Impact of Peroxynitrite Modification on Structure and Immunogenicity of H2A Histone

Peroxynitrite is an extremely reactive entity and has in vivo existence. Its interaction with biomolecules may cause oxidation and nitration. In this study, commercially available H2A histone was exposed to peroxynitrite generated in vitro. The peroxynitrite-mediated structural changes in histone were studied by ultraviolet & fluorescence spectroscopy, high performance liquid chromatography, anilinonaphthalene-8-sulfonic acid binding and polyacrylamide gel electrophoresis. Analysis of results revealed that carbonyl, nitrotyrosine and dityrosine contents were significantly increased in peroxynitrite-modified H2A compared with native H2A. Rabbits challenged with peroxynitrite-modified H2A induced high titre antibodies. The immunogenicity of peroxynitrite-modified H2A was directly proportional to protein nitrotyrosine content and induced antibodies showed specificity for the immunogen and good cross-reaction with nitrated epitopes of other modified proteins. Formation of high molecular weight immune complex with retarded mobility further supports the specificity of induced anti-100 mu M peroxynitrite-modified H2A antibodies for the immunogen. It may be concluded that induction of anti-H2A histone antibodies could be due to protection of peroxynitrite-modified histone from proteolytic breakdown and its subsequent recognition by immunoregulatory cells as foreign molecule.