Comparative studies of the chemiluminescent horseradish peroxidase-catalysed peroxidation of acridan (GZ-11) and luminol reactions: effect of pH and scavengers of reactive oxygen species on the light intensity of these systems

In this study, the chemiluminescent horseradish peroxidase/H2O2-catalysed o xidation of acridan (GZ-11) substrate was compared with the well-characteri zed light-producing luminol reaction. p-Iodophenol and p-phenylphenol were used as enhancers, respectively, for the luminol and acridan reactions. The se two light-producing systems showed significant differences in relation t o the effect of pH, as well as the effect of scavengers of reactive oxygen species, on the light intensity. Light production measured could be as low as pH 2.6 in the acridan reaction, whereas light emission was not detected in the luminol system below pH 5.6. In contrast with the luminol system, it was found that superoxide dismutase does not inhibit the light intensity o f the acridan system. This suggests that superoxide anion does not particip ate in the mechanism of the light-emitting steps of the acridan reaction. A ddition of hydroxyl radical scavengers, mannitol and benzoate, to the acrid an reaction medium had no appreciable effect on the chemiluminescent intens ity, indicating that hydroxyl radicals do not interfere in light-emitting s teps. In addition, the peroxidation of the acridan substrate was found to b e very slow at pH 5.6 in the absence of the enhancer, p-phenylphenol, where as in its presence a rapid degradation of the acridan substrate was observe d. Therefore, it is suggested that the enhancer might be initially oxidized by the HRP/H2O2 system, resulting in the formation of the enhancer radical , which could be the actual oxidizing agent of the acridan substrate. Toget her, the data presented in this paper indicate that the chemiluminescent ho rseradish peroxidase-catalysed peroxidation of acridan (GZ-11) is more spec ific than the luminol reaction for the reactive oxygen species involved in the light-emitting steps, i.e, H2O2. Copyright (C) 2000 John Wiley & Sons, Ltd.