Votyakova Tatyana V, Reynolds Ian J
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Arch Biochem Biophys. 2004 Nov 1;431(1):138-44. doi: 10.1016/j.abb.2004.07.025.
We report here that reduced pyridine nucleotides and reduced glutathione result in an oxidation of Amplex Red by dioxygen that is dependent on the presence of horseradish peroxidase (HRP). Concentrations of NADH and glutathione typically found in biological systems result in the oxidation of Amplex Red at a rate comparable to that produced, for example, by respiring mitochondria. The effects of NADH and glutathione in this assay system are likely to be the result of H(2)O(2) generation via a superoxide intermediate because both catalase and superoxide dismutase prevent the oxidation of Amplex Red. These results suggest caution in the assay of H(2)O(2) production in biological systems using the Amplex Red/HRP because the assay will also report the mobilization of NADH or glutathione. However, the interruption of this process by the addition of superoxide dismutase offers a simple and reliable method for establishing the source of the oxidant signal.
我们在此报告，还原型吡啶核苷酸和还原型谷胱甘肽会导致双氧对Amplex Red的氧化，该氧化过程依赖于辣根过氧化物酶（HRP）的存在。生物系统中常见的NADH和谷胱甘肽浓度会导致Amplex Red以与例如呼吸线粒体产生的速率相当的速度被氧化。在该检测系统中，NADH和谷胱甘肽的作用可能是通过超氧化物中间体产生H₂O₂的结果，因为过氧化氢酶和超氧化物歧化酶都能阻止Amplex Red的氧化。这些结果表明，在使用Amplex Red/HRP检测生物系统中H₂O₂产生时需谨慎，因为该检测也会报告NADH或谷胱甘肽的动员情况。然而，通过添加超氧化物歧化酶来中断这一过程，为确定氧化信号的来源提供了一种简单可靠的方法。
