McGeehan John, Ravelli Raimond B G, Murray James W, Owen Robin Leslie, Cipriani Florent, McSweeney Sean, Weik Martin, Garman Elspeth F
EMBL, 6 rue Jules Horowitz, 38042 Grenoble, France.
J Synchrotron Radiat. 2009 Mar;16(Pt 2):163-72. doi: 10.1107/S0909049509001629. Epub 2009 Feb 25.
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.
X射线可在低温冷却的大分子晶体中产生高浓度的自由基。一些自由基在电磁光谱的可见光（VIS）范围内具有较大的消光系数，可通过光学和光谱方法进行观测。现已构建了一台具有高时间分辨率的在线显微分光光度计，它能够在X射线数据收集过程中测量紫外/可见吸收光谱（200 - 1100纳米）。在多种低温条件下典型的X射线诱导产生的蓝色已被确定为捕获的溶剂化电子。含二硫键的蛋白质在毫摩尔浓度下会形成二硫自由基，其吸收最大值在400纳米左右。在100 K时，溶剂化电子和二硫自由基的寿命似乎在数秒至数分钟的范围内。与二硫自由基相比，溶剂化电子的X射线诱导自由基形成动力学的温度依赖性有所不同。在线显微分光光度计为分析和表征蛋白质及酶的中间体和氧化还原状态提供了一种与X射线衍射互补的技术。
