DNA-protein crosslinks induced by protein peroxides

In the study reported here, the authors investigate the action of protein peroxides on DNA, using BSA or insulin as model proteins. Solutions of BSA or insulin were irradiated with gamma rays to produce protein peroxide adducts. After complete reduction of the radiation generated H202 with a small amount of catalase, the protein solutions were incubated at 37 deg C with DNA . This treatment led to retarded migration and band broadening during agarose gel electrophoresis. The extent of retardation was proportional to incubation time and protein peroxide concentration. It was deduced that incubation of DNA with peroxidised proteins led to formation of covalent crosslinks between the molecules. A DNA gel shift could also be prevented by complete reduction of the protein peroxide groups before incubation. When the peroxide groups were allowed to partially autodecompose over a period of days, the gel shift was progressively decreased. To determine whether secondary radicals arising from the decomposition of the protein peroxide groups were responsible for the crosslinks, free radical scavengers were included during incubation. Of the scavengers DMSO, sodium formate, mannitol, tert-butanol, Trolox and ascorbate, only sodium formate was able to prevent a gel shift, and this was probably due to its salt effect rather than to any radical scavenging