STRUCTURAL CHARACTERIZATION OF THE PRODUCTS OF HYDROXYL-RADICAL DAMAGE TO LEUCINE AND THEIR DETECTION ON PROTEINS

We have previously reported the formation of valine hydroperoxides and aldehydes from hydroxyl-radical attack on free valine and protein mol ecules. We have also demonstrated that the major degradation products of valine hydroperoxides by several biochemical and cellular systems a re the corresponding hydroxides, and therefore proposed that hydroxyva lines may serve as useful in vivo markers for studying protein oxidati on. Here we have undertaken the structural elucidation of the oxidatio n products of leucine, another amino acid which is very susceptible to peroxidation. Hydroxyl-radical (HO.) attack on L-leucine in the prese nce of oxygen, followed by NaBH4 reduction, gave rise to Eve major oxi dation products which have been isolated and identified. On the basis of chemical and spectroscopic evidence, the five products have been id entified as (2S)-gamma-hydroxyleucine, (2S,4S)-delta-hydroxyleucine, ( 2S,4R)-delta-hydroxyleucine, (2S,4R)-4-methylproline (trans-4-methyl-L -proline) and (2S,4S)-4-methylproline (cis-4-methyl-L-proline). The th ree hydroxyleucines have been confirmed to be the reduction products o f the corresponding hydroperoxyleucines, while the two proline analogu es are from reduction of their corresponding cyclic Schiff bases. By H PLC analysis using post-column omicron-phthaldialdehyde derivatization , we have detected hydroxyleucines in the hydrolysates of tripeptides and proteins which had been gamma-radiolysed and treated with NaBH4. F urthermore, we demonstrate the occurrence of the hydroxyleucines on pr oteins in physiological and pathological samples. Hydroxyleucines, lik e hydroxyvalines, may provide useful in vivo markers for studying prot ein oxidation. In the present study we also investigated the competiti on between leucine, valine and phenylalanine for HO., and proposed a p ossible radical-transfer process in such free-radical reactions.