Structural characterization of the products of hydroxyl-radical damage to leucine and their detection on proteins

Abstract

We have previously reported the formation of valine hydroperoxides and aldehydes from hydroxyl-radical attack on free valine and protein molecules. We have also demonstrated that the major degradation products of valine hydroperoxides by several biochemical and cellular systems are the corresponding hydroxides, and therefore proposed that hydroxyvalines may serve as useful in vivo markers for studying protein oxidation. Here we have undertaken the structural elucidation of the oxidation products of leucine, another amino acid which is very susceptible to peroxidation. Hydroxyl-radical (HO•) attack on l-leucine in the presence of oxygen, followed by NaBH4 reduction, gave rise to five major oxidation products which have been isolated and identified. On the basis of chemical and spectroscopic evidence, the five products have been identified as (2S)-γ-hydroxyleucine, (2S,4S)-δ-hydroxyleucine, (2S,4R)-δ-hydroxyleucine, (2S,4R)-4-methylproline (trans-4-methyl-l-proline) and (2S,4S)-4-methylproline (cis-4-methyl-l-proline). The three hydroxyleucines have been confirmed to be the reduction products of the corresponding hydroperoxyleucines, while the two proline analogues are from reduction of their corresponding cyclic Schiff bases. By HPLC analysis using post-column o-phthaldialdehyde derivatization, we have detected hydroxyleucines in the hydrolysates of tripeptides and proteins which had been γ-radiolysed and treated with NaBH4. Furthermore, we demonstrate the occurrence of the hydroxyleucines on proteins in physiological and pathological samples. Hydroxyleucines, like hydroxyvalines, may provide usefulin vivo markers for studying protein oxidation. In the present study we also investigated the competition between leucine, valine and phenylalanine for HO•, and proposed a possible radical-transfer process in such free-radical reactions.