circular dichroism, or CD, is defined as the differential absorption of left and right hand circularly polarizedlight:
At a given wavelength:
ΔA = (AL - AR)
ΔA is the difference between absorbance of left circularly polarized and right circularly polarized light
(this is what is usually measured). it can also be expressed as:
ΔA = (εL - εR) C l
where C is the molar concentration and l is path length and εL and εR are the molar extinction coefficients for RCP and LCP light then
Δε = (εL - εR) - is the molar circular dichroism
this is what is usually meant by the circular dichroism of the substance
Although ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity. The molar ellipticity is:
[θ] = 3298Δε
In general, this phenomenon will be exhibited in absorption bands of any optically active molecule. As a consequence, circular dichroism is exhibited by biological molecules, because of the dextrorotary (e.g. some sugars) and levulorotary (e.g. some amino acids) molecules they contain. Noteworthy as well is that secondary structure will also impart a distinct CD to their respective molecules. Therefore, the alpha helix of proteins and the double helix of nucleic acids have CD spectral signatures representative of their structures.
The ultraviolet CD spectrum of proteins can predict important characteristics of their secondary structure. CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha-helix conformation, the beta-sheet conformation, the beta-turn conformation, or some other (random) conformation. These fractional assignments place important constraints on the possible secondary conformations that the protein can be in. CD can not, in general, say where the alpha helices that are detected are located within the molecule or even completely predict how many there are. Despite this, CD is a valuable tool, especially for showing changes in conformation. It can, for instance, be used to study how the secondary structure of a molecule changes as a function of temperature or of the concentration of denaturing agents. In this way it can reveal important thermodynamic information about the molecule that can not otherwise be easily obtained. Anyone attempting to study a protein will find CD a valuable tool for verifying that the protein is in its native conformation before undertaking extensive and/or expensive experiments with it. Also, there are a number of other uses for CD spectroscopy in protein chemistry not related to alpha-helix fraction estimation.
CD spectroscopy is usually used to study proteins in solution, and thus it complements methods that study the solid state. This is also a limitation, in that many proteins are embedded in membranes in their native state, and solutions containing membrane structures are often strongly scattering. CD is sometimes measured in thin films.
CD has also been studied in carbohydrates, but with limited success due to the experimental difficulties associated with measurement of CD spectra in the vacuum ultraviolet (VUV) region of the spectrum (100-200 nm), where the corresponding CD bands of unsubstituted carbohydrates lie. Substituted carbohydrates with bands above the VUV region have been successfully measured.
It may be of interest to note that the protein CD spectra used in secondary structure estimation are related to the π to π* orbital absorptions of the amide bonds linking the amino acids. These absorption bands lie partly in the so-called vacuum ultraviolet (wavelengths less than about 200 nm). The wavelength region of interest is actually inaccessible in air because of the strong absorption of light by oxygen at these wavelengths. In practice these spectra are measured not in vacuum but in an oxygen-free instrument (filled with pure nitrogen gas).
