Amino acid hydroperoxides, structure

Another derivatization approach is reduction of the hydroperoxide, followed by structural characterization of the corresponding alcohol, which is usually easier to handle. Thus, the structure of amino acid hydroperoxides can be characterized more easily if, after having ascertained the hydroperoxide nature of the compound, it is reduced to the alcohol with NaBH4. The structure of three valine hydroperoxides obtained on y-radiation of bovine serum albumin, a tripeptide (31) or valine (34) was elucidated after reduction, hydrolysis (if necessary), chromatographic separation, and application of the usual MS and NMR methods on the individual hydroxy derivatives of valine. ... 

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. 

Peroxidases are heme iron-containing proteins similar in structure to that of cytochromes P450. The major difference is that peroxidases have histidine as the axial ligand instead of cysteine, and there are also other polar amino acids close to the heme iron that help to catalyze the peroxidase function of the enzyme (41). The result is that the peroxidases very rapidly catalyze the reduction of hydroperoxides to alcohols (or water in the case of... 

Knowledge about the chemical structure of the antioxidative MRP is very limited. Only a few attempts have been made to characterize them. Evans, et al. (12) demonstrated that pure reductones produced by the reaction between hexoses and secondary amines were effective in inhibiting oxidation of vegetable oils. The importance of reductones formed from amino acids and reducing sugars is, however, still obscure. Eichner (6) suggested that reductone-like compounds, 1,2-enaminols, formed from Amadori rearrangement products could be responsible for the antioxidative effect of MRP. The mechanism was claimed to involve inactivation of lipid hydroperoxides. 

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). 

F21. Fu, S., Hick, L. A., Sheil, M. M., and Dean, R. T., Structural identification of valine hydroperoxides and hydroxides on radical-damaged amino acid, peptide, and protein molecules. Free Radicals Biol. Med. 19, 281-292 (1995). 

When the crystal structure of a protein is not available, other techniques can be employed to identify the amino acids that are involved in its structure and function. Commonly used techniques include chemical cross-linking, site-specific chemical modifications, and mutagenesis. Chemical modifications of Met residues using oxidizing agents such as hydrogen peroxide, t-butyl hydroperoxide, chloramine T, and sodium periodate have been useful in identifying structure and function relationships in many proteins (1-7). 

The most popular method involves 2-thiobarbituric acid (TBA) two molecules of 2-thiobarbituric acid are condensed with malonaldehyde. The emergent chromogen — the two tautomeric structures of the red TBA-malonaldehyde adduct — is determined at 532 nm, and also often at 450 nm, to determine aUcenals and aUcanals, respectively. The qualitative Kreis test was based on a similar principle it involved detection of the epihydrine aldehyde — a tautomeric malondialdehyde — in a color reaction with resorcine or phloroglucinol. The popularity of the TBA test stems from a correlation between the results and sensory evaluations. Paradoxically, this is related to the most important drawback of the TBA technique — its lack of specificity. In addition to the reaction with malonaldehyde, TBA forms compounds of identical color with other aldehydes and ketones, products of aldehyde interaction with nitrogen compounds, and also with saccharides, ascorbic acid, creatine, creatinine, trimethylamine oxide, trimethylamine, proteins, and amino acids. For this reason, the TBA test may even be treated as a proteolysis indicator (Kolakowska and Deutry, 1983). Recently, TBA-reactive substances (TEARS) were introduced, primarily to stress that the reaction involves hydroperoxides in addition to aldehydes. Due to the nonspecificity of the TEARS test, its results reflect the rancidity of food better than other conventional methods, especially off-flavor, which is caused by volatiles from lipids as well as being affected by products of lipids interaction with nitrogenous compounds. 

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.

Schiff base compounds formed by the interaction of oxidation products with proteins, phospholipids and nucleic acids produce chromophores showing characteristic fluorescence spectra. The Schiff base formed between malonaldehyde and amino acids is attributed to the conjugated structure -NH=CH-CH=CH-NH-. Lipid-soluble fluorescence chromophores are produced from oxidized phospholipids and from oxidized fatty acid esters in the presence of phospholipids. These chromophores have fluorescence emission maxima at 435-440 nm and excitation maxima at 365 nm. The Schiff base of malonaldehyde and phospholipids has a higher wavelength maximum for emission (475 nm) and excitation (400 nm). The interaction between oxidized arachidonic acid and dipalmityl phosphatidylethanolamine produce similar fluorescence spectra (maximum excitation at 360-90 nm and maximum emission at 430-460 nm). The products from oxidized arachidonic acid and DNA have characteristic fluorescence spectra, with excitation maximum at 315 nm and emission maximum at 325 nm. Similar fluorescence spectra, with excitation maximum at 320 mn and emission maximum at420 nm, are obtained from the interactions of either lipid hydroperoxides or secondary oxidation products with DNA in the presence of metals and reducing agents, or different aldehydes, ketones and dimeric compounds from oxidized linolenate. Therefore, the Schiff base produced from various oxidized lipids and phospholipids and DNA may be considered to be due to a mixture of closely related chromophores. 

The structural features of 11b are noteworthy even in the presence of excess amino acid, only the 1 1 complex of 12 and Co was detected by NMR spectroscopy in sharp contrast to complex 4 (Fignre 4.4). This is probably due to the steric bulk of ligand 12 and explains its increased reactivity and lower stability. Unfortunately, we were unable to obtain reproducible results using complex 11b, as yields (40-70%) and reaction time (8 8 h) were batch-dependent. In many cases, an initiation time was observed before the reaction started. Mukaiyama and co-workers used ferf-butyl hydroperoxide as a cobalt-catalyst for the hydration of certain olefins when initiation of the reaction was difficult. A similar effect was observed in the hydroazidation reaction when using catalyst 11 with ethanesulfonyl azide (7) for the hydroazidation of 4-phenylbut-l-ene (3), complete conversion was observed after 2-8 h using 30% of ferf-butyl hydroperoxide. In situ formation of complex 11b in the reaction mixture leads to reproducible reaction times (2h) and yields (70%). Co(BF4)2-6H20 was the best Co salt for this procedure, as complex formation was faster than with other salts and quick oxidation to the Co(III) complex occurred in the presence of tert-butyl hydroperoxide. 

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