Photoinduced Protein Nitration Method by Sensitizer Tris(bipyridine)-Ruthenium (II) Chloride Complex

GIMÉNEZ, EZEQUIEL; THOMAS M. JOVIN; CAVAZZUTTI, GIAN FRANCO; HENNING URLAUB; ANDRÉS MARTÍN TOSCANI; FALOMIR-LOCKHART, LISANDRO JORGE

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

The addition of a -NO2 group to the side chain of proteins is a well-known posttranslational modification (PTM) related, not only to physiological processes, but also to awide range of pathologies triggered or promoted by oxidative stress, including the agingprocess. The -NO2 group is mainly added to Tyrosine (Tyr) residues, but can also beincorporated to side chains of Tryptophan, Phenylalanine and Cysteine. Since this PTM isa common feature in several chronic diseases and disorders, in vitro studies haveemployed chemical reagents such as tetranitromethane and peroxynitrite as nitrativeagents to model oxidative conditions. However, photochemical methods have aroused aspowerful alternatives, in which different photosensitizers, such as rivoflavin and pterins,together with light, trigger radical reactions involved in oxidative processes but in a morecontrollable way. In this regard, Tris(bipyridine)-Ruthenium(II) Chloride complex(Ru(bpy)3Cl2) has been successfully used as sensitizer in photoinduced crosslinkingreactions of unfolded proteins (PICUP), particularly with alpha-Synuclein (Borsarelli et al,Free Rad. Biol. Med., 2012). Despite itsproven potential as photosensitizer, it has not been tested for protein nitration.Therefore, we have adapted PICUP protocols to favors nitration over crosslinking inphotochemical reactions induced by Ruthenium complexes, ammonium persulfate andlight in order to systematically produce recombinant proteins with these types of PTMsfor further characterization.Our work first focused on the characterization and optimization of conditions for free Tyrresidues nitration, including putative side reactions and interference by other aminoacids. Then we evaluated the modification of different model proteins and its effect overtheir activity employing spectroscopic techniques and mass spectrometry. Supported byour results, we propose a novel method to massively produce nitrated proteins, whichcould be employed to investigate the consequences of introducing -NO2 on proteins as amodel for oxidative stress-related pathologies in vitro.