I am doing some quenching studies of a synthesized compound by titrating it with DNA. Since the excitation wavelength of the compound is around 260 nm, which is the absorption wavelength of DNA itself, how do I account for the percentage of light which is not used to excite the compound ? I am getting lower emission of the compound but not entirely due to a quenching mechanism, some is due to the absorption of DNA and I need to take that contribution.
It becomes easy if there are no changes in the absorption spectrum (i.e., no extinction coefficient changes or spectral shifts) on interaction of the dye with DNA. Here are some suggestions. You could do an absorbance or an absorption spectrum experiment on a spectrophotometer if your concentrations permit.
Method I
Absorbance is an additive property i.e., if there are two species then the total absorbance of the sample is the sum of the respective individual absorbance of the species involved. This is only true under the assumption that species don't interact i.e., no dimer or aggregate formation occurs.
You could do an independent experiment with just the DNA alone (control), measure the absorbance. This should give you the amount of light intensity that is being absorbed by DNA and the rest of the light intensity is absorbed by your molecule.
ATotal = ADye + ADNA
If the above relation holds good then you could separate out the absorbance due to DNA, that solves your problem.
Method II
Alternatively, you could record the absorption spectrum of the sample containing both DNA and dye and resolve the two spectra by fitting it to their respective individual spectra and use it to estimate the absorbance of individual components at a particular wavelength.
Now if you know the absorbance contribution of DNA then that can be accounted in your fluorescence expts too.
You should meausre the DNA sample alone, and estimate its absorption coefficient at the excitation wavelength. Then, lambert-beer law will tell you the fraction of light which is absorbed by DNA transition even in the presence of the dye, hence the remaining part only is absorbed by the dye, giving rise to a reduction in the emission even if no quenching occurs.
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