Hydroxyl Radical-Induced Hydrogen/Deuterium Exchange in Amino Acid Carbon-Hydrogen Bonds

Hydroxyl radicals produced by radiolysis under anaerobic conditions in the presence of dithiothreitol and D2 O have been shown to be capable of inducing hydrogen/deuterium (^{1}{\rm H}/{}^{2}{\rm H}$) exchange in carbon-hydrogen bonds of amino acids. When the solution is saturated with N2 O, a ^{1}{\rm H}/{}^{2}{\rm H}$ exchange efficiency of 38% (based on the G value of $5.6\times 10^{-7}\ {\rm mol}\ {\rm J}^{-1}$ for hydroxyl radical) was determined by measuring the amino acid isotope ratio $[{\rm M}+{\rm H}+1]^{+}/[{\rm M}+{\rm H}]^{+}$ using electrospray ionization-mass spectrometry. The incorporation of 2 H was proportional to the amount of hydroxyl radical generated and required the presence of dithiothreitol. Using standard anaerobic reaction conditions with dithiothreitol and N2 O, incorporations of 2 H of 3% and 8% into L-valine (100 μM, 35 μM DTT) and L-leucine (100 μM, 31 μM DTT), respectively, were achieved after a dose of 89 Gy and, using $d_{8}\text{-}{\rm DL}\text{-valine}$ (100 μM, 35 μM DTT) with H2 O as the solvent, approximately 2% incorporation of protium was detected. Additionally, ^{1}{\rm H}/{}^{2}{\rm H}$ exchange into the peptide $({\rm Ala}^{2})\text{-leucine}$ enkephalin produced 6% incorporation of 2 H. These results directly demonstrate the ability of sulfhydryl groups to mediate the chemical repair of proteins through hydrogen-atom donation to an amino acid carbon-centered radical, thus providing a means of isotopically labeling solvent-accessible amino acid residues of peptides and proteins.