Nakai Kozo, Mason Ronald P
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, MD F0-01, Research Triangle Park, NC 27709, USA.
Free Radic Biol Med. 2005 Oct 15;39(8):1050-8. doi: 10.1016/j.freeradbiomed.2005.05.019.
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.
我们在此证明，一氧化氮（NO）和二氧化氮（NO₂）均能与抹香鲸肌红蛋白（swMb）中因与过氧化氢反应而形成的酪氨酸自由基发生反应。使用一种新型的抗5,5-二甲基-1-吡咯啉N-氧化物（DMPO）多克隆抗血清通过蛋白质印迹法检测酪氨酸自由基，该抗血清能特异性识别源自蛋白质自由基的DMPO硝酮加合物。在DMPO存在的情况下，过氧化氢与swMb反应形成DMPO酪氨酸自由基，这在电子自旋共振和免疫自旋捕获研究中均有报道。在30 mmHg的生理氧张力下，NO和NO₂⁻均显著抑制DMPO-Mb的形成。如果酪氨酸自由基捕获的这种抑制作用至少部分是由于酪氨酸自由基与NO和NO₂的反应，那么应该会形成硝基酪氨酸。与这一预期相符的是，通过蛋白质印迹法检测发现，在30 mmHg氧张力下添加过氧化氢时，用低浓度NO或NO₂⁻处理的swMb会形成硝基酪氨酸。NO产生的硝基酪氨酸量高于NO₂⁻，这意味着存在两种不同的与过氧亚硝酸盐无关的硝基酪氨酸形成机制，并且NO不仅仅是*NO₂的来源。
