Sawa T, Akaike T, Maeda H
Department of Microbiology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan.
J Biol Chem. 2000 Oct 20;275(42):32467-74. doi: 10.1074/jbc.M910169199.
Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)NN(O)NO (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.
过氧亚硝酸根（ONOO⁻）是一种强效的硝化和氧化剂，它由一氧化氮（NO）与超氧阴离子（O₂⁻）快速反应形成。它似乎参与了许多炎症和神经退行性疾病的病理生理过程。最近有报道（Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280 - 27285）称，在中性pH条件下由NO和O₂（NO/O₂）生成的ONOO⁻在硝化酪氨酸方面的效率远低于预先形成的ONOO⁻。在此，我们采用更短的孵育时间和更灵敏的电化学检测系统，重新评估了NO/O₂对酪氨酸的硝化作用。在含有0.1 mM L - 酪氨酸的磷酸盐缓冲液（pH 7.0）中，由丙胺亚硝基铁氰化钠（CH₃N[N(O)NO]⁻(CH₂)₃NH₂⁺CH₃）和黄嘌呤氧化酶以蝶呤为底物产生的NO/O₂通量原位生成的ONOO⁻（5分钟内产生2.9微摩尔；10纳米/秒）能产生可观量的硝基酪氨酸。该NO/O₂通量产生的硝基酪氨酸产量约为相同通量预先形成的ONOO⁻（2.9微摩尔/5分钟）产生量的70%。当使用次黄嘌呤作为底物时，由于次黄嘌呤氧化过程中产生的尿酸的抑制作用，NO/O₂对酪氨酸的硝化作用基本消除。正如预期的那样，NO/O₂引起酪氨酸硝化作用被ONOO⁻清除剂依布硒啉抑制，而被NaHCO₃增强了2倍，因为CO₂促进酪氨酸硝化。NO/O₂通量（2.9微摩尔/5分钟）产生的硝基酪氨酸和二酪氨酸形成的情况与预先形成的ONOO⁻产生的情况一致。在生理浓度的游离酪氨酸（<0.5 mM）下，与低纳摩尔通量的ONOO⁻引起的二酪氨酸形成相比，酪氨酸硝化占主导。总之，我们的结果表明，与O₂一起产生的NO硝化酪氨酸的反应性和效率与化学合成的ONOO⁻相似，这表明ONOO⁻可能是体内生理和病理事件中酪氨酸硝化的重要来源。
