Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase.
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.
Ranguelova
Kalina
K
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, MD F0-01, 111 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709, USA. ranguelovak@niehs.nih.gov
Suarez
Javier
J
Magliozzo
Richard S
RS
Mason
Ronald P
RP
D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry*; D002374:Catalase / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007538:Isoniazid / Q000494:pharmacology; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009589:Nitrogen Oxides; D016877:Oxidants / Q000494:pharmacology; D011817:Rabbits; D013113:Spin Labels; D019097:Spin Trapping
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003497:Cyclic N-Oxides; D005609:Free Radicals; D009589:Nitrogen Oxides; D016877:Oxidants; D013113:Spin Labels; C015388:nitrones; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid