Osman A M, Zomer G, Laane C, Hilhorst R
Department of Biomolecular Sciences, Wageningen University, The Netherlands.
Luminescence. 2000 May-Jun;15(3):189-97. doi: 10.1002/1522-7243(200005/06)15:3<189::AID-BIO585>3.0.CO;2-A.
In this study, the chemiluminescent horseradish peroxidase/H(2)O(2)-catalysed oxidation of acridan (GZ-11) substrate was compared with the well-characterized light-producing luminol reaction. p-Iodophenol and p-phenylphenol were used as enhancers, respectively, for the luminol and acridan reactions. These two light-producing systems showed significant differences in relation to 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 of the acridan system. This suggests that superoxide anion does not participate in the mechanism of the light-emitting steps of the acridan reaction. Addition of hydroxyl radical scavengers, mannitol and benzoate, to the acridan reaction medium had no appreciable effect on the chemiluminescent intensity, indicating that hydroxyl radicals do not interfere in light-emitting steps. In addition, the peroxidation of the acridan substrate was found to be very slow at pH 5.6 in the absence of the enhancer, p-phenylphenol, whereas in its presence a rapid degradation of the acridan substrate was observed. Therefore, it is suggested that the enhancer might be initially oxidized by the HRP/H(2)O(2) system, resulting in the formation of the enhancer radical, which could be the actual oxidizing agent of the acridan substrate. Together, the data presented in this paper indicate that the chemiluminescent horseradish peroxidase-catalysed peroxidation of acridan (GZ-11) is more specific than the luminol reaction for the reactive oxygen species involved in the light-emitting steps, i. e, H(2)O(2).
在本研究中，将化学发光的辣根过氧化物酶/H₂O₂催化吖啶（GZ - 11）底物的氧化反应与特征明确的发光鲁米诺反应进行了比较。对碘苯酚和对苯基苯酚分别用作鲁米诺和吖啶反应的增强剂。这两种发光体系在pH值的影响以及活性氧清除剂对光强度的影响方面表现出显著差异。在吖啶反应中，测得的发光在pH 2.6时可能低至很低水平，而在鲁米诺体系中，pH低于5.6时未检测到发光。与鲁米诺体系相反，发现超氧化物歧化酶不会抑制吖啶体系的光强度。这表明超氧阴离子不参与吖啶反应发光步骤的机制。向吖啶反应介质中添加羟基自由基清除剂甘露醇和苯甲酸盐对化学发光强度没有明显影响，表明羟基自由基不干扰发光步骤。此外，发现在没有增强剂对苯基苯酚的情况下，吖啶底物在pH 5.6时的过氧化非常缓慢，而在其存在下观察到吖啶底物迅速降解。因此，建议增强剂可能最初被HRP/H₂O₂体系氧化，导致形成增强剂自由基，这可能是吖啶底物的实际氧化剂。总之，本文给出的数据表明，化学发光的辣根过氧化物酶催化的吖啶（GZ - 11）过氧化反应对于发光步骤中涉及的活性氧物种，即H₂O₂，比鲁米诺反应更具特异性。
