REVISED PROTOCOLS FOR USE OF DISPERSIBLE RADIOACTIVITY AT X 9A

ydroxyl radicals formed by radiolysis can function as a chemical probe since their reactivity with proteins and DNA-protein complexes is influenced by solvent accessibility within these molecules. In contrast to other methods with reaction time scales of a few seconds where bonds of the peptide backbone are cleaved our studies of proteins have revealed that amino acids side chains are modified by synchrotron radiolysis. In order to apply this technology to characterize transient intermediates of a protein folding process and protein/ligand interactions, it is necessary to understand the nature and reactivity order of radiolytic modifications to amino acids. This investigation reports the mass spectrometric identification of radiolysis-induced modification of amino acids: Phe, Tyr, Pro, Met, Cys and Trp. The additional use of O18-labeled water under aerobic experimental conditions reveals that amino acid side chain modifications occur through two pathways involving both hydroxyl radicals from water and molecular oxygen from air. The order of reactivity for these amino acids has been examined by tandem mass spectrometry.