Dosimetry Determines the Initial OH Radical Concentration in Fast Photochemical Oxidation of Proteins (FPOP)

Abstract

Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally. We report here an approach using isotope dilution gas chromatography/mass spectrometry (GC/MS) to determine quantitatively the initial •OH concentration (we found ~ 0.95 mM from 15 mM H2O2) from laser photolysis and to investigate the quenching efficiencies for various •OH scavengers.

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…The ⋅OH concentration profile calculated under pseudo-first order conditions for [Gln] = 20 mM drops close to zero within 1 μs (blue line, Figure 1b) [30]. This plot represents the foundation of the claim that FPOP is a microsecond covalent labeling technique [30][31][32].…”

“…Photochemical cleavage of hydrogen peroxide by a nanosecond laser pulse generates radicals according to H 2 O 2 → 2 ⋅OH. In purely aqueous solution and for an initial ⋅OH concentration of 1 mM [32], the recombination (2 ⋅OH → H 2 O 2 , k = 6 × 10 9 M -1 s -1 ) [46] will follow the profile of Figure 1a, where a significant ⋅OH concentration persists over a few μs and beyond. FPOP employs scavengers (usually Gln) to shorten the ⋅OH lifetime.…”

“…This extraordinarily short time frame has important implications. First, under single-hit conditions, FPOP should be immune to labeling-induced artifacts because protein tagging will be complete before conformational changes can take place [30][31][32]. Secondly, FPOP holds promise for examining short-lived protein folding intermediates [33].…”

“…Instead, the assertion of a~1 μs labeling pulse is based on kinetic estimates [30], indirect statistical data [31], and stationary (not timeresolved) dosimetry [32]. It is instructive to revisit the key arguments [30].…”

“…It cannot be ruled out that secondary radicals contribute to analyte oxidation within the FPOP flow tube. In other words, the claim that FPOP labeling is restricted to ã 1 μs time scale [30][31][32] has to be carefully re-examined.…”