Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting

We noted differences in the antibody response to 3-nitrotyrosine (NO(2)Tyr) in fixed and non-fixed tissues, and studied therefore potential problems associated with non-fixed tissues in Western blot analyses. Three different monoclonal anti-nitrotyrosine antibodies in Western blot analysis of inflammatory stimulated rat abdominal, liver and lung tissue homogenates caused no immunoreactivity, in contrast to a polyclonal nitrotyrosine antibody applied in fixed and non-fixed tissues. Western blot studies using both mono- and polyclonal antibodies showed a temperature- and heme group-dependent reduction of NO(2)Tyr in nitrated rat and bovine serum albumin incubated with dithiothreitol. Mass spectrometric analyses of a nitrated peptide angiotensin II revealed under similar conditions a positive temperature effect between 56 and 70 degrees C on reduction of NO(2)Tyr to 3-aminotyrosine which is not detected by anti-NO(2)Tyr antibodies. Western blot analysis may therefore underestimate the level of tissue nitration, and factors causing a reduction of NO(2)Tyr during sample preparation might conceal the actual nitration of proteins.