Studies on the interactions of nogalamycin with duplex DNA

Abstract

1. (1) Electron micrographs of T7 DNA molecules complexed with nogalamycin at r = 0.15, when prepared for electron microscopy by using ammonium acetate as a hypophase, were remarkably free from the “microkinks” which were present in abundance in free-DNA micrographs prepared under similar conditions. Microkinks could be removed by the stretching resulting from the intercalation of nogalamycin between the adjacent base pairs of T7 DNA. The average length extension of the complexed T7 DNA molecules at r = 0.12–0.17 was 23% of the length of the native T7 DNA. This gives an upper limit of intercalative binding sites of nogalamycin in T7 DNA as 11 nogalamycin molecules intercalating per 100 nucleotides. Calf thymus DNA molecules complexed with nogalamycin at P/D = 0.05–0.10 were thickened structures, with intra- and inter-molecular aggregation, probably indicating the stacking of polymeric nogalamycin molecules on the external surface of native DNA. 2. (2) The relative viscosity of the nogalamycin · DNA complexes in phosphate/EDTA/saline buffer (0.2 M Na +) rose sharply with increasing r, up to r = 0.12 or up to D/P = 0.22 and up to r = 0.12 in 0.001 M Tris · HCl plus 1 M NaCl. This proves that nogalamycin intercalates in native DNA and indicates that the upper limit of intercalating sites is nearly 12 per 100 nucleotides in high molar solvents. 3. (3) The binding of nogalamycin in native calf thymus DNA, as shown by Scatchard plots, seems to be dependent on the ionic strength of the environment. There was a 27% reduction of the total binding sites as the environment was changed from 0.001 M Tris · HCl, pH 7.4 to that plus 1 M NaCl. However, the strong binding sites, as obtained from the binding isotherms, were 0.10 per nucleotide and did not show variation in the above range of ionic strengths. 4. (4) Thermal absorbance profiles of the complexes of nogalamycin, daunomycin and ethidium bromide with native DNA at P/D = 14 showed the release of the bound drug molecules in the helix-coil transition zone of the complexes. However, the profiles of those at P/D = 4 showed that a fraction of the bound drug molecules were released at earlier temperatures, suggesting that this fraction was weakly bound. The results, as a whole, indicated (i) a close similarity between the strong binding sites and the intercalative sites of nogalamycin in native DNA and (ii) the involvement of nogalamycin in weak interactions with native DNA.