Propagation of free-radical reactions in concentrated protein solutions

Abstract

The reactions of proteins with biologically relevant oxidants have been widely studied, although most of the work has been performed in diluted homogenous solutions conditions that differ from those in intracellular environments. Cellular compartments represent highly crowded milieu in which high concentrations of biomolecules are present, unspecific intermolecular interactions are promoted, and physicochemical properties of constituents are modified. In this work, we propose that the high concentration at which proteins are present inside cells favours radical oxidative reactions between polypeptides which propagate in an oxygen-dependent process similar to membrane lipid peroxidation. The results presented herein show that highly concentrated solutions of bovine serum albumin (BSA) exposed to peroxynitrite, or metmyoglobin/H₂O₂, initiate the formation and propagation of protein peroxyl radicals, as evidenced by oxygen consumption, fluorescence spectroscopy, chemiluminescence, and electron paramagnetic resonance studies. Moreover, peroxyl radicals are capable of converting nitrite to nitrogen dioxide, which can oxidise amino acid residues to further assist radical-mediated protein oxidation. In addition, we also show that nitrone spin traps stop these propagation reactions in proteins, in line with the previously reported antioxidant role of these compounds in vivo. In summary, our results suggest that in crowded environments such as cellular compartments radical chain reactions propagate protein oxidative damage, highlighting a previously under recognised mechanism of cellular nitroxidative stress.

Keywords:

• Protein radicals
• nitrones spin traps
• oxidative stress
• peroxyl radicals
• peroxynitrite

Disclosure statement

Neither of the authors have conflict of interest.

Additional information

Funding

A.A. and M.M. were partially supported by fellowships from Agencia Nacional de Investigación e Innovación (ANII, Uruguay) and Universidad de la República (CAP/CSIC), respectively. This work was supported by grants of Universidad de la República (CSIC and Espacio Interdisciplinario) and Agencia Nacional de Investigación e Innovación [Fondo Clemente Estable, FCE_2014_104233]. Additional funding was provided by PEDECIBA and Ridaline, the latter through Fundación Manuel Pérez, Facultad de Medicina, Universidad de la República, Uruguay.