Michalski Radoslaw, Zielonka Jacek, Gapys Ewa, Marcinek Andrzej, Joseph Joy, Kalyanaraman Balaraman
From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
the Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
J Biol Chem. 2014 Aug 8;289(32):22536-53. doi: 10.1074/jbc.M114.553727. Epub 2014 Jun 13.
Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7-23 M(-1) s(-1)) were significantly higher than that measured for H2O2 (1.5 M(-1) s(-1)). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1-1.5 × 10(3) M(-1) s(-1). Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.
蛋白质中氨基酸及其残基（酪氨酸、半胱氨酸、色氨酸和组氨酸）的氢过氧化物是在活性氧诱导的氧化修饰过程中形成的。氨基酸氢过氧化物是不稳定的中间体，可进一步引发蛋白质中的氧化损伤。现有的检测方法（亚铁阳离子氧化法和碘量法）无法用于实时测量。在本研究中，我们发现，荧光香豆素硼酸（CBA）探针与氨基酸和蛋白质氢过氧化物反应会形成相应的荧光产物7-羟基香豆素。7-羟基香豆素的形成不依赖于过氧化氢酶。基于这一发现，我们开发了一种适用于多孔板形式的荧光实时检测方法。这是第一份使用基于CBA的检测方法实时监测氨基酸和蛋白质氢过氧化物的报告。该方法用于检测从暴露于可见光和光敏剂（孟加拉玫瑰红）的巨噬细胞中获得的细胞裂解物中的蛋白质氢过氧化物。我们还测量了氨基酸氢过氧化物（酪氨酸、色氨酸和组氨酸氢过氧化物）与CBA之间反应的速率常数，这些值（7 - 23 M⁻¹ s⁻¹）显著高于过氧化氢（1.5 M⁻¹ s⁻¹）的测量值。使用基于CBA的竞争动力学方法，还测定了氨基酸氢过氧化物与谷胱甘肽过氧化物酶模拟物依布硒啉反应的速率常数，其值在1.1 - 1.5 × 10³ M⁻¹ s⁻¹范围内。依布硒啉和硼酸盐都可用作氨基酸和蛋白质氢过氧化物的小分子清除剂。在此我们还展示了色氨酸暴露于一氧化氮和超氧化物的共生成通量时会形成色氨酸氢过氧化物。这一发现揭示了生物系统中氨基酸和蛋白质氢过氧化物形成的新机制。
