Monitoring nitrotyrosinylation of a synthetic peptide by capillary zone electrophoresis.
Proteins may be nitrated on tyrosyl residues (nitrotyrosinylated) by the action of reactive nitrogen species in inflamed tissues. Capillary electrophoresis was used to monitor this reaction in a model system with tetranitromethane as the nitrotyrosinylating reagent and a synthetic pentapeptide containing one tyrosine as the target molecule. The reaction was readily followed by capillary electrophoresis performed at pH 8 and, using an absorption wavelength of 436 nm, the signature spectral characteristics of the nitrotyrosinylated peptide were verified on-line. The peak appearance time for the nitrotyrosinylated peptide was more than 1 min longer than that of the starting material and a single main product was observed in contrast to the case when peroxynitrite was used as the nitrotyrosinylating reagent. Capillary electrophoresis appears to be a convenient method for the optimization of nitrotyrosinylation, examination of reaction inhibitors, and for studies of the consequences of nitrotyrosinylation, e.g., for antibody binding and for the function of the target protein or peptide.
Bakhøj
A
A
Department of Autoimmunology, Statens Serum Institute, Copenhagen, Denmark.
Heegaard
N H
NH
D019075:Electrophoresis, Capillary / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis; D013774:Tetranitromethane; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D010455:Peptides; C002744:3-nitrotyrosine; D014443:Tyrosine; D013774:Tetranitromethane