Pfeiffer S, Mayer B
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
J Biol Chem. 1998 Oct 16;273(42):27280-5. doi: 10.1074/jbc.273.42.27280.
Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of .NO and O-2 at physiological pH. In the present study, we measured the nitration of free tyrosine by .NO and O-2 generated at well defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]- dia zen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite. Bolus addition of peroxynitrite (1 mM) to tyrosine (1 mM) at pH 7.4 yielded 36.77 +/- 1.67 microM 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from .NO and O-2, which were generated at equal rates ( approximately 5 microM x min-1) from 1 mM spermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mM hypoxanthine was much less efficient (0.67 +/- 0.01 microM; approximately 0.07% of total product flow). At O-2 fluxes exceeding the .NO release rates, 3-nitrotyrosine formation was below the detection limit of the high performance liquid chromatography method (<0.06 microM). Nitration was most efficient (approximately 0.3%) with the .NO donor alone, i.e. without concomitant generation of O-2. Nitration by .NO had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1-2 mM), and was not enhanced by NaHCO3 (up to 20 mM), indicating that it was mediated by .NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from .NO and O-2 as a mediator of tyrosine nitration in vivo.
蛋白质酪氨酸残基的硝化作用被认为是炎症条件下过氧亚硝酸盐介导的组织损伤的一个标志物。通过大剂量添加纯过氧亚硝酸盐，人们已在体外对硝化反应进行了广泛研究，这种实验方法很难反映体内情况，而过氧亚硝酸盐的产生被认为是在生理pH值下由一氧化氮（.NO）和超氧阴离子（O₂⁻）持续生成所致。在本研究中，我们分别测定了由供体化合物（Z）-1-[N-[3-氨丙基]-N-[4-(3-氨丙基铵基)丁基]-氨基]-重氮-1,2-二醇盐（精胺亚硝酸盐）以明确速率产生的一氧化氮（.NO）和超氧阴离子（O₂⁻）以及黄嘌呤氧化酶反应所导致的游离酪氨酸的硝化作用。将结果与纯过氧亚硝酸盐引发的既定硝化反应进行了比较。在pH 7.4条件下，向酪氨酸（1 mM）中一次性添加过氧亚硝酸盐（1 mM）产生了36.77±1.67 μM的3-硝基酪氨酸，回收率约为4%。然而，由1 mM精胺亚硝酸盐、28毫单位/毫升黄嘌呤氧化酶和1 mM次黄嘌呤以相等速率（约5 μM·min⁻¹）产生的一氧化氮（.NO）和超氧阴离子（O₂⁻）所形成的过氧亚硝酸盐效率要低得多（0.67±0.01 μM；约占总产物流量的0.07%）。当超氧阴离子（O₂⁻）通量超过一氧化氮（.NO）释放速率时，3-硝基酪氨酸的形成低于高效液相色谱法的检测限（<0.06 μM）。仅使用一氧化氮（.NO）供体时硝化效率最高（约0.3%），即没有伴随超氧阴离子（O₂⁻）的生成。一氧化氮（.NO）引发的硝化作用的最适pH值为8.2，随着酪氨酸浓度（0.1 - 2 mM）的增加而逐渐增加，并且不受碳酸氢钠（高达20 mM）的增强，这表明它是由二氧化氮（.NO₂）而非过氧亚硝酸盐介导的。我们的结果表明，由一氧化氮（.NO）和超氧阴离子（O₂⁻）产生的过氧亚硝酸盐并非体内酪氨酸硝化作用的介质。
