Equipment available: Jasco J-815 Spectropolarimeter
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £50/day
Sample requirements: Path length 1mm to 0.05mm (thin cuvettes), Protein concentrations- 0.1 - 0.05mg/ml, approx 200μl sample, for 1mm cuvette, Buffers- low buffer concentration preferable. Usually 10mM phosphate buffer + 20mM NaCl
Circular Dichroism Spectrophotometry
Circular Dichroism Spectrophotometry (CD) measures the interaction of circularly polarised light with molecules. Circularly polarised light comes from the sum of two waves left- and right-handed that are at right angles to each other and phase shifted. Circular dichroism results from differential absorption of the beams by a sample with an electric and a magnetic transition dipole moment that can either be intrinsic e.g. a chiral centre or induced from the local environment of the chromophore e.g. DNA bases in a helix. In proteins, the chiral arrangement of peptide bonds in secondary structures such as α-helix and β-sheet leads to characteristic CD spectra at UV wavelengths. Alteration of the relative orientations, due to conformational or structural variations, causes changes in the CD spectrum.
- Checking the integrity of a protein – e.g. comparing a mutant with the wild type CD spectrum to see if the structure is unchanged. It should be noted that the presence of secondary structure does not necessarily prove that a protein is correctly folded.
- Studying stability - it is possible to unfold proteins by heating and monitor the process by the CD spectrum. A cooperative transition over a narrow temperature range is indicative of a folded protein.
- 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 analyse a given CD spectrum for contributions from α-helix, β-sheet and other elements.
Jasco J-815 CD Spectrophotometer
The J-815 is a modern CD spectrometer. It was installed in November 2013 and is serviced annually. The circular polarisation is generated by a photoelastic modulator (PEM). This generates left- and right-circular polarised 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 synchronised to the PEM, allowing the difference in absorption for the left- and right-polarised light to be quantified. The optical path and sample compartment are continuously purged with nitrogen, as oxygen 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 optics. The nitrogen is supplied by cylinders that should be checked before the start of the experiment.
- The machine is fitted with a computer-controlled Peltier temperature control unit, allowing measurements at temperatures from 10C to 100C.
- The machine is equipped with a 90 degree light port, allowing for CD to be measured in a fluorescence mode.
CD cuvettes are made of high-grade quartz for good light transmission and should be specially annealed to reduce strains in the quartz which can give rise to background signals. For observing protein secondary we recommend 1mm path lengths and expect users to buy their own (e.g. Starna Scientific "21/Q/1 cells for CD"). We also have a quartz 1cm cuvette for near UV work. Cuvettes should be cleaned with a 2% Hellmanex solution directly after use to prevent protein films building up on the walls.
We recommend protein concentrations of 0.1 - 0.05mg/ml, and approx 200μl, for 1mm cuvettes for working in the far-UV (190 - 230 nm), and 1 mg/ml and 2-3mls for 1cm path length in the near-UV (240-300nm).
Many buffers absorb in the far UV. Some to avoid are: chloride, citrates, acetate, certain "Good" buffers e.g. MOPS, DTT and imidazole. A 10 mM sodium or potassium phosphate buffer is often recommended for CD. We recommend 10mM phosphate buffer + 20mM NaCl If salt is essential NaF can substitute for NaCl. Tris and TEA are reasonable and can be pH'd with sulphuric or phosphoric acid if the chloride is a problem. Glycerol should not interfere.
http://dichroweb.cryst.bbk.ac.uk/html/home.shtml An on-line server for protein CD; DichroWeb incorporates five popular and effective algorithms to calculate protein secondary structure content.
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