JASCO Circular Dichroism Spectrometer
CD can be used to monitor changes in the conformation of biopolymers and is used mainly for studying changes in the secondary and tertiary structure of proteins. CD can also be used for any molecules containing a chromophore in a chiral environment that absorbs radiation in an accessible region of the UV/Vis spectrum, including nucleic acids, proteins, metal-ion chelates and porphyrins.
To exhibit a CD spectrum a molecule must contain a chromophore that absorbs radiation by virtue of the electronic configuration of its ground state at room temperature. The energy absorbed results in a transition to a higher-energy excited state. The excited state chromophore can interact with its environment in a way that differs from the ground state. In proteins, tryptophan, tyrosine, and phenylalanine are the main chromophores that absorb in the near-UV (240-320-nm) region, and the peptide bond is the main chromophore absorbing in the far-UV (180-240-nm) region. Disulfide bonds and histidine residues can also contribute to a CD spectrum. In addition, to observe a CD spectrum, the chromophore needs to be in, or associated with a chiral environment. The chromophores in the side chains of amino acids (e.g. Phe, Tyr, and Trp) are not chiral however, they are integrated into chiral amino acids, oligopeptides and proteins.
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 with respect to secondary structure. As for fluorescence spectroscopy or any spectroscopic method, the sample needs to be chemically pure and homogeneous.
Current Protocols in Protein Science, Unit 7.6: Determining the CD Spectrum of a Protein, 2004. John Wiley & Sons, Inc.
Sreerama, N., et. al. 1999b. Estimation of the number of a-helical and b-strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 8:370-380.
Johnson, W. C. 1990. Protein secondary structure and circular dichroism: A practical guide. Proteins: Struct., Funct., Genet. 7:205-214.
VIDEO SHOWING CD OPERATION
