Grupo de Biofisicoquímica, Departamento de Ciencias Biológicas, CENUR Litoral Norte, Universidad de la República, Rivera 1350, 50000, Salto, Uruguay.
Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800, Montevideo, Uruguay.
Free Radic Biol Med. 2022 Nov 20;193(Pt 1):474-484. doi: 10.1016/j.freeradbiomed.2022.10.317. Epub 2022 Nov 2.
Guanine (Gua), among purines, is a preferred oxidation/nitration target because of its low one-electron redox potential. The reactive oxygen/nitrogen species peroxynitrite (ONOO), produced in vivo by the reaction between nitric oxide (NO) and superoxide radical (O), is responsible for several oxidative modifications in biomolecules, including nitration, nitrosation, oxidation, and peroxidation. In particular, the nitration of Gua, although detected, as well as its reaction kinetics have been seldom investigated. Thus, we studied the concentration- and temperature-dependent formation of 8-nitroguanine (8-NitroGua) in phosphate buffer (pH 7.40) using stopped-flow spectrophotometry. Traces showed a biexponential behavior, with best-fit rate constants: k = 4.4 s and k = 0.41 s (30 °C, 400 μM both Gua and ONOO). k increased linearly with the concentration of both reactants whereas k was concentration-independent. Linear regression analysis of k as a function of Gua and ONOO concentration yielded values of 2.5-6.3 × 10 Ms and 1.5-3.5 s for the second-order (slope) and first-order (ordinate) rate constants, respectively (30 °C). Since ONOO is a short-lived species, its decay kinetics was also taken into account for this analysis. The 8-NitroGua product was stable for at least 4 h, so no spontaneous denitration was observed. Stopped-flow assays using antioxidants and free-radical scavengers suggested a mixed direct/indirect reaction mechanism for 8-NitroGua formation. Gua nitration by ONOO was also observed in the presence of physiologically relevant CO concentrations. The reaction product identity, its yield (∼4.2%, with 400 μM ONOO and 200 μM Gua), and the reaction mechanism were unequivocally determined by HPLC-MS/MS experiments. In conclusion, 8-NitroGua production at physiologic pH reached significant levels in a few hundred milliseconds, suggesting that the process might be kinetically relevant in vivo and can likely cause permanent nitrative damage to DNA bases.
鸟嘌呤（Gua）是嘌呤中的一种，由于其单电子还原电势较低，因此是优先氧化/硝化的靶标。过氧亚硝酸盐（ONOO）是由一氧化氮（NO）和超氧自由基（O）反应生成的活性氧/氮物种，它负责生物分子中的几种氧化修饰，包括硝化、亚硝化、氧化和过氧化。特别是，鸟嘌呤的硝化虽然已经被检测到，但它的反应动力学却很少被研究。因此，我们使用停流分光光度法研究了在磷酸盐缓冲液（pH 7.40）中浓度和温度依赖性的 8-硝基鸟嘌呤（8-NitroGua）的形成。迹线显示出双指数行为，最佳拟合速率常数为：k = 4.4 s 和 k = 0.41 s（30°C，400 μM 鸟嘌呤和 ONOO 均）。k 随反应物浓度呈线性增加，而 k 则与浓度无关。k 作为 Gua 和 ONOO 浓度的函数的线性回归分析得出了二阶（斜率）和一阶（纵轴）速率常数的值分别为 2.5-6.3×10 M s 和 1.5-3.5 s（30°C）。由于 ONOO 是一种短寿命的物质，因此也考虑了其衰减动力学来进行此分析。8-NitroGua 产物在至少 4 小时内稳定，因此没有观察到自发的脱硝化。使用抗氧化剂和自由基清除剂的停流测定表明，8-NitroGua 形成的混合直接/间接反应机制。在生理相关 CO 浓度存在下，也观察到了 ONOO 对 Gua 的硝化。通过 HPLC-MS/MS 实验，明确确定了反应产物的身份、产率（在 400 μM ONOO 和 200 μM Gua 时约为 4.2％）和反应机制。总之，在生理 pH 下，8-NitroGua 的产生在几百毫秒内达到了显著水平，这表明该过程在体内可能具有动力学相关性，并可能对 DNA 碱基造成永久性硝化损伤。
