The scDNA produced two clear bands representing the super-coiled (Form-I) and relaxed circular form (Form-II) of scDNA in the absence of the M(bpy)2 complex, as expected. The presence of the Cu(bpy)2 complex resulted in the disappearance of both bands corresponding to Forms-I and -II (lanes 2–3, Fig. 2). The bands were smeared, suggesting that scDNA cleavage occurred at more than one place. On the other hand, the Zn(bpy)2 and Cd(bpy)2 complexes showed very small or no cleavage activity under the conditions adopted in this study. A range of reactive oxygen species (ROS) might be involved in the cleavage process. To identify

find more the nature of ROS species, the effect of ROS scavengers was tested [28], [29], [30] and [31]. Fig. 3 shows the effect of the scavengers on scDNA cleavage by the Cu(bpy)2 complex detected by electrophoresis.

The presence of 5 mM sodium azide and 50 mM DMSO (lanes 4 and 5, Fig. 3) showed very little inhibition for the cleavage process. Similar to that in the absence of scavengers check details which produced a predominantly smeared band (lane 1), the presence of either sodium azide or DMSO resulted in a smeared band. Considering that sodium azide and DMSO are scavengers for singlet oxygen (1O2) and hydroxyl radicals (·OH), these two oxygen species did not participate in the cleavage process. Tiron, a superoxide radical, ·O2−, scavenger, had a large inhibition effect on the cleavage of scDNA (lane 3). The smeared band did not appear whereas the amount of scDNA decreased with increasing Form-II band (nicked open circular form). The presence of catalase, a H2O2 scavenger, also resulted in the disappearance of the smeared band whereas the bands correspond to nicked circular and linear forms were apparent. This suggested that the role of the oxygen radical is essential for the Cu(bpy)2-induced scDNA cleavage. LD is an excellent tool for probing the cleavage of sc and dsDNA [15], [16], [17], [18] and [32]. This real-time LD technique is based

on the fact that the magnitude of LD in the DNA absorption region solely reflects the flexibility and length of DNA if the other factors including the optical density, viscosity, temperature and flow rate (in the flow orientation case) are kept constant. Fig. 4 shows the LD spectrum of the crotamiton DNA in the presence and absence of the Cu(bpy)2 complex at the time of mixing and 20 min after mixing. In the absence of the Cu(bpy)2 complex, the LD spectrum of dsDNA was negative and its shape resembled the negative of the absorption spectrum as expected from the set-up adopted in this study [17], [18] and [19]. Both the shape and intensity of the LD spectrum of dsDNA remained in the absence of the Cu(bpy)2 complex. The maximum intensity was observed at 258 nm. The presence of the Cu(bpy)2 complex caused a decrease in intensity, a 5 nm red-shifted maximum and a positive band above 300 nm at the time of mixing.