Calf thymus dna

To check whether tested compounds may interact with DNA, an EtBr displacement assay was carried out according to the method described by Cain et al. [35 (link)]. A 3 mL buffer containing 2 mM Hepes, 0.01 mM EDTA, 9.4 mM NaCl 1.26 µM EtBr, pH 7.0 was mixed in a fluorescence cuvette with 1 µL of 1 mg/mL solution of calf thymus DNA (Worthington, Lakewood, NJ, USA). The cuvette was placed in a fluorescence spectrophotometer (Perkin-Elmer LS55, Waltham, MA, USA). Then 1 µL portions of compound solutions (20 mM in DMSO) were added step by step and after thorough mixing the intensity fluorescence was read (Ex 546 nm, Em 595 nm). This method is based on the competition of an added compound with EtBr for DNA intercalation sites. The fluorescence intensity of EtBr increases upon DNA binding. The addition of a compound displacing intercalated EtBr leads to quenching the fluorescence caused by the EtBr/DNA complex. The percent of fluorescence decrease was plotted against the concentration (µM) of each compound and C₅₀ value of each was determined. C₅₀ is defined as the concentration of the added compound required to reduce the fluorescence of the EtBr/DNA complex to 50%. C₅₀ values were used to calculate apparent binding constants (K_app) of tested compounds to DNA [36 ].