Investigation on radical-based damage of RNase A in aqueous solution and lipid vesicles.

The y-irradiation of bovine pancreatic ribonuclease A (RNase A) in aqueous solution were investigated at different doses by vibrational spectroscopy as well as enzymatic assay, electrophoresis, and HPLC analysis. Both functional and structural changes of the protein were caused by attack of H atoms and OH radicals. In particular, Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and amino acidic residues that are preferential sites of the radical attack (i.e., tyrosine and methionine). After partial structural changes by the initial radical attack, the internal sulfur-containing amino acid residues were rendered susceptible to transformation. By using the biomimetic model of dioleoyl phosphatidyl choline vesicle suspensions containing RNase A, the damage to methione residues could be connected to a parallel alteration of membrane unsaturated lipids. In fact, thiyl radical species formed from protein degradation can diffuse into the lipid bilayer and cause isomerization of the naturally occurring cis double bonds. As a consequence, trans unsaturated fatty acids are formed in vesicles and can be considered to be markers of this protein damage.