Evaluating Cu(II)-Peptide DNA Damage

In vitro DNA damage brought about by the Cu^II–peptide complexes were assessed by agarose gel electrophoresis. Reaction mixtures contained 10 μm base pair pUC19, 100 nm Cu^II–ATCUN–AMP with 1 mm H₂O₂ and 1 mm sodium ascorbate in 20 mm HEPES, 100 mm NaCl, at pH 7.40. Reaction was incubated at room temperature and quenched by adding 3X loading dye containing 1 mm EDTA at two time points—30 min and 2 h. Then, a 15 μL aliquot was loaded on a 1 % agarose gel containing ethidium bromide (EtBr) and run at 80 V for 90 min. Gels were imaged using a Bio-Rad GelDoc XR+ Imager, and bands were quantified using the accompanying Image Lab 5.0 software. A correction factor or 1.47 was applied to the intensity of the supercoiled form to account for its decreased ability to intercalate EtBr.^{[25 (link)]} Normalized DNA cleavage activity was calculated using the initial and final amounts of supercoiled DNA according to the formula: [(initial–final)/initial] × 100. Quantified supercoiled DNS is shown as mean ± standard deviation of three independent measurements.