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Spectroscopy
Volume 19, Issue 1, Pages 43-51
http://dx.doi.org/10.1155/2005/263649

Calibration and standardisation of synchrotron radiation and conventional circular dichroism spectrometers. Part 2: Factors affecting magnitude and wavelength

Andrew J. Miles,1 Frank Wien,1 Jonathan G. Lees,1,3 and B.A. Wallace1,2

1Department of Crystallography, Birkbeck College, University of London, London WC1E 7HX, United Kingdom
2Centre for Protein and Membrane Structure and Dynamics, Daresbury Laboratory, Warrington WA4 4AD, United Kingdom
3School of Biological Sciences, Queen Mary, University of London, London E1 4NS, United Kingdom

Copyright © 2005 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Circular dichroism (CD) spectroscopy is an important tool in structural biology, especially for protein secondary structure analyses. Synchrotron radiation circular dichroism (SRCD) spectroscopy is a modified version of the technique that uses the intense light from a synchrotron source to enable the collection of data to much lower wavelengths than possible on conventional circular dichroism (cCD) instruments. There is a need for standardization of calibration methods amongst and between cCD and SRCD instruments to ensure consistency and the ability to use common reference databases for empirical secondary structural analyses. In a previous study (Spectroscopy17 (2003), 653–661), we compared optical rotation measurements on several cCD and SRCD instruments, whilst holding constant other experimental factors. In this study, other experimental parameters which contribute to the spectral magnitude, such as cell pathlength and protein concentration determinations, are examined. In addition, the extent of wavelength calibration variations between instruments and their effects on secondary structure calculations have been examined. Hence, this paper provides additional practical guidance for “good practice” in the measurement of CD data.