Chirality and Circular Dichroism Spectroscopy

Many amino acids exist as chiral molecules

Chiral molecules are mirror images or enantiomers. There is no symmetry operation in 3D-space that can be performed on one enantiomer to make it overlay the other. Enantiomerism is a special form of isomerism. The physical properties of enantiomers are identical in every way except two: how the molecules interact with polarized light and how they interact with other chiral molecules.

Circular dichroism (CD), measured as a function of wavelength, is the difference in absorbance of left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL). This difference can be detected when a molecule contains one or more light-absorbing groups - so-called chiral chromophores.

Circular dichroism (CD) = ΔA(λ) = A(λ)L-CPL - A(λ)R-CPL, where λ is the wavelength

Generating a CD spectrum

When chiral chromophores are present, one state of circularly polarized light will be absorbed to a greater or lesser extent than the other. Over corresponding wavelengths, a CD signal can therefore be positive or negative, depending on whether L-CPL is absorbed to a greater extent than R-CPL (CD signal positive) or to a lesser extent (CD signal negative).

Chirascan CD spectrometers measure alternately the absorbance of L- and R-CPL and then calculate the CD signal.

CD spectra vary according to differences in absorbance of
L- and R-CPL.

CD spectrum of vitamin B12 (cobalamin). Multiple peaks show how CD varies as a function of wavelength and that a CD spectrum may exhibit positive and negative peaks.

Typical protein CD spectra

The chiral center is the α-carbon atom in the protein backbone.

Disulphide bonds, often found in proteins, may influence both far- and near-UV CD spectra.

The principle chiral chromophore that dominates in the far-UV spectrum of proteins is the peptide carbonyl bond (C=O): π → π* at ~ 190 nm; n → π* at ~ 210-230 nm.

In the near-UV, aromatic amino acids are the predominant chromophore.