Photo-oxidation of proteins

Abstract

Photo-induced damage to proteins occurs via multiple pathways. Direct damage induced by UVB (λ 280–320 nm) and UVA radiation (λ 320–400 nm) is limited to a small number of amino acid residues, principally tryptophan (Trp), tyrosine (Tyr), histidine (His) and disulfide (cystine) residues, with this occurring via both excited state species and radicals. Indirect protein damage can occur via singlet oxygen (¹O₂¹Δ_g), with this resulting in damage to Trp, Tyr, His, cystine, cysteine (Cys) and methionine (Met) residues. Although initial damage is limited to these residues multiple secondary processes, that occur both during and after radiation exposure, can result in damage to other intra- and inter-molecular sites. Secondary damage can arise via radicals (e.g. Trp, Tyr and Cys radicals), from reactive intermediates generated by ¹O₂ (e.g. Trp, Tyr and His peroxides) and via molecular reactions of photo-products (e.g. reactive carbonyls). These processes can result in protein fragmentation, aggregation, altered physical and chemical properties (e.g. hydrophobicity and charge) and modulated biological turnover. Accumulating evidence implicates these events in cellular and tissue dysfunction (e.g.apoptosis, necrosis and altered cell signaling), and multiple human pathologies.

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Introduction

Most forms of life on Earth are exposed to light on a daily basis, and in most cases this exposure is essential to viability and health. As the Earth's upper atmosphere absorbs UV radiation with wavelengths below ca. 295 nm (i.e. all of the UVC band, λ 200–280 nm, and a considerable proportion of the UVB band, λ 280–320 nm), the majority of exposure at the Earth's surface consists of UVA radiation (λ 320–400 nm; 95% of all UV exposure) and visible light, as well as the infra-red wavelengths. Despite the positive effects of light exposure, UV radiation and visible light in the presence of suitable chromophores can induce protein damage. This review discusses the mechanisms of these processes and its consequences.

Mechanisms of photo-induced damage to proteins

Photo-induced damage to proteins can occur via two major pathways.^1–4 The first of these is mediated predominantly by UVB (and to a lesser extent UVA) radiation, where direct absorption by particular amino acid residues (e.g.tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), histidine (His), methionine (Met), cysteine (Cys) and disulfide (cystine) bonds) results in the formation of electronically-excited states and photo-ionization reactions. In addition to direct absorption of light, photosensitized reactions also occur through the absorption of UV and/or visible light by a sensitizing compound. These sensitizers can be endogenous species (e.g.porphyrins,^5,6 aromatic lens filter compounds,^7,8vitamins such as riboflavin⁹) or exogenous species (e.g.drugs, polyaromatic compounds and dye molecules^10–15). The sensitizers are typically excited to a short lived excited singlet state that rapidly undergoes intersystem crossing to give a longer lived triplet. The latter either undergoes decay processes to the ground state, or reacts with other species by one of two pathways (known as Type I and Type II mechanisms) that can result in damage to target molecules (e.g.proteins). Type I damage results in free radical formation arising from either one electron oxidation or hydrogen atom abstraction from the substrate, whereas the Type II mechanism involves energy transfer from the excited sensitizer to molecular oxygen, to yield the first excited state of singlet oxygen (¹O₂¹Δ_g; reviewed in ref. 2, 16, and 17) which readily oxidizes surrounding targets. Sensitizers rarely react solely by a Type I or Type II mechanism due to variations in energy transfer efficiency, thus photosensitized reactions typically proceed via a mixture of ¹O₂ and radical species (e.g.O₂⁻˙ and substrate radicals).¹⁶ The protein damage profile is typically affected by the proportion of each reactive species generated through photosensitization, and therefore depends critically on the nature of the sensitizer, the excitation wavelength and the reaction conditions.¹⁶

Damage to proteins induced by UV/visible irradiation

Proteins are major cellular targets for direct photo-oxidation due to their high abundance and the presence of endogenous chromophores within the protein structure (certain amino acid side-chains as well as bound chromophores, such as flavins and porphyrins). A major pathway for oxidation of amino acids, peptides and proteins by UV light is via the absorption of the incident radiation by the protein. The major intrinsic chromophoric species present in proteins are Trp, Tyr, Phe, His, Cys and cystine side chains, with all other amino acid side chains and peptide bonds only absorbing radiation with λ < 230 nm. Upon absorption of UV light the chromophoric residues are typically converted to their first excited singlet states that have only short lifetimes.² These states readily lose energy by inter-system crossing to the triplet state, and energy transfer to other groups through collisional deactivation. The resulting triplet states typically have much longer lifetimes (on the order of microseconds) than the singlet states and are able to undergo electron transfer and hydrogen abstraction mechanisms, rather than solely energy transfer processes. The (photo)physical properties of these triplets are well characterised (reviewed in ref. 2 and 4) and will only be reviewed briefly below. The chemistry of the (hydrated) electron resulting from direct photo-ionisation is also well characterized,^18–20 with addition to O₂, to give O₂⁻˙, being a major and fast reaction (k = ca. 10¹⁰ dm³ mol⁻¹ s⁻¹).¹⁸ Hydrated electrons can also add to free carboxyl groups (e.g. the C-terminus, or Asp/Glu side-chains) and amine groups (e.g. the N-terminus, or Lys side-chains) in processes that result in deamination and elimination of H˙ respectively. Addition can also occur at other sites, particularly with cystine (to give a radical-anion, RSSR⁻˙), and protonated His side-chains. In peptides, the hydrated electron can also add to the carbonyl groups of the peptide backbone to give a radical-anion that can subsequently give rise to backbone cleavage. These reactions have been reviewed.^18–21

Indirect photo-oxidation of proteins occurs through the formation and subsequent reactions of ¹O₂ with Trp, His, Tyr, Met and Cys side-chains. These reactions are facile as ¹O₂ reacts with these protein side-chains more rapidly than with most other cellular targets at pH 7.4.^3,4,16,17,22 Furthermore, proteins are also present in most biological systems at much higher concentrations than other potential targets for ¹O₂ (reviewed in ref. 3, 4, and 17). Under alkaline conditions (typically at pH > 10) ¹O₂ also reacts with Arg and Lys in their neutral (unprotonated) forms.²³ Whilst direct ¹O₂-mediated damage to proteins primarily occurs at these residues, consumption of other amino acids can also occur through reaction of ¹O₂-induced intermediates (such as hydroperoxides) with these residues.

The following sections discuss the nature and mechanism of UV damage to each of these susceptible amino acids in more detail.

Tryptophan side-chains

The indolic side-chain of Trp is the strongest UV chromophore in proteins (with the absorbance band exhibiting a high molar absorptivity which extends to greater wavelengths than any of the other residues), but the overall significance of Trp photo-oxidation is attenuated by its relatively low abundance in many proteins. Nevertheless, Trp exhibits a rich photochemistry. The first triplet state of Trp (³Trp) readily undergoes electron transfer reactions with suitable electron acceptors²⁴ to give, for example, with disulfides the corresponding radical anion (RSSR⁻˙) and a Trp radical-cation (Trp⁺˙; reviewed in ref. 2). The Trp radical-cations formed via these mechanisms rapidly deprotonate to the neutral indolyl radical (Trp˙; Fig. 1).²⁵ Subsequent reactions of the indolyl (Trp˙) radicals include reaction with O₂ to give a ring C-3 peroxyl radical that can undergo further hydrogen atom abstraction reactions (Fig. 1).^21,25–27 Evidence has been presented for the repair of both the indolyl and peroxyl radicals by antioxidants such as ascorbate, selenocysteine and GSH (e.g.^28–32).

Fig. 1 Mechanisms of oxidation of Trp by direct UV absorption and ¹O₂-mediated pathways. Key to structures (where the α-amino acid moiety is represented by R = CH(CO₂H)NH₂): 1, indolyl radical; 2, C-3 peroxyl radical; 3, C-3 hydroperoxide; 4, dioxetane intermediate; 5, C-3 alcohol; 6, 3α-hydroperoxypyrroloindole; 7, 3α-hydroxypyrroloindole; 8, N-formylkynurenine; 9, kynurenine; 10, 3α-dihydroxypyrroloindole.

Trp residues can also undergo direct photo-ionization processes. There has been some debate over whether these are mono- or bi-photonic processes,^24,33 but recent studies suggest that both mono- and bi-photonic mechanisms occur simultaneously,^34,35 and extensive research continues on the multi-photon chemistry of Trp.

In addition to direct photo-oxidation, Trp residues are also highly susceptible to ¹O₂-mediated damage. Oxidation of Trp by ¹O₂ initially results in the formation of either a dioxetane intermediate across the C2–C3 double bond of Trp, or a hydroperoxide at the C3 position (Fig. 1).^36,37 These intermediates can subsequently decompose to N-formylkynurenine (and hence kynurenineviahydrolysis) or can undergo ring closure reactions to form 3α-hydroperoxypyrroloindoles and 3α-dihydroxypyrroloindoles.^38–41Decomposition of the peroxide intermediates may involve non-radical reactions to yield, for example, 3α-hydroxypyrroloindoles, or homolysis of the –O–O– bond to give radicals. Metal ions, heat and UV light have been shown to catalyze Trp peroxide decomposition to radicals,³⁸ and the mechanism and products of such reactions has been reviewed.³⁷ These processes have recently been reinvestigated in detail using isotopically ¹⁸O-labeled ¹O₂ in H₂O to generate the various intermediates; these studies have allowed the origin of incorporated oxygen atoms to be accurately assessed.^40,41 Similar reactions appear to occur with both the free amino acid and N-blocked Trp residues, and hence analogous materials are formed on Trp-containing peptides and proteins, though the yields of some of the intermediates is modulated.^38,39

Two of the major products of Trp photo-oxidation, N-formylkynurenine and kynurenine, are more effective photosensitizing agents than the parent amino acid⁴² and can produce a variety of reactive species that further degrade these and other structures. Prolonged exposure (over several hours) of Trp to UV/visible light results in extensive Trp photo-degradation leading to products arising from dehydrogenation and cyclisation reactions^39,43 as well as hydroxylated Trp and kynurenine derivatives that implicate hydroxyl radicals (or radical-cations, with subsequent rapid hydration to give the hydroxylated species) in the photo-oxidation process.^43,44 A recent study has reported the detection of nitrotryptophan in such systems, suggesting that reactive nitrating species are formed as a result of UVA photolysis of Trp-containing peptides;^43,44 the mechanism underlying this surprising and interesting observation, has not been elucidated. A number of oxidation/degradation products of free Trp have been shown to react with proteins, and particularly lens proteins, with this resulting in the formation of covalently-bound adducts at Cys, His and Lys residues.^8,45–48 These materials have been shown to act as sensitizers of further oxidative damage.^48,49 Thus, the formation of N-formylkynurenine, kynurenine and related materials on UV-exposed proteins may lead to an enhancement in the photo-oxidation of the target proteins due to further generation of reactive species.

Tyrosine side-chains

The first triplet state of Tyr undergoes similar electron transfer processes to those described above for Trp, leading to formation of Tyr⁺˙ and phenoxyl radicals following (rapid) deprotonation of the radical-cation (Fig. 2).^2,25 As for Trp, the radical-cation of Tyr can also be generated via direct photo-ionization reactions.^2,50 The Tyr phenoxyl radicals generated by these mechanisms can undergo a wide variety of reactions, including oxidation of susceptible substrates^51–53 and dimerization (via C–O and C–C linkages) to yield di-tyrosine products and hydrogen atom abstraction reactions (see Fig. 2; reviewed in ref. 21). The Tyr phenoxyl radical can also be efficiently repaired by a range of low-molecular-mass antioxidants including ascorbate, nitric oxide, glutathione and selenocysteine.^{28–32,54,55}

Fig. 2 Oxidation pathways for Tyrvia direct UV absorption or ¹O₂ production. Key to structures (where the α-amino acid moiety is represented by R = CH(CO₂H)NH₂): 1, tyrosyl radical; 2, a C–O linked isomer of a dityrosine crosslink; 3, Tyr endoperoxide; 4, HOHICA (3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indol-2-carboxylic acid).

Photo-oxidation of free Tyr residues by ¹O₂ initially yields unstable endoperoxides (Fig. 2). These rapidly decompose via ring opening to give a C1 hydroperoxide, and cyclized products involving nucleophilic addition of the α-amino group.^56–59 Thermal decay of these intermediates gives rise to a cyclized indolic product, 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indol-2-carboxylic acid (HOHICA; Fig. 2),^56–59 which can be oxidized further to a decarboxylated keto compound under basic conditions.^56,57 The decomposition of the initial peroxides and cyclized products can be accelerated by exposure to metal ions or UV light resulting in radical production, but the identities of the decomposition products generated via these radical pathways are unknown.^58,60

In peptides and proteins, the nucleophilic ring closure reactions of the α-amino group are less favorable (due to its incorporation into a peptide bond), and consequently reaction with other nucleophiles can compete with this cyclization (A. Wright, C. L. Hawkins and M. J. Davies, unpublished data). Such addition reactions could play an important role in protein cross-linking if neighbouring Lys, Arg or Cys residues were to provide the nucleophilic centre. Previous studies have reported that di-tyrosine is not a major product of ¹O₂-mediated oxidation of Tyr on proteins,^61,62 and it has been suggested that another Tyr-derived product, 3,4-dihydroxyphenylalanine (DOPA) is not generated by reaction of ¹O₂.^61,62 However, more recent data have shown that photolysis of proteins in the absence or presence of a ¹O₂-generating photosensitizer (e.g. Rose Bengal) generates both of these materials in significant yields.^43,44,63,64 Further oxidation and hydroxylation of DOPA residues to form dopaquinone and trihydroxyphenylalanine (TOPA) derivatives has also been demonstrated on prolonged exposure of Tyr solutions to UVA light.^43,44

Phenylalanine side-chains

UV-mediated oxidation of Phe is relatively straightforward when compared to the other aromatic amino acids. Excitation of Phe to the first triplet state results in a simple photo-dissociation reaction that yields a benzyl radical (Fig. 3).⁶⁵ Direct photo-ionization of Phe results in radical-cation formation, which undergoes rapid hydration to yield hydroxylated ring products (o-, m- and p-Tyr; Fig. 3; reviewed in ref. 50), though there is also evidence for deprotonation to yield benzyl radicals.²⁵ These processes are however only significant with short wavelength UV radiation.

Fig. 3 Direct UV absorption by Phe leads to formation of the triplet state and subsequent radical formation, or photo-ionization followed by hydration to form Tyr isomers.

Histidine side-chains

Direct absorption of UV or visible light by His is not a major mechanism, and the majority of damage observed at such residues occurs via sensitized processes. Recent studies with model His compounds in the presence of flavins have demonstrated the formation of radicals on the imidazole function,⁶⁶ and this has also been demonstrated with other sensitizers.⁶⁷ The direct interaction (via a Type I mechanism) between the triplet state flavin and His (as determined by EPR spin trapping techniques) is markedly pH dependent,⁶⁶ with this attributed to electronic effects arising from the differential protonation state of the imino group.⁶⁶ The majority of His oxidation mediated by photo-excited flavins appears to occur via production of ¹O₂, consistent with studies with other sensitizers where evidence for ¹O₂ as the reactive intermediate has been obtained.⁶⁷

His oxidation, both free and in peptides, by ¹O₂ has been studied extensively, and occurs via the initial formation of one or more endoperoxides (Fig. 4).^67,68 The structure of these endoperoxides has been determined by low temperature NMR in organic solvents,^69,70 but at higher (ambient) temperatures they undergo rapid reactions that result in the formation of multiple decomposition products including aspartic acid, asparagine and urea (Fig. 4).^68,71,72 These products arise from opening of the imidazole ring, and may occur via radical processes.^67,73 In aqueous solutions, free His also yields bicyclic products generated by nucleophilic attack of the α-amino group onto the oxidized, but still intact, imidazole ring, as characterized by NMR.⁶⁷ Many of these initial His-derived products can undergo further reactions, with this resulting in the formation of dimers from N-acetyl-His,⁶⁷ and His-His and His-Lys cross-links following nucleophilic addition of a (non-oxidized) His or Lys side-chain, to a keto group on an oxidized His.^74,75

Fig. 4 Reaction pathways for ¹O₂-mediated oxidation of His residues (where the α-amino acid moiety is represented by R = CH(CO₂H)NH₂).

In addition to ¹O₂-mediated photo-oxidation of His residues, it has been shown that light exposure of proteins in the presence of stilbene-derived fluorescent whitening agents (FWA; commonly used in the textile and clothing industries) can lead to the formation of 2-oxo-His and nitrated His derivatives.⁷⁶ These products suggest that radical species may be involved, and the formation of peroxynitrite (ONOO⁻/ΟNΟΟH) has been suggested,⁷⁶ leading in turn to the formation of HO˙ and NO₂. viaONOOH decomposition (or CO₃⁻˙ and NO₂. from the corresponding CO₂ adduct of ONOO⁻). The mechanism of this photo-induced nitration of protein-bound His residues has not been definitively elucidated.

Cysteine and cystine groups

The direct absorption of UV radiation by disulfide bonds, and ensuing reactions, has primarily been studied with shorter UV wavelengths (λ < 260 nm) than those under consideration here. Under these conditions there is evidence that the initial singlet state undergoes homolytic cleavage of the disulfide bond to form two short-lived thiyl radicals that can recombine to regenerate the parent disulfide (reviewed in ref. 77). However, there is controversy over the identities of the radicals formed, with suggestions that the key intermediates are RSS˙, or even [RS-(SR)-SR]˙ formed via adduction of RSS˙ to a further cysteine moiety (see ref. 78). Short wavelength UV irradiation of cystine can also yield the radical-cation (RSSR⁺˙) and cystine S-monoxide (among other products), in the presence of O₂.⁷⁸ Evidence for homolytic cleavage of the C–S bond has also been presented following irradiation with UVC wavelengths.⁷⁹ Recent studies using longer wavelength photolysis (λ ca. 300 nm) of a model peptide containing an intrachain disulfide bond, have demonstrated the formation of a thiyl radical pair that can rapidly recombine or undergo a series of complex rearrangement reactions leading to the formation of products such as cyclic thioethers and H₂S.⁸⁰

A common mechanism of photo-oxidation of disulfide bonds is viaelectron transfer from ³Trp or ³Tyr species to cystine, with this resulting in the formation of disulfide radical anions (RSSR⁻˙).^{2,24,77,81–84} This species can readily dissociate, in a reversible reaction, to give the anion (RS⁻) and a radical (RS˙) or can undergo rapid electron transfer with O₂ to give O₂⁻˙.^4,21Thiyl radicals formed via such dissociation have been shown to undergo multiple reactions including hydrogen abstraction from suitable C–H bonds (and hence propagation of damage and chain reactions, both within and between proteins and peptides, and with other biomolecules),^85–89 recombination with the thiyl anion to regenerate the disulfide radical anion, or reaction with O₂ to give a thiyl peroxyl radical (RSOO˙) (reviewed in ref. 21). The resulting thiyl peroxyl radicals can eliminate O₂ thereby reverting to thiyl radicals, or can isomerize to a sulfonyl radical (RS(=O)O˙) and hence give rise to sulfinic (RSO₂H) and sulfonic (RSO₃H) acids (reviewed in ref. 90,91).

Production of ¹O₂ through photosensitization reactions is a major pathway to Cys oxidation, with this postulated to occur via formation of a zwitterion (RS⁺–OO⁻);^92,93 subsequent decomposition of this short-lived intermediate yielding cystine, thiosulfinates and other species.⁹²Cystine, is a major reaction product^16,94 when further Cys residues are able to react with the initial intermediate. However, other products including cysteic acid (RSO₃H) can also be formed, when such reactions are slow or unfavourable.¹⁶

Methionine side-chains

Photosensitized oxidation of Met residues has been studied extensively, and has been reviewed recently.⁹⁵ Many of these studies have utilized aromatic ketones as sensitizers, excited with laser wavelengths in the 300–400 nm region. The triplet states that are formed undergo charge-transfer with Met to form the radical cation MetS⁺˙,^96,97 which can undergo a range of reactions depending on the pH, including dimerization (to give the corresponding dimeric radical-cation with a two-centre, three-electron bond) and deprotonation reactions to form carbon-centred radicals from adjacent C–H bonds on the side-chain.^98,99

Met can also be oxidized by ¹O₂ arising from sensitizers.^4,95,100 This leads to formation of a zwitterionic species (R₂S⁺-OO⁻) which, depending on the pH, either oxidizes a second Met (to give two moles of Met sulfoxide; MetSO) or eliminates H₂O₂ to form a single mole of MetSO¹⁰¹ (reviewed in ref. 16). Stable cyclic intermediates involving the α-amino nitrogen and the sulfur atom of the thioether have also been reported (reviewed in ref. 16), with subsequent hydrolysis again yielding a 1 : 1 ratio of MetSO and H₂O₂.¹⁰¹

There is little evidence for direct UV/visible damage to Met residues, although a recent study has demonstrated, by X-ray absorption spectroscopy, that Met is converted to MetSO and MetS⁺˙ in the presence or absence of O₂, respectively.¹⁰² As these studies have been carried out in the absence of a sensitizer, the formation of these products is attributed to UV absorption by weak bands at 262 and 285 nm that tail into the UVA region. However, these studies used a Xe arc lamp as the light source, and it is unclear as to what wavelengths are responsible for the observed oxidation products.

Damage transfer reactions within proteins

Transfer of damage between the initial sites of oxidation in peptides and proteins and other residues is well characterised and primarily involves aromatic and sulfur-containing residues. Examination of these processes has yielded important information about the transfer of oxidizing and reducing equivalents through proteins (reviewed in ref. 103–106). The reduction potentials of side-chain and backbone-derived radicals indicate that the ultimate sink for oxidising equivalents in proteins are Tyr and Cys residues (or Trp is the absence of these side-chains).¹⁰⁴ Some of these reactions are equilibria and hence damage transfer can occur in both directions and the overall effects can be readily reversed. Thus Tyr phenoxyl radicals can both be generated by thiyl radicals and repaired by Cys reactions,^104,107 with the balance of these processes determined, at least in part, by the concentration of thiol anions, as thiyl radicals can be removed via reaction with these species (to give the disulfide radical anion).

Oxidation of Trp residues can yield indolyl radical, but it has been established that if such species are generated on proteins that also contain Tyr residues, rapid electron transfer can occur between these species to yield Tyr phenoxyl radicals;^103,105 such transfer can occur over long distances within (and between) proteins, but the rate of these processes is critically dependent on the protein structure. Transfer can be slow even if the residues are close in distance, if they are present in unfavourable and rigid conformations.¹⁰⁸ In contrast, transfer can occur rapidly over very long distances if the structure is flexible, or if there are suitable intervening residues that can act as a conduit for electron migration.^104,108,109 In many proteins the exact transfer pathway is unclear due to the presence of multiple alternative pathways. These processes may result in a different pattern and rates of oxidation than might be initially expected on the basis of the reactivity of the initiating oxidant.¹⁰⁴

The cystine disulfide bond is the major sink for photo-ejected electrons and reducing equivalents, with this competing with direct addition to O₂ in some cases.¹⁰³ Transfer to disulfide bonds can occur via both hydrogen bonding networks and via the backbone.^106,110 The formation of disulfide radical-anions via such reactions can be of particular importance, as thiyl radicals formed from cleavage of the radical-anion can in turn oxidise Tyr residues, thereby providing a point of convergence of oxidation and reduction pathways.

Evidence has also been obtained for radical transfer from side-chain sites to the α-carbon, and for repair of α-carbon radicals by Cys residues. It has been shown that thiyl radicals generated from the Cys residue in GSH can abstract a hydrogen atom from backbone α-carbon sites,^111–113 due to the stability of the α-carbon radical. Analogous thiyl to α-carbon transfer reactions have also been detected with the thiyl radical from homocysteine (when the amino group is deprotonated),¹¹⁴ and Cys itself, despite the apparent involvement of an (unfavourable) 1,4 rearrangement in the latter reaction.^89,115,116

Structural and functional effects of photo-induced protein oxidation

The data reviewed above illustrate the diversity of damage that can be generated on proteins on exposure to UV/visible light in the presence or absence of a sensitizing molecule and O₂. However, the consequences of photo-induced protein oxidation generally fall into a few discreet categories that are outlined below.

Protein unfolding

Photo-oxidation of some proteins can result in an increased extent or susceptibility to unfolding and conformational changes, enhanced exposure of hydrophobic residues, and altered light scattering and optical rotation properties (e.g. ref. 16, 61, and 117–122). Studies on apo (i.e. non-heme containing) horseradish peroxidase have provided some insight into the mechanisms of unfolding, as this protein is highly resistant to photo-oxidation, with this attributed, at least in part, to the tertiary protein structure protecting susceptible residues from damage.¹²³ Studies on milk proteins have demonstrated that random coil proteins (such as caseins) are more susceptible to individual amino acid damage than more densely-packed globular proteins (e.g. lactalbumin, lactoglobulin),¹²⁰ consistent with this hypothesis. However in the study of milk proteins, the proteins displaying the greatest changes in secondary and/or tertiary structure were globular proteins that exhibited less measurable damage at the individual amino acid level.¹²⁰ In contrast, photo-oxidation of lactoferrin resulted in a more compact protein structure, rather than protein unfolding.¹²⁰ These data are consistent with low levels of damage to particular residues in globular proteins having major effects on protein structure, particularly if these are buried within a compact structure. In contrast, damage to exposed residues (e.g. in random coil structures) appears to have much less marked structural impact. These limited data indicate that there are multiple factors that contribute to photo-induced protein unfolding, with the contribution of each process likely to be dependent on the structure and conformation of individual proteins; the contribution of individual factors remain to be elucidated.

Protein aggregation

The formation of high-molecular-mass aggregates (dimers and higher species) is a common and well-documented consequence of protein photo-oxidation.^{121,122,124–126} Aggregates may arise from radical–radical termination reactions, such as reaction of two Tyr-derived phenoxyl radicals to give di-tyrosine (alternatively notated as bi-tyrosine) (e.g. ref. 63, 120, and 127), but multiple other reactions have also been shown to lead to cross-link formation, with some of these occurring via “dark” reactions after the cessation of light exposure.¹²⁸ Evidence has been presented for a role for oxidized His residues in cross-link formation,^{61,67,74,75,129,130} with this potentially involving His oxidation products that contain carbonyl functions which can undergo addition reactions with other nucleophiles.^67,68 An increase in the yield of (unspecified) protein carbonyls has been reported on photo-oxidation of albumin by porphyrin sensitizers.^6,131 It is likely that at least some of the reported cross-links arise from reaction of carbonyl compounds (on His and other residues) formed by photo-oxidation, with nucleophilic Lys, Arg and Cys side chains. The formation of such cross-links may explain the observed loss of Lys and Arg residues in some photo-oxidized proteins (reviewed in ref. 16). His residues have been suggested to be of particular importance in cross-link generation, as it has been reported that cross-links are not formed with proteins subject to photo-oxidation that lack His residues;^61,62 the number of proteins that fall in to this category are however low, and hence other factors may also be important.

Protein fragmentation

Photo-induced fragmentation of proteins appears to be of lesser importance than aggregation with many proteins, although there is evidence that it can occur under some circumstances.^16,132,133Lysozyme has been shown to be fragmented by photolysis in the presence of a sensitizer due to backbone cleavage, with Trp oxidation and radical formation implicated in this process.¹³⁴ Radicals formed at the α-carbon site on the peptide backbone are known to be key intermediates in chain cleavage in the presence of O₂ (reviewed in ref. 4, 19, and 135), and peroxides formed on His and Tyr residues have been shown to be capable of generating such radicals at least in small peptides.^58,60,67 Thus initial oxidation at side-chain sites may result in subsequent backbone cleavage via damage transfer within a protein (or possibly on other proteins, though this has not been examined in detail), with radicals as key intermediates. These transfer reactions may occur to nearby residues within a protein sequence, or to remote locations in the protein sequence, if the sites are close in space within the protein 3-D structure.

Enzyme inactivation

Enzymes have long been known to be inactivated on exposure to UV light,¹³⁶ and a considerable number of studies on such loss of activity have been published (reviewed in ref. 2, 16, and 17). Much of the photochemistry that occurs with free amino acids also appears to occur with proteins (except where cyclization reactions involving the free α-amino group are involved) and hence enzymes with active site (or otherwise critical) Cys, Met, Trp, His or Tyr residues are likely to be most susceptible to photo-inactivation. Direct photo-oxidation of such residues may only be one cause of enzyme inactivation, as a number of studies have shown that reactive protein peroxides, formed principally on His, Trp and Tyr residues, can give rise to secondary inactivation of other enzymes, at least in vitro (reviewed in ref. 3). Similar effects have recently been reported in cells exposed to visible light and sensitizers, with enzyme inactivation occurring both during, and after the cessation of light exposure.^137–139 Photo-generated peroxides have been shown to be capable of oxidizing susceptible residues that lead to enzyme inactivation in crucial intracellular enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH),¹⁴⁰ caspases,¹⁴¹proteintyrosine phosphatases (PTP),¹⁴² cathepsins and related proteases,¹⁴³ as well as multi-enzyme complexes such as the 26S proteasome.¹⁴⁴ Furthermore, protein peroxides also react rapidly with, and hence deplete, low-molecular-mass antioxidants such as GSH¹⁴⁵ which may have important ramifications for the function of other antioxidant enzymes (e.g. the glutathione peroxidase family; GPxs) via depletion of crucial co-factors.^{138,145–147} Recent studies have shown that these protein- and peptide-derived peroxide reactions may be modulated by peroxiredoxins (Prx2/Prx3) via rapid competitive reaction of the peroxide with the Cys residues in the peroxiredoxins.¹⁴⁸

Consequences of protein photo-oxidation in biological systems

Modulation of protein structure and function, whether occurring via direct or indirect processes as outlined above, can affect a wide range of cellular and biochemical responses. The consequences of such modulation will be discussed in this section, in particular in the context of cellular signalling, redox homeostasis, proteolytic turnover of damaged molecules, and cell survival or death. This discussion is not exhaustive and only highlights some particular examples; further data in some of these effects can be found in other articles within this issue.

Effects of photo-oxidation on cellular signalling

Multiple reactive intermediates (e.g.¹O₂, O₂˙⁻, HO˙, H₂O₂, other peroxides) generated by light exposure can modulate various cellular signalling pathways including the mitogen-activated protein kinase family (MAPK), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK1/2) and nuclear factor-κB (NFκB).^149–152

Experimental studies with isolated proteins, cells, and tissues have documented that reactive species generated as a result of exposure to UV or visible light are capable of both activating or inactivating kinases, and inactivating phosphatases.^153–155 This is likely to result in changes in the overall extent of protein phosporylation as a result of the complimentary action of these enzyme families in adding and removing phosphate groups respectively. The effect of exposure is likely to be system-specific due to the large number of kinases and phosphatases within cells/tissues. Furthermore the different members of these families are probably differentially susceptible to oxidative damage and changes in activity. It is therefore impossible to generalize as to what effect UV and/or visible light will have on phosphorylation status of any particular system. Phosphatase and tensin homolog (PTEN), protein tyrosine phosphatase 1B (PTP 1B), MAPK phosphatase, calcineurin, and low-molecular-weight (LMW) tyrosine phosphatase are a few examples of phosphatase enzymes whose activity is affected by UV and/or visible light exposure, with this reported to be due to oxidation of redox-sensitive Cys residues, particularly that present in the active sites of the phosphatases.¹⁵⁶ This Cys is critical for enzyme activity, and is readily oxidised to multiple species (see above) some of which are reversible (e.g. formation of sulfenic acid or disulfide species), and some not (e.g. sulfinic and sulfonic acid formation). The former results in a temporary inhibition of activity, with this postulated to be a redox switch and potential mechanism to prevent irreversible oxidation that may occur under severe exposure.¹⁵⁷ Such Cys residues have been shown to be susceptible to oxidation by both primary oxidant species (e.g.¹O₂)^{139,158–160} and secondary oxidation products such as aldehydes and hydroperoxides/H₂O₂.^38,161 The latter reactions may account for secondary (dark phase) effects after the cessation of initial UV/visible light exposure.

Studies on the activity of a range of kinases have provided evidence of a more complex biphasic (i.e.activation–inhibition) regulatory mechanism,¹⁶² with the overall effect dependent on the nature and extent of exposure; this again is dependent on the particular system examined. This complex biphasic response has been proposed to arise from the presence of multiple redox-sensitive residues within these proteins.¹⁶² In the src family of non-receptor tyrosine kinases, and protein kinase C, these redox-sensitive residues appear to be present in regulatory and catalytic domains of the protein. With low levels of oxidant exposure, redox-sensitive residue(s) in the regulatory domain appear to be targeted with oxidation generally resulting in kinase activation and/or an enhancement in activity. In the case of protein kinase C, this activation/enhancement appears to be due to protein conformational changes, which lead to an “opening” of the kinase active site. On exposure to greater extents of oxidation, an inhibition or reduction in kinase activity has been detected with protein kinase C and creatine kinase.^163,164 This inactivation may occur as a result of direct oxidation of the catalytic cysteine by oxidants, when present at high levels.

Zinc is a redox-inactive metal ion that is abundant in living organisms. It plays a critical role in the structure and function of many proteins and enzymes, including many transcription factors where it is often chelated by Cys or His residues, in Zn₁Cys₂His₂ clusters; related clusters (e.g.Zn₁Cys₂His₁) are also present in the active sites of some enzymes (e.g.alcohol dehydrogenases and matrix metalloproteinases, MMPs). Given the propensity of Cys and His to undergo photo-oxidative damage (see above) it is not surprising that UVA irradiation has been shown to increase the level of free Zn²⁺ in fibroblast cells,¹⁶⁵ with the release of this metal ion linked with the oxidation of the Cys ligands. In mast cells, Zn²⁺ release was accompanied by a reduction in protein tyrosine phosphatase activity, and activation of stress-signalling ERK and JNK-MAPK pathways.¹⁶⁶ It has been proposed that this Zn²⁺-mediated phosphatase inhibition is due to interaction of the active site Cys of the phosphatase with the released Zn²⁺,¹⁶⁷ though the studies discussed above suggest that direct oxidant effects on the phosphatase enzymes may also occur.

In each of these cases it is clear that oxidative processes arising from UV irradiation, or visible light exposure in the presence of a sensitizer can result in modulation of cell signalling pathways as evidenced by changes in intracellular protein phosphorylation levels.¹⁶⁸ In some cases antioxidants have been shown to attenuate these effects,^150,169,170 but whether this arises from scavenging of radicals, and/or other oxidants (e.g.¹O₂, H₂O₂, other peroxides), modulation of oxidant formation (e.g. as a result of the absorption of the incident radiation), or changes in repair rates (e.g.reduction of sulfenic acids and disulfides by added thiols, either directly or enzymatically), is not always clear.

Effects of photo-oxidation on redox homeostasis

Alterations in redox status, and particularly changes in the ratio of reduced to oxidized thiols, is a well-documented consequence of exposure to UVA or visible light in the presence of a sensitizer. Whilst such alterations during the period of exposure are well characterized, it is clear from a number of studies that this phenomenon can occur after cessation of photo-oxidation and persist for a considerable period. Direct oxidation of thiols (both protein-bound and low molecular mass, e.g.GSH) is likely to account, at least in part, for the immediate changes in redox status. Thus a simultaneous increase in oxidizing species, detected using a redox-sensitive fluorescent probe, and depletion of intracellular reduced glutathione (GSH) have been detected immediately following UVA irradiation in multiple studies (e.g. ref. 146). Long-lived oxidants such as peroxides may contribute to subsequent changes and prolong the oxidative stress.^138,139

Changes in the levels of various intracellular low-molecular-mass antioxidants, and the expression and activity of antioxidant and repair enzyme systems, are also likely to play a key role in determining the overall effect of UVA/visible light plus sensitizer exposure. Effects of such changes on cell survival and mutagenicity have been studied in detail, but relatively little work has been carried out on understanding the mechanistic details of the effect of irradiation on intracellular antioxidant capacity. The data obtained to date, suggest that UVA irradiation, or visible light/sensitizer combinations, may impair intracellular antioxidant capacity by: (a) directly or indirectly activating or inactivating critical enzymes; (b) changing the expression of these species; (c) depleting the availability of intracellular reductants and co-factors; (d) inhibiting dehydrogenase enzymes responsible for the maintenance of NADPH/NADP⁺ and NADH/NAD⁺ ratios; (e) directly consuming antioxidant species; and (f) by giving rise to further reactive intermediates (e.g.peroxides).

It is well established that UVA irradiation can decrease the availability of cellular reductants, including GSH and NADPH, but whether this arises from direct oxidation of these species, or as a result of indirect depletion through enzymatic activity (e.g. via the GSH/GSH peroxidase/glutaredoxin or thioredoxin reductase/thioredoxin/peroxiredoxin systems) is unclear. Thus the intracellular availability of NADPH, a co-factor for many antioxidant enzymes, has been shown to be influenced by changes in the activity of dehydrogenase enzymes, with both glucose-6-phosphate dehydrogenase and isocitric dehydrogenase reported to be inhibited by ¹O₂in vitro.^139,171,172 Direct inhibition of the protective activities of superoxide dismutase,^173,174catalase,^173,175glutathione reductase,^147,176methionine sulfoxide reductase,¹⁷⁷thioredoxin,¹³⁹glutathione peroxidase,^176,178 and thioredoxin reductase (A. Suryo Rahmanto and M. J. Davies, unpublished data) has been reported. In some cases, inhibition appears to involve oxidation of amino acids critical for conformational integrity,¹⁷⁵ or the redox activity of the proteins,^179,180 or both. For instance, Maresca et al. have reported that oxidation of Trp and Met residues (to 5-hydroxyTrp and MetSO respectively) in catalase on exposure to UVA radiation, is associated with changes in electrophoretic mobility and antioxidant activity.¹⁷⁵ Recently, a correlation between the inhibition of glutathione peroxidase with protein fragmentation, and thioredoxin reductase with aggregation, has been detected following exposure of these enzymes to ¹O₂ (A. Suryo Rahmanto and M. J. Davies, unpublished data).

Recent studies have indicated that some of the secondary effects of photo-generated oxidants can be modulated by protective enzymes. Thus peroxiredoxins react with long-lived amino acid-, peptide- and protein hydroperoxides generated by ¹O₂, as well as their well-established substrate H₂O₂,^181,182 with this resulting in thiol oxidation, formation of peroxiredoxin dimers, and ultimately loss of peroxidase activity.¹⁴⁸ Similar evidence has emerged from studies with selenoproteins,^183–185 with H₂O₂-mediated oxidation of the selenocysteine (Sec) residues in thioredoxin reductase and glutathione peroxidase resulting in the (reversible) formation of a selenenic acid, and thiodiselenide.^184,186 Rhee et al. have also shown however that reaction at the active site Sec residue in glutathione peroxidase can result in irreversible loss of activity due to conversion of the Sec residue to dehydroalanine, with release of the selenium centre from the protein.¹⁸⁵

UVA-induced oxidative stress can also result in the release of redox active iron from heme groups (either free on bound to proteins) and other sites of iron sequestration. The release of iron may contribute to secondary damage as a result of the activity of this metal ion in catalyzing oxidant formation,^187,188 though upregulation of heme-degrading (e.g.heme oxygenase-1¹⁸⁹) and iron-binding proteins (e.g. ferritin¹⁹⁰) may minimize such effects with any increase in “free” iron (e.g. in human fibroblasts) being transient.¹⁹⁰ These processes appear to involve, at least in part, the inactivation of iron regulatory protein-1 (IRP-1), which modulates the level of both the iron-binding protein transferrin, and the iron-sequestering protein ferritin.¹⁹¹ This inactivation of IRP-1 has been ascribed to oxidation of the Cys-437 residue in this protein.¹⁹¹ Release of redox-active iron from ferritin-iron complexes as a result of proteolytic degradation following oxidation has also been reported.^190,192

A number of studies have also highlighted the importance of plasma membrane oxidoreductases, in particular NADPH oxidase-1 (Nox-1), in generating oxidants post-cessation of UVA irradiation. It has been reported that UVA irradiation increases NADPH oxidase activity, and that reduction of Nox1 activity by either pharmacological approaches (diphenyleneiodonium) or siRNA, reduces the level of oxidant formation in human keratinocytes significantly.¹⁹³ Schauen et al. have also shown that inhibition of Nox-1 activity by diphenyleneiodonium improves the viability of human fibroblasts subject to UVA irradiation significantly.¹⁹⁴

Amino acid-, peptide-, and protein hydroperoxides, common products generated by both radicals in the presence of O₂, and by ¹O₂, can inhibit both the activity of various thiol-containing enzymes (e.g.glutathione reductase and peroxiredoxins^145,148) and deplete reducing co-factors such as GSH.¹³⁸

Effects of photo-oxidation on cellular proteolytic pathways

A number of studies have attempted to quantify protein damage in cells, and in skin, arising from photo-oxidation (e.g. ref. 195–198). This has been most commonly addressed by measurement of protein carbonyls, a generic marker of protein damage. It should however be noted that carbonyls are a relatively minor product of photo-oxidation, and that the resulting values reflect only the difference between the rates of generation and removal of this oxidation product; the total extent of formation may be markedly different, especially if protein catabolic systems are not significantly impaired. Two of the major systems that remove modified materials are the proteasomal system and the lysosomal pathway, both of which are intracellular systems (the latter can however remove damaged extracellular material after internalization). Turnover of extracellular protein damage is therefore less rapid and efficient. The proteasomal pathway has been reported to account for 70–80% of the turnover of oxidized proteins.¹⁹⁹ A recent study has reported that UVA irradiation decreases the proteolytic activity of the proteasome in human fibroblasts and skin tissue, with ¹O₂ believed to be the mediator of damage and inactivation.²⁰⁰ However, studies with human keratinocytes and isolated proteasome preparations have indicated that the 20S core of the proteasome is resistant to oxidative inactivation.^196,201 Studies with both the 20S and 26S forms using H₂O₂ suggest that redox modulation of proteasomal activity occurs via the 19S regulatory subunit present in the 26S (but not 20S) particle.^196,201 Recent work has shown that oxidized proteins, and particularly those containing hydroperoxide groups generated by ¹O₂, can inhibit some of the proteolytic activities of the 26S proteasome,¹⁴⁴ with this associated with the loss of proteasomal thiols.¹⁴⁴ Thus both initial reactive intermediates, such as ¹O₂, and photo-products such as hydroperoxides, appear to be capable of modulating the catabolism of both oxidized materials (via the 20S proteasome) and normal proteins (via the 26S proteasome). The latter inhibition may contribute to alterations in cell signalling as a result of the key role of the 26S system in regulating cellular signalling systems.

Oxidative damage may also impair proteasome activity by other mechanisms, including via the formation of large protein aggregates that are poorly digested for multiple reasons, including access into the catalytic core of the 20S proteasome that requires complete unfolding of the protein structure. Large protein aggregates, or aggresomes, which may result from extensive protein oxidation with consequent conformational changes, intra- and/or inter-molecular cross-links and enhanced hydrophobicity, appear to be particularly resistant to degradation. Lipofuscin (sometimes also known as age pigment) which contains modified proteins²⁰² has been shown to inhibit proteasomal activity.^195,200,203 This material may also act as a photo-sensitizer of further damage in some cell types possibly as a result of the presence of chromophoric structures generated by oxidation.²⁰⁴UVA radiation may also affect the proteasomal pathway by targeting the ubiquitinylation system, which is critical for the signalling and trafficking of modified proteins for turnover. A number of key ubiquitin-conjugating enzymes are Cys-dependent, which is likely to make these species susceptible to photo-oxidation by both initial oxidants and downstream reactive intermediates (e.g.hydroperoxides). Direct evidence for modulation of this pathway by UVA radiation, however, has yet to be reported.

The activity of the second major intracellular pathway for turnover of modified proteins, the lysosomal pathway is also susceptible to oxidative damage, though this has been less well studied than the proteasomal system. Photo-oxidation of human fibroblast using acridine orange, a sensitizer that accumulates in lysosomes, results in disruption of lysosomal membranes.²⁰⁵ Wan et al. reported that this loss of membrane integrity increased the lysosomal proton permeability and its sensitivity to osmotic damage.^206,207 Disruption of lysosomal membranes has been associated with both membrane protein thiol oxidation and protein cross-linking.²⁰⁶ In addition to these structural changes, it has been shown that peptide and protein hydroperoxides generated by ¹O₂, can inhibit some of the major protease enzymes present in lysosomes; the activity of the Cys-dependent cathepsins B, L and S are particularly affected (both in the isolated form, and when present in cell lysates and in intact macrophage cells^138,139,143). The activity of the Asp-dependent protease cathepsin D was less markedly inhibited.^138,139,143 Such inhibition of the cellular protein catabolic systems may result in an accumulation of damaged materials within cells and enhanced cellular dysfunction.¹⁴³

UVA radiation can also damage extracellular materials both directly and indirectly. It has been reported that extracellular matrix proteins including collagen and elastin can both potentiate the formation of H₂O₂ (possibly as a result of the presence of chromophoric materials on these proteins arising from oxidation and/or glycation), and be targets of damage.²⁰⁸ While the species formed on these proteins are yet to be characterized, subsequent incubation of human fibroblast cells with these oxidized proteins induced intracellular oxidant generation, inhibition of cell proliferation, and damage to DNA.²⁰⁸

Matrix metalloproteinases (MMP) are key extracellular proteases that can be up-regulated by UV irradiation; these enzymes play a key role in both the degradation of extracellular matrix proteins and matrix remodelling. In vitro and ex vivo studies have demonstrated that MMP up-regulation (both at transcriptional and translational levels) can occur via a redox-controlled process of activation/inhibition of phosphatases and kinases in the Ras-JNK phosphorylation signalling pathway.²⁰⁹ Koch et al. reported a redox-dependent negative regulation of MMP-1 activity by GSH and various low molecular mass thiols (Cys, homocysteine, and N-acetyl Cys), in addition to the well-established regulation by the endogenous tissue inhibitors of matrix metalloproteinases (TIMPs);²¹⁰ this inhibition has been postulated to occur as a result of the interaction of the thiols with the active site Zn²⁺ of the MMPs. Photo-oxidation may however also directly inhibit these enzymes as a result of oxidation of the ligands at the Zn²⁺ active site (see above), though this has not been explored in detail.

Photo-oxidation and cell death

Oxidative stress caused by UVA irradiation initiates a series of cellular responses that can result in cell death either viaapoptosis or necrosis. Apoptosis appears to be the predominant mechanism with modest UV doses, with this process detected for both keratinocytes and fibroblastsin vivo (e.g. in sunburnt skin²¹¹).

Extensive studies have examined the mechanism(s) of apoptosis following UVA irradiation. This has been detected as early as 30 min post-exposure in vitro, and within 8 h in skin tissue; long-wave UVA (320–400 nm) has been reported to be more effective than UVB and UVC in inducing early apoptosis.²¹² The pathways believed to be involved in such apoptosis have been reviewed elsewhere.²¹³ Zhuang and Kochevar reported the activation of death receptor Fas, independent of Fas ligand, in human leukaemia HL-60 cells by UVA irradiation;²¹⁴ this ligand-independent activation may occur as a result of ¹O₂-mediated protein oxidation and cross-linking, and thus may be responsible for the observed clustering of Fas receptors.²¹⁴ Such oxidation and receptor cross-linking have also been demonstrated in ligand-activated TNF receptor; binding of CD95 and TRAIL to their respective TNF receptors stimulated the formation of inter-protein disulfide bonds and stabilisation of the active receptor trimer.²¹⁵

Initiation of apoptosis commonly involves the activation of caspases, which are Cys-Asp proteases. Modulation of caspase activity may therefore affect the fate of exposed cells. Caspases in UVA-irradiated cells may be favoured targets for oxidants generated by photo-oxidation as a result of their active site Cys residues, and a number of studies have provided evidence for ¹O₂, H₂O₂, and/or amino acid and peptide hydroperoxides as potent caspase inhibitorsin vitro and in intact cells.^141,216,217

A number of studies have suggested that UVA irradiation has only minimal direct effects on mitochondria with regard to the triggering of cell apoptosis.^194,218 Thus loss of mitochondria membrane potential and release of the mitochondrially-derived apoptosis inducing factor cytochrome c, after UV irradiation, may be mediated by cellular responses rather than direct effects.

Protein photo-oxidation in human disease: deleterious effects

Protein photo-oxidation occurs primarily in tissues routinely exposed to sunlight, with skin and eyes being major target organs. Long-term photo-exposure of skin is known to result in decreased elasticity, an increased leathery texture and changes in pigmentation (reviewed in ref. 152, 197, and 219–221). These macroscopic effects are believed to be consequences of changes at the molecular level, with at least some of these due to photo-damage to structural proteins such as collagen and elastin, and/or repetitive induction of the inflammatory response.^{152,197,219–222} These processes may also be integral in the development of skin cancers upon prolonged exposure to UVA radiation (e.g. ref. 223–225). DNA damage is clearly a major potential driving force, but epigenetic factors and UV-induced damage to critical proteins (e.g. inactivation of DNA repair enzymes, altered cell signalling, inhibition of caspases and hence inhibition of apoptosis in damaged cells) may also play a major role (reviewed in ref. 50).

Photosensitization reactions have been postulated to contribute to the development of lens cataracts and macular degeneration in the eye (reviewed in ref. 4 and 226–229). Many of the UV filters in lenses, which appear to act by absorbing incident radiation with subsequent physical quenching of the excited states without significant formation of reactive species, are derivatives of Trp (e.g.kynurenine, 3-hydroxykynurenine, 3-hydroxykynurenine-O-β-D-glucoside and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-β-D-glucoside^7,8). The first step in the production of these filter compounds is oxidation of free Trp mediated by the enzyme indoleamine 2,3-dioxygenase.²³⁰ Despite the efficiency of physical quenching processes, some chemical oxidation of the filter compounds occurs through continued exposure to UV/visible light, and this can result in the formation of oxidation products, including deaminated unsaturated aldehydes. These can become covalently attached to lens proteins, such as crystallins, viaMichael addition reactions and other processes (e.g. ref. 8, 45, and 231–233). The modified filters, both free and protein-bound, can act as photosensitizers of further protein damage, with formation of ¹O₂, peroxides and radicals implicated in these mechanisms.^{48,64,234–236}

Protein photo-oxidation in human disease: beneficial effects

In addition to being implicated as a causative feature in numerous pathologies, photo-oxidation of proteins has been utilized beneficially in a variety of medical treatments. The technique of photodynamic therapy (PDT) has been available for several decades, with much research and development continuing into this technique (reviewed in ref. 10–15). The basic premise of PDT is to administer a photosensitizing drug (or a precursor that is metabolized into a photosensitizer), before illuminating with a light source such as a lamp, laser or light emitting diode (LED) in the presence of O₂. The excitation of the drug leads to ¹O₂ and radical formation (as described above) that damages neighbouring biomolecules. This controlled production of oxidants has applications, for example, in treating a wide range of cancers, in bacterial inactivation (e.g. in dentistry), in photoangioplasty and treatment of a variety of skin disorders, including psoriasis and acne.^10–15,237 An exciting related development is that of “photochemical tissue bonding” in which a sensitizer and light can be used to suture together pieces of tissue with little scarring of tissue; recent success for this technique has been demonstrated in the repair of peripheral nerves and other tissue damage.^238–240 This process appears to occur without significant photo-toxicity.²³⁹

Protein photo-oxidation in food, agriculture and commercial materials

The photo-oxidation of proteins occurs in a wide variety of non-medical or health-related scenarios and can have beneficial or adverse effects. In all cases gaining further insight into the photo-induced mechanisms involved, is essential to promote or prevent these reactions as required, and some of these research areas are highlighted briefly below.

Over recent years food scientists have taken increasing interest in the effect of photo-induced protein oxidation on food quality and preservation (reviewed in ref. 241). Dairy products such as milk and cheese have received much attention as riboflavin, which is present at significant levels in many of these products, sensitizes the production of reactive oxygen species in the presence of light. The resulting photo-oxidation that can occur during processing and/or storage can result in the production of “off-flavours” or discolouration and can also inhibit enzymes involved in clotting (e.g. discussed in ref. 9, 120, 242, and 243). The effects of protein oxidation have also been investigated in muscle tissues such as red meat (reviewed in ref. 244), with photo-oxidation leading to changes in tenderness, palatability and colour.

Photo-oxidation of proteins also has implications in the textile industry, with the fluorescence whitening agents (FWA) that are frequently used in textile processing, and in washing powders, acting as photosensitizers. Recent research has shown that photo-oxidation of wool mediated by these compounds results in undesirable yellowing through oxidation of Trp and His residues on keratin and other proteins.^78,245–247

Conclusions

The majority of protein damage initiated by UVA and visible light is mediated by photo-sensitization reactions and the production of ¹O₂ and other reactive oxygen species. ¹O₂ reacts preferentially with the aromatic and sulfur-containing side-chains of Trp, His, Tyr, Met, Cys and cystine residues, with endoperoxides and zwitterionic species the main primary products. However, these species are typically moderately reactive intermediates that undergo further reactions, including via radical mechanisms and ring opening reactions that result in disruption of protein structure. Cross-linking and aggregation of proteins predominates over fragmentation processes for ¹O₂-mediated oxidation. In addition to photosensitized reactions, direct photo-oxidation reactions can occur with some protein residues (e.g. Trp), resulting in the formation of excited states and radical species that can also play a role in photo-induced protein degradation and its biological sequelae.

Knowledge of the mechanisms involved in photo-degradation of proteins is increasingly being utilized to our benefit in the areas of food science. Whilst the chemical modifications and resulting structural ramifications of UV/visible radiation on proteins are increasingly well characterized, much remains to be done in unraveling the biological consequences of these changes.

Acknowledgements

This work was supported by grants from the Australian Research Council under the ARC Centres of Excellence (CE0561607) and Discovery (DP0988311) programs.

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Footnote

† Contribution to the themed issue on the biology of UVA.

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