 空气中只消耗NADH，并且吸光度没有变化 Lc++。这可能意味着Lc++In的光激发 NADH的存在导致了Lcz+的形成，Lcz+ 与氧相互作用再生LC++并形成超氧阴离子 激进的。因此，光泽精不能用来测量超氧化物 光生物学中的队形。

Lucigenin (LC++) is frequently used for the luminescent detection of superoxide. To detect superoxide, lucigenin must be reduced to the lucigenin cation radical (LCz+) which forms a dioxetane after reaction with superoxide. Spontaneous decomposition of the dioxetane intermediate gives rise to the chemilumunescence. In the present study we show, using the ESR spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), that photoexcitation of lucigenin in the presence and absence of reducing agent NADH produces DEPMPO-superoxide and hydroxyl radical adducts. In the presence of SOD the signal due to DEPMPO-superoxide is not observed and only the ESR spectrum of the hydroxyl radical adduct is observed. The combination of SOD and catalase inhibits formation of both superoxide and hydroxyl adducts. Generation of superoxide was also demonstrated by cytochrome-c reduction which was partially inhibitable by SOD. The anaerobic photolysis of LC++ in the presence of reducing agent NADH leads to the formation of reduced lucigenin with spectral characteristics in the visible region. In the presence of air only NADH is consumed and there is no change in the absorbance of LC++. This probably implies that the photoexcitation of LC++ in the presence of NADH results in the formation of LCz+, which on interaction with oxygen regenerates LC++ and forms superoxide anion radical. Consequently, lucigenin cannot be used to measure superoxide formation in photobiology.