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![[Figure 1]](ig5034fig1mag.jpg)
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Figure 1
Major reactions scenarios for X-ray radiolysis in dilute protein samples. (a) Schematic showing the position of bulk-, surface- and bound-water (cavity- and buried-water) (light blue) in a protein molecule (dark blue). (b) Radiolysis of water and the timescale of sequence of events. Reproduced from Gupta et al. (2014  ) and Liljenzin (2002). (c) The location of hydroxyl radicals (red) generated in situ from ionization or activation of water by X-ray irradiation. The ![[^\bullet]](teximages/ig5034fi1.gif) OH radicals react with nearby side chains in close proximity, and yield covalent modifications on the protein side chains (yellow). Radiolysis of bulk water starts with the ionization of water on the time scale of 10−16 s. The key product, OH, diffuses (10−7 s) out in the bulk (red arrows) and undergoes reactions with other OH, buffer molecules and protein side chains (bimolecular reactions are indicated by black arrows, and approximate values of the rate constants for different reactions are shown). The rapid counterproductive reactions, such as OH—OH recombination, as well as various other reactions, scavenge OH and reduce the concentration of OH in the bulk. Thus a sufficient X-ray dose or high-flux-density beam is needed to maintain a steady concentration of OH that will lead to a detectable yield of side-chain modification on the protein in solution. In contrast, OH radicals formed from activation of a bound water can react faster with side chains in proximity because of the translational and rotational ordering of water and because fewer scavenging reactions by other OH or highly reactive buffer constituents are available. Radiolysis of water also produces electrons, which rapidly become solvated and react with O2 to produce superoxide radicals. In general, the reactivity of side chains to solvated electrons is lower than to hydroxyl radicals (Xu & Chance, 2007). Peroxides and superoxides undergo slow reactions with protein side chains and are quenched as described in §2.1 and §2.3. Solvated electrons consume O2, which is also required for side-chain labeling by OH radicals (Fig. 2); thus short irradiation time as well as high flux density are the key factors for success of the XF-MS experiment. Reproduced in part from Gupta et al. (2014). |
![[Figure 1]](ig5034fig1.jpg)
 | JOURNAL OF SYNCHROTRON RADIATION |
ISSN: 1600-5775
