Cross-linking protein hydroperoxides

Nucleic adds, protein cross-links, 974-5 Nucleobase hydroperoxides, decomposition, 918, 975-7 Nucleophiles... 

Radicals generated during peroxidation of lipids and proteins show reactivity similar to that of the hydroxyl radical however, their oxidative potentials are lower. It is assumed that the reactive alkoxyl radicals rather than the peroxyl radicals play a part in protein fragmentation secondary to lipid peroxidation process, or protein exposure to organic hydroperoxides (DIO). Reaction of lipid radicals produces protein-lipid covalent bonds and dityrosyl cross-links. Such cross-links were, for example, found in dimerization of Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum. The reaction was carried out in vitro by treatment of sarcoplasmic reticulum membranes with an azo-initiator, 2,2/-azobis(2-amidinopropane) dihydrochloride (AAPH), which generated peroxyl and alkoxyl radicals (V9). 

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). 

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). 

Some chemicals and also oxidants have been shown to cause the covalent cross-linking of heme to apo-P450 [197], Correia s group has characterized the products of the destruction of P450 3A4 with cumene hydroperoxide the information is consistent with a dipyrrolic fragment of heme bonnd to fragment of the protein [1428]. 

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). 

Polyunsaturated fatty acyl groups (or acids) can become oxidized by chemical or enzymic means (section 3.3). Lipid hydroperoxides (ROOH) are stable intermediates of peroxidation and accumulate in peroxidized lipids. These are capable of reacting with transition metals to generate RO and ROO- radicals. The latter can then cause structural changes in proteins including cross-linking and fragmentation. 

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