Lysine, hydroperoxide formation

Hi. Lysine. Gamma radiolysis of aerated aqueous solution of lysine (94) has been shown, as inferred from iodometric measurements, to give rise to hydroperoxides in a similar yield to that observed for valine and leucine. However, attempts to isolate by HPLC the peroxidic derivatives using the post-column derivatization chemiluminescence detection approach were unsuccessful. This was assumed to be due to the instability of the lysine hydroperoxides under the conditions of HPLC analysis. Indirect evidence for the OH-mediated formation of hydroperoxides was provided by the isolation of four hydroxylated derivatives of lysine as 9-fluoromethyl chloroformate (FMOC) derivatives . Interestingly, NaBILj reduction of the irradiated lysine solutions before FMOC derivatization is accompanied by a notable increase in the yields of hydroxylysine isomers. Among the latter oxidized compounds, 3-hydroxy lysine was characterized by extensive H NMR and ESI-MS measurements whereas one diastereomer of 4-hydroxylysine and the two isomeric forms of 5-hydroxylysine were identified by comparison of their HPLC features as FMOC derivatives with those of authentic samples prepared by chemical synthesis. A reasonable mechanism for the formation of the four different hydroxylysines and, therefore, of related hydroperoxides 98-100, involves initial OH-mediated hydrogen abstraction followed by O2 addition to the carbon-centered radicals 95-97 thus formed and subsequent reduction of the resulting peroxyl radicals (equation 55). 

As already mentioned, one of the products of action of hydroxyl radicals on proteins is protein hydroperoxides (G6). Valine and lysine residues are particu-larily susceptible to hydroperoxide formation. Reduction of hydroperoxides produces respective hydroxy derivatives of amino acids. Three valine hydroxides derived from hydroperoxides of this amino acid have been characterized structurally as p-hydroxyvaline [(2S)-2-amino-3-hydroxy-3-methyl-butanoic acid], (2S,3S)-y-hydroxyvaline [(2S,3S)-2-amino-3-hydroxymethyl-butanoic acid], and (2S,3R)-y -hydroxyvaline [(2S,3R)-2-amino-3-hydroxymethyl-butanoic acid (Fig. 12). They are suggested to be possible markers of protein peroxidation (F21). 

Previously, we have examined the formation of amino acid hydroperoxides following exposure to different radical species [100]. We observed that valine was most easily oxidised, but leucine and lysine are also prone to this modification in free solution. Scheme 12 illustrates the mechanism for formation of valine hydroperoxide. However, tertiary structure becomes an important predictor in proteins, where the hydrophobic residues are protected from bulk aqueous radicals, and lysine hydroperoxides are most readily oxidised. Hydroperoxide yield is poor from Fenton-derived oxidants as they are rapidly broken down in the presence of metal ions [101]. Like methionine sulphoxide, hydroperoxides are also subject to repair, in this case via glutathione peroxidase. They can also be effectively reduced to hydroxides, a reaction supported by the addition of hydroxyl radical in the presence of oxygen. Extensive characterisation of the three isomeric forms of valine and leucine hydroxides has been undertaken by Fu et al. [102,103], and therefore will not be discussed further here. 

Fig. 7. Oxidation products of proteins. The vertical structure in the middle represents the main peptide chain with amino acid side groups extending horizontally (M2). The a-carbons in the primary chain can be oxidized to form hydroperoxides. Reactions on the right side near the top exemplify oxidation of the primary chain leading to a peroxyl radical. Side chains represented are lysine, methionine, tyrosine, cysteine, and histidine, top to bottom, respectively. Modifications of the side chains and primary chain lead to carbonyl formation and charge modifications. If these reactions are not detoxified by antioxidants, they may propagate chain reactions within the primary chain, leading to fragmentation of the protein. See the text for details, o, represents reaction with oxygen RNS, reactive nitrogen species ROS, reactive oxygen species. Dense dot represents unpaired electron of radical forms.

Lipoxygenase (LOX) converts polyunsaturated fatty acids, such as linoleic and linolenic acids, to lipid hydroperoxides (Figure 2)(52,73,74). The lipid hydroperoxides then form hydroperoxide radicals, epoxides, and/or are degraded to form malondialdehyde. These products are also strongly electrophilic, and can destroy individual amino acids by decarboxylative deamination (e.g., lysine, cysteine, histidine, tyrosine, and tryptophan) cause free radical mediated cross-linking of protein at thiol, histidinyl, and tyrosinyl groups and cause Schiff base formation (e.g., malondialdehyde and lysine aldehyde) (39,49,50,74-78). 

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