Structural damages of sulfur-containing proteins caused by radical stress: assessment by Raman spectroscopy and tandem radical potential

By escaping the cell's antioxidant defences, free radicals can damage biomolecules, including proteins, leading to modifications involved in many degenerative diseases. In fact, the exposure of proteins to free radicals may cause structural and functional changes. In particular, a single radical event that leads to an initial damage involving sulfur-containing amino acid residues could produce a reactive species able to damage another cell compartment such as lipid domains. In this context the radical-induced damages on sulfur-containing proteins, such as ribonuclease A (RNase A), were evaluated as well as the possible post-translational mechanism of the damage to another cell compartment, such as lipid domain. Free radical generation, mimicking an endogenous radical stress, was obtained by ?-irradiation of aqueous solutions. By changing the appropriate conditions of irradiation, a selection of the reacting radical species was carried out. In particular, the separate contribution of a reducing species such as Ho was investigated more in detail in the present work.1,2 Neutral hydrogen atom is a simple one-electron equivalent reducing agent and its reactions are of great interest since they can shed light on basic problems of one-electron equivalent reaction kinetics. RNase A is a small protein (MW 13700) cross-linked by four disulfide bonds and consisting of 124 amino acid residues of which six are Tyr, four Hys and four Met; Trp and free Cys are not present. The protein degradation due to radical exposure was evaluated by Raman spectroscopy. In fact, Raman spectrum can provide valuable information on amino acid side chains (i.e. S-S, Tyr, Trp, Cys-Metal) and conformational changes in the protein secondary structure Specific damages occur at sensitive amino acid sites, selectively, rather than indiscriminately, leading to the structure modification of the protein. Sulfur-containg residues (Met and Cys) and aromatic residues are appreciably attacked. In particular, exposure of the protein even at the lowest irradiation dose produces significant changes in the C-S bonds of the Met residues, as indicated by the splitting of the 724 cm-1 band (731 and 725 cm-1), as well as in Cys, indicating that the action of Ho atoms gives rise to a profound change in the sulfur-containing residues.