The generation of ferryl or hydroxyl radicals during interaction of haemproteins with hydrogen peroxide.

The oxidation of 2-keto-4-thiomethyl butyric acid (KTBA) and methionine to ethylene has been used to evaluate generation of ferryl species or hydroxyl radicals by H2O2-activated haemproteins or free ferric ions. Hydrogen peroxide was generated by a glucose oxidase-glucose system at a rate of 1 microM/min. Free ferric in the presence of H2O2 oxidizes KTBA, and this was highly inhibited by hydroxyl radical scavengers, caeruloplasmin, superoxide dismutase (SOD) and EDTA. However, when metmyoglobin, methaemoglobin (MtHb) or horseradish peroxidase (HRP) were tested in the same model system, hydroxyl radical scavengers suppressed partially KTBA oxidation and caeruloplasmin, SOD and EDTA failed to inhibit the reaction. Cytochrome-c was found to be a weak promoter of KTBA oxidation in the presence of H2O2. Methionine was oxidized to ethylene by an active system which generates hydroxyl radicals, but not by H2O2-activated metmyoglobin. Ferric ions chelated to membranes or ADP in the presence of H2O2 generated enzymatically, initiated membranal lipid peroxidation only in the presence of ascorbic acid, and this was inhibited by EDTA. In contrast, metmyoglobin and methaemoglobin activated by H2O2 generated by the same system, initiated membranal lipid peroxidation and this was not inhibited by EDTA. It is concluded that ferryl and not HO. is the main oxidant in systems containing myoglobin and haemoglobin activated by low concentrations of H2O2.