Proteins oxidative modifications

Table 21.7 Influence of Enterosgel administration on the content of products of protein oxidative modification in blood plasma of patients with severe burns. Carbonyl groups were determined using 2,4-dinitrophenylhydrazine...

Protein cross-links may be also produced in reaction of 4-hydroxynonenal with lysine, histidine, serine, and cysteine residues, primarily via Michael addition (J5, R7, U8). These reactions occur spontaneously, but also may be catalyzed by certain glutatione 5-transferases. The glutathione transferase A4-4, which unlike other alpha-class glutathione transferases, shows high catalytic activity toward lipid peroxidation products such as 4-hydroxynon-2-enal, is the key enzyme for these reactions (B31). Products of protein coupling with aldehydes secondary to lipid peroxidation have a specific fluorescence, which can herald the protein oxidative modification process (CIO). 

Pentosidine is determined by HPLC with spectrofluorimetric detection (excitation and emission wavelengths of 335 and 385 nm, respectively) (S14), although immunochemical and ELISA assays for determination of various protein oxidative modification products have become increasingly popular (08). Protein-aldehyde adducts can be estimated using adduct-specific antibodies (U2, Wl). Another approach requires stabilization of adducts, producing derivatives resistant to conditions used in protein acid hydrolysis and quantification of hydrolysis products by gas chromatography-mass spectrometry (R7). 

Sophorastilbene A (637) and (+)-a-viniferin (635) isolated from Sophora moorcroftiana BENTH ex BAKER inhibited Cu -induced protein oxidative modification (ICsos 1.3 and 3.5 pM, respectively), lipid peroxidation and generation of superoxide anion [505,506]. Paeoninol (661) from Paeonia emodi displayed potent inhibitoiy potential against the enzyme lipoxygenase in a concentration-dependent fashion with the ICsoof0.77 M [316]. 

Diselenolane-3-pentanoic acid, in which the sulphur atoms of a-lipoic acid are replaced with selenium, displayed markedly different antioxidant properties when compared to a-lipoic acid (Matsugo et al. 1997). l,2-Diselenolane-3-pentanoic acid was unable to inhibit protein oxidative modification of human low density hpoprotein and bovine serum albumin induced by copper ion or hydroxyl radical, whereas a-lipoic acid showed significant protection. However, l,2-diselenolane-3-pentanoic acid was able to inhibit the formation of Hpid peroxidation products in low density hpoprotein after oxidation by copper, while a-hpoic acid did not. 

Gupta SK, Dua A, Vohra BP (2003) Withania somnifera (Ashwagandha) attenuates antioxidant defense in aged spinal cord and inhibits copper induced lipid peroxidation and protein oxidative modifications. Drug Metab Drug Interact 19 211-222... 

In order to understand the potential for haem proteins to mediate the oxidative modification of LDLs, the interaction between ruptured erythrocytes (Paganga et al., 1992) and ruptured myocytes (Bourne etal., 1994) with LDL has been explored. Previous studies from this group have demonstrated that ferryl myoglobin radicals and ruptured cardiac myocytes, which generate ferryl myoglobin species on activation (Turner et al., 1990,... 

Copper salts such as CuS04 are potent catalysts of the oxidative modification of LDL in vitro (Esterbauer et al., 1990), although more than 95% of the copper in human serum is bound to caeruloplasmin. Cp is an acute-phase protein and a potent inhibitor of lipid peroxidation, but is susceptible to both proteolytic and oxidative attack with the consequent release of catalytic copper ions capable of inducing lipid peroxidation (Winyard and... 

Liu, J. Li, Q. Yang, X. van Breemen, R. B. Bolton, J. L. Thatcher, G. R. Analysis of protein covalent modification by xenobiotics using a covert oxidatively activated tag raloxifene proof-of-principle study. Chem. Res. Toxicol. 2005, 18, 1485-1496. 

From a broader perspective, protein oxidation can result in covalent modification at many sites other than just at cysteine thiols. The earliest reports on protein oxidation date from the first decade of the twentieth century, but it took many more years to characterize these reactions and their products (Dakin, 1906). 

Unfortunately, there are no universal methods to detect all types of protein oxidation, because the products formed can be so diverse in nature. However, some forms of protein oxidation can be assayed using chemical modification (Davies et al., 1999 Shacter, 2000). In particular, the formation of carbonyl groups on proteins can be targeted using the reagent 2,4-dinitrophenyl-hydrazine (DNPH). This compound reacts with aldehydes to form 2,4-dinitrophenylhydrazone derivatives, which create chromogenic modifications that can be detected at high sensitivity in microplate assays or Western blot analysis (Buss et al., 1997 Winterbourn et al., 1999). 

The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine (DNPH Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modification produces detectable complexes, but it does not provide information as to what amino acids are involved. O-ECAT modifies carbonyl end products of protein oxidation and in addition, it can provide exact information as to the amino acids that were oxidized. Mass spec analysis of modified proteins performed after proteolysis gives the exact amino acid sequences including the sites of O-ECAT reagent modification. The same antibody that is specific for the metal chelate portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT... 

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 earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

In contrast to numerous literature data, which indicate that protein oxidation, as a rule, precedes lipid peroxidation, Parinandi et al. [66] found that the modification of proteins in rat myocardial membranes exposed to prooxidants (ferrous ion/ascorbate, cupric ion/tert-butyl-hydroperoxide, linoleic acid hydroperoxide, and soybean lipoxygenase) accompanied lipid peroxidation initiated by these prooxidant systems. 

Cervera and Levine [81] studied the mechanism of oxidative modification of glutamine synthetase from Escherichia coli. It was found that active oxygen species initially caused inactivation of the enzyme and generated a more hydrophilic protein, which still was not a substrate for the protease. Continuous action of oxygen species resulted in the formation of oxidized protein subjected to the proteolytic attack of protease. 

Another important characteristic of oxidative stress in thalassemia is LDL oxidative modification. Livrea et al. [388] showed that the concentration of hydroperoxides in LDL of thalassemia patients was equal to 22.60+ 12.84 nmol/mg LDL protein compared to 6.25 +3.04 nmol/mg in control LDL. These authors proposed that the enhanced LDL oxidation in thalassemia was connected with the depletion of vitamin E in LDL. Interestingly, these findings contradict the suggestion about the prooxidant role of vitamin E (a-tocopherol) in LDL oxidation (Chapter 25). It was proposed that LDL oxidation could be the origin of atherogenetic risk in thalassemic patients. 

Fredriksson, A., Ballasteros, M., Dukan, S., and Nystrom, T. (2006). Induction of the heat shock regulaon in response to increased mistranslation requires oxidative modification of the malformed proteins. Mol. Microbiol. 59, 350-359. 

Mechanism of Action A tetracyclic compound that blocks reuptake norepi nephri ne by CNS presynaptic neuronal membranes, increasing availability at postsynaptic neuronal receptor sites, and enhances synaptic activity. Therapeutic Effect Produces antidepressant effect, with prominent sedative effects and low anticholinergic activity. Pharmacokinetics Slowly and completely absorbed after PO administration. Protein binding 88%. Metabolized in liver by hydroxylation and oxidative modification. Excreted in urine. Unknown if removed by hemodialysis. Half-life 27-58 hr. 

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