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

GIMENEZ E; CAVAZZUTTI, GF; TOSCANI AM; JOVIN TM; URLAUB H; FALOMIR LOCKHART LJ

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 a wide range of pathologies triggered or promoted by oxidative stress, including the aging process. The -NO2 group is mainly added to Tyrosine (Tyr) residues, but can also be incorporated to side chains of Tryptophan, Phenylalanine and Cysteine. Since this PTM is a common feature in several chronic diseases and disorders, in vitro studies have employed chemical reagents such as tetranitromethane and peroxynitrite as nitrative agents to model oxidative conditions. However, photochemical methods have aroused as powerful alternatives, in which different photosensitizers, such as rivoflavin and pterins, together with light, trigger radical reactions involved in oxidative processes but in a more controllable way. In this regard, Tris(bipyridine)-Ruthenium(II) Chloride complex (Ru(bpy)3Cl2) has been successfully used as sensitizer in photoinduced crosslinking reactions of unfolded proteins (PICUP), particularly with alpha-Synuclein (Borsarelli et al, Free Rad. Biol. Med., 2012). Despite its proven potential as photosensitizer, it has not been tested for protein nitration.Therefore, we have adapted PICUP protocols to favors nitration over crosslinking in photochemical reactions induced by Ruthenium complexes, ammonium persulfate and light in order to systematically produce recombinant proteins with these types of PTMs for further characterization.Our work first focused on the characterization and optimization of conditions for free Tyr residues nitration, including putative side reactions and interference by other amino acids. Then we evaluated the modification of different model proteins and its effect over their activity employing spectroscopic techniques and mass spectrometry. Supported by our results, we propose a novel method to massively produce nitrated proteins, which could be employed to investigate the consequences of introducing -NO2 on proteins as amodel for oxidative stress-related pathologies in vitro.AcknowldegmentsThe present work has been possible thanks to the support of CONICET, UNLP, ANPCyT and, particularly, to the Foundations Bunge y Born, Williams and Max Planck