Wardman Peter
University of Oxford, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom.
Methods Enzymol. 2008;441:261-82. doi: 10.1016/S0076-6879(08)01214-7.
The commonest probes for "reactive oxygen and nitrogen species" are reduced fluorescein and rhodamine dyes that fluoresce when oxidized. The reduced dyes are reactive toward peroxynitrite, although probably not directly but via free radical oxidants derived from it: hydroxyl, carbonate, and nitrogen dioxide free radicals. The reaction with peroxynitrite can be monitored by rapid mixing and stopped-flow spectrophotometry, but reliable measurement of reactivity of the peroxynitrite-derived radicals requires specialized techniques such as flash photolysis or pulse radiolysis to monitor the fast reactions in real time. A key feature of oxidation by radicals is that the reaction produces an intermediate fluorescein or rhodamine radical, which normally is oxidized further by oxygen to yield the fluorescent, stable product. Susceptibility of the yield of fluorescence to interference by antioxidants can be assessed from kinetic parameters, which reflect reactivity. This chapter outlines methods for estimation of key rate constants involving peroxynitrite-derived oxidants.
用于检测“活性氧和氮物种”最常用的探针是还原型荧光素和罗丹明染料，它们在被氧化时会发出荧光。还原型染料对过氧亚硝酸盐具有反应活性，不过可能不是直接反应，而是通过由过氧亚硝酸盐衍生的自由基氧化剂：羟基自由基、碳酸根自由基和二氧化氮自由基起作用。与过氧亚硝酸盐的反应可以通过快速混合和停流分光光度法进行监测，但要可靠地测量过氧亚硝酸盐衍生自由基的反应活性，需要采用诸如闪光光解或脉冲辐解等专门技术来实时监测快速反应。自由基氧化的一个关键特征是反应会产生中间产物荧光素或罗丹明自由基，该自由基通常会被氧气进一步氧化，生成荧光稳定产物。可以根据反映反应活性的动力学参数评估荧光产率受抗氧化剂干扰的敏感性。本章概述了估算涉及过氧亚硝酸盐衍生氧化剂的关键速率常数的方法。
