Calibration and Standardisation of Synchrotron Radiation Circular Dichroism and Conventional Circular Dichroism Spectrophotometers英文文献资料.docVIP

Spectroscopy 17 (2003) 653–661 IOS Press 653 Calibration and Standardisation of Synchrotron Radiation Circular Dichroism and Conventional Circular Dichroism Spectrophotometers Andrew J. Miles , Frank Wien , Jonathan G. Lees , A. Rodger , Robert W. Janes and a a a b c B.A. Wallace a,? aDepartment of Crystallography, Birkbeck College, University of London, London WC1E 7HX, UK bDepartment of Chemistry, Warwick University, Coventry CV4 7AL, UK cSchool of Biological Sciences, Queen Mary, University of London, London E1 4NS, UK Abstract. Synchrotron radiation circular dichroism (SRCD) is an emerging technique in structural biology with particular value in protein secondary structure analyses since it permits the collection of data down to much lower wavelengths than conventional circular dichroism (cCD) instruments. Reference database spectra collected on different SRCD instruments in the future as well as current reference datasets derived from cCD spectra must be compatible. Therefore there is a need for standardization of calibration methods to ensure quality control. In this study, magnitude and optical rotation measurements on four cCD and three SRCD instruments were compared at 192.5, 219, 290 and 490 nm. At high wavelengths, all gave comparable results, however, at the lower wavelengths, some variations were observable. The consequences of these differences on the spectrum, and the calculated secondary structure, of a representative protein (myoglobin) are demonstrated. A method is proposed for standardising spectra obtained on any CD instrument, conventional or synchrotron-based, with respect to existing and future databases. 1. Introduction Synchrotron radiation circular dichroism (SRCD) spectroscopy is a method that was ?rst developed more than 20 years ago [1,2] but which is just beginning to realise its full potential in