JASCO Model J-1500 Circular Dichroism Spectrometer
Circular dichroism (CD) can monitor changes in the conformation of biopolymers and is used mainly for studying changes in the secondary and tertiary structure of protein... the methods can, in principle, be used for any molecules containing a chromophore that absorbs radiation in an accessible region of the spectrum, such as nucleic acids, carbohydrates and porphyrins.
There are two requirements for a molecule or group of atoms in a molecule to exhibit a circular dichroism (CD) spectrum. The first is the presence of a chromophore - i.e., a group that can absorb radiation by virtue of the electronic configuration of its resting or ground state at room temperature. The energy absorbed results in a transition to a higher-energy or excited state, which has a different distribution of electrons around the nucleus. It can therefore interact with its environment in a way that differs from the ground state. In proteins, tryptophan, tyrosine, and phenylalanine are the main chromophores in the near-UV (240- to 320-nm) region; the peptide bond is the main chromophore in the far-UV (180- to 240-nm) region. Disulfide bonds and histidine residues are two other chromophores whose contribution to CD are, in general, less marked. Most chromophores exhibit more than one transition and their spectra are a composite of several absorption bands. It is an important feature of CD that, unlike optical rotation, the wavelength region within which spectra are observed is limited strictly to the wavelength region of the individual absorption bands ...
The second requirement for CD is that the chromophore be in, or closely associated with, an optically asymmetric environment. The chromophores in proteins are themselves not chiral and exhibit no optical activity. The phenolic group of tyrosine, for example, exhibits a CD spectrum only because it is connected to an optically asymmetric carbon atom. However, when the same group is packed in a folded protein in an environment that is asymmetric with respect to polarity, or is interacting through the phenolic hydroxyl, it exhibits a different CD spectrum that is specific to the particular environmental influences acting on the chromophore.
Plane-polarized radiation comprises two circularly polarized vectors of equal intensity, one right-handed and the other left-handed, which are separately measured in the CD spectrometer by means of a photoelastic modulator. A chromophore situated in an optically symmetrical environment will normally absorb the two components equally so that, when recombined after passing through a solution of the chromophore, they result once again in radiation oscillating in a single plane. A chromophore situated in an optically asymmetric environment, however, will absorb each of the two components to a different extent, the difference being DA. When recombined, the resultant vector describes an ellipse, the ratio of whose major and minor axes determines the ellipticity. The value of DA, and hence that of ellipticity, can be positive or negative depending on the nature of the asymmetric environment ...
It is a general consequence of the above principles that CD spectra of molecules in solution are located in the same wavelength region as their absorption bands. For proteins this means the far-UV and near-UV regions, as well as regions extending into the visible and near infrared. These regions have their origin in and provide information about the polypeptide backbone and its conformation (far-UV), the aromatic amino acid residues and their environments (near-UV) and bound ligands such as heme and cofactors (visible and near infrared). Experimentally, the near-UV, visible, and near-infrared regions can be treated together ...
In both the far-and near-UV regions, CD spectra can be used empirically as "fingerprints" of a particular protein, with the spectrum resulting from the aromatic residues being rather more specific and hence diagnostic. The far-UV spectra, however, can provide information about the protein conformation in terms of its secondary structure. As for fluorescence spectroscopy or any spectroscopic method, the sample needs to be chemically pure and homogeneous.
From "Determining the CD Spectrum of a Protein", Currents Protocols in Protein Science (2004) 7.6.1-7.6.24, John Wiley & Sons, Inc.