EPR studies on the selectivity of hydroxyl radical attack on amino acids and peptides

Direct rapid-flow EPR experiments together with computer simulations have been used to examine the selectivity of hydroxyl radical (generated using a Ti3+/H2O2 redox couple) attack on a number of aliphatic amino acids, amino acid derivatives and small peptides. For glycine, glycine derivatives and glycine peptides attack at the alpha-carbon position predominates under all conditions; in peptides attack at the C-terminal site is preferred over mid-chain sites, which in turn are favoured over the N-terminal position. This behaviour is rationalised in terms of the destabilising effect of the protonated alpha-amino group, which can exert both short- and long-range effects. With alanine peptides hydrogen atom abstraction at the side-chain methyl group predominates with free amino acid; significant levels of attack at the alpha-carbon position are however observed with peptides. In contrast, with valine and leucine peptides side-chain attack always predominates irrespective of whether the backbone amino group is derivatized or not; the ratio of side-chain species is also only marginally affected. The preference for attack at tertiary side-chain sites over primary side-chain methyl groups in such peptides is small. These results support the hypothesis that the selective fragmentation of large proteins as a result of exposure to hydroxyl radicals in the presence of oxygen may occur primarily as a result of attack at the alpha-carbon position of Surface-exposed glycine and alanine residues.