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

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 NO is not just a source of NO2.