Formation of protein-derived electrophiles in ribonuclease A by biologically relevant oxidants

Oxidative modifications in proteins have been extensively studied and found to increase in diabetes, cardiovascular diseases, neurodegenerative diseases, and aging. Some of the most studied modifications include the nitration of tyrosine and the formation of carbonyls in proteins. Tyrosine can also be oxidized to 3-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)-L-alanine (HOCHDA) by several biologically relevant systems, a product that is electrophilic and reactive to biological nucleophiles such as glutathione. Herein, we characterized the reaction of a peptide containing HOCHDA with fluorescein-tagged glutathione by HPLC and mass spectrometry. To explore the possibility that the formation of oxidation-derived electrophiles occurs in proteins, we oxidized the tyrosine-rich, small protein, ribonuclease A, by different biologically relevant oxidizing systems and used fluorescein-tagged glutathione as the nucleophilic reagent. Oxidation of ribonuclease A with singlet oxygen, known to generate HOCHDA efficiently, generated an electrophile that reacted with fluorescein-tagged glutathione and was resistant to reduction by dithiothreitol. The amount of fluorescein-glutathione attached to the protein was quantified by gel filtration HPLC. Other oxidants such as peroxyl radical (from AAPH), ferryl (from hydrogen peroxide reaction with Fe(II):EDTA), and peroxynitrite, also generated a modified protein that reacted with fluorescein-glutathione. Analysis by LC-MS/MS indicated the formation of mono-oxygenated tyrosyl residues and di-oxygenated histidyl residues after exposure of the protein to AAPH which are good candidates to be the electrophilic centers. The formation of electrophiles was a common feature in the reactions of oxidants with ribonuclease A and may constitute an underappreciated mechanism of protein oxidative modification.