Data Collection

CD data can be collected as a function of wavelength (scan) or time (assay). To collect a scan, the wavelength range, number of data points, and the integration mode must first be selected. Note that choosing an exact wavelength range is essential in order to apply a secondary structure analysis (see Secondary Structure Analysis of Proteins for details; 260-190 nm is the standard for application of most algorithms.

Two integration modes are available. The first is a constant integration time mode, in which the time spent collecting data is constant and user defined for each data point. Because light levels often vary enormously across a spectral range in a CD scan, a second integration time mode is available in which the integration time is a function of the PMT detector high volts (thus, it is a function of the intensity of light). At regions of the spectrum in which light levels are high, a short integration time is used. When light levels are low, a longer time is spent collecting data. The integration time increases as an exponential function that can be adjusted by the user. The goal of this function mode is to achieve a reasonably constant signal/noise ratio throughout the spectrum (Figure 5).

Fixed Time/Datum Note insufficient time for best S/N below 200 nm

Variable Time/Datum Equal S/N along entire range

Fig. 5

To collect a baseline, place a cuvette containing all components except the sample (e.g. for a protein the baseline will be the cuvette containing buffer only). It is important to include the cuvette in the baseline because birefringence of the cuvette can cause an apparent CD signal. It is also beneficial to place the cuvette in the same orientation it will be during the actual measurement. Once a scan is collected, it must be assigned as the baseline. The baseline will be subtracted from all subsequent data until this baseline is deselected. In the assay mode, data is collected as a function of time. The total time of the assay as well as the number of points can be selected. In addition, a baseline value of the cuvette and buffer can be subtracted from the data.

Scans (or assays) can be made as a function of time, temperature, or concentration of titrant. To accomplish this, a script function is employed in which scans are automatically collected as a function of temperature or concentration. The script file is chosen in the repeated scans mode. The type of script (temp or titration), number of scans to be taken, the temperatures (or volumes to be delivered), and incubation times are set in this file. Data collection results in a 3-dimensional data file in which the axes are wavelength, temperature (or concentration), and CD signal. The data can be fit globally using GlobalWorks (see Thermal or Chemical Denaturation of Proteins) or 2D data can be extracted and fit.

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