Reiter C D, Teng R J, Beckman J S
Departments of Anesthesiology, Biochemistry and Molecular Genetics, and Pediatrics, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233, USA.
J Biol Chem. 2000 Oct 20;275(42):32460-6. doi: 10.1074/jbc.M910433199.
Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.
酪氨酸硝化是一氧化氮与超氧化物反应产生的过氧亚硝酸根（ONOO⁻）的一种广泛使用的标志物。Pfeiffer和Mayer（Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280 - 27285）报道，次黄嘌呤加黄嘌呤氧化酶产生的超氧化物与精胺NONOate产生的一氧化氮相结合，在中性pH条件下不会使酪氨酸硝化。他们认为，在中性pH条件下，一氧化氮和超氧化物形成了一种反应性较低的中间体，与预先形成的碱性过氧亚硝酸根不同，该中间体不会使酪氨酸硝化。使用停流分光光度计在pH 7.4条件下将超氧化钾与一氧化氮快速混合，我们报道以100%的产率形成了一种在光谱和动力学上与预先形成的碱性顺式过氧亚硝酸根相同的中间体。此外，该中间体使酪氨酸硝化的产率和速率与预先形成的过氧亚硝酸根相同。在没有超氧化物的有氧条件下，等量浓度的一氧化氮不会产生可检测浓度的硝基酪氨酸。二氧化碳将一氧化氮加超氧化物的硝化效率提高到与过氧亚硝酸根相同的程度。在使用黄嘌呤氧化酶作为超氧化物来源的实验中，次黄嘌呤氧化形成的尿酸显著抑制了酪氨酸硝化，这足以解释先前报道的酪氨酸硝化缺乏的现象。我们得出结论，在生理pH条件下，一氧化氮与超氧化物反应形成的过氧亚硝酸根仍然是体内负责酪氨酸硝化的重要物质。
