ESR spin-trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide
ESR spin-trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide
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DOI：
10.1074/jbc.271.26.15498
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发表时间：
1996-06-28
影响因子：
4.8
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Barr, DP;Gunther, MR;Mason, RP
用ESR自旋捕获技术和亚硝基自旋捕获剂3，5-二溴-4-亚硝基苯磺酸（DBNBS）和2-甲基-2-亚硝基丙烷（MNP）研究了马心细胞色素c与过氧化氢的反应。用两种自旋捕集器得到的ESR谱都是典型的固定化氮氧自由基，表明加合物是一种高分子。ESR谱的强度与DBNBS/（.）在厌氧条件下进行的反应大大增强了细胞色素c自由基加合物，这表明自旋捕获器与O-2竞争与自由基位点的反应。DBNBS和MNP加合物的非特异性蛋白水解显示各向同性的三线谱。此外，蛋白酶处理的MNP细胞色素c蛋白自由基加合物获得了高分辨率的ESR谱。在该谱图中检测到的超精细偶联与真实的MNP/酪氨酰加合物中检测到的超精细偶联完全相同。C-13标记的酪氨酸芳环位置产生了额外的超精细偶联，证明自由基位点确实位于酪氨酸环上。细胞色素c衍生的加合物来自细胞色素与H2 O2的反应。因此，在反应期间似乎形成了四个自由基位点，其中至少一个是酪氨酸。
The reaction of horse heart cytochrome c with hydrogen peroxide was investigated using the ESR spin-trapping technique and the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP). The ESR spectra obtained using both spin traps were typical of an immobilized nitroxide and indicated that the adduct was a macromolecule, The intensity of the ESR spectrum corresponding to the DBNBS/(.)cytochrome c radical adduct was greatly enhanced by performing the reaction under anaerobic conditions, which suggested that the spin trap was competing with O-2 for reaction with the radical site(s), Nonspecific proteolysis of either the DBNBS or the MNP adducts revealed isotropic three-line spectra, In addition, a high resolution ESR spectrum for the protease-treated MNP cytochrome c-derived protein radical adduct was obtained. The superhyperfine couplings detected in this spectra were identical to those detected from an authentic MNP/tyrosyl adduct, Carbon-13 labeling of the aromatic ring positions of tyrosine yielded additional hyperfine coupling, demonstrating that the radical site was definitely located on the ring of tyrosine, Mass spectrometry detected as many as four DBNBS/(.)cytochrome c-derived adducts from the reaction of cytochrome with H2O2, Thus, it would appear four radical sites are formed during the reaction, at least one of which is tyrosine.