Ronsein Graziella E, Oliveira Mauricio C B, Miyamoto Sayuri, Medeiros Marisa H G, Di Mascio Paolo
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
Chem Res Toxicol. 2008 Jun;21(6):1271-83. doi: 10.1021/tx800026g. Epub 2008 May 6.
Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O2(1Deltag)]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from W18O18OH decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.
蛋白质被认为是活性氧的重要作用靶点。事实上，色氨酸（W）已被证明是对包括单线态分子氧[O₂(¹Δg)]在内的许多氧化剂高度敏感的氨基酸。在本研究中，通过高效液相色谱/质谱联用和核磁共振分析，对两种顺式和反式色氨酸氢过氧化物（WOOH）异构体进行了全面表征，它们是主要的W氧化光产物。这些光产物经热分解生成相应的醇。此外，WOOHs在加热或碱化条件下会分解，导致N-甲酰基犬尿氨酸（FMK）的形成。使用¹⁸O标记的氢过氧化物（W¹⁸O¹⁸OH），可以确认由W¹⁸O¹⁸OH分解产生的两个氧标记的FMK分子的形成。这一结果表明，FMK中的两个氧原子均来自氢过氧化物基团。此外，这些反应是化学发光（CL）的，表明存在二氧杂环丁烷裂解途径。由于WOOH分解的CL光谱与FMK荧光光谱匹配，明确将FMK鉴定为发光物质，因此证实了这一机制。
