650 spectrophotometer

The UV/Vis spectroscopy studies were performed using a JASCO 650 spectrophotometer provided by the PROTEOMASS-BIOSCOPE facility. A Bruker TENSOR (REQUIMTE-Chemistry Department, FCT-UNL) spectrophotometer was used to obtain the FT-IR spectra; All FT-IR experiments were performed in KBr disks.

The UV–Vis spectra of 1.8 mL samples containing 20 µM of the examined compounds and escalating volumes of DNA stock solution (0–40 µM with a 5 µM increment) were recorded for the DNA binding investigations. The samples had been prepared in Tris-HCl buffer (5 mM Tris-HCl/ 50 mM NaCl, pH 7.4 adjusted with a 0.1 M HCl solution).
The concentration of the DNA stock solution was calculated by measuring the absorption intensity at 260 nm and using a molecular extinction coefficient of 6600 M⁻¹·cm⁻¹. We established that the DNA stock solution was sufficiently free of protein (the ratio between the absorption at 260 nm and 280 nm, respectively, was found to be in the range 1.8–2) [84 (link)]. Using a Jasco 650 spectrophotometer (Jasco, Tokyo, Japan), these experiments were carried out in 1 cm quartz cells at room temperature.
Equation (2) was used to calculate the binding constant (K_b).
$$\frac{\left[\mathrm{DNA}\right]}{{\mathsf{\epsilon }}_{\mathrm{a}}-{\mathsf{\epsilon }}_{\mathrm{f}}}=\frac{\left[\mathrm{DNA}\right]}{{\mathsf{\epsilon }}_{\mathrm{b}}-{\mathsf{\epsilon }}_{\mathrm{f}}}+\frac{1}{K_b\left({\mathsf{\epsilon }}_{\mathrm{b}}-{\mathsf{\epsilon }}_{\mathrm{f}}\right)}$$
where [DNA] is the nucleobase concentration of DNA in the sample, ε_a, ε_b, ε_f are the apparent absorption coefficient, the extinction coefficient for the completely bound complex, and the extinction coefficient for the free complex, respectively, and K_b is the intrinsic binding constant. The slope (1/(ε_b − ε_f)) to the intercept (1/K_b(ε_b − ε_f)) ratio was used to calculate K_b [103 (link)].