Interaction of bleomycin A2 with DNA studied by resonance Raman spectroscopy: intercalation or groove binding?

A systematic approach toward the analysis of drug–DNA interactions using Raman spectroscopy: The binding of metal‐free bleomycins A 2 and B 2 to calf thymus DNA

“…Concentrations were determined using uv spectrophotometry, ⑀ ct-DNA ϭ 13.2 cm Ϫ1 mM Ϫ1 /base pair. 11 Bleomycin sulfate was purchased from Sigma (St. Louis, MO) and was used as the mixture of Blm A 2 ϩ B 2 without purification for some of the studies. Blm B 2 was purified using high performance liquid chromatography using a C18 reversed phase column, a flow rate of 2 mL/min and a linear gradient that is achieved using two mobile phases: A ϭ 0.2% trifluoroacetic acid, B ϭ methanol.…”

“…7,11 Samples were prepared such that the ratio of drug:ct-DNA base pairs was 1:10. The final concentration of Blm was ϳ3-3.5 mM while that of ct-DNA was 30 -35 mM base pairs (⑀ ϭ13.0 cm Ϫ1 mM Ϫ1 ).…”

“…The final concentration of Blm was ϳ3-3.5 mM while that of ct-DNA was 30 -35 mM base pairs (⑀ ϭ13.0 cm Ϫ1 mM Ϫ1 ). 11 The ct-DNA sample was scanned first, and then the appropriate amount of Blm was added and the previously scanned DNA, this time drug bound, was scanned. Addition of Blm to DNA was accomplished using small 5-10 L addition volumes containing a relatively concentrated drug solution, such that the concentration of solvent remained close to constant for both samples, ct-DNA and Blm/DNA complex.…”

“…This often accompanies changes in base pair stacking interactions 22 and is evidence for some intercalation of Blm between the bases. 11,34,35 In the spectral region 1160 -1290 cm Ϫ1 there are numerous downshifts in frequency. In particular, one band at 1215 cm Ϫ1 ; assigned to G, with significant contributions from the C8 -N9 coordinate of the imidazole ring has an intensity loss of ϳ50%, downshifts by 3 cm Ϫ1 , and also becomes narrower by 6 cm Ϫ1 .…”

“…A recent uv resonance Raman study (UVRR) 11 focused on the interactions of Blm A 2 with calf thymus DNA, poly(dA-dT)-poly(dA-dT), and poly(dGdC)-poly(dG-dC). Mixtures of Blm A 2 with poly(dAdT)-poly(dA-dT) and with calf thymus DNA resulted in marked Raman hypochromism of a thymidine marker band while no hypochromism was seen for any of the resonances in poly(dG-dC)-poly(dG-dC).…”