Hydroperoxide-mediated fragmentation of proteins

Abstract

1. Chemiluminescence and benzoic acid hydroxylation were used to detect oxygen-centred free-radical production by 2.5 mM-H2O2 and 100 microM-Cu2+. Free radicals could not be detected by these methods when H2O2 was replaced with 10 mM-t-butyl hydroperoxide (TBH) or 10 mM-cumene hydroperoxide (CH). The inclusion of the thiol compound dithioerythritol (DTET; 100 microM) increased radical production by H2O2 and Cu2+ as judged by both assays. Mannitol scavenged radicals in the chemiluminescence system in a dose-dependent manner. 2. H2O2, TBH and CH, each with Cu2+, gave rise to substantial fragmentation of the protein bovine serum albumin (BSA). This fragmentation could be increased by the inclusion of DTET. Omission of Cu2+ or the addition of the chelator DETAPAC (diethylenetriaminepenta-acetic acid; 1 mM) lead to virtual abolition of fragmentation. Autoxidized lipid in the presence of Cu2+ caused protein fragmentation by reactions of lipid hydroperoxides. 3. Polyacrylamide-gel electrophoresis in the presence of SDS confirmed that production of fragments had occurred. 4. Susceptibility of BSA to enzymic hydrolysis by two different proteinases acting at pH 5 and pH 7.2 was increased after a limited exposure to hydroperoxides in the presence of Cu2+. 5. These results may have biological significance, particularly for proteins in lipid environments (e.g. membrane proteins and lipoproteins).