As CD spectroscopy can analyze only optically active species, it is convenient to start the module with a brief introduction of optical activity. In nature almost every life form is handed, meaning that there is certain degree of asymmetry, just like in our hands. One cannot superimpose right hand on the left because they are non-identical mirror images of one another. So are the chiral (handed) molecules, they exist as enantiomers, which mirror images of each other (Figure 1). One interesting phenomena related to chiral molecules is their ability to rotate plane of polarized light. Optical activity property is used to determine specific rotation, [ α ]^T_λ, of pure enantiomer. This feature is used in polarimetery to find the enantiomeric excess, (ee), present in sample.

Figure 1: Schematic depiction of chirality/handedness of an amino acid.

Circular dichroism (CD) spectroscopy is a powerful yet straightforward technique for examining different aspects of optically active organic and inorganic molecules. Circular dichroism has applications in variety of modern research fields ranging from biochemistry to inorganic chemistry. Such widespread use of the technique arises from its essential property of providing structural information that cannot be acquired by other means. One other laudable feature of CD is its being a quick, easy technique that makes analysis a matter of minutes. Nevertheless, just like all methods, CD has a number of limitations, which will be discussed while comparing CD to other analysis techniques.

CD spectroscopy and related techniques were considered as esoteric analysis techniques needed and accessible only to a small clandestine group of professionals. In order to make the reader more familiar with the technique, first of all, the principle of operation of CD and its several types, as well as related techniques will be shown. Afterwards, sample preparation and instrument use will be covered for protein secondary structure study case.

Depending on the light source used for generation of circularly polarized light, there are:

In the CD spectrometer the sample is places in a cuvette and a beam of light is passed through the sample. The light (in the present context all electromagnetic waves will be refer to as light) coming from source is subjected to circular polarization, meaning that its plane of polarization is made to rotate either clockwise (right circular polarization) or anti-clockwise (left circular polarization) with time while propagating, see Figure 2.

Figure 2: Schematic representation of (a) right circularly polarized and (b) left circularly polarized light. Adapted from L. Que, Physical Methods in Bioinorganic Chemistry – Spectroscopy and Magnetism, University Science Books, Sausalito (2000).

The sample is, firstly irradiated with left rotating polarized light, and the absorption is determined by Equation 1. A second irradiation is performed with right polarized light. Now, due to the intrinsic asymmetry of chiral molecules, they will interact with circularly polarized light differently according to the direction of rotation there is going to be a tendency to absorb more for one of rotation directions. The difference between absorption of left and right circularly polarized light is the data, which is obtained from Equation 2, where ε_L and ε_R are the molar extinction coefficients for left and right circularly polarized light, c is the molar concentration, l is the path length, the cuvette width (in cm). The difference in absorption can be related to difference in extinction, Δε, by Equation 3.

(1)

(2)

(3)

Usually, due to historical reasons the CD is reported not only as difference in absorption or extinction coefficients but as degree of ellipticity, [θ]. The relationship between [θ] and Δε is given by Equation 4.

(4)

Since the absorption is monitored in a range of wavelengths, the output is a plot of [θ] versus wavelength or Δε versus wavelength. Figure 3 shows the CD spectrum of Δ–[Co(en)₃]Cl₃.

Figure 3: CD spectrum of Δ–[Co(en)₃]Cl₃.

Magnetic circular dichroism (MCD) is a sister technique to CD, but there are several distinctions:

MCD is powerful method for studying magnetic properties of materials and has recently been employed for analysis of iron-nitrogen compound, the strongest magnet known. Moreover, MCD and its variation, variable temperature MCD are complementary techniques to Mossbauer spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Hence, these techniques can give useful amplification to the chapter about Mossbauer and EPR spectroscopy.

Linear dicrosim (LD) is also a very closely related technique to CD in which the difference between absorbance of perpendicularly and parallelly polarized light is measured. In this technique the plane of polaliration of light does not rotate. LD is used to determine the orientation of absorbing parts in space.

Most of proteins and peptides will require using buffers in order to prevent denaturation. Caution should be shown to avoid using any optically active buffers. Clear solutions are required. CD is taken in high transparency quartz cuvettes to ensure least interference. There are cuvettes available that have path-length ranging from 0.01 cm to 1 cm. Depending on UV activity of buffers used one should choose a cuvette with path-length (distance the beam of light passes through the sample) that compensates for UV absorbance of buffer. Solutions should be prepared according to cuvette that will be used, see Table 2.

Besides, just like salts used to prepare pallets in FT-IR, the buffers in CD will show cutoffs at a certain point in low wavelength region, meaning that buffers start to absorb after certain wavelengh. The cutoff values for most of common buffers are known and can be found from manufacturer. Oxygen absorbs light below 200 nm. Therefore, in order to remove interference buffers should be prepared from distilled water or the water should be degassed before use. Another important point is to accurately determine concentration of sample, because concentration should be known for CD data analysis. Concentration of sample can be determined from extinction coefficients, if such are reported in literature also for protein samples quantitative amino acid analysis can be used.

Many CD instrument come bundled with a sample compartment temperature control unit. This is very handy when doing stability and unfolding/denaturation studies of proteins. Check to make sure the heat sink is filled with water. Turn the temperature control unit on and set to chosen temperature.

UV source in CD is very powerful lamp and can generates large amounts of Ozone in its chamber. Ozone significantly reduces the life of the lamp. Therefore, oxygen should be removed before turning on the main lamp (otherwise it will be converted to ozone near lamp). For this purpose nitrogen gas is constantly flushed into lamp compartment. Let Nitrogen flush at least for 15 min. before turning on the lamp.

After saving the data for both the spectra of the sample and blank is smoothed using built-in commands of controller software. The smoothed baseline is subtracted from the smoothed spectrum of the sample. The next step is to use software bundles which have algorithms for estimating secondary structure of proteins. Input the data into the software package of choice and process it. The output from algorithms will be the percentage of a particular secondary structure conformation in sample. The data shown in Figure 7 lists commonly used methods and comparers them for several proteins. The estimated secondary structure is compared to X-ray data, and one can see that it is best to use several methods for best accuracy.

Figure 7: Comparison of secondary structure estimation methods. Adapted by permission from N. Greenfield, Nat. Protoc., 2006, 1, 6.