Abstract

Hydroxyl radicals produced by radiolysis under anaerobic conditions in the presence of dithiothreitol and D2O have been shown to be capable of inducing hydrogen/deuterium (1H/2H) exchange in carbon-hydrogen bonds of amino acids. When the solution is saturated with N2O, a 1H/2H exchange efficiency of 38% (based on the G value of 5.6 x 10(-7) mol J(-1) for hydroxyl radical) was determined by measuring the amino acid isotope ratio [M+H+1]+/[M+H]+ using electrospray ionization-mass spectrometry. The incorporation of 2H was proportional to the amount of hydroxyl radical generated and required the presence of dithiothreitol. Using standard anaerobic reaction conditions with dithiothreitol and N2O, incorporations of 2H of 3% and 8% into L-valine (100 microM, 35 microM DTT) and L-leucine (100 microM, 31 microM DTT), respectively, were achieved after a dose of 89 Gy and, using d8-DL-valine (100 microM, 35 microM DTT) with H2O as the solvent, approximately 2% incorporation of protium was detected. Additionally, 1H/2H exchange into the peptide (Ala2)-leucine enkephalin produced 6% incorporation of 2H. 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.

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