The quantity of material required for far-UV CD experiments is typically in the order of 1 – 50 µM and volumes are in the range of 60 – 500 µl. The purity of the sample should be as high as possible and at least greater than 95%. Samples should be equilibrated into the same buffer as the reference (gel filtration or dialysis should be used to match the reference buffer). For better results, the buffer alone should not have a high absorbance in the region of the spectrum (far or near-UV). For instance, high concentrations of dithiothreitol, histidine or imidazole cannot be used in the far-UV region. The chloride ion as buffer component is not recommended with CD experiments and should therefore be kept as low as possible within the limit of acceptability for the molecule stability.
The far-UV CD spectrum recorded between 260 nm and the lowest wavelength as possible is sensitive to the peptide bond conformation and as a consequence reflects the secondary structure of the protein (a-helix, b-sheet, b-turn and unordered content). In the case of nucleix acids and oligonucleotides, the aromatics bases are the main chromophores with absorption below 300 nm. Therefore protein stability can be assessed using CD by following changes in the spectrum with increasing temperature (thermal stability) or denaturing agent concentration (chemical stability). Far-UV CD experiments should take 15 to 90 min. for a complete spectrum (including sample preparation and control) whereas stability experiments last 3 to 8 hours.
The near-UV CD spectrum (between 320 and 250 nm) leads to a signal sensitive to the tertiary structure derived from Trp, Tyr, Phe and Cys residues. Since the signal strength in he near-UV CD region is much weaker than that in the far-UV CD region, such near-UV spectra require about 1 ml of protein solution with an optical density at 280 nm of 0.5 to 1.0.
In addition, the CD instrument is equipped with a 2 syringe stopped-flow device, a very useful approach to measure kinetic process and specially characterize protein folding. Please not these measurements may be tasking because multiple shot accumulation is necessary to extract valuable result.
The CD output may include up to four channel, including CD signals, total absorbance and photomultiplier voltage as well as a fourth one dedicated to an external probe (pH, …). The multiple position Peltier cuvette holder may hold up to 6 cells and enables CD measurements with Peltier temperature control. A stopped-flow accessory equipped with 2 syringes is installed on the instrument as well as a titration unit. Software allows direct determination of secondary structure contents in situ.
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