Protein photo-oxidation

Riboflavin absorbs light maximally at about 450nm and in doing so can be excited to a triplet state. This excited form of riboflavin can interact with triplet 02 to form a superoxide anion OJ (or H202 at low pH). Excited riboflavin can also oxidize ascorbate, a number of amino acids and proteins and orotic acid. Riboflavin-catalysed photo-oxidation results in the production of a number of compounds, most notably methional (11.1) which is the principal compound responsible for the off-flavour in milk exposed to light. 

Oxidation. The side chains of cysteine (Cys), histidine (His), methionine (Met), tryptophan (Trp), and tyrosine (Tyr) residues are susceptible to oxidation. Oxidation can result in loss of protein activity. Met is the most reactive residue, oxidizing even with atmospheric oxygen to form Met-sulfoxide, which is frequently observed in proteins (Fig. 135). Additional sources of oxidation include oxidizing agents (peroxides in excipients), metal-catalyzed oxidation and photo-oxidation. Oxidation can be detected... 

Scoffone, E., Jori, G., Galiazzo, G. Selective photo-oxidation of amino acids in protein, in Chemical Reactivity and Biological Role of Functional Groups in Enzymes (. Smellie, R. M. S.) Academic Press, London and New York 1970... 

Group counting may be used of course to study any chemical modification of proteins. A recent example is provided by the use of difference counting in a study of the effect of photo-oxidation on proteins (Vodrazka et al., 1961). 

Other reagents which have been used for the modification of histidyl residues in proteins include l-fluoro-2,4-dinitrobenzene, diazonium-IH-tetrazole (see Andres and Atassi 1973), iodine (Covelli and Wolff 1966 Wolff and Covelli 1969), N-bromosuccinimide, ethoxyformic anhydride (Melchior and Fahrney 1970), and bromoacetone (Beeley and Neurath 1968). These reagents appear to offer little advantage over alkylation with a-haloacids, or dye-sensitized photo-oxidation. Indeed, some are far less specific. 

Reagents reactive solely toward the methionyl side-chain in native proteins have not been described to date. Selective conversion of the thioether to the sulfoxide derivative by photosensitized oxidation has been reported in a protein devoid of free thiol groups. The selective photosensitized oxidation employed methylene blue or hema-toporphyrin as sensitizers, and aqueous acetic acid (30-90% v/v), or acidic buffers at pH 2-6.5, as solvents (Scoffone et al. 1970). Anaerobic photo-oxidation in 4 M aqueous acetone has been reported to lead to the same result (Gennari and Jori 1970). Methionine can be regenerated from the sulfoxide by incubation with thiols (5 % aqueous j9-mercapto-ethanol at pH 8.0, for 24 hr under Nj Jori et al. 1968). 

Vanadate-induced photo-oxidation of serine and subsequent cleavage of the protein were also observed for ribulose-l,5-diphoshate carboxylase/oxygenase from spinach leaves. Activity could be restored by treatment with sodium boronate, which reduces formylglycine back to serine.Similarly, vanadate-induced UV oxidation of threonine (Thr353, at the phosphorylation site) in the sarcoplasmatic reticulum (SR) Ca +-ATPase can be restored by reduction with Na[BH4]. For monovanadate to become catalyti-cally active it is essential that Mg + and ADP bind to the phosphorylation site of the enzyme in its Ca +-receptive conformation, suggesting the formation of an active MgADPV as proposed for the myosin fragmentation. In contrast, decavanadate catalyses the photo-oxidation of a serine (Seri 86) residue in the SR ATPase in the absence of ADP.P bl... 

Roberts J.E., Finley, E.L., Patat, S.A., and Schey, K.L. (2001) Photo-oxidation of lens proteins with xanthurenic acid a putative chromophore for cataractogenesis, Photochem. Photobiol., 74, 740-744. 

Siefermann-Harms D (1990c) Protective function of the apoprotein of the light-harvesting chlorophyll-a/b-protein complex in pigment photo-oxidation. J Photochem Photobiol B Biol 4 283-295... 

The development of a blue/green fluorescence in aged human lenses [129-130] is well known. This rise in fluorescence was reproduced in vitro by incubating HK with crystallins under aerobic conditions [116]. Subsequent protein digestion and aminoacid analysis confirmed the covalent modification. The extent and pattern of protein modification of these experiments are quite similar to those obtained with cataractous lenses. A photo-oxidative mechanism operating in age-related cataract development [123, 131-134] is therefore confirmed [116]. Further ex-... 

The photosensitized oxidation of certain amino-acids in proteins plays an important part in photodynamic action. A study has been made of the oxidation of amino-acids by microwave-generated 102.173 The photo-oxidation of tryptophan-containing peptides174 and of histidine (sensitized by flavin)175 have been noted. It is reported that the crystal violet-sensitized photo-oxidation of cysteine to cysteic acid does not involve 102 production.178... 

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