Protein radical formation on thyroid peroxidase during turnover as detected by immuno-spin trapping.
Thyroid peroxidase (TPO) is a 933 amino acid residue, heme-containing, integral membrane glycoprotein that catalyzes two steps in the maturation of the thyroid hormone precursor. As with other peroxidases, these reactions require hydrogen peroxide and initial enzyme oxidation. Previous researchers studied the oxidative state of the TPO heme moiety using spectrophotometric and catalytic analyses. We use a novel antiserum to 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to detect radical-derived DMPO spin-trapped TPO. Our work reveals that TPO generates radical adducts in the presence of H2O2, but that the generation of these adducts can be suppressed by the addition of substrates and inhibitors. Chemical alteration of the tyrosine residues of TPO greatly reduces the generation of TPO-DMPO adducts. Iodide strongly suppresses the H2O2-generated production of TPO radical adducts and protects the enzyme from loss of enzyme activity. Because the normal catalytic mechanism of TPO involves the production of radical species, TPO is potentially more susceptible to oxidative damage than most enzymes which do not require H2O2 as a substrate. We hypothesize that oxidatively damaged TPO may trigger the production of anti-TPO autoantibodies, resulting in the development of autoimmune thyroid disorders. Evidence that correlates iodine deficiencies with development of thyroid autoimmune disorders supports this conjecture.
Ehrenshaft
Marilyn
M
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. ehrensh1@niehs.nih.gov
Mason
Ronald P
RP
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004105:Diiodotyrosine / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007453:Iodide Peroxidase / Q000378:metabolism*; D007470:Monoiodotyrosine / Q000494:pharmacology; D009589:Nitrogen Oxides / Q000378:metabolism; D019097:Spin Trapping / Q000379:methods*; D014443:Tyrosine / Q000494:pharmacology
D000596:Amino Acids; D003497:Cyclic N-Oxides; D005609:Free Radicals; D009589:Nitrogen Oxides; C015388:nitrones; D014443:Tyrosine; D004105:Diiodotyrosine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide; D007453:Iodide Peroxidase; D007470:Monoiodotyrosine