Lucigenin demonstration

It is extremely important that the interaction of quinones with XO (Reaction (3)) is reversible that can lead to receiving erroneous results at the measurement of superoxide production by SOD-inhibitable cytochrome c reduction [28,29] (see also Chapter 27). Lusthof et al. [30] demonstrated that 2,5-bis(l-aziridinyl)-l,4-benzoquinones are directly reduced by XO. Interestingly at quinone concentrations greater than 25pmol I 1, quinones entirely suppressed one-electron reduction of dioxygen, and cytochrome c was completely reduced by the semiquinones formed. It is well known that cytochrome c and lucigenin are effective superoxide scavengers and due to that, these compounds are widely used in the quantitative assays of superoxide detection. Nonetheless, under certain experimental conditions they can be directly reduced by XO [31]. 

However, a more discouraged fact is that benzoquinone accelerated SOD-inhibitable part of cytochrome c reduction, which is usually considered as a reliable proof of superoxide formation. Such a phenomenon has been first shown by Winterbourn [7], who suggested that SOD may shift the equilibrium of Reaction (4) to the right even for nonredox cycling quinones. The artificial enhancement of superoxide production by SOD in the presence of quinones was demonstrated in the experiments with lucigenin-amplified CL, in which benzoquinone was inhibitory [6],... 

It should be mentioned that Spasojevic et al. [57] recently determined the two-electron reduction potential of lucigenin in water as —0.14 V. As this value is close to the one-electron reduction potential of dioxygen °[02 702] = — 0.16 V, these authors regarded their finding as a support for lucigenin redox cycling. However, it has been demonstrated long ago that two-electron reduction potentials cannot be used for the calculation of equilibrium for one-electron transfer processes [58]. 

The different sensitivity of lucigenin and luminol to reactive oxygen species is reflected by some opposite results with certain compounds demonstrated by a low correlation coefficient for lucigenin- versus luminol-amplified chemiluminescence (Muller-Peddinghaus and Wurl 1987). 

Hepatocarcinogenic N-nitrosodimethylamine known to enhance microsomal hpid peroxidation (Jose and Slater 1973) produced oxidative damage in male Wistar rats, demonstrated by increased lipid peroxidation in vivo, by increased lucigenin-... 

Nonetheless, a dioxetan decomposition mechanism for lucigenin chemiluminescence, based on the exergonic processes described in Chap. V, seems well established [3]. A direct demonstration of the intermediacy of this dioxetane was first made [4] in 1969 by treating 10,10 -dimethyl-9,9 -biacrylidene (4) with singlet oxygen from several sources. Emission from N-methyl acridone was unequivocally shown. The lifetime of the intermediate was characteristic of the supposed dioxetane. Intramolecular electron transfer has been suggested as the excitation mechanism in the decomposition of this and other electron-rich dioxetans. 

Several lucigenin byproducts were identified, and ESR (at pH 13) demonstrated the presence of radicals formed independently of the presence of oxygen. Such signals disappeared rapidly on the addition of H2O2. Reaction of O2 with the lucigenin radical cation (i. e. lucigenin plus one electron) apparently gave N-methyl acridone. 

This reaction can be used to demonstrate energy transfer by adding fluorescen or rhodamine B (about 20 mg of each on the scales given in 10 ml. H2O and ethanol respectively). However, lucigenin (see later) is much more suitable for this purpose since it lasts longer. 

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