β-Scission of C-3 (β-Carbon) Alkoxyl Radicals on Peptides and Proteins: A Novel Pathway Which Results in the Formation of α-Carbon Radicals and the Loss of Amino Acid Side Chains

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Abstract
Exposure of proteins to radicals in the presence of O2 brings about multiple changes in the target molecules. These alterations include oxidation of side chains, fragmentation, cross-linking, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes, and formation of new reactive groups, including hydroperoxides. These processes can result in the loss of structural or enzymatic activity. Backbone fragmentation is known to occur via a number of mechanisms, most of which involve hydrogen abstraction from the α-carbon site on the backbone. In this study, we demonstrate that initial attack at a side chain site, the β-position (C-3), can give rise to formation of α-carbon radicals, and hence backbone cleavage, via the formation and subsequent β-scission of C-3 alkoxyl radicals. This β-scission reaction is rapid (k estimated to be >107 s-1) even with primary alkoxyl radicals derived from Ala residues, and occurs when the alkoxyl radicals are generated from a variety of precursors, including hydroperoxides and nitrate esters. These reactions release the former side chain as a reactive aldehyde or ketone; thus, Ala peptides release high yields of methanal (formaldehyde). This product has been quantified with a number of oxidized peptides and proteins, and can account for up to 64% of the initial attacking radicals with some Ala peptides. When quantified together with the hydroperoxide precursors, these species account for up to 80% of the initial radicals, confirming that this is a major process. Methanal causes cell toxicity and DNA damage and is an animal carcinogen and a genotoxic agent in human cells. Thus, the formation and subsequent reaction of alkoxyl radicals formed at the C-3 position on aliphatic amino acid side chains on peptides and proteins can give rise to both backbone fragmentation and the release of further reactive species which can cause cell toxicity and mutagenicity.