Overcoming protein denaturation caused by irradiation in a high-flux synchrotron radiation circular dichroism beamline.
It has been established that the new circular dichroism beamline CD12 has sufficiently high flux at low wavelengths to cause apparent irradiation problems with protein samples while their synchrotron radiation circular dichroism (SRCD) spectra are being collected. The cause of this effect has been extensively investigated and is reported in an accompanying paper [Wien et al. (2005). J. Synchrotron Rad. 12, 517-523.]. Experiments suggest that localized heating of the protein sample, leading to denaturation, is the probable cause. Methods to circumvent this problem by limiting the beam flux are reported. This was achieved using either an attenuation cell of water placed beam-side of the sample cell, or limiting the beam cross-sectional area hitting the sample. Such methods are shown to result in substantial reduction or apparent complete removal of this protein denaturation over the course of collecting three successive spectra. Elimination of this denaturation problem enables multiple SRCD scans for protein samples to be collected, which are vital both for good practice and for statistically valid results.
Janes
Robert W
RW
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK. r.w.janes@qmul.ac.uk
Cuff
Alison L
AL
D002942:Circular Dichroism / Q000295:instrumentation / Q000379:methods*; D003461:Crystallography / Q000295:instrumentation / Q000379:methods; D004307:Dose-Response Relationship, Radiation; D009113:Muramidase / Q000032:analysis / Q000737:chemistry* / Q000528:radiation effects*; D011487:Protein Conformation / Q000528:radiation effects; D011489:Protein Denaturation / Q000528:radiation effects; D011829:Radiation Dosage; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry* / Q000528:radiation effects*; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods*; D017356:Synchrotrons; D014965:X-Rays
D012709:Serum Albumin; D009113:Muramidase