UVVisible Spectroscopy

In a complex with DNA the guest molecule is positioned in an environment which is different from that of the uncomplexed molecule in solution. The guest molecules, especially solvatochromic compounds such as organic dyes [23], usually have different absorption properties in the complexed and uncomplexed forms. Thus, on addition of DNA to a solution of an intercalator or groove binder, a shift of the absorption maximum to longer wavelengths (bathochromic shift or red shift) and a decrease of the absorbance (hypochromicity) occurs. In practice the association process is monitored by a spectrophotometric titration, during which aliquots of DNA solution are added to a solution of the guest molecule. The absorption spectra at each dye-to-DNA ratio are determined and superimposed [24]. A representative spectrophotometric titration is shown for 9-aminoacridizinium bromide (5a) with calf thymus DNA (Figure 2.5.2). The long-wavelength absorption maximum of 5a is shifted to longer wavelength by 12 nm on addition of DNA, and a significant decrease of the absorbance takes place; this is indicative of an associative interaction between the dye and DNA.

Further information might be extracted from spectrophotometric titrations if isosbestic points are observed. An isosbestic point appears when each absorption spectrum of the titration has the same absorbance at a particular wavelength, i.e. a

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Wavelength I nm Fig. 2.5.2. Spectrophotometry titration of acridizinium salt 5a with ct DNA in phosphate buffer (10 mM, pH 7.0); thin arrows indicate the increasing or decreasing absorption bands during the course ofthe titration; thick arrows indicate isosbestic points.

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Wavelength I nm Fig. 2.5.2. Spectrophotometry titration of acridizinium salt 5a with ct DNA in phosphate buffer (10 mM, pH 7.0); thin arrows indicate the increasing or decreasing absorption bands during the course ofthe titration; thick arrows indicate isosbestic points.

point of intersection in all superimposed absorption spectra (see Figure 2.5.2, thick arrow). Such isosbestic points reveal that each absorption spectrum arises from almost exclusively two different absorbing species (e.g. complexed and uncomplexed dye). Because it is likely that each binding mode results different absorption properties, an isosbestic point indicates that mainly one particular binding mode between the DNA and the guest molecule occurs. It should, nevertheless, be noted that a second, but minor, binding mode only contributes marginally to the overall absorption spectrum and might not have a significant influence on the isosbestic points.

Data from spectrophotometric titrations may also be used to determine the association constant (K) between the dye and DNA [25]. The data from spectrophotometric titrations, i.e. absorbance data (Aobs) at a fixed wavelength, are used to determine the concentration of bound dye (Cb), the concentration of uncomplexed dye (c), and the number of bound dye molecules per base pair (r) according to Eqs

where Af is the absorbance of the uncomplexed dye, Ab is the absorbance of the bound dye, Aobs is the absorbance of a mixture of the free and bound compounds,

Tab. 2.5.1. Binding constants and binding-site size of acridizinium bromides 5a and 5b as determined from spectrophotometry titrations with st DNA, (poly[dA-dT]-poly[dA-dT]), and (poly[dG-dC]-poly[dG-dC]).
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