Specific electrochemical nitration of horse heart myoglobin

Earlier findings on electronitration of hen egg-white lysozyme demonstrated a product which was mononitrated at Tyr23, by ion-exchange chromatography, absorbance at 430 nm, dithionite reduction, and Edman sequencing of a nitr ated proteolytic peptide. However, the whole protein was not sequenced; the refore, although the enzyme remained active upon nitration, reaction at oth er residues could not be completely eliminated. This study has now been ext ended to the redox protein myoglobin. We demonstrate the novel electronitra tion (electrooxidation in the presence of nitrite) of a specific tyrosine r esidue in horse heart myoglobin and also in apomyoglobin. Production of the yellow chromophore, 3-nitrotyrosine (3-NT), was apparent in apomyoglobin f rom A(430) but was masked in holomyoglobin by the Soret band. In both cases ' the presence of 3-NT in the electronitrated samples was further indicated by the binding of antibody to 3-NT in Western blots. High-resolution elect rospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry revealed a reaction product at [M + 45] (consistent with substitution of NO, for H), indicating that the nitration reaction is the o nly reaction occurring which gives rise to a change I. Ln mass in the elect rooxidation. Fragmentation mass spectrometry identified the nitration site as Tyr103, with no nitration at Tyr146. The procedure may be useful in prep aring model nitrated proteins for the study of disease mechanisms. (C) 2001 Academic Press.