Radicals protein hydroperoxide formation

Gebicki JM (1997) Protein hydroperoxides as new reactive oxygen species. Redox Report 3 99-110 Gebicki S, Gebicki JM (1993) Formation of peroxides in amino acids and proteins exposed to oxygen free radicals. Biochem J 289 743-749... 

Fig. 1. a-Oxidation of amino acids. Hydroxyl radical (or other reactive radical) abstracts hydrogen atom from the a-carbon. The C-centered free radical formed may react with other amino acid residues or dimerize in the absence of oxygen, which leads to protein aggregation. In die presence of oxygen the carbon-centered radical forms peroxyl radical. Reduction of peroxyl radical leads to protein hydroperoxide. Decomposition of hydroperoxide leads to formation of carbonyl compounds via either oxidative deamination or oxidative decarboxylation. Oxidation of the new carbonyl group forms a carboxyl group. 

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). 

Lipid oxidation in muscle foods is one of the major deteriorative reactions causing losses in quality during processing and storage. The oxidation of unsaturated fatty acids leads to formation of free radicals and hydroperoxide. These intermediary compounds are unstable and cause the oxidation of pigments, flavors, and vitamins. Oxidized unsaturated lipids bind to protein and form insoluble lipid-protein complexes. This accounts for toughened texture and poor flavor of frozen seafoods (Khayat and Schwell, 1983). 

Figure 11.6. Protein-protein crosslink formation by interaction of protein radicals (P ) with lipid hydroperoxides.

Free peroxyl radicals react with proteins with the formation of protein free radicals, which react with other protein free radicals to yield protein dimers, or with lipid free radicals to yield copolymers. The occurrence of a lipoprotein with covalent bonds and protein oligomers during oxidation of proteins (PH) by alkoxyl (RO ) and peroxyl (ROO ) radicals is shown in the following equations. A protein radical (P ) forms most frequently by splitting off the labile hydrogen atom on C , an alkoxyl radical yields a hydroxy acid and a peroxyl radical yields hydroperoxide ... 

Reactions of proteins with Hpid hydroperoxides and other oxidised Hpids give rise to different types of Hpoproteins, in which Hpids and proteins are bound by physical bonds (as in natural Hpoproteins), but also by covalent bonds. Reactions with hydroperoxides, free radicals, epoxides and aldehydes lead to the fission of some protein bonds, formation of protein radicals and oligomers, cross links between protein chains and some sensitive functional groups of amino acids are oxidised (see Section 2.5.1.1). 

Inhibition and stimulation of LOX activity occurs as a rule by a free radical mechanism. Riendeau et al. [8] showed that hydroperoxide activation of 5-LOX is product-specific and can be stimulated by 5-HPETE and hydrogen peroxide. NADPH, FAD, Fe2+ ions, and Fe3+(EDTA) complex markedly increased the formation of oxidized products while NADH and 5-HETE were inhibitory. Jones et al. [9] also demonstrated that another hydroperoxide 13(5)-hydroperoxy-9,ll( , Z)-octadecadienoic acid (13-HPOD) (formed by the oxidation of linoleic acid by soybean LOX) activated the inactive ferrous form of the enzyme. These authors suggested that 13-HPOD attached to LOX and affected its activation through the formation of a protein radical. Werz et al. [10] showed that reactive oxygen species produced by xanthine oxidase, granulocytes, or mitochondria activated 5-LOX in the Epstein Barr virus-transformed B-lymphocytes. 

Schnurr et al. [22] showed that rabbit 15-LOX oxidized beef heart submitochondrial particles to form phospholipid-bound hydroperoxy- and keto-polyenoic fatty acids and induced the oxidative modification of membrane proteins. It was also found that the total oxygen uptake significantly exceeded the formation of oxygenated polyenoic acids supposedly due to the formation of hydroxyl radicals by the reaction of ubiquinone with lipid 15-LOX-derived hydroperoxides. However, it is impossible to agree with this proposal because it is known for a long time [23] that quinones cannot catalyze the formation of hydroxyl radicals by the Fenton reaction. Oxidation of intracellular unsaturated acids (for example, linoleic and arachidonic acids) by lipoxygenases can be suppressed by fatty acid binding proteins [24]. 

In the field of enzyme catalysis, heme-proteins such as cytochrome P450, for example, exhibit both types of 0-0 bond cleavages in organic hydroperoxides and peroxy acids (178). Heterolytic cleavage of HOOH/ROOH yields H20 or the corresponding alcohol, ROH and a ferryl-oxo intermediate (Scheme 4). Homolytic 0-0 bond cleavage results in the formation of a hydroxyl (HO ) or an alkoxyl (RO ) radical and an iron-bound hydroxyl radical. 

The health impairing and toxic elfects of oxidation of lipids are due to loss of vitamins, polyenoic fatty acids, and other nutritionally essential components formation of radicals, hydroperoxides, aldehydes, epoxides, dimers, and polymers and participation of the secondary products in initiation of oxidation of proteins and in the Maillard reaction. Dilferent oxysterols have been shown in vitro and in vivo to have atherogenic, mutagenic, carcinogenic, angiotoxic, and cytotoxic properties, as well as the ability to inhibit cholesterol synthesis (Tai et ah, 1999 Wpsowicz, 2002). 

In subsequent work, Hamberg has reported that anerobic oxidation of lineolic acid by cumene hydroperoxide catalyzed hy sperm whale Mb results in formation of five products, the two major products being ll(i ,S)-hydroylinoleic acid (29% 5deld) and ( )cis-9,10-epoxy-(12Z)-octadecenoic acid (16%) (235). In this work, it was proposed that the second oxidizing equivalent required for substrate hydroxylation was probably provided by a protein-centered radical. 

There are no available data on the formation of hydroperoxides derived from DNA within cells. This is likely explained, at least partly, by the fact that DNA is a poorer target than proteins for OH radical as observed upon exposure of mouse myeloma cells to ionizing radiation . However, indirect evidence for DNA peroxidation within cells may be inferred from the measurement of final degradation products that may derive from thymine and guanine hydroperoxidation as the result of oxidation reactions mediated by OH radical and O2, respectively (Sections n.A.2 and n.E.2). It may be pointed out that the measurement of oxidized bases and nucleosides within DNA has been the subject of intense research during the last decade and accurate methods are now available . This includes DNA extraction that involves the chaotropic Nal precipitation step and the use of desferrioxamine to chelate transition metals in order to prevent spurious oxidation of overwhelming nucleobases to occur . HPLC coupled to electrospray ionization... 

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