Abstract
The binding parameters of ethidium bromide (EtBr) with DNA of various GC/AT ratios were determined using absorption and fluorescence spectroscopy. Our experimental data clearly demonstrate the co-existence of fluorescing and non-fluorescing types of “strong” binding at low concentration of EtBr. The fluorescent complex corresponds to the ordinary intercalative model. The non-fluorescent complex is referred to semi-intercalative binding of EtBr. The binding constant (K) and the number of base pairs corresponding to a binding site (n) of the fluorescent (K f and n f ) and non-fluorescent (K nf, nnf) types of interactions were determined. The average size of binding site (n) is equal to 1.5 bp (absorption spectroscopy), and nf ca. 2 bp (fluorescence spectroscopy). It was shown that nnf is dependent on GC-content and total n is independent of it.
Highlights
Large number of biologically active small molecules in cells interact with DNA in a variety of ways, one of which is the intercalation.[1,2,3,4,5,6,7] Majority of intercalators might form more than one type of complexes with DNA, the prominent representative of which is ethidium bromide (EtBr)
When EtBr forms a complex with DNA, the electronic distribution of the ligand changes, which is perceptible in the absorbance spectrum with a simultaneous shift to longer wavelength and in the decreasing of value of the molar extinction coefficient,[7] whereas in fluorescence the signal amplitude increases after the formation of the DNA‐EtBr complex.[11]
One of the reliable means to investigate the binding process and to distinguish the binding modes is the combination of absorption spectroscopy detecting the total concentration of adsorbed on DNA molecules and fluorescence method showing the amount of intercalated in the double helix molecules.[11,17]
Summary
Large number of biologically active small molecules in cells interact with DNA in a variety of ways, one of which is the intercalation.[1,2,3,4,5,6,7] Majority of intercalators might form more than one type of complexes with DNA (multimodal ligands), the prominent representative of which is ethidium bromide (EtBr). Investigations showed that at small values of drug/DNA ratio (r) this ligand may form two types of “strong” complexes with DNA, one of which has been proposed to be a stably bound external binding and second type is the well described intercalation. Intercalation as the primary binding mode is observed by absorption spectroscopy supported by a red spectrum shift corresponding to the spectrum of free EtBr.[13] In fluorescence this event is detected because of the enhancement of the fluorescence amplitude after intercalation.[11] the employment of these two techniques separately is not able to distinguish the external binding mode.[14].
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