Immunochemical detection of nitric oxide and nitrogen dioxide trapping of the tyrosyl radical and the resulting nitrotyrosine in sperm whale myoglobin

Free Radical Biology & Medicine
Kozo Nakai, Ronald P Mason

Abstract

We demonstrate herein that nitric oxide (*NO) and nitrogen dioxide (*NO2) both react with the tyrosyl radical formed in sperm whale myoglobin (swMb) by reaction with hydrogen peroxide. The tyrosyl radical was detected by Western blotting using a novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts. In the presence of DMPO, hydrogen peroxide reacts with swMb to form the DMPO tyrosyl radical as is known from both electron spin resonance and immuno-spin trapping investigations. Both *NO and NO2- significantly suppressed DMPO-Mb formation under the physiological oxygen tension of 30 mm Hg. If this inhibition of DMPO trapping of the tyrosyl radical is due, at least in part, to the reaction of the tyrosyl radical with *NO and *NO2, then nitrotyrosine should be formed. In line with this expectation, swMb treated with low concentrations of *NO or NO2- formed nitrotyrosine when hydrogen peroxide was added under 30 mm Hg oxygen tension as detected by Western blotting. The amount of nitrotyrosine generated with *NO was higher than with NO2-, implying that there are two different peroxynitrite-independent nitrotyrosine formation mechanisms and that...Continue Reading

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Citations

Jan 22, 2008·The Journal of Biological Chemistry·Agnes KeszlerNeil Hogg
Mar 16, 2006·Proceedings of the National Academy of Sciences of the United States of America·Kozo NakaiRonald P Mason
Jun 28, 2008·Free Radical Biology & Medicine·Eleanor C Kennett, Michael J Davies
Mar 29, 2014·IUBMB Life·Marcelo G BoniniAlyssa M Master
Mar 6, 2010·Journal of the European Academy of Dermatology and Venereology : JEADV·K NakaiY Kubota
Apr 10, 2013·Biochimica Et Biophysica Acta·Clare L Hawkins, Michael J Davies
Apr 4, 2014·Chemical Reviews·Luisa B Maia, José J G Moura
Sep 1, 2009·The Journal of Immunology : Official Journal of the American Association of Immunologists·Saurabh ChatterjeeRonald P Mason
Sep 1, 2016·Porto Biomedical Journal·Daniel Filipe Cruz, Margarida Fardilha

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