Consider now a more complex case, like the binding of a protein to an oligonucleotide.

In this case, both the ligand (say the protein) an the receptor (the oligonucleotide) contribute to the CD spectrum.

Moreover, in the far UV region (l < 240 nm) they contribute SIMULTANEOUSLY.

By visual inspection of the above spectra, obtained at increasing protein concentration, it is possible to note that the protein binding causes a change in the oligonucleotide structure, because the oligonucleotide band centered at 260 nm, where the protein contribution is unimportant, changes in intensity as the protein concentration is  increased.

The percentage change in the oligonucleotide 260 nm band is a measure of the quantity of complex formed, so it can be plotted versus the protein concentration to give a curve like the one reported in the preceding page.

Moreover, the decrease of the oligonucleotide 260 nm band is in general an evidence of unstacking, so that, in the particular case presented above, it is possible to relate a structural change in the oligonucleotide to the occurring binding.

However, due to the change in structure of the oligonucleotide NO STRUCTURAL INFORMATION CAN BE OBTAINED FOR THE PROTEIN, because both the protein and the oligonucleotide contributes to the observed spectra below 240 nm. Subtraction of the spectra of the free protein and of the free oligonucleotide from the complex spectrum CANNOT HELP, because the spectrum of at least the oligonucleotide is different in presence of the protein.

In those cases where no changes at 260 nm are observed, so that evidence is provided that the oligonucleotide contribution to the CD spectrum is constant at increasing protein concentration, you can subtract the oligonucleotide contribution from the complex spectrum and then build a binding curve based on the protein structural changes. ONLY IN THESE CASES, you can obtain the protein structure after complex formation.