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X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200-1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.

Original publication

DOI

10.1107/S0909049509001629

Type

Journal article

Journal

J Synchrotron Radiat

Publication Date

03/2009

Volume

16

Pages

163 - 172

Keywords

Cold Temperature, Color, Computer-Aided Design, Crystallization, Equipment Design, Equipment Failure Analysis, Online Systems, Reproducibility of Results, Sensitivity and Specificity, Spectrophotometry, Ultraviolet, X-Ray Diffraction