Abstract
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.