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 nitrated proteolytic peptide. However, the whole protein was not sequenced; therefore, although the enzyme remained active upon nitration, reaction at other residues could not be completely eliminated. This study has now been extended to the redox protein myoglobin. We demonstrate the novel electronitration (electrooxidation in the presence of nitrite) of a specific tyrosine residue in horse heart myoglobin and also in apomyoglobin. Production of the yellow chromophore, 3-nitrotyrosine (3-NT), was apparent in apomyoglobin from A430 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 electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry revealed a reaction product at [M + 45] (consistent with substitution of NO2 for H), indicating that the nitration reaction is the only reaction occurring which gives rise to a change in mass in the electrooxidation. Fragmentation mass spectrometry identified the nitration site as Tyr103, with no nitration at Tyr146. The procedure may be useful in preparing model nitrated proteins for the study of disease mechanisms.
Kendall
G
G
Centre for Molecular and Biomolecular Electronics, School of Science and the Environment, Coventry University, Coventry, CV1 5FB, United Kingdom.
Cooper
H J
HJ
Heptinstall
J
J
Derrick
P J
PJ
Walton
D J
DJ
Peterson
I R
IR
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005583:Fourier Analysis; D006736:Horses; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; C002744:3-nitrotyrosine; D014443:Tyrosine; D009584:Nitrogen