Circular Dichroism in Analysis of Biomolecules
Published Online: 15 SEP 2006
Copyright © 2000 John Wiley & Sons, Ltd. All rights reserved.
Encyclopedia of Analytical Chemistry
How to Cite
Pančoška, P. 2006. Circular Dichroism in Analysis of Biomolecules. Encyclopedia of Analytical Chemistry.
- Published Online: 15 SEP 2006
Circular dichroism (CD) spectroscopy belongs to the family of chiroptical methods. These methods utilize the interaction of circularly polarized light (CPL) with chiral molecules and molecular systems to obtain more detailed information about their structure and electronic or vibrational states. CD spectrum Δɛ(ν) is, in fact, the difference of two absorbance spectra: one measured with the left circularly polarized light (LCPL) and the other one recorded with the right circularly polarized light (RCPL) Δɛ(ν) = ɛL(ν) − ɛR(ν). CD spectra can be measured as electronic circular dichroism (ECD) in spectral regions of electronic transitions (ultraviolet (UV)) through visible (VIS) and as vibrational circular dichroism (VCD) in the infrared (IR) spectral regions. An ECD spectrum can be also recorded as the difference excitation spectrum for fluorescence spectra excited by LCPL and RCPL, respectively (fluorescence-detected circular dichroism (FDCD)). The Raman optical activity (ROA) and circularly polarized luminescence spectroscopies complete the family of currently developed chiroptical methods. The differences Δɛ(ν) measured as CD are typically ∼10−5 of sample absorbance. Special modifications of dispersive (for ECD and VCD) or Fourier transform infrared (FTIR) (for VCD) spectrometers are needed to measure CD with a reasonable signal-to-noise ratio (S/N). Polarization modulation of the incident light by a photoelastic modulator and synchronous electronic processing of the resulting photoelectric signal in the spectrometers are typically used for this purpose.
Molecular structure is encoded in the CD spectra because the chiral field of the CPL wave that induces a spectral transition in the chiral molecule can be observably altered both by the transition electron density redistribution (as in the conventional absorption spectroscopy) and by the transition magnetic field accompanying the molecular charge redistribution. The structure and conformation of the studied molecule define the relative orientation of the characteristic directions of these two effects. This relative orientation affects the probability of absorption of photons of RCPL and LCPL and, in turn, determines the CD sign, the primary information that is unique for CD spectroscopy. In the absolute value, CD intensity (“secondary” unique information) is related to the angle of electric and magnetic transition moments and can, therefore, be converted into the molecular conformation once the suitable theoretical model is available. For complicated molecules (biomolecules, proteins, nucleic acids) where the corresponding theoretical calculations are too complex, the empirical interpretive methods based on reference sets of spectra measured for molecules with known structures (from X-ray or nuclear magnetic resonance (NMR) experiments) are used with success. For example, using these empirical methods, the fractions of regular secondary structures (secondary structure fraction (SSF)) in globular proteins can be determined with a relative error of 3–7% and the number of secondary structure segments in proteins with a typical error of one to three segments per protein fold.
CD spectroscopy is highly sensitive to the polarization artifacts that can be related to the optical imperfections of the optical parts of the spectrometer. To achieve an acceptable S/N, the sample total absorbance A should be also controlled (typically A < 0.8). This, in effect, restricts the selection of usable solvents and largely determines the concentration ranges of studied samples.