Methods for Generating Hydroxyl Radicals

Hydroxyl radicals can be generated chemically by using the Fenton reagent [117]: Fe2+ reduces H2O2 to hydroxide and hydroxyl radical, but this process is slow. The radicals can also be generated by radiation: for example, synchrotron radiation cleaves water into a proton, electron and a hydroxyl radical [118], whereas UV light homolytically cleaves H2O2 into two hydroxyl radicals [119, 120]. As these methods require tens of milliseconds to minutes [121], we utilized a UV laser, which should have advantages for the fast photolysis of hydrogen peroxide into hydroxyl radicals [30]. No significant oxidation should occur prior to the laser pulse (traces of prior oxidation can easily be confirmed by performing a simple peroxide-plus-protein control; Fig. 11.8A). We expect the laser-produced hydroxyl radicals to react with the protein side-chains or recombine to reform H2O2. Owing to these two pathways, the radical concentration drops to below 1 mM within approximately 100 ms as determined by kinetic calculations using the known rate constant for hydroxyl-radical recombination [122]. The protein oxidation profile that is achieved in that timeframe shows considerable protein oxidation (note the peaks separated by 16 Da in Fig. 11.8B).

By adding excess chemical quencher to the system prior to irradiation, the radicals should react with the quencher according to first order kinetics. If 20 mM phenylalanine were added to the system, the radicals would be consumed within 70 ns of the laser pulse, and the use of 20 mM glutamine results in complete reaction of all radicals within 1 ms of the laser pulse (Fig. 11.8C). Given that protein secondary structure packing does not unfold faster than 10 ms, for even the fastest systems studied thus far [30, 123-125], a 1-ms reaction timescale eliminates nearly all concerns about protein unfolding as a result of oxidation. The other way of mitigating this concern is to conduct the footprinting under ''single hit''

Fig. 11.8 (A) Laser irradiation of 10 mM apomyoglobin in 10 mM NaH2PO4, pH 7.8, and 20 mM phenylalanine as a scavenger. (B) Oxidation of 10 mM apomyoglobin in 10 mM NaH2PO4, pH 7.8, 15 mM H2O2, and 20 mM glutamine as a scavenger, limiting the reaction to 1 ms. (C) Oxidation of 10 mM apomyoglobin in 10 mM NaH2PO4, pH 7.8, 15 mM H2O2 with no scavenger, resulting in up to 100 ms reaction duration.

Fig. 11.8 (A) Laser irradiation of 10 mM apomyoglobin in 10 mM NaH2PO4, pH 7.8, and 20 mM phenylalanine as a scavenger. (B) Oxidation of 10 mM apomyoglobin in 10 mM NaH2PO4, pH 7.8, 15 mM H2O2, and 20 mM glutamine as a scavenger, limiting the reaction to 1 ms. (C) Oxidation of 10 mM apomyoglobin in 10 mM NaH2PO4, pH 7.8, 15 mM H2O2 with no scavenger, resulting in up to 100 ms reaction duration.

conditions, where each protein molecule reacts only once with the radical. ''Single hit'' conditions, whereby each protein contains only one oxidation site, are difficult to achieve while still affording good coverage and sensitivity, however, and concerns linger when any method deviates from being fast and/or ''single hit'' [126].

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