In Figure 3c, fluorescence tells us that the tryptophans in α-lactalbumin unfold in two phases, the first being slow and the second from 55 oC to 65☌. However, one does need to take care as to what information is being displayed. The use one makes of a CD spectrum depends on what question is being asked: is an absolute measure of helix and sheet content of a protein required? Or does one simply want to know whether a peptide folds in a lipidic environment or a nucleic acid or protein unfolds when heated? The latter questions can often be answered by visual inspection of spectra ( Figure 3). The d-d bands arise from a very different mechanism: the perturbation of magnetic dipole allowed d-d transitions by in-ligand transitions arranged in a helical geometry about the metal centre.įor biomacromolecules, such as proteins and DNAs, we usually know the chirality of the component amino acids or nucleosides and we are more interested in what the CD spectrum tells us about how they are arranged into secondary or even tertiary structures ( Figure 3). The fact that the zero CD point corresponds with the absorbance maximum confirms that the CD is dominated by the coupled oscillator model. For example, the coupling of electric dipole transition moments in the aromatic phenanthroline ligands of 2+ gives rise to the sharp in-ligand bands of Figure 2. Alternatively, if a molecule can be divided into achiral chromophores whose transition polarizations are known, then one of a number of models for calculating the CD spectrum may be appropriate and the handedness of the molecule may be able to be determined quite simply.
For small molecules, fairly high-quality CD spectra can be calculated for known (or proposed) geometries and compared with experiment. A non-zero spectrum tells you that the system being studied is chiral, with helical rearrangements of electrons during electronic transitions.
Assuming that the above pitfalls have been avoided and one has measured a good CD spectrum, we ask what can be deduced from it.