The technique CD measures the interaction of circularly polarized light with molecules. Circularly polarized light comes in left- and right-handed forms, rather like screw threads. These forms interact equally with non-chiral chromophores, but differently when the chromophore has a right- or left-handed form. The result is a small difference in the extinction coefficients for left- and right-polarized light, which varies with wavelength.
In proteins, the chiral arrangement of peptide bonds in secondary structures such as a-helix and b-sheet leads to characteristic CD spectra at UV wavelengths. Alteration of the relative orientations, due to confirmational or structural variations, causes changes in the CD spectrum.
Other molecules such as nucleic acids also have CD spectra at wavelengths where they absorb light. Theory of measurements is described elsewhere. A typical CD spectrum is shown at the right for 10 mg/mL polypeptide solution in a 0.01 mm cell. A step wise procedure gives operational details.
Some Protein Applications
Checking the integrity of a protein - if there is a mutated a conserved residue in an enzyme and it has lost activity, is this because the residue was critical for the mechanism, or because its absence has caused the protein to misfold. If the structure is unchanged then the CD spectrum should be very similar, if not the spectrum should be different.
Studying stability - it is possible to unfold proteins by adding reagents such as guanidinium chloride, H2N+=C(NH2)2.Cl- , or heating them above ambient temperature. The CD spectrum is a useful way of monitoring the structure changes that occur during this denaturation, and of telling whether it is reversible. Titration experiments can be used to quantify stability changes in a protein with and without ligand or modified in other ways.
Under favorable circumstances the CD spectrum can give a quantitative indication of the proportion of different secondary structure types in a protein. A number of programs are available that will analyze a given CD spectrum for contributions from a-helix, b-sheet, and other elements. It is necessary to pay very careful attention to sample purity, accuracy of concentration, and buffer conditions for reliable results.
JASCO's J-810 CD Spectropolarimeter -- The J-810 is the latest model CD spectropolarimeter. It is located in the BRIN laboratory W186.
The light source is a 150 w Xenon lamp, which covers the full spectral range (180 - 800 nm). The optics use a prism monochromator to disperse the light into different wavelengths.
The circular polarization is generated in a device called a photoelastic modulator (PEM). This generates left- and right-circular polarized light alternately by stressing a crystal at high frequency. After the light passes through the sample it is detected by a photomultiplier tube. The signal electronics are synchronized to the PEM, allowing the difference in absorption for the left- and right-polarized light to be quantified. The optical path and sample compartment are continuously swept with nitrogen. Oxygen in air absorbs short wavelength UV light, which would not only degrade the signal but also create ozone which is toxic and damaging to the mirrors in the optical path.
Accessories
The instrument is fitted with a computer-controlled Peltier temperature control unit, allowing measurements at temperatures from 0 oC to 100 oC. At the right is a picture of a Julabo temperature control maintains water temperatures for the Peltier device.
A constant flow of ultra high purity nitrogen is supplied from a nitrogen generator. Ultra hight purity nitrogen is separated from atmospheric air by the generator.
CD Autotitrator is shown below at the left. Automated Titration Systems allow long term kinetic, chemical denaturation, and ligand binding studies in a completely unattended mode. Titration experiments can also be used to quantify stability changes in a protein with and without ligand or modified in other ways. Mixtures of solvents, such as chloroform– methanol titrations can be carried out by increasing the concentration of methanol from 0 to 80% and expressing the data in terms of molar ellipticity.
Cuvettes
CD cuvettes are made of high-grade quartz for good light transmission. Additionally they may be specially annealed to reduce strains in the quartz which can give rise to background signals. The BRIN Laborator has cell of several path lengths available - 1 cm (volume required 2.5 - 3.5 uL), 1 mm (300 - 400 uL) and a "demountable window" cell comprising two precision made quartz plates which are pushed together in a holder making a cell with a path length of 0.1 mm. This requires only about 50 - 60 uL of sample, but it is difficult to recover after use. The 1 cm cells will accommodate a small stirrer which is useful when doing titrations or temperature ramping. Cuvettes must be kept scrupulously clean for best results - films of protein adsorbed on the sides can cause baseline distortions. It is always a good idea to run a "buffer blank" before measuring a sample (see Sample Requirements and Choice of Buffers).
Sample Requirements
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