Hydroxyl radical oxidative modification

Hydroxyl radical oxidative modification, one of the footprinting approaches, utilizes hydroxyl radicals to oxidize certain amino acid side chains followed by extent of modification measurement by MS. For example, fast photochemical oxidation of proteins (FPOP) [35] involves irradiation of protein sample solutions containing hydrogen peroxide by a KrF excimer laser and generation of hydroxyl radicals in the solution. FPOP occurs on the microsecond timescale for the labeling... 

It should be noted that Reaction (4) is not a one-stage process.) Both free radical N02 and highly reactive peroxynitrite are the initiators of lipid peroxidation although the elementary stages of initiation by these compounds are not fully understood. (Crow et al. [45] suggested that trans-ONOO is protonated into trans peroxynitrous acid, which is isomerized into the unstable cis form. The latter is easily decomposed to form hydroxyl radical.) Another possible mechanism of prooxidant activity of nitric oxide is the modification of unsaturated fatty acids and lipids through the formation of active nitrated lipid derivatives. 

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]. 

The common motif shared by non-heme iron oxygenases contains an active site, where two histidines and one carboxylate occupy one face of the Fe(ll) coordination sphere. These enzymes catalyze a variety of oxidative modification of natural products. For example, in the biosynthesis of clavulanic acid, clavaminic acid synthase demonstrates remarkable versatility by catalyzing hydroxylation, oxidative ring formation and desaturation in the presence of a-ketoglutarate (eq. 1 in Scheme 7.22) [80]. The same theme was seen in the biosynthesis of isopenicillin, the key precursor to penicillin G and cephalosporin, from a linear tripeptide proceeded from a NRPS, where non-heme iron oxygenases catalyze radical cyclization and ring expansion (eq. 2 in Scheme 7.22) [81, 82]. 

Dipyridamole has also been shown to have antioxidant effects (19). Antioxidants act to remove harmful reactive-oxygen species and protect low-density lipoproteins (LDL) from oxidation oxidized LDL plays a key role in the development and propagation of atherosclerosis. The antioxidant effects of dipyridamole may be both direct (by scavenging oxygen and hydroxyl radicals, inhibiting lipid peroxidation and oxidative modification of LDL) (20-22) and indirect (via adenosine, which reduces superoxide anion generation). Dipyridamole has been shown to be a more effective anioxidant than ascorbic acid, alpha-tocopherol, or probucol (22). 

Another interesting topic, yet not well understood, is how the oxidation of nucleic acid bases influences the stability of DNA, and in particular, to what extent it changes the nature of intermolecular interactions. The biological consequences of damage to nucleic acids have been the subject of numerous experimental studies [39, 40], DNA may be exposed in vivo to hydroxyl radicals produced during endogenic cellular processes [41,42], Increased concentration of modified nucleic acid base derivatives (i.e., 8-oxo-guanine, 2-oxo-adenine) in cancer cells has been observed. For this reason, the analysis of the influence of modification of nucleic acid bases by hydroxyl radical on the nature of intermolecular interactions seems to be very advisable. The results of calculations presented in Fig. 20.2 show that... 

Babusikova, E., Kaplan, P., Lehotsky, J., Jesenak, M., and Dobrota, D. 2004. Oxidative modification of rat cardiac mitochondrial membranes and myofibrils by hydroxyl radicals. Gen. Physiol. Biophys. 23 327-335. 

Radical attack of nucleic bases in DNA and RNA results in their hydroxylation, disorders their regular package and decreases stability of the macromolecules with subsequent fragmentation. Actually, in cells with marked oxygen metabolism oxidative modification of nucleic acids takes place in amount exceeding ten thousand hits a day [5]. However, most of them have no after-effects for cell viability demonstrating the presence of specific cellular antioxidant defense repare system. 

Effects of ionizing radiation on lipid molecules have been understood by studying model systems which are simpler than the real biological membranes, such as PUFA micelles and liposomes. The formation of lipid oxidative modifications of PUFAs appears as a dynamic process initiated by hydroxyl free radicals generated by water radiolysis, amplified by a propagating-chain mechanism involving alkyl and peroxyl free radicals, and leading not only to hydroperoxides but also to a lot of other lipidic oxidized end-products. Kinetic data, such... 

The stepwise reduction of oxygen produces hydrogen peroxide, and finally, a hydroxyl radical, which is a strong oxidant implicated in cellular oxidative stress. This oxidative stress causes glutathione depletion, a disruption of the cellular calcium regulation and modifications of cellular proteins, thus... 

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. 

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