2.1. Measuring CD spectra

To optimise the amount of high quality and reproducible CD data obtained from a given sample, it is essential to follow good practice protocols for data collection (see Table 1 for example).^8,9 To accurately determine the secondary structure of a protein based on CD data, the data obtained must include a spectral range covering, at least, the wavelengths between 240 and 190 nm; more accurate results are obtained if data collected includes even lower wavelengths, because more electronic transitions (peaks) will be included, increasing the information content of the data. To achieve such measurements, conditions must be used so that the total absorbance of the sample (due to protein, buffer and other added components) is below ∼1.2 at all wavelengths. This may be challenging for wavelengths below 210 nm, where the absorbance tends to rise, sometimes precipitously, due to the cumulative effects of the buffer and the peptide chromophores along with contributions from water (or other solvents) and the composition material of the optical cell. In addition, light scattering effects from any undissolved protein or suspended particles in the solution, such as lipids present in membrane samples,⁹ may also contribute to this challenge.

The overall absorbance of the sample can be monitored by the dynode voltage or high tension (HT) signal produced during data collection. This is a measure of the voltage applied to the detector to amplify the small circular dichroism signal. The maximum HT cutoff values for individual CD instruments differ, but they correspond to the maximum dynode voltage reading above which the sample absorbance is too high for sufficient light to penetrate. Above this value the apparent CD signal and the intensity of the unpolarised light signal that emerges from the sample decrease to a level where the spectrum becomes noisy, and lead to distortions in both the magnitude and the shape of the measured peaks. Hence the maximum HT value for a given CD instrument needs to be determined.

For a given sample, minimisation of the total absorbance can be achieved by choosing buffer constituents with low absorbance in both the near and far UV wavelength region, and where this is not possible, using the lowest possible concentration of buffer and salts without compromising the stability of the protein. In addition a combination of protein and buffer concentrations, optical cell pathlength, optical cell material, and instrument parameters such as slit width and averaging time can be used to optimise the CD signal.^8–10

Secondary structure analyses are also significantly affected by the accuracy of the protein concentration measurement, since this will have an effect on the magnitude of the CD spectrum when scaled to standard units.^8,9 The most widely used colorimetric methods for determining protein concentrations, including Biuret, Lowry, bicinchoninic acid assays, and Coomassie blue staining; all produce different values/accuracies for proteins (depending on their amino acid compositions). Measuring the absorption of the sample at 280 nm (A₂₈₀) is the most convenient and reliable method (although its accuracy can depend on the number of aromatic amino acids and to a lesser extent, their location (surface or buried) in the protein). Such measurements can be achieved (without wasting a great deal material) using micro-UV spectrometers such as Nanodrops.

Another important consideration is if there is a time lag between sample preparation, concentration determination, and CD measurements, or even during the course of a long series of CD measurements, there may be a change in the protein concentration, due to aggregation or precipitation, especially if the sample is unstable or sensitive to light. This issue can be obviated in part by measuring the concentration of the protein immediately before (or as near in time as possible to) measuring the CD spectrum. It can also be monitored by examining the HT measurements obtained during the course of the CD measurements. If the HT values decrease, this could be indicative of protein precipitation/aggregation, bubble formation, or even sample leakage, during the course of the measurement, and should indicate that a new sample needs to be used.

Using an inaccurate value for the optical cell pathlength will also have significant effects on spectral magnitude and therefore the accuracy of secondary structure analysis. This can be an issue when using demountable cells with pathlengths less 0.01 cm, as the manufacturer-reported values can have a wide margin of error. In addition, the loading and assembly of such cells can be non-reproducible.¹⁰ However, accurate pathlength measurements can be obtained for these cells using the interference fringe method,¹⁰ which requires use of a standard benchtop UV/Vis spectrophotometer.

Finally, as CD instruments are comprised of a number of optical components, there can be variations between instruments which lead to small differences in spectra of the same sample measured on two different instruments. Such disparities can be reduced or mitigated by obtaining calibration measurements with a standard reference material such camphorsulphonic acid (CSA) or ammonium camphour sulfonate (ACS) measured on the same instrument used for the sample measurements.^7,11,12 Both of these compounds have two well-defined and well-separated peaks of known absolute magnitude, so comparisons between the calculated and the experimental values can be used to create a calibration curve over the wavelength range of the spectrum. Multiplying the measured CD values of the protein spectrum by the CD values of this curve at each wavelength, will adjust the spectrum so that it better matches the spectra measured on any other instrument where this procedure is carried out.