Spin Trapping Investigation of Peroxide- and Isoniazid-Induced Radicals in Mycobacterium tuberculosis Catalase-Peroxidase

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• 作者：Kalina Ranguelova ; Javier Suarez ; Richard S. Magliozzo ; Ronald P. Mason
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：October 28, 2008
• 年：2008
• 卷：47
• 期：43
• 页码：11377-11385
• 全文大小：268K
• 年卷期：v.47,no.43(October 28, 2008)
• ISSN：1520-4995

文摘

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.