Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase.
Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase.
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结核分枝杆菌过氧化氢酶-过氧化物酶中过氧化物和异烟肼诱导的自由基的自旋捕获研究。
DOI：
10.1021/bi800952b
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发表时间：
2008-10-28
期刊：
影响因子：
2.9
通讯作者：
Mason, Ronald P.
中科院分区：
文献类型：
--
作者：
Ranguelova, Kalina;Suarez, Javier;Magliozzo, Richard S.;Mason, Ronald P.
一种新的方法，免疫自旋捕获试验，使用一种新的兔多克隆抗DMPO（5，5-二甲基-1-吡咯啉N-氧化物）抗血清检测蛋白质自由基衍生的DMPO硝酮加合物的血红素蛋白结核分枝杆菌过氧化氢酶过氧化物酶（KatG）。这项工作表明，蛋白质硝酮加合物的形成是依赖于叔丁基OOH和DMPO的浓度所示的蛋白质印迹和酶联免疫吸附试验（ELISA）。我们还检测到在由叔丁基过氧化物（tert-BuOOH）或过氧化氢的酶促生成驱动的Mtb KatG周转期间形成的蛋白质-蛋白质交联。DMPO由于其捕获负责交联的氨基酸自由基的能力而抑制这种二聚化。通过酪氨酸和色氨酸阻断的化学修饰表明酪氨酸残基是二聚体形成的一个位点。结核病药物异烟肼（INH）的存在也阻止了交联，因为它与蛋白质自由基竞争。蛋白质DMPO硝酮加合物也产生了连续流量的过氧化氢。这些发现表明，蛋白质基自由基不仅在Mtb KatG周转与烷基过氧化物，而且在过氧化氢的存在下形成。此外，在异烟肼的存在下，蛋白质-DMPO硝酮加合物的形成加速。
A new approach, the immuno-spin trapping assay, used a novel rabbit polyclonal anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antiserum to detect protein radical-derived DMPO nitrone adducts in the hemoprotein Mycobacterium tuberculosis catalase-peroxidase (KatG). This work demonstrates that the formation of protein nitrone adducts is dependent on the concentrations of tert-BuOOH and DMPO as shown by Western blotting and an enzyme-linked immunosorbent assay (ELISA). We have also detected protein-protein cross-links formed during turnover of Mtb KatG driven by tert-butyl peroxide (tert-BuOOH) or enzymatic generation of hydrogen peroxide. DMPO inhibits this dimerization due to its ability to trap the amino acid radicals responsible for the cross-linkage. Chemical modifications by tyrosine and tryptophan blockage suggest that tyrosine residues are one site of formation of the dimers. The presence of the tuberculosis drug isoniazid (INH) also prevented cross-linking as a result of its competition for the protein radical. Protein-DMPO nitrone adducts were also generated by a continuous flux of hydrogen peroxide. These findings demonstrated that the protein-based radicals were formed not only during Mtb KatG turnover with alkyl peroxides but also in the presence of hydrogen peroxide. Furthermore, the formation of protein-DMPO nitrone adducts was accelerated in the presence of isoniazid.
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影响因子：
4.8
作者：
Chen, YR;Gunther, MR;Mason, RP
通讯作者：
Mason, RP
影响因子：
4.8
作者：
Barr, DP;Gunther, MR;Mason, RP
通讯作者：
Mason, RP
影响因子：
4.8
作者：
Chouchane, S;Girotto, S;Magliozzo, RS
通讯作者：
Magliozzo, RS
影响因子：
3.3
作者：
Dikalov, S;Jiang, JJ;Mason, RP
通讯作者：
Mason, RP
影响因子：
4.8
作者：
Lardinois, OM;de Montellano, PRO
通讯作者：
de Montellano, PRO