Peroxynitrite-dependent tryptophan nitration
Peroxynitrite-dependent tryptophan nitration
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DOI：
10.1021/tx950133b
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发表时间：
1996-03-01
影响因子：
4.1
通讯作者：
Radi, R
中科院分区：
文献类型：
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作者：
Alvarez, B;Rubbo, H;Radi, R
过氧亚硝酸盐（ONOO-），是超氧化物（O-2）和一氧化氮（(NO)- n -）的反应产物，硝酸盐酪氨酸和其他酚类物质。我们在此报告色氨酸在没有过渡金属的情况下也被过氧亚硝酸盐硝化为一种主要的硝基色氨酸异构体，这是由光谱特征和液相色谱-质谱分析确定的。在高过氧亚硝酸盐与色氨酸的比例下，也检测到其他氧化产物。酸性pH下，过氧亚硝酸盐生成的硝基色氨酸增加，表观pK(a)为7.8。高浓度的Fe3+-EDTA可以促进过氧亚硝酸盐诱导的硝基色氨酸的形成，而添加15 μ M的Cu/Zn超氧化物歧化酶对色氨酸硝化的影响最小。半胱氨酸、抗坏血酸和蛋氨酸降低硝基色氨酸产量的程度与它们与基态过氧亚硝酸盐的反应速率预测的程度相似，典型的羟基自由基清除剂部分抑制硝化。观察到的硝基色氨酸形成速率常数与色氨酸浓度的关系曲线呈向下弯曲。因此，不依赖金属的硝化反应动力学被解释为两个平行的机制。其中，基态过氧亚硝酸在37℃下硝化色氨酸，二级速率常数为184 +/- 11 M(-1) s(-1)，激活焓为9.1 +/- 0.3 kcal mol(-1)，激活熵为-19 +/- 1 cal mol(-1) K-1。在第二种机制中，ONOOH*是一种由反式过氧亚硝酸在稳定状态下形成的活化中间体，是硝化剂。
Peroxynitrite (ONOO-), the reaction product of superoxide (O-2(.-)) and nitric oxide ((NO)-N-.), nitrates tyrosine and other phenolics. We report herein that tryptophan is also nitrated by peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography-mass spectrometry analysis. At high peroxynitrite to tryptophan ratios, other oxidation products were detected as well. The amount of nitrotryptophan formed from peroxynitrite increased at acidic pH, with an apparent pK(a), of 7.8. High concentrations of Fe3+-EDTA were required to enhance peroxynitrite-induced nitrotryptophan formation, while addition of up to 15 mu M Cu/Zn superoxide dismutase had a minimal effect on tryptophan nitration. Cysteine, ascorbate, and methionine decreased nitrotryptophan yield to an extent similar to that predicted by their reaction rates with ground-state peroxynitrite, and typical hydroxyl radical scavengers partially inhibited nitration. Plots of the observed rate constant of nitrotryptophan formation vs tryptophan concentration presented downward curvatures. Thus, the kinetics of metal-independent nitration reactions were interpreted in terms of two parallel mechanisms. In the first one, ground-state peroxynitrous acid nitrated tryptophan with a second-order rate constant of 184 +/- 11 M(-1) s(-1) at 37 degrees C. The activation enthalpy was 9.1 +/- 0.3 kcal mol(-1), and the activation entropy was -19 +/- 1 cal mol(-1) K-1. In the second mechanism, ONOOH*, an activated intermediate derived from trans-peroxynitrous acid formed in a steady state, was the nitrating agent.