Identification of the sites of hydroxyl radical reaction with peptides by hydrogen/deuterium exchange: Prevalence of reactions with the side chains

Hydroxyl radical-effected protium/deuterium (H-1/H-2) exchange into the C-H bonds present in peptides has been used to identify the site of hydrogen a tom abstraction by hydroxyl radical. Radiolysis of anaerobic, N2O-saturated D2O solutions containing peptide and dithiothreitol generates a hydroxyl r adical that mediates H-1/H-2 exchange into the side chains of peptides of u p to 66 atom % excess H-2. The H-1/H-2 exchange is determined by measuring the isotope ratio, [M + H + 1](+)/[M + H](+), of the peptide using electros pray ionization-mass spectrometry. The H-1/H-2 exchange within each residue of the peptide was determined by measuring the isotope ratio of each isola ted dansyl amino acid following hydrolysis and derivatization. Generation o f 0.40 mM hydroxyl radical effected H-1/H-2 exchange into each of the five different residues of (Ala(2))-leucine enkephalin (YAGFL). The propensity o f the residues to undergo exchange was L > Y > A congruent to F > G, indepe ndent of whether they were radiolyzed separately or as the peptide. The min imal exchange into glycine suggests that reaction of hydroxyl radical with the side chain hydrogens predominates over reaction with the polypeptide al pha-hydrogens. The ability of radiolysis to effect H-1/H-2 exchange into a larger peptide, SNEQKACKVLGI, was also demonstrated.