Differential abilities of nitrogen dioxide and nitrite to nitrate proteins in thylakoid membranes isolated from Arabidopsis leaves.

Exposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in nitration of specific chloroplast proteins. To determine whether NO2 itself and/or nitrite derived from NO2 can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO2-bubbled or potassium nitrite (KNO2) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO2 concentrations in the NO2-bubbled buffer were calculated by numerically solving NO2 dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the 2 treatments was designated nitration by NO2. Both NO2 and nitrite mediated nitration of proteins, and the nitration ability per unit NO2 concentration was ∼100-fold greater than that of nitrite.