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The damage caused by ionizing radiation to biomolecules, especially DNA, is the reason to treat cancer via radiation therapy. A better understanding of the molecular processes and the quantification of the various damaging mechanisms is the prerequisit to develope efficient therapies. Hereby the understanding of the processes involved in the damage to DNA are of key interest due to its central role in reproduction and mutation. Due to the high amount of water in biological tissue, most of the damage is caused by the secondary particles produced by the interaction of the IR with water. Thereby a multitude of species are produced, e.g. kinetic low energy electrons, prehydrated electrons, OH-radicals and ions. The quantification of the contribution to DNA damage of the various species is of interest. Here we present an experimental approach to disentangle their relative DNA strand break yield. Plasmid DNA is irradiated in water with electrons under the presence of different scavengers. With the presented method it will be possible to reveal the relative contributions of OH-radicals, low energy electrons and prehydrated electrons to the DNA single and double strand break yield.
Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.