Abstract
The fact that proteins are the main target of reactive species formed in the cells and extracellular fluids has led to the realization of a great deal of research devoted to revealing the molecular and biological consequences associated with the presence of intermediary protein radicals. This review article describes and comments upon the main chemical pathways involving primary proteic radicals.
Introduction
Reactive species, such as free radicals and oxidants, have been proposed as an important source of oxidative damage to biological molecules. This deleterious effect has been directly associated with the development of human diseases, such as Alzheimer (1–3), autoimmune diseases (4, 5), cardiovascular diseases (6–8), Crohn’s disease (9), kidney diseases (10, 11) and Parkinson’s disease (12–14).
Among all biological molecules, proteins are the main target of oxygen- and nitrogen-derived reactive species owing to their abundance in cells and extracellular fluids. In fact, it has been estimated that these macromolecules scavenge between 50% and 75% of the primary reactive species produced in vivo (15). Proteins can react with different in vivo-generated reactive species such as alkoxyl, hydroxyl and peroxyl radicals, as well as with carbonate radical anion and nitrogen-centered reactive species, among others (16). It has been proposed that, after the reaction of these reactive species with proteins, a carbon-centered free radical (P·) can be generated either in the backbone or in the side chain of amino acid residues. P· constitutes one of the first intermediaries formed in proteins when they are exposed to oxidative environments. There are different fates for P· (17). The major pathway, and probably the most important in an aerobic medium, is its reaction with molecular oxygen to give a protein-derived peroxyl radical (POO·). This pathway prevails over most other reactions even at low oxygen tensions in metabolically active tissues. This does, however, take into account that the reaction between the carbon-centered radical and O2 generally occurs at, or near, the diffusion-controlled limit (i.e., k ca. 109 l mol-1 s-1) (18). A secondary fate for P· is its dimerization, a process probably likely to be uncommon in the presence of oxygen, but its occurrence has been reported in some special cases (19–24). The third fate corresponds to the possibility that P· can be repaired, although at a relatively slow rate compared to the high reactivity towards O2 (vide infra).
The mechanism of P· and POO· formation has been extensively described by different research groups (17, 25, 26), and, when the attack involves the main backbone of the protein, it can be represented by reactions 1 and 2, respectively:
where R· represents a free radical such as an alkoxyl, hydroxyl, peroxyl or carbonate free radical, among others.
As is inferred from reaction 1, the chemical environment of the α-carbon strongly modulates the ability of the hydrogen to be subtracted by a particular reactive species. Since glycine residues contain only hydrogens in this position, the lack of steric or electronic effects explains its large capacity to form P· and POO· (17). Conversely, for most amino acid residues, the presence of side chains makes the possible abstraction of a hydrogen atom from the α-carbon by reactive species difficult. New oxidizable targets are also incorporated. In fact, the oxidation of amino acid side chains and the corresponding formation of P· and peroxyl radicals have been widely studied. Studies on γ radiolysis of valine, leucine and lysine aqueous solutions have demonstrated the formation of peroxyl radicals on their side chains (25). In the case of tyrosine, one of the pathways of its reaction with hydroxyl radical is that it forms a phenoxyl secondary free radical that reacts with oxygen, forming the corresponding peroxyl radical derivative. After the elimination of HO2· and ketol intermediate formation, a 3,4-dihydroxyphenylalanine is generated as a final product (Scheme 1) (25, 27).
Scheme 1
Mechanism of POO· formation during tyrosine oxidation induced by hydroxyl radicals.
Tryptophan residues can also be oxidized by mechanisms involving peroxyl radical intermediates. Reactive species can abstract the hydrogen atom at position 1 of tryptophan, generating a N· free radical. After delocalization and interaction with oxygen, it forms a peroxyl radical, leading to N-formyl kunurenine (NKy) and kynurenine (Ky) as final products (Scheme 2) (25, 28–30). This mechanism has been frequently proposed, in spite of the fact that Candeias et al. (31) reported a low kinetic rate constant (lower than 106m-1 s-1) for the reaction between tryptophanyl radical and oxygen.
Scheme 2
Mechanism of tryptophan oxidation induced by reactive species.
Another way to generate peroxyl radicals is related to the initial attack toward cysteine residues. They are present in most human proteins, and their intracellular content is comparable to that of the most abundant intracellular low-molecular-mass thiol, glutathione (32, 33). Primary thiyl radicals (RS·) are formed in the reaction of thiol compounds with free radicals. Secondary reactions of RS· result in half-lives of the order of microseconds, hindering its analysis in biological systems. However, in recent years and by using electron paramagnetic resonance spin trapping and other experimental protocols, it has been possible to assess its presence (34). RS· can undergo several reactions; some of them are represented below (35, 36):
Reaction 3 corresponds to a radical-radical reaction, and reaction 4 shows the reaction with molecular oxygen to give a thiyl peroxyl radical whose secondary reactions can lead to sulfinic, sulfenic and sulfonic acids. Reaction 5 represents a S-nitrosation process, and reaction 6 corresponds to a S-glutathiolation, where GS- denotes the thiolate form of glutathione.
The abstraction of a hydrogen atom is indicated in reaction 7. The hydrogen abstraction by thiyl radicals from the side group or C-H bonds in model peptides (37) and proteins (38, 39) has been a matter of increasing interest in recent years owing to the biological significance of these kinds of reactions. The intermolecular reactions of thiyl radicals with amino acids within model peptide structures, N-acetylamino acid amides and diketopiperazines, proceed with rate constants of the order of 103–105m-1 s-1 (40) and have been reported as a reversible process in some systems (41, 42). The hydrogen abstraction by thiyl radicals from C-H bonds in proteins takes place intramolecularly, with a control of selectivity that depends on the protein secondary structure (38). The α-C-H bonds of amino acids located inside of α-helices and β-sheets have higher homolytic C-H bond dissociation energies compared to the α-C-H bonds of amino acids in extended conformations (38). The set of reactions 3–7 supports the fact that POO· can also be formed through secondary processes following the initial interaction of free radicals with SH bonds in the proteins.
In spite of the lower reactivity of peroxyl radicals than that of other reactive species such as hydroxyl, carbonate or alcoxyl radicals (43), once POO· is generated (either in the backbone or in the side chains of amino acid residues) it is able to induce processes such as chain radical reactions, dimerization, triggering scission or hydrogen abstraction. Therefore, POO· can be considered as a reactive species able to increase the initial damage inflicted by other reactive species. Attention is increasingly given to these processes since they lead to irreversible protein modifications and can be implicated in the pathological conditions mentioned above.
Role of peroxyl radicals in chain reactions
A simple mechanism for protein modification mediated by peroxyl radicals produced from the thermolysis of an azocompound [such as AAPH, 2,2′-azo-bis(2-amidinopropane) dihydrochloride] under aerobic conditions is depicted in the following set of reactions 8 to 13:
where R· and ROO· are the azocompound-derived alkyl and peroxyl radicals, respectively.
These reactions lead to a global process whose rate law depends on the solute (protein) concentration. In particular, this oversimplified mechanism predicts that the rate law of protein modification should depend on the protein concentration.
In this context, it has been reported that, in systems involving azocompounds and catalase, superoxide dismutase, or acid phosphatase, at low protein concentration, proteins follow a first-order kinetic and the kinetic order of the initiator (azoderivative) is equal to 0.5. In contrast, at high protein concentrations, proteins follow a zero-order kinetic and the initiator kinetic order (azoderivative) is 1 (44, 45). Furthermore, in reactions 8–13, which rely on conditions of quantitative capture of the primary radicals (ROO·) by proteins, up to one amino acid can be modified by each radical introduced into the system. In spite of the fact that these speculations are proven in several systems such as the oxidation of horseradish peroxidase, sticholysin II hemolytic toxin and yeast alcohol dehydrogenase (46–48), some apparent contradictions have been found in other systems:
stoichiometric values (n, defined as the moles of proteins oxidized for each peroxyl radical introduced into the system) larger than 1 (44, 45, 49–51)
kinetic order in proteins at high protein concentrations considerably higher than 0 (50).
These anomalies have been attributed (50–52) to the occurrence of chain reactions driven by peroxyl radicals:
P· reacts with oxygen in agreement with reaction 11, extending the chain reactions until the occurrence of a termination process according to reaction 13 or to alternate reaction 15:
Chain reactions have been evidenced in some studies such as that carried out by Lissi and Clavero (50), employing lysozyme as substrate, where nearly three tryptophan groups were modified per radical introduced into the system. Also, Arenas et al. (19) reported that other amino acid residues, such as tyrosine and methionine, were also modified by peroxyl radicals, up to an average of 4.5 amino acids oxidized by each AAPH-derived peroxyl radical produced (19). These chain reactions operate independently of the free radical produced in the initiation step. For example, it has been noted that, following the reaction between bovine serum albumin and hydroxyl radicals in aerobic conditions, nearly 30 amino acids are modified per radical introduced into the system (51). Similar chain reactions take place employing other proteins (lysozyme, n=7) and polypeptides (melittin, n=15) (51).
These radical-mediated chain reactions can involve side chain-to-side chain, backbone-to-backbone, side chain-to-backbone and/or, finally, backbone-to-side chain processes (52), and can be promoted by the vicinity of the free unpaired electron and the target amino acid residue. The process can take place by electron and/or hydrogen atom transfer, and tunnelling effects could contribute to a high reaction rate (53).
Another peculiar aspect of these types of data is the large (average) number of amino acid residues that are modified by each inactivated protein (48, 54). These low inactivation efficiencies have also been observed in protein oxidation processes initiated by radiation or Fenton-like mechanisms (51, 55). For example, lysozyme lost half of its activity only after the modification of 42 amino acids per protein when γ radiation was employed as a radical source. Interestingly, when a similar degree of inactivation was promoted by Fenton’s reaction, a smaller number (13.5) of damaged amino acids were observed (55). The most likely explanation for these extensive damages was the occurrence of intra-protein chains (51, 52). The difference between both results can be explained in terms of the differences in the rate of input of radicals. Fenton’s reaction provides a faster rate of radical production and, hence, shorter free-radical chains. In fact, if this type of process is taking place, the oxidized ensemble should comprise intact proteins (those that have not been damaged) and proteins with a large number of modifications that (probably) have completely lost their activity as an enzyme, toxin or carrier. In other words, damaged amino acids are not randomly distributed over the whole oxidized ensemble but are concentrated in those proteins that have suffered intra-protein oxidation chains. This oxidized ensemble will comprise a few heavily damaged (inactive) protein molecules and ‘intact’ (active) proteins. This implies that the chain reaction must be mainly intramolecular and hence nearly independent of the protein concentration. This appears as contradictory to Lissi and Clavero’s (50) results obtained in the AAPH-lysozyme system, where a chain process appears as operating only at high protein concentrations. However, if because of the low reaction rate of termination steps involving two POO· radicals, the chains are terminated by reactions involving the primary radicals, such as
the decrease in ROO· steady-state concentration associated with an increase in protein concentration would favor the number and length of the intramolecular chains at high protein concentrations.
Role of peroxyl radicals in protein aggregation and fragmentation
Several reactions confirm the relevant role that POO· plays in the chain of reactions where proteins can undergo when they are exposed to radicals (52). Among these reactions, POO· can undergo dimerization (reaction 17) or give rise to hydroperoxides (reactions 18 and 19).
In addition, the tetroxide derivative generated in reaction 17 can undergo a decomposition process producing alkoxyl radicals (PO·) in agreement with reaction 20:
Both protein hydroperoxides (vide infra) and PO· are able to participate in other reactions, extending the initial damage inflicted by reactive species. In particular, PO· is more reactive than POO· (43) and can undergo rapid hydrogen abstraction reactions, as well as facile unimolecular fragmentation and rearrangements (52), leading to the formation of carbonyl groups. In fact, PO· formation in the main polypeptide chain, followed by β scission, has been considered as an important source of carbonyl groups in oxidized proteins (56). However, results have recently shown that carbonyls are present in non-fragmented or aggregated proteins, which contradicts the former possibility (19). Fragmentation and oligomerization are typical processes present in free-radical reactions of proteins triggered by oxygen- and nitrogen-derived reactive species. These processes are mostly irreversible (with the exception of association through the formation of S-S bonds). In spite of the relatively low quantum yields, both processes, and particularly oligomerization, are very important since they change the solubility of the proteins, leading to insoluble aggregates with deleterious effects, such as those present in cataracts (57). Dimerization can follow reactions in the side groups and/or in the polypeptide chains. The last process is particularly important for very reactive free radicals, such as hydroxyl radicals. In contrast, it can be expected that the less reactive radicals, such as POO·, selectively attack more reactive targets whose secondary reactions lead to aggregation. A typical example is di-tyrosine whose formation starts with a hydrogen abstraction from the phenol group. This dimerization is typical of free-radical reactions and has been frequently used as a marker of the extent of protein oxidation and also as a urinary biomarker of oxidative stress conditions (17, 58, 59).
In the context of protein fragmentation, glycine residues have been shown to be the most relevant amino acid residues in this process (17). The mechanism is associated with the formation of a carbon-centered free radical, with the consecutive POO· formation, and with PO· through reactions 17 and 20. Recently, tryptophan-derived peroxyl radicals have also been proposed as an alternative pathway of protein fragmentation initiated by oxidation in the reactive side group of this amino acid (Scheme 3) (19).
Scheme 3
Proposed mechanism for protein fragmentation through tryptophan-derived peroxyl radicals (19).
It is interesting to note that the side chain-to-backbone damage transfer (as presented in Scheme 3) is similar to that observed when a carbon-centered free radical is formed by the action of hydroxyl radicals on aliphatic side chains (53, 56). Additionally, the same hydrogen abstraction from the tryptophan-indole ring (Scheme 3) has been put forward as the initial step in covalent dimerization of human superoxide dismutase promoted by carbonate radicals (22).
It has been demonstrated that protein fragmentation and aggregation are extremely dependent on the target protein. For example, as is depicted in Table 1, the damage to lysozyme and human serum albumin (HSA) inflicted by AAPH-derived peroxyl radicals in similar experimental conditions led to different yields of such processes (60).
Table 1
Quantum yields (Φ) of lysozyme and HSA modifications elicited by AAPH-derived peroxyl radicals.
| Target protein | Φoligomer×103 | Φfragmentation×103 | Φfragmentation/Φcarbonyl |
|---|---|---|---|
| Lyso | 140±40 | 300±10 | 2.3±0.4 |
| HSA | 21±8 | 10±4 | 0.06±0.01 |
Values represent the rate of protein modification/rate of peroxyl radical production (60).
The data presented in Table 1 would suggest that
carbonyl groups in the protein are a poor predictor of protein damage;
the yield (Φ) of carbonyl group formation in HSA is considerably smaller than that during fragmentation. This implies that this process is not regulated by β scission of protein-derived alkoxyl radicals, leading to carbonyl formation and main backbone cleavage.
As mentioned previously, the relevance of fragmentation and aggregation processes strongly depends on the protein target. Particularly, reactions between two different proteins to crosslinking by reactions triggered by free radicals arise as a plausible process in biological environments. Oxidation of protein mixtures gives the opportunity to assess such kinds of reactions. Data obtained using a mixture containing lysozyme and HSA, two proteins characterized by different charges at physiological pH (7.4), could lead to the production of non-covalent aggregates (prior to oxidation), as shown in Table 2.
Table 2
Quantum yields (Φ) of dimer formation in the oxidation of lysozyme (Lyso) and HSA mixtures (60).
| ROS source | ΦLyso-Lyso×103 | ΦHSA-HSA×103 | ΦLyso-HSA×103 |
|---|---|---|---|
| AAPH pyrolysisa | 2.0±0.3 | <0.2 | 40±6 |
| Riboflavinb | 0.9±0.1 | 0.04±0.005 | 2.3±0.6 |
aValues represent the rate of protein modification/rate of peroxyl radical production. bPhotosensitized processes induced by riboflavin irradiation.
These data would indicate that physical association prior to oxidation favors the covalent binding of the proteins, when the process is mediated either by AAPH-derived peroxyl radicals or by riboflavin photolysis.
An interesting aspect of protein fragmentation and oligomerization promoted by peroxyl radicals is the non-random cleavage and dimerization, indicating that the initial attack and/or the cleavage takes place at selected positions along the main chain and/or in specific lateral groups (19). These reactions could be associated with the selectivity of peroxyl radicals on amino acid residues and also by the selectivity of secondary reactions between protein-protein (P-P), protein-fragments (P-F) and/or fragments-fragments (F-F). Data obtained employing AAPH as the free-radical source are consistent with the dimerization of lysozyme by the P-P type, whereas in the case of glucose-6-phosphate dehydrogenase (G6PD), particularly at low radical doses, it seems to be of the P-F type. In addition, an efficient reaction such as
would explain the presence of aggregates with molecular weights lower than that of P-P dimer and the lack of carbonyls in the fragments (19).
Protein hydroperoxides as intermediaries in the development of biological damage initiated by ROS
In 1992, it was reported (61) that protein hydroperoxides (POOH) can consume key cellular reductants. The potential biological significance of such reactions is that it may cause depletion of ascorbate and glutathione, and thus constitute a source of oxidative stress (61). Simultaneously to this oxidizing capacity (61), a second type of reactive species, protein-bound reducing moieties (PBRedM), was detected, which were recognized by their capacity to reduce cytochrome c. PBRedM species are even preserved in proteins from which hydroperoxides have been removed by the reduction by ascorbate or glutathione. This long-lived reducing capacity was attributed to a tyrosine hydroxylation pathway that produces catechols, such as 3,4-dihydroxyphenylalanine-like molecules, which are known to be able to reduce transition metals (62, 63).
The hydroperoxides generated when amino acids, peptides and proteins are exposed to radicals in the presence of O2 are stable in the absence of exogenous catalysts (e.g., redox-active transition metal ions). However, they decompose rapidly in the presence of these agents to give a variety of free radicals including alkoxyl, peroxyl and carbon-centered free-radical species. The formation of these free radicals allows the propagation of oxidative damage to other biomolecules (64) including lipids, proteins (65, 66), antioxidants (67, 68) and DNA (69–73).
Peroxidized proteins react with DNA molecules giving rise to strong intermolecular crosslinks whose extension depends on the number of POOH present in the oxidized proteins (74). The mechanism proposed for this crosslink reaction would require the initial formation of alkoxyl radicals in a reaction mediated by a DNA-bound metal (M) (74):
followed by
to give the covalent crosslink. It is stated that any protein peroxidized by ROS in a cell can potentially form such crosslinks if it comes into close proximity to DNA by diffusion or during functional association. The DNA-protein crosslinks have received increasing attention in the last decade in regard to their induction (72, 73), repair (71) and biological consequences (69, 71).
Repair of protein radicals
There is general agreement that proteins are the major target for reactive species, and increased levels in protein oxidation products have been measured in aging and some diseases (75–80). As mentioned previously, the precursors of these products are P· and POO·, two free radicals that could be reduced by biologically relevant derivatives such as glutathione and ascorbate. Studies employing THE pulse radiolysis technique have demonstrated the formation of tryptophan and tyrosine carbon-centered radicals during irradiation with nanosecond pulses (and fast transient spectra analysis) of dilute tryptophan and/or tyrosine-containing peptides and protein solutions (81, 82). In dilute solutions, such irradiation generates several decomposition products according to
In spite of this, for the absorption of a particular energy, the amount of these products can be seen by the wide range of species able to initiate the oxidation processes, which implies a highly complex system, especially in the presence of such complex targets as proteins. For this reason, experiments are usually carried out in the presence of N2 O (to produce HO·) and N3 (to produce N3·), according to
In contrast to HO·, azide radical (N3·) is more selective, inflicting oxidative damage only to aromatic amino acids such as tryptophan and tyrosine (82). Tryptophan and tyrosine carbon-centered radicals are characterized by transient spectra with maxima at 405 and 510 nm, respectively. Following the formation and decay of the absorption intensity at these wavelengths, in the absence and presence of antioxidants, the kinetic rate constants have been determined for the repair of P· in peptides and proteins mediated by glutathione (near 103–105m-1 s-1), ascorbate and urate (near 107–109m-1 s-1 for both compounds) (81–83). Glutathione (43), a well-known low-molecular-weight antioxidant, repairs lysozyme tryptophan radical and N-acetyltryptophan amide radicals with kinetic rate constants (7.8±0.4×104 and 1.2±0.2×105m-1 s-1, respectively) considerably higher than that of its reaction towards N-acetyl-tyrosine amide radicals (3±2×103m-1 s-1) (82). The kinetic rate constants of the reactions between ascorbate and tyrosyl (TyrO·) or tryptophanyl (Trp·) radicals in the free amino acids, as well as those included in model peptides or proteins, are between two- and three orders of magnitude higher (ca. 107–108m-1 s-1) than that found in the presence of glutathione (67). Interestingly, both ascorbate and glutathione inhibit the transfer of electrons from tyrosine to Trp· in proteins in vitro, but glutathione does not prevent the formation of POO· or hydroperoxides. In this context, although the reaction between O2 and the radicals located in most amino acids, to form peroxyl radicals, is fast [k=(3±1)×109m-1 s-1] (84), it is necessary to take into consideration that the O2 concentration in most mammalian tissues is commonly below 50 μmol/l, whereas that of ascorbate is at least 10 times higher. In this sense, the repair of protein-centered radicals by ascorbate, which are characterized by higher reaction rates than those of glutathione, should be competitive with the reaction with molecular oxygen (81).
The repair of Trp· and TyrO· in proteins by urate has been studied by pulse radiolysis in solutions saturated with N2 O (83). The electrode potential at pH 7 [E°′ (urate radical/urate)≈+0.59 V, relative to the normal hydrogen electrode] (85) is lower than those of amino acid radicals and makes the reaction of urate with these radicals thermodynamically favorable. Domazou et al. (83) observed in chymotrypsin that urate efficiently repairs Trp· with a kinetic rate constant of 2.7×108m-1 s-1, ca. 14 times higher than that derived for N-acetyltryptophan amide, 1.9×107m-1 s-1. In pepsin, urate repairs TyrO· with a rate constant of 3×108m-1 s-1, ca. 12 times smaller than that reported for free tyrosine. The efficiency of urate at repairing protein-bound TyrO· or Trp· varies strongly with the protein (83, 86, 87), with no relation to the protein size. In conclusion, the fact that the amino acid radical is bound to the protein does not constitute a barrier for its reaction with urate (83).
According to the values of the kinetic rate constants for the reactions between carbon-centered free radicals of the proteins and ascorbate (81) and urate (83), it would be expected that urate repairs protein radicals faster than ascorbate (83): (a) in tissues or compartments that contain urate at higher concentrations than ascorbate, such as plasma, heart and saliva (88–92); and (b) under conditions where ascorbate is unstable, e.g., at the acidic pH of the stomach.
In the case of the repair of P· and POO· mediated by phenols, early works developed by Jovanovic and Simic (93) studied the ability of phenols to repair Trp· (as well as other indole analogues) generated by a reaction with Br2·- during pulse radiolysis experiments in aqueous media at different pH values. Since the equilibrium
is a displacement to the right (pKa of Trp·+=4.3) at physiological pH, the neutral form of Trp· should be the species present in biological environments. Interestingly, no reaction of Trp· with oxygen was evidenced; therefore POO· was not generated in these experimental conditions. Because of its resonance, Trp· slowly reacts with hydrogen donors such as phenols and thiols (93). In contrast, Trp· readily reacts, by electron transfer, with good electron donors as ascorbate (93).
The ability of catechin, quercetin and rutin for repairing neutral Trp· and TyrO· in buffer, micelles and in the presence of human serum albumin has been reported. In agreement with ascorbate, glutathione and urate, the repair of Trp· mediated by these flavonoids is faster than for TyrO· (81–83, 86). In addition, from experiments carried out in SDS, CTAB and Triton X-100 micelles, the role that the environment plays in modulating the rate of the repair reaction was evidenced (86). In the presence of HSA, the ability of the flavonoids to repair Trp· and TyrO· was considerably decreased, a result probably associated with the limited accessibility of HSA-bound flavonoids to free Trp· and TyrO· radicals (86). In similar studies, but generated by pulse radiolysis, free radicals in ApoB present in LDL particles (94), or more recently in apoferritin (95), quercetin was shown to be highly efficient in repairing protein radicals (Trp· and TyrO·).
The ability of phenols to repair P· depends on their redox potential; phenols with low reduction potential values could be considered as better ‘repairers’ of protein radicals. However, the relevance of the repair of P· radicals by natural polyphenols (flavonoids) could be minimized by the low concentrations that they reach in plasma after an intake of a polyphenolic-rich meal (mostly fruit, vegetables and some beverages) (96). In spite of the fact that the biological chemistry of different oxidants is becoming well understood, the actual relevance of the polyphenol-free radical reactions studied in in vitro conditions is often unclear (97). Taking into account that, physiologically, it is unlikely that polyphenol and carotenoid concentrations approach those of ascorbate, glutathione and urate, particularly in tissues or compartments, it is also unlikely that polyphenols have a biological role involving direct reaction with reactive species or protein-derived free radicals. Nonetheless, it has been suggested that the strong binding of flavonoids to proteins, in particular HSA, could increase the local concentration of these compounds, possibly favoring a protein free-radical repair process (86). In contrast, it has been postulated that the in vivo role of ascorbate, glutathione and urate is closely related to their ability to repair macromolecules. These processes are mostly driven by P·, since POO· radicals do not play a relevant role in repair reactions. In fact, the reaction between POO· and antioxidants (including phenols, ascorbate, glutathione and urate) should not be considered as a repair reaction, since the generated products (hydroperoxides) also have the capacity to trigger protein damage. In addition, it should also be considered that repair reactions on P· are not always a means of restoring the biological function of proteins (67).
Concluding remarks and future perspectives
Proteins are the main target of oxygen-, sulfur- or carbon-centered radicals.
Secondary reactions of protein-centered radicals can lead to different modifications that include the formation of carbonyl and hydroperoxide derivatives, fragmentation and covalent aggregation.
These protein modifications are not necessarily located at the site of the initial free radical attack; they can involve the occurrence of chain radical reactions and extend the damage to other biological components.
In tissues with low protein turnover (e.g., the eye lens and also organs during the senescence), the effect of chemical modifications induced by free radicals is accumulative and has significant functional and conformational consequences. As it is presented in this review, protein-derived free radicals, in particular carbon-centered and peroxyl radicals, play a pivotal role in such processes. These free radicals can be formed by multiple pathways, implying that proteins under oxidative conditions follow a complex behavior. This subject has shown increasing interest from research studies and will stimulate new research works on this field.
Acknowledgments
This work was supported by FONDECYT no. 1141142.
References
1. Ansari MA, Scheff SW. Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol 2010; 69: 155–67.10.1097/NEN.0b013e3181cb5af4Search in Google Scholar
2. Markesbery WR. Oxidative stress hypothesis in Alzheimer’s disease. Free Radic Biol Med 1997; 23: 134–47.10.1016/S0891-5849(96)00629-6Search in Google Scholar
3. Perry G, Cash AD, Smith MA. Alzheimer disease and oxidative stress. J Biomed Biotechnol 2002; 2: 120–3.10.1155/S1110724302203010Search in Google Scholar
4. Buttke TM, Sandstrom PA. Oxidative stress as amediator of apoptosis. Immunol Today 1994; 15: 7–10.10.1016/0167-5699(94)90018-3Search in Google Scholar
5. Shah AA, Sinha AA. Oxidative stress and autoimmune skin disease. Eur J Dermatol 2013; 23: 5–13.10.1684/ejd.2012.1884Search in Google Scholar
6. Ceriello A, Motz E. Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler Thromb Vasc Biol 2004; 24: 816–23.10.1161/01.ATV.0000122852.22604.78Search in Google Scholar
7. Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 2000; 18: 655–73.10.1097/00004872-200018060-00002Search in Google Scholar
8. Lakshmi SVV, Padmaja G, Kuppusamy P, Kutala VK. Oxidative stress in cardiovascular disease. Ind J Biochem Biophys 2009; 46: 421–40.Search in Google Scholar
9. Iborra M, Moret I, Rausell F, Bastida G, Aguas M, Cerrillo E, Nos P, Beltrán B. Role of oxidative stress and antioxidant enzymes in Crohn’s disease. Biochem Soc Trans 2011; 39: 1102–6.10.1042/BST0391102Search in Google Scholar
10. Oberg BP, McMenamin E, Lucas FL, McMonagle E, Morrow J, Ikizler TA, Himmelfarb J. Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney Int 2004; 65: 1009–16.10.1111/j.1523-1755.2004.00465.xSearch in Google Scholar
11. Small DM, Coombes JS, Bennett N, Johnson DW, Gobe GC. Oxidative stress, anti-oxidant therapies and chronic kidney disease. Nephrology 2012; 17: 311–21.10.1111/j.1440-1797.2012.01572.xSearch in Google Scholar
12. Henchcliffe C, Beal MF. Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis. Nat Clin Pract Neurol 2008; 4: 600–9.10.1038/ncpneuro0924Search in Google Scholar
13. Jenner P. Oxidative stress in Parkinson’s disease. Ann Neurol 2003; 53: S26–36.10.1002/ana.10483Search in Google Scholar
14. Zhou C, Huang Y, Przedborski S. Oxidative stress in Parkinson’s disease amechanism of pathogenic and therapeutic significance. Ann N Y Acad Sci 2008; 1147: 93–104.10.1196/annals.1427.023Search in Google Scholar
15. Davies MJ. Radical-mediated protein oxidation: from chemistry to medicine. New York, USA: Oxford University Press; 1997.Search in Google Scholar
16. Davies MJ. Oxidative damage to proteins. In: Chatgilialoglu C, Studer A, editors. Encyclopedia of radicals in chemistry, biology and materials. New Jersey: Wiley, 2012: 1425–58.Search in Google Scholar
17. Davies MJ. The oxidative environment and protein damage. Biochim Biophys Acta 2005; 1703: 93–109.10.1016/j.bbapap.2004.08.007Search in Google Scholar
18. Neta P, Grodkowski J, Ross AB. Rate constants for reactions of aliphatic carbon-centered radicals in aqueous solution. J Phys Chem Ref Data 1996; 25: 709–1050.10.1063/1.555978Search in Google Scholar
19. Arenas A, Lopez-Alarcon C, Kogan M, Lissi E, Davies MJ, Silva E. Chemical modification of lysozyme, glucose 6-phosphate dehydrogenase, and bovine eye lens proteins induced by peroxyl radicals: role of oxidizable amino acid residues. Chem Res Toxicol 2013; 26: 67–77.10.1021/tx300372tSearch in Google Scholar
20. Buder I, Schwitzgebel G, Samsoniya S, Gogritchiani E, Chikvaidze I. Electrochemical behaviour of indoles with methyl, benzyl,and dibenzyl-4-yl groups. Khim Geterotsikl Soedin 2005; 9: 1323–31.Search in Google Scholar
21. Karaaslan C, Suzen S. Electrochemical behaviour of biologically important indole derivatives. Int J Electrochem 2011; 2011: 1–10 (ID 154804).10.4061/2011/154804Search in Google Scholar
22. Medinas DB, Gozzo FC, Santos LFA, Iglesias AH, Augusto O. A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity. Free Radic Biol Med 2010; 49: 1046–53.10.1016/j.freeradbiomed.2010.06.018Search in Google Scholar
23. Silva E, Ugarte R, Andrade A, Edwards AM. Riboflavin-sensitized photoprocesses of tryptophan. J Photochem Photobiol B 1994; 23: 43–8.10.1016/1011-1344(93)06984-BSearch in Google Scholar
24. Udum YA, Dudukcu M, Koleli F. Electrochemical polymerization and spectroscopic investigation of 2-methylindole. React Funct Polym 2008; 68: 861–7.10.1016/j.reactfunctpolym.2007.12.006Search in Google Scholar
25. Cadet J, Di Mascio P. Peroxides in biological systems. In: Rappoport Z, editor. The chemistry of peroxides. West Sussex: Wiley, 2006: 915–99.Search in Google Scholar
26. Stadtman ER. Oxidation of free amino-acids and amino-acid-residues in proteins by radiolysis and by metal-catalyzed reactions. Ann Rev Biochem 1993; 62: 797–821.10.1146/annurev.bi.62.070193.004053Search in Google Scholar
27. Gieseg SP, Simpson JA, Charlton TS, Duncan MW, Dean RT. Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins. Biochemistry 1993; 32: 4780–6.10.1021/bi00069a012Search in Google Scholar
28. Friedman M, Cuq JL. Chemistry, analysis, nutritional-value, and toxicology of tryptophan in food – A review. J Agric Food Chem 1988; 36: 1079–93.10.1021/jf00083a042Search in Google Scholar
29. Aspee A, Lissi EA. Kinetics and mechanism of the chemiluminescence associated with the free radical-mediated oxidation of amino acids. Luminescence 2000; 15: 273–82.10.1002/1522-7243(200009/10)15:5<273::AID-BIO591>3.0.CO;2-MSearch in Google Scholar
30. Simat T, Meyer K, Stover B, Steinhart H. Oxidation of free and peptide bound tryptophan. Adv Exp Med Biol 1996; 398: 655–9.10.1007/978-1-4613-0381-7_106Search in Google Scholar
31. Candeias LP, Wardman P, Mason RP. The reaction of oxygen with radicals from oxidation of tryptophan and indole-3-acetic acid. Biophys Chem 1997; 67: 229–37.10.1016/S0301-4622(97)00052-5Search in Google Scholar
32. Calcutt G. The sulphydryl groups of some normal tissues and some animal tumours. Br J Cancer 1964; 18: 197–207.10.1038/bjc.1964.22Search in Google Scholar
33. Ying J, Clavreul N, Sethuranam M, Adachi T, Cohen RA. Thiol oxidation in signaling and response to stress: detection and quantification of physiological and pathophysiological thiol modifications. Free Radic Biol Med 2007; 43: 1099–108.10.1016/j.freeradbiomed.2007.07.014Search in Google Scholar
34. Stoyanovsky DA, Maeda A, Atkins JL, Kagan VE. Assessments of thiyl radicals in biosystems: difficulties and new applications. Anal Chem 2011; 83: 6432–8.10.1021/ac200418sSearch in Google Scholar
35. Schöneich C. Cysteine residues as catalysts for covalent peptide and protein modification: a role for thiyl radicals. Biochem Soc Trans 2011; 39: 1254–9.10.1042/BST0391254Search in Google Scholar
36. Takashima M, Shichiri M, Hagihara Y, Yoshida Y, Niki E. Reactivity toward oxigen radicals and antioxidant action of thiol compounds. BioFactors 2012; 38: 240–8.10.1002/biof.1014Search in Google Scholar
37. Mozziconaccci O, Sharov V, Williams TD, Kerwin BA, Schöneich C. Peptide cysteine thiyl radicals abstract hydrogen atoms from surrounding amino acids: The photolysis of a cystine containing model peptide. J Phys Chem B 2008; 112: 9250–7.10.1021/jp801753dSearch in Google Scholar
38. Mozziconaccci O, Williams TD, Kerwin BA, Schöneich C. Reversible intramolecular hydrogen transfer between protein cysteine thiyl radicals and αC-H bonds in insulin: control of selectivity by secondary structure. J Phys Chem B 2008; 112: 15921–32.10.1021/jp8066519Search in Google Scholar
39. Zhou S, Mozziconaccci O, Kerwin BA, Schöneich C. The photolysis of disulfide bonds in IgG1 and IgG2 leads to selective intramolecular hydrogen transfer reactions of cysteine thiyl radicals, probed by covalent H/D exchange and RPLC-MS/MS analysis. Pharm Res 2013; 30: 1291–9.10.1007/s11095-012-0968-1Search in Google Scholar
40. Nauser T, Schöneich C. Thiyl radicals abstract hydrogen atoms from the aC-H bonds in model peptides: absolute rate constants and effect of amino acid structure. J Am Chem Soc 2003; 125: 2042–3.10.1021/ja0293599Search in Google Scholar
41. Nauser T, Casi G, Koppenol W, Schöneich C. Reversible intramolecular hydrogen transfer between cysteine thiyl radicals and glycine and alanine in model peptides: absolute rate constants derived from pulse radiolysis and laser flash photolysis. J Phys Chem B 2008; 112: 15034–44.10.1021/jp805133uSearch in Google Scholar
42. Nauser T, Koppenol W, Schöneich C. Reversible hydrogen transfer reactions in thiyl radicals from cysteine and related molecules: absolute kinetics and equilibrium constants determined by pulse radiolysis. J Phys Chem B 2012; 116: 5329–41.10.1021/jp210954vSearch in Google Scholar
43. Buettner GR. The pecking order of free-radicals and antioxidants – lipid-peroxidation, α-tocopherol, and ascorbate. Arch Biochem Biophys 1993; 300: 535–43.10.1006/abbi.1993.1074Search in Google Scholar
44. Escobar JA, Rubio MA, Lissi EA. Sod and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic Biol Med 1996; 20: 285–90.10.1016/0891-5849(95)02037-3Search in Google Scholar
45. Aspee A, Lissi EA. Acid phosphatase reaction with peroxyl radicals: inactivation mechanism and behavior of the partially modified ensemble. Arch Biochem Biophys 2000; 379: 245–51.10.1006/abbi.2000.1887Search in Google Scholar
46. Lissi EA, Salim-Hanna M, Faure M, Videla LA. 2,2′-Azo-bis-amidinopropane as a radical source for lipid peroxidation and enzyme inactivation studies. Xenobiotica 1991; 21: 995–1001.10.3109/00498259109039539Search in Google Scholar
47. Videla LA, Salim-Hanna M, Lissi EA. Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals. Characteristics and influence of nicotinamide-adenine dinucleotides. Biochem Pharmacol 1992; 44: 1443–52.10.1016/0006-2952(92)90547-VSearch in Google Scholar
48. Pazos IF, Alvarez C, Lanio ME, Martinez D, Morera V, Lissi EA, Campos AM. Modification of sticholysin II hemolytic activity by free radicals. Toxicon 1998; 36: 1383–93.10.1016/S0041-0101(98)00016-6Search in Google Scholar
49. Campos AM, Lissi EA, Vergara C, Lanio ME, Alvarez C, Pazos I. Kinetics and mechanism of St I modification by peroxyl radicals. J Protein Chem 1999; 18: 297–306.10.1023/A:1021087312176Search in Google Scholar
50. Lissi EA, Clavero N. Inactivation of lysozyme by alkylperoxyl radicals. Free Radic Res Commun 1990; 10: 177–84.10.3109/10715769009149886Search in Google Scholar
51. Neuzil J, Gebicki JM, Stocker R. Radical-induced chain oxidation of proteins and its inhibition by chain-breaking antioxidants. Biochem J 1993; 293: 601–6.10.1042/bj2930601Search in Google Scholar
52. Hawkins CL, Davies MJ. Generation and propagation of radical reactions on proteins. Biochim Biophys Acta 2001; 1504: 196–219.10.1016/S0005-2728(00)00252-8Search in Google Scholar
53. Houee-Levin C, Bobrowski K. The use of the methods of radiolysis to explore the mechanisms of free radical modifications in proteins. J Proteomics 2013; 92: 51–62.10.1016/j.jprot.2013.02.014Search in Google Scholar
54. Kang JH, Kim KS, Choi SY, Kwon HY, Won MH, Kang TC. Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu,Zn-superoxide dismutase modification. Biochim Biophys Acta 2002; 1570: 89–96.10.1016/S0304-4165(02)00158-7Search in Google Scholar
55. Edwards AM, Ruiz M, Silva E, Lissi E. Lysozyme modification by the fenton reaction and gamma radiation. Free Radic Res 2002; 36: 277–84.10.1080/10715760290019291Search in Google Scholar
56. Headlam HA, Mortimer A, Easton CJ, Davies MJ. Beta-scission of C-3 (beta-carbon) alkoxyl radicals on peptides and proteins: a novel pathway which results in the formation of α-carbon radicals and the loss of amino acid side chains. Chem Res Toxicol 2000; 13: 1087–95.10.1021/tx0001171Search in Google Scholar
57. Stefek M, Kyselova Z, Rackova L, Krizanova L. Oxidative modification of rat eye lens proteins by peroxyl radicals in vitro: protection by the chain-breaking antioxidants stobadine and trolox. Biochim Biophys Acta 2005; 1741: 183–90.10.1016/j.bbadis.2005.01.006Search in Google Scholar
58. Giulivi C, Traaseth NJ, Davies KJ. Tyrosine oxidation products: analysis and biological relevance. Amino Acids 2003; 25: 227–32.10.1007/s00726-003-0013-0Search in Google Scholar
59. Il’yasova D, Scarbrough P, Spasojevic I. Urinary biomarkers of oxidative status. Clin Chim Acta 2012; 413: 1446–53.10.1016/j.cca.2012.06.012Search in Google Scholar
60. Arenas A, Vasquez R, Lopez-Alarcon C, Lissi E, Silva E. Oxidative damage of lysozyme and human serum albumin and their mixtures: a comparison of photosensitized and peroxyl radical promoted processes. Protein J 2011; 30: 359–65.10.1007/s10930-011-9341-1Search in Google Scholar
61. Simpson JA, Narita S, Gieseg S, Gebicki S, Gebicki JM, Dean RT. Long-lived reactive species on free-radical-damaged proteins. Biochem J 1992; 282: 621–4.10.1042/bj2820621Search in Google Scholar
62. Halliwell B, Gutteridge J. Free radicals in biology and medicine. New York, USA: Oxford University Press; 1989.Search in Google Scholar
63. Pierpont C, Lange C. The chemistry of transition metal complexes containing catechol and semiquinone ligands. New Jersey, USA: Wiley, 2007.10.1002/9780470166420.ch5Search in Google Scholar
64. Ostdal H, Davies MJ, Andersen HJ. Reaction between protein radicals and other biomolecules. Free Radic Biol Med 2002; 33: 201–9.10.1016/S0891-5849(02)00785-2Search in Google Scholar
65. Headlam HA, Gracanin M, Rodgers KJ, Davies MJ. Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides. Free Radic Biol Med 2006; 40: 1539–48.10.1016/j.freeradbiomed.2005.12.036Search in Google Scholar
66. Gracanin M, Lam MA, Morgan PE, Rodgers KJ, Hawkins CL, Davies MJ. Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome. Free Radic Biol Med 2011; 50: 389–99.10.1016/j.freeradbiomed.2010.11.019Search in Google Scholar
67. Gebicki JM, Nauser T, Domazou A, Steinmann D, Bounds PL, Koppenol WH. Reduction of protein radicals by GSH and ascorbate: potential biological significance. Amino Acids 2010; 39: 1131–7.10.1007/s00726-010-0610-7Search in Google Scholar
68. Liu CC, Gebicki JM. Intracellular GSH and ascorbate inhibit radical-induced protein chain peroxidation in HL-60 cells. Free Radic Biol Med 2012; 52: 420–6.10.1016/j.freeradbiomed.2011.10.450Search in Google Scholar
69. Barker S, Weinfeld M, Murray D. DNA-protein crosslinks: their induction, repair, and biological consequences. Mutat Res 2005; 589: 111–35.10.1016/j.mrrev.2004.11.003Search in Google Scholar
70. Barker S, Murray D, Zheng J, Li L, Weinfeld M. A method for the isolation of covalent DNA-protein crosslinks suitable for proteomics analysis. Anal Biochem 2005; 344: 204–15.10.1016/j.ab.2005.06.039Search in Google Scholar
71. Ide H, Shoulkamy MI, Nakano T, Miyamoto-Matsubara M, Salem AM. Repair and biochemical effects of DNA-protein crosslinks. Mutat Res 2011; 711: 113–22.10.1016/j.mrfmmm.2010.12.007Search in Google Scholar
72. Luxford C, Dean RT, Davies MJ. Radicals derived from histone hydroperoxides damage nucleobases in RNA and DNA. Chem Res Toxicol 2000; 13: 665–72.10.1021/tx000053uSearch in Google Scholar
73. Luxford C, Dean RT, Davies MJ. Induction of DNA damage by oxidised amino acids and proteins. Biogerontology 2002; 3: 95–102.10.1023/A:1015228001561Search in Google Scholar
74. Gebicki S, Gebicki JM. Crosslinking of DNA and proteins induced by protein hydroperoxides. Biochem J 1999; 338: 629–36.10.1042/bj3380629Search in Google Scholar
75. Beal MF. Oxidatively modified proteins in aging and disease. Free Radic Biol Med 2002; 32: 797–803.10.1016/S0891-5849(02)00780-3Search in Google Scholar
76. Davies MJ, Fu SL, Wang HJ, Dean RT. Stable markers of oxidant damage to proteins and their application in the study of human disease. Free Radic Biol Med 1999; 27: 1151–63.10.1016/S0891-5849(99)00206-3Search in Google Scholar
77. Dean RT, Fu SL, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 1997; 324: 1–18.10.1042/bj3240001Search in Google Scholar
78. Linton S, Davies MJ, Dean RT. Protein oxidation and ageing. Exp Gerontol 2001; 36: 1503–18.10.1016/S0531-5565(01)00136-XSearch in Google Scholar
79. Smith CD, Carney JM, Satrk-Reed PE, Oliver CN, Stadtman ER, Floyd RA, Markesbery WR. Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci USA 1991; 88: 10540–3.10.1073/pnas.88.23.10540Search in Google Scholar
80. Stadtman ER, Oliver CN. Metal-catalyzed oxidation of proteins – physiological consequences. J Biol Chem 1991; 266: 2005–8.10.1016/S0021-9258(18)52199-2Search in Google Scholar
81. Domazou AS, Koppenol WH, Gebicki JM. Efficient repair of protein radicals by ascorbate. Free Radic Biol Med 2009; 46: 1049–57.10.1016/j.freeradbiomed.2009.01.001Search in Google Scholar
82. Nauser T, Koppenol WH, Gebicki JM. The kinetics of oxidation of GSH by protein radicals. Biochem J 2005; 392: 693–701.10.1042/BJ20050539Search in Google Scholar
83. Domazou AS, Zhu HP, Koppenol WH. Fast repair of protein radicals by urate. Free Radic Biol Med 2012; 52: 1929–36.10.1016/j.freeradbiomed.2012.02.045Search in Google Scholar
84. Neta P, Huie RE, Ross AB. Rate constants for reactions of peroxyl radicals in fluid solutions. J Phys Chem Ref Data 1990; 19: 413–513.10.1063/1.555854Search in Google Scholar
85. Simic MG, Jovanovic SV. Antioxidation mechanisms of uric-acid. J Am Chem Soc 1989; 111: 5778–82.10.1021/ja00197a042Search in Google Scholar
86. Filipe P, Morliere P, Patterson LK, Hug GL, Maziere JC, Maziere C. Mechanisms of flavonoid repair reactions with amino acid radicals in models of biological systems: a pulse radiolysis study in micelles and human serum albumin. Biochim Biophys Acta 2002; 1572: 149–62.10.1016/S0304-4165(02)00304-5Search in Google Scholar
87. Hoey BMBJ. The repair of oxidized amino acids by antioxidants. Biochim Biophys Acta 1984; 791: 212–8.10.1016/0167-4838(84)90011-6Search in Google Scholar
88. Sorensen LB. Extrarenal disposal on uric acid. In: Kelley WN, Weiner IM, editors. Uric acid handbook of experimental pharmacology. New York: Springer, 1978: 325–36.Search in Google Scholar
89. Moore S, Calder KAC, Miller NJ, Riceevans CA. Antioxidant activity of saliva and periodontal-disease. Free Radic Res 1994; 21: 417–25.10.3109/10715769409056594Search in Google Scholar
90. Wayner DD, Burton GW, Ingold KU, Barclay LR, Locke SJ. The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma. Biochim Biophys Acta 1987; 924: 408–19.10.1016/0304-4165(87)90155-3Search in Google Scholar
91. Frei B, Stocker R, Ames BN. Antioxidant defenses and lipid-peroxidation in human-blood plasma. Proc Natl Acad Sci USA 1988; 85: 9748–52.10.1073/pnas.85.24.9748Search in Google Scholar
92. Nagler RM, Klein I, Zarzhevsky N, Drigues N, Reznick AZ. Characterization of the differentiated antioxidant profile of human saliva. Free Radic Biol Med 2002; 32: 268–77.10.1016/S0891-5849(01)00806-1Search in Google Scholar
93. Jovanovic SV, Simic MG. Repair of tryptophan radicals by antioxidants. J Free Radic Biol Med 1985; 1: 125–9.10.1016/0748-5514(85)90016-9Search in Google Scholar
94. Filipe P, Morlière P, Patterson LK, Hug GL, Mazière JC, Mazière C, Freitas JP, Fernandes A, Santus R. Repair of amino acid radicals of apolipoprotein B100 of low-density lipoproteins by flavonoids. A pulse radiolysis study with quercetin and rutin. Biochemistry 2002; 41: 11057–64.10.1021/bi026133+Search in Google Scholar
95. Morlière P, Mazière JC, Patterson LK, Conte MA, Dupas JL, Ducroix JP, Filipe P, Santus R. On the repair of oxidative damage to apoferritin: a model study with the flavonoids quercetin and rutin in aerated and deaerated solutions. Free Radic Res 2013; 47: 463–73.10.3109/10715762.2013.791024Search in Google Scholar
96. O’Reilly JD, Mallet AI, McAnlis GT, Young IS, Halliwell B, Sanders TAB. Consumption of flavonoids in onions and black tea: lack of effect on F-2-isoprostanes and autoantibodies to oxidized LDL in healthy humans. Am J Clin Nutr 2001; 73: 1040–4.10.1093/ajcn/73.6.1040Search in Google Scholar
97. Winterbourn CC. Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol 2008; 4: 278–86.10.1038/nchembio.85Search in Google Scholar
©2014 by Walter de Gruyter Berlin/Boston
This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.