[17] Detection and quantitation of nitrotyrosine residues in proteins: In vivo marker of peroxynitrite

This chapter discusses the detection and quantitation methods of nitrotyrosine residues in proteins. Nitrotyrosine is detected in human diseases associated with oxidative stress and is visualized using immunological techniques in atherosclerotic plaques of human coronary vessels, in lungs of infants with acute lung injury and sepsis, and in adult respiratory distress syndrome (ARDS). High-performance liquid chromatography (HPLC) analysis is used to detect nitrotyrosine in synovial fluid from patients with rheumatoid arthritis. Peroxynitrite can be synthesized from sodium nitrite and acidified hydrogen peroxide. Selective tyrosine nitration can be accomplished by titrating protein with tetranitromethane (TNM) at neutral or alkaline conditions (pH 7–8). TNM is a potent carcinogen, which must be handled carefully. The residual TNM and trinitromethane must be removed prior to nitrotyrosine quantitation. Nitrotyrosine is essentially nonfluorescent whereas aminotyrosine is highly fluorescent and has a characteristic emission spectrum. Thus, fluorescent detection of aminotyrosine can be used as an alternative to direct detection of nitrotyrosine. Quantitation of nitrotyrosine using the solid-phase immunoradiochemical method has the advantage of high sensitivity and does not require sample manipulation.