Oxidative damage to lysozyme by the hydroxyl radical: comparative effects of scavengers

The hydroxyl radical (OH.) is a highly-damaging reactive oxygen species, given its high reactivity and the consequent generation of secondary free radicals. This study was aimed at determining the qualitative and quantitative aspects of OH. scavenging by pentoxifylline (Ptx, a methylxanthine), uric acid and thymine on the OH.-induced alterations of a protein, lysozyme. Lysozyme was inactivated by OH. with a yield of 6.5 mol OH./mol lysozyme; moreover, SDS-PAGE showed a loss of native lysozyme (14.4 kDa), the presence of dimer and trimer aggregates and characteristic fragmentation. Tryptophan fluorescence was lost before aggregation became detectable in terms of bityrosine formation. Increasing concentrations of OH. scavengers gave increasing protection of lysozyme activity. Although all three compounds scavenge OH. with high rate constants, their effects were different: uric acid and Ptx prevented aggregation and preserved enzyme activity, whereas thymine preserved activity but did not prevent aggregation. These differences appear to be related to the formation of reducing secondary radicals, underlining the importance of this mechanism in the effects of scavengers.