SUPEROXIDE ANION FORMATION DURING PHOTOCHEMICAL EXCITATION OF LUCIGENIN

光泽精（LC++）常用于发光检测 超氧化物。 为了检测超氧化物，光泽精必须被还原到 光泽精阳离子自由基（LC？+）反应后形成二氧杂环丁烷 超氧化物。 二氧杂环丁烷的自发分解 中间体引起化学发光。 本 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-吡咯啉氧化物（DEPMPO）， 在存在和不存在还原剂的情况下光泽精的光激发 试剂NADH产生DEPMPO-超氧化物和羟基自由基加合物。 在SOD的存在下，由于DEPMPO-超氧化物的信号不是 观察到的，只有羟基自由基加合物的ESR谱是 观察 SOD和过氧化氢酶的组合抑制了 超氧化物和羟基加合物。 超氧化物的产生是 细胞色素C减少也证明了这一点， 由SOD提供。 LC ~（++）的厌氧光解 还原剂NADH的加入导致还原光泽精的形成 具有可见光区的光谱特性。 存在下 空气中只有NADH被消耗，吸光度没有变化 关于LC++ 这可能意味着LC++在 NADH的存在导致LC？+的形成，其在 与氧的相互作用使LC++再生并形成超氧阴离子 激进 因此，光泽精不能用于测量超氧化物 光生物学中的形成

Lucigenin (LC++) is frequently used for the luminescent detection of superoxide. To detect superoxide, lucigenin must be reduced to the lucigenin cation radical (LC?+) 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 Boxide (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 r educ ing 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 LC?+, which on interaction with oxygen regenerates LC++ and forms superoxide anion radical. Consequently, lucigenin cannot be used to measure superoxide formation in photobiology.