Circular Dichroism
A circular dichroism (CD) spectrum is an example of a difference spectrum.
It records the difference in the absorbance of left- and right-handed circularly polarized light.
This difference can be nonzero only when the sample is chiral.
Many biopolymers exhibit circular dichroism.
The CD exhibited by a particular polymer is strongly affected by its conformation, and especially so when changes in periodic ordered structures are involved.
Therefore CD is often used to monitor order-disorder transitions of biopolymers, such as the helix-coil transition.
CD spectra are reported in two ways in the literature:
Note that Delta e can be either positive or negative, while the extinction coefficients themselves must be positive.
This signed property of CD often makes it easy to recognize the existence of several distinct electronic transitions with nearly the same energy.
Illustration with Polypeptides
Poly(L-lysine) can exist in three conformations in dilute aqueous solution, depending on the pH (degree of ionization of the amino groups in the side chains) and temperature.
For this reason, its CD in the ultraviolet region of the spectrum has been extensively studied (see Greenfield, N.; Fasman, G. D. "Computed Circular Dichroism Spectra for the Evaluation of Protein Conformation" Biochemistry 1969, 8, 4108-4116).
The three conformations adopted by poly(L-lysine) are found in many other polypeptides, although not every polypeptide can form all of them as easily as does poly(L-lysine).
The three conformations are readily distinguishable by their CD spectra in the spectral region where the amide unit in the backbone is the chromophore.
- Alpha helix (high pH, ambient temperature): Three recognizable bands:
- Delta e = -11 at 222 nm
- Delta e = -12 at 208 nm
- Delta e = +30 at 190 nm
- Beta sheet (high pH, elevated temperature): Two recognizable bands
- Delta e = -5.5 at 217-218 nm
- Delta e = +9 at 195 nm
- Random coil (low pH): Two recognizable bands
- Delta e = +1 at 217 nm
- Delta e = -12 at 197 nm
These three conformations are less easily distinguished in the absorption spectra than in the CD spectra.
All three conformations show only one maximum in the absorbance in this spectral region, with the maximum at 190-197 nm.
The extinction coefficients for these three conformations differ by no more than a factor of 2.
However, if one were to look for helices in an equimolar solution of poly(L-lysine) and poly(D-lysine), CD would be useless.
The CD spectrum of the right-handed helices of the former polymer would exactly cancel the CD spectrum of the left-handed helices of the latter polymer.
Absorption spectra, however, could still be used.
The shapes and intensities of the CD spectra may be strongly perturbed if the polypeptide contains an additional chromophore in its side chain.
For example, the CD spectrum of the alpha helix of poly(L-tyrosine) (Peggion, E.; Cosani, A.; Terbojevich, M. "Solution Properties of Synthetic Polypeptides. Assignment of the Conformation of Poly(L-tyrosine) in Water and in Ethanol-Water Solutions" Macromolecules 1974, 7, 453-459.) is very much different from the result cited above for poly(L-lysine), because the phenolic unit in the tyrosine side chain also contributed to the CD in this region of the spectrum.
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July 1, 1999
Wayne L. Mattice: wlm@polymer.uakron.edu