2.5. DNA-Binding Studies

Absorption spectra of the complexes in the presence of calf thymus DNA were recorded, subtracting the DNA absorption. Six solutions in 5 mM Tris-HCl (pH = 7.4) and 50 mM NaCl were prepared by maintaining the concentration of copper complexes at 20 µM (5% v/v DMSO was used to enhance the solubility of the complexes as they are not soluble in aqueous mediums) and varying the ratio of [DNA]/[Cu complex] (from 0 to 8.0). To compare the quantitative affinity of the complexes bound to DNA, the intrinsic-binding constant K_b can be calculated from the following Equation (1) based on the titration process:

where εA, εB and εF correspond to the extinction coefficient for the copper complex at the specific addition of DNA, before the addition of DNA, and at the fully bound mode, respectively. K_b is obtainable from plotting [DNA]/(εA − εF) versus [DNA] [33].

A solution containing 100 µM ct-DNA and 10 µM ethidium bromide (EtBr) was prepared in aqueous Tris-HCl/NaCl buffer system (pH = 7.2). The ct-DNA-EtBr solutions were incubated for at least 24 h. The solution was treated with different concentrations of each one of the copper complexes in DMSO (the amount of DMSO was ca. 5% v/v), maintaining the ct-DNA and EtBr concentrations. The changes in the emission spectra of the solutions were followed between 500 nm and 750 nm upon excitation at 390 nm after incubating the complexes for four min. The Stern-Volmer quenching constants (K_SV) were calculated using the Equation (2) below:

where F_o and F are the emission intensities in the absence and the presence of the samples, respectively. The [Cu complex] was plotted against [F_o/F]; the K_SV value was equal to the slope [33,34].

The viscosity measurements were conducting using an Ostwald viscometer. Micro volumes (10 µL) of copper complexes were added to a solution of ct-DNA in buffer where the [Cu complex]/[DNA] ratio was maintained in the range 0.02 to 0.2. The solutions were allowed to incubate for four min at 25 °C in a water bath before measurements. The flow times for the solutions were recorded and replicated at least four times. The relative viscosity (η/η_o)^1/3 were plotted against [Cu complex]/[DNA], where η_o and η represent the specific viscosity of the ct-DNA alone and the ct-DNA-Cu, respectively. The specific viscosity η and η_o were calculated using the formulation [(t − t_b)/t_b] where t is the observed flow time and t_b is the buffer flow time [33,34].