Bityrosine Formation Research Articles

A mechanistic approach towards the formation of bityrosine in proteins by ionizing radiation – GYG model peptide

One of the methods of protein crosslinking used in the synthesis of protein-based nanoparticles is the formation of bityrosine bridges, which may be achieved by the recombination of phenoxyl-type radicals in irradiated protein solutions. Radiation-induced formation of phenoxyl radicals in tyrosine is promoted in presence of H+. In this work, kinetics of this process and the influence of pH were studied by pulse radiolysis measurements of a model peptide H-Gly-Tyr-Gly-OH (GYG), which may help to solve questions raised for bigger, more complex systems in comparable conditions. The main route of phenoxyl radicals formation consists of the addition reaction of hydroxyl radical to the phenol ring on the tyrosine side-chain and proton catalyzed water molecule elimination. A similar effect was observed in studies of tripeptide solutions containing phosphate buffer. The presented data include time-resolved optical spectroscopy of transient species formed under pulse electron beam irradiation and a comparison of the kinetics of the phenoxyl radicals formation in samples at various pH and in presence of phosphate buffer. The postulated mechanism and obtained values of rate constants of the formation and decay of transient species were additionally checked by simple probabilistic simulations.

Protection by ethyl pyruvate against gamma radiation induced damage in bovine serum albumin

Environmental factors like ionizing radiation induced generation of reactive oxygen species (ROS) cause macromolecular damage under physiological conditions. Proteins are the potential targets of ROS induced oxidative damage because of their abundance and their critical functions in the biological systems. The present study investigates the protective potential of ethyl pyruvate (EP) against ionizing radiation induced oxidative damage of bovine serum albumin (BSA) using spectroscopic, biochemical and SDS-PAGE techniques. Spectroscopic data shows that EP prevents the build up of protein damage markers like bityrosine formation and oxidation of tryptophan. Protein melting studies shows that the melting temperature (Tm) of the irradiated protein does not change significantly in the presence of EP. Biochemical assays indicate that ionizing radiation causes the generation of carbonyls and malondialdehyde and the loss of thiol content in proteins that is prevented by EP. The SDS-PAGE profile of gamma irradiated BSA shows the radioprotective effect of EP. These results indicate the radiation induced oxidative and molecular changes in the protein and that the EP protected the BSA from these modifications. Therefore, these results imply that EP has a good antiradical property and hence it can be proposed as a good radioprotective agent.

The effects of radiation and experimental conditions over papain nanoparticle formation: Towards a new generation synthesis

Papain is a natural enzyme extracted from the fruit of Carica papaya Linnaeus, successfully applied in the pharmaceutical area as a drug carrier and debridement agent for wounds. In recent studies papain nanoparticles were synthesized and crosslinked with the use of ionizing radiation in the search for biopharmaceutical advantages as well as the development of bioactive nanocarriers. This study addresses the effects of buffer molarity and irradiation conditions on papain nanoparticles formation. Nanoparticles were synthesized on ice bath using ethanol (20%, v/v) as a cosolvent and crosslinked by gamma radiation using a 60Co source. Experimental variables included the synthesis in deionized water and in 1, 10, 25 and 50 mM phosphate buffer, under different temperatures of − 20 °C, 0 °C and 20 °C before and throughout the irradiation period, and using radiation dose rates of 0.8, 2, 5 and 10 kGy h−1 to reach the dose of 10 kGy. Proteolytic activity was quantified using Nα-benzoyl-DL-arginine-p-nitroanilide hydrochloride. Nanoparticle size and crosslinking by means of bityrosine were evaluated by dynamic light scattering and fluorescence measurements, respectively. Buffer molarity and radiation dose rate were identified to influence bityrosine formation and proteolytic activity without impacting nanoparticle size. Variations in temperature impacted bityrosine formation exclusively. Optimized conditions for papain nanoparticle synthesis were achieved using 50 mM phosphate buffer at the dose rate of 5 kGy h−1 and temperature of 0 °C throughout the process.

Potential role of ferric hemoglobin in MS pathogenesis: Effects of oxidative stress and extracellular methemoglobin or its degradation products on myelin components

There is a well-documented relationship between cerebral vasculature and multiple sclerosis (MS) lesions: abnormal accumulations of iron have been found in the walls of the dilated veins in cerebral MS plaques. The source of this iron is unknown, but could be related to the recognized phenomenon of capillary and venous hemorrhages leading to blood extravasation. In turn, hemorrhaging leading to hemolysis results in extracellular release of hemoglobin, a reactive molecule that could induce local oxidative stress, inflammation, and tissue damage. Our previous studies with a reduced form of hemoglobin (oxyHb) have demonstrated its ability to cause extensive lipid and protein oxidation in vitro, which would result in membrane destabilization. Here, we investigated in further detail the mechanism by which the more abundant oxidized form of extracellular hemoglobin (metHb), and dissociated hemin, cause direct oxidative damage to myelin components, specifically membrane-mimetic lipid vesicles and myelin basic protein (MBP), a highly-abundant protein in the CNS. Oxidation of lipids was assessed by the formation of conjugated diene/triene and malondialdehyde, and oxidation of MBP was demonstrated by the bityrosine formation and by the change in protein mass. Our results show that metHb causes oxidative damage to MBP and myelin lipids, partly by transferring its hemin moiety to protein and lipid, but mostly as an intact protein possibly via formation of a ferryl radical. These results elucidating the mechanism of extracellular hemoglobin-induced oxidative damage to myelin components support the need for further research into vascular pathology in MS pathogenesis, to gain insight into the role of iron deposits and/or in stimulation of different comorbidities associated with the disease.

Chemical oxidation decreases proteolytic susceptibility of skeletal muscle myofibrillar proteins

The objective of this study was to investigate the effect of chemical oxidation on proteolysis susceptibility of myofibrillar proteins. Myofibrils were prepared from pig M. longissimus dorsi and oxidised by a hydroxyl radical generating system. Protein oxidation level was measured by the carbonyl content, free thiol group content and bityrosine formation. Oxidised or non-oxidised myofibrillar proteins were exposed to papain and proteolysis was estimated by fluorescence using fluorescamine. Oxidation of myofibrillar proteins was dependent upon the oxidising agent concentration. Disulfide bridge and bityrosine formation indicated that oxidation by OH° can induce protein polymerization. Electrophoretic study showed that myosin was the protein most sensitive to oxidation. Results showed a direct and quantitative relationship between protein damages by hydroxyl radical and decreased proteolytic susceptibility. Electrophoretic observations suggest that polymerization and aggregation may explain in part decreased susceptibility of myofibrillar proteins to proteolysis.

Free radical-induced protein modification and inhibition of Ca2+-ATPase of cardiac sarcoplasmic reticulum.

The effect of oxidative stress on the Ca2+-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO4/EDTA (0.2 micromol Fe2+ per mg of protein) at 37 degrees C for 1 h in the presence or absence of antioxidants. FeSO4/EDTA decreased the maximum velocity of Ca2+-ATPase reaction without a change of affinity for Ca2+ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca2+-ATPase and decrease in tryptophan fluorescence, while the loss of -SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca2+-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca2+-ATPase activity.

Cigarette smoke-induced protein oxidation and proteolysis is exclusively caused by its tar phase: prevention by vitamin C

We have reported before that whole phase cigarette smoke (CS) contains stable oxidants that cause oxidative damage and increased proteolysis of proteins [Free Radic. Biol. Med. 27 (1999) 1064]. Here, we demonstrate that these oxidants are exclusively present in the tar phase of the CS and not its gas phase and can almost wholly account for the observed whole phase CS-induced oxidation of human plasma proteins as well as extensive oxidative proteolysis of guinea pig lung and heart microsomal proteins in vitro. The mechanism of the tar phase CS-induced proteolysis of microsomal proteins involves two-steps: (i) initial oxidation of the proteins by oxidants present in the tar extract followed by (ii) rapid proteolytic degradation of the oxidized proteins by proteases present in the microsomes. Like the whole phase CS, the oxidative damage of proteins caused by the tar phase CS, as evidenced by the formation of protein carbonyl and bityrosine as well as loss of tryptophan residues and thiol groups, is also almost completely prevented by ascorbic acid and only partially by glutathione. Other antioxidants, including superoxide dismutase, catalase, vitamin E, β-carotene and mannitol are ineffective. This again leads us to suggest that adequate intake of vitamin C may help smokers to evade the CS-induced degenerative diseases associated with oxidative damage. The revelation of the acute toxicity of the tar phase with respect to CS-induced oxidative damage also urges the necessity of trapping it more effectively by suitable cigarette filters to reduce the health damage caused to smokers.

Novel inter-protein cross-link identified in the GGH-ecotin D137Y dimer.

In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl-glycyl-histidine (GGH) mediates efficient and specific oxidative protein cross-linking. The fusion of GGH to the N terminus of a protein allows for the cross-linking reagent to be delivered in a site-specific fashion, making this system extremely useful for analyzing protein-protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross-linking efficiency. Cross-linking increased four-fold for GGH-ecotin D137Y in comparison to wild-type GGH-ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross-linked GGH-ecotin D137Y dimer. Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross-link between the N-terminal glycine residues and the engineered tyrosine at position 137 has been characterized. The dimer cross-link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products. We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue. This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross-linking yields, and suggests that the cross-linking reagent may be of more general utility than previously thought.

Vitamin C prevents cigarette smoke induced oxidative damage of proteins and increased proteolysis

Aqueous extract of cigarette smoke (CS) contains some stable oxidants, which oxidize human plasma proteins, bovine serum albumin, amino acid homopolymers, and also cause extensive oxidative degradation of microsomal proteins. Similar observations are made when the aqueous extract of cigarette smoke is replaced by whole phase CS solution or whole phase cigarette smoke. CS-induced microsomal protein degradation is a two step process: (i) oxidation of proteins by the oxidants present in the CS and (ii) rapid proteolytic degradation of the oxidized proteins by proteases present in the microsomes. Using aqueous extract of CS equivalent to that produced from one-twentieth of a cigarette, the observed initial and postcigarette smoke treated values of different parameters of oxidative damage per milligram of microsomal proteins are respectively: 0.24 and 1.74 nmoles for carbonyl formation, 125.4 and 62.8 fluorescence units for tryptophan loss, 10.2 and 33.4 fluorescence units for bityrosine formation, and 58.3 and 12.2 nmoles for loss of protein thiols. When compared with sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of untreated microsomal proteins, the extent of microsomal protein degradation after treatment with whole phase CS solution or aqueous extract of CS is above 90%. Ascorbate (100 μM) almost completely prevents cigarette smoke–induced protein oxidation and thereby protects the microsomes from subsequent proteolytic degradation. Glutathione is partially effective, but other antioxidants including superoxide dismutase, catalase, vitamin E, probucol, β-carotene, mannitol, thiourea, and histidine are ineffective. The gas phase cigarette smoke contains unstable reactive oxygen species such as superoxide (O 2 −•) and hydrogen peroxide (H 2O 2) that can cause substantial oxidation of pure protein like albumin but is unable to produce significant oxidative damage of microsomal proteins. Gas phase cigarette smoke–induced albumin oxidation is not only inhibited by ascorbate and glutathione but also by superoxide dismutase, catalase and mannitol. The stable oxidants in the cigarette smoke are not present in the tobacco and are apparently produced by the interaction of O 2 −•/H 2O 2/OH of the gas phase with some components of the tar phase during/following the burning of tobacco.

Irradiation Dose and Calcium Effect on the Mechanical Properties of Cross-Linked Caseinate Films

The effect of irradiation dose on the formation of cross-links in calcium caseinate solutions was investigated. The formation of bityrosine was found to increase with the irradiation dose for the basic formulation containing calcium caseinate, carboxymethylcellulose, and glycerol. Addition of CaCl2 to the basic formulation increased the fluorescence signal of bityrosine for all irradiation doses suggesting that CaCl2 can have an indirect effect on the formation of bityrosine by shortening molecular distances between polypeptides, thus making easier the formation of bityrosine. Furthermore, gels were formed for solutions containing CaCl2 at irradiation doses ≥16 kGy. The formation of gels was found to be accountable for the increased mechanical strength in the corresponding films. A maximum gel strength was obtained at an irradiation dose of 64 kGy for all formulations, suggesting a maximum cross-linking density. Such a feature was also noted when poly(ethylene glycol), mannitol, and sorbitol were added to...

Formation of Sterilized Edible Films Based on Caseinates: Effects of Calcium and Plasticizers.

gamma-Irradiation was used to produce free-standing sterilized edible films based on caseinate. The effects of calcium ions (Ca(2+)) and two plasticizers, namely propylene glycol (PG) and triethylene glycol (TEG), were investigated, as was the effect of the irradiation on both the gel formation and mechanical properties of the resulting films. gamma-Irradiation provoked formation of bityrosine, i.e., cross-links, accounting for the increase of the puncture strength of films. The presence of PG or TEG enhanced the formation of cross-links, leading to an improved mechanical strength of films. TEG was found to interact more favorably with the caseinate than PG, being responsible for the improved film extensibility. Addition of Ca(2+) caused the formation of gels. The breaking strength of gels was directly related to the concentration of Ca(2+), while the puncture strength of films was found to be almost independent of the calcium concentration. Moreover, a high irradiation dose seemed to affect the protein structure, accounting for the decrease of the breaking strength of gels and for the depreciation of the mechanical behavior of films.

The permselectivity of glomerular basement membrane can be compromised by glycation or by exposure to low levels of hypochlorite

Earlier studies indicated that chemically crosslinking glomerular basement membrane (GBM) rendered it more permeable to water and to macromolecules. Here possible mechanisms for the introduction of crosslinks into GBM under pathological conditions were explored. Glycation with glucose and with fructose over periods of 2 wk (fructose) and 6 weeks (glucose) rendered the GBM more permeable to water and myoglobin as judged from in vitro ultrafiltration behaviour. The membranes were also made more permeable to serum following glycation. The permeation changes were shown to be dependent on glycoxidative reactions judging by their inhibition by EDTA and DTPA. Aminoguanidine also prevented glycation from altering the permeability of GBM. Fluorescence studies indicated the formation of bityrosine in glycated GBM. Studies with oxidants showed that while hydrogen peroxide superoxide and peroxynitrite had little effect on GBM, hypochlorite anion was capable of increasing GBM permeability to water, myoglobin, albumin and serum. Changes in permeation were induced by very low quantities of hypochlorite, well within the range of the amounts of hypochlorite formed by activated neutrophils. Thus glycoxidation, or oxidation by hypochlorite, are chemical mechanisms by which GBM permeability can be increased.

Identification of oxidation-sensitive peptides within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+-ATPase.

We have examined the oxidative sensitivity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) membranes, exposing isolated SR membranes to the thermolabile water soluble free radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Incubation with up to 702 microM AAPH-derived radicals results in a concentration- and time-dependent inhibition of calcium-dependent ATPase activity correlating with the loss of monomeric Ca2+-ATPase polypeptides, and the concomitant appearance of higher molecular weight species. However, no oxidant-induced protein fragmentation is detected. The observed formation of oxidant-induced bityrosine accounts for the intermolecular Ca2+-ATPase cross-links, as well as intramolecular cross-links. The oxidation of sulfhydryl groups to disulfides as another possible source of intermolecular cross-links has been ruled out after examination of SDS -PAGE performed under both reducing and non-reducing conditions. Exposure of the SR membranes to AAPH-derived radical species results in a small degree of lipid peroxidation that is not correlated with enzyme inactivation, suggesting that modification of membrane-spanning peptides is not related to enzyme inactivation. Six cytoplasmic peptides have been identified that are modified by exposure to AAPH or, alternatively, to hydrogen peroxide, suggesting that these regions of the Ca2+-ATPase are generally sensitive to oxidants. These oxidized peptides were identified after separation by reversed-phase HPLC followed by N-terminal sequencing and amino acid analysis as corresponding to the following sequences of the Ca2+-ATPase: (i) Glu121 to Lys128, (ii) His190 to Lys218, (iii) Asn330 to Lys352, (iv) Gly432 to Lys436, (v) Glu551 to Arg604, and (vi) Glu657 to Arg671. The Glu551 to Arg604 peptide, located within the nucleotide binding domain, was found to participate in the formation of intermolecular bityrosine cross-links with the identical Glu551 to Arg604 peptide from a neighboring Ca2+-ATPase polypeptide chain.

Reduction of ferrylmyoglobin by beta-lactoglobulin.

Reduction of iron (IV) in ferrylmyoglobin in the presence of beta-lactoglobulin in aqueous solution is the result of two parallel reactions: (i) a so-called autoreduction, and (ii) reduction by beta-lactoglobulin in a second-order-reaction resulting in bityrosine formation in beta- lactoglobulin. In the pH-region investigated (5.4-7.4), the rate of reduction increased for both reactions with decreasing pH. The second order-reaction had for non-denatured beta-lactoglobulin the activation parameters: delta H* = 45 kJ.mol-1 and delta S not equal to = -93 J.mol-1.K-1 at pH = 7.0 and ionic strength 0.16 (NaCl). Reduction of ferrylmyoglobin by beta-lactoglobulin denatured by heat (86 degrees C for 3 min) or by hydrostatic pressure (300 MPa for 15 min) resulted in formation of higher molecular weight species as detected by size-exclusion chromatography and by SDS-PAGE. No molecular weight changes were observed for reduction of ferrylmyoglobin by native beta-lactoglobulin. Detection of bityrosine in the native beta-lactoglobulin fraction after oxidation with ferrylmyoglobin indicated intra-molecular bityrosine formation. In heat-denatured beta-lactoglobulin bityrosine formation could be of intra-molecular and/or of inter-molecular origin, the latter being confirmed by size-exclusion chromatography.

NADPH-initiated cytochrome P450-mediated free metal ion-independent oxidative damage of microsomal proteins. Exclusive prevention by ascorbic acid.

In this paper we demonstrate that NADPH-initiated oxidative damage of microsomal proteins occurs in the absence of free metal ions and that this protein oxidation is mediated by cytochrome P450 (cyt P450). Oxidized proteins are rapidly degraded by proteases. Ascorbate (AH2) specifically inhibits free metal ion-independent cyt P450-mediated protein oxidation and thereby prevents subsequent proteolytic degradation. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, and glutathione are ineffective. This is in variance with free metal ion-catalyzed protein oxidation, which is accelerated by AH2 and inhibited by catalase. Oxidative damage of proteins has been assessed by the production of carbonyl groups, bityrosine formation, and tryptophan loss. The mechanism of protein oxidation has been studied using a reconstituted system comprised of purified NADPH-cyt P450 reductase, cyt P450, and isolated microsomal proteins as well as model polypeptides, e.g. poly-L-proline and poly-L-lysine. Cyt P450 Fe3+ is reduced by NADPH-cyt P450 reductase to cyt P450 Fe2+, which consumes oxygen in a stoichiometric proportion to produce cyt P450 Fe2+ O2, the resonance form of which is a perferryl moiety, cyt P450 Fe3+.O2-.. It is proposed that cyt P450 Fe3+.O2-. abstracts hydrogen from amino acid side chains leading to the production of carbonyl derivatives. Tentatively, AH2 prevents protein oxidation by interacting with cyt P450 Fe3+.O2-..

Hypochlorite oxidation causes cross-lingking of Lp(a)

When purified low density lipoprotein (LDL) or lipoprotein(a) (Lp[a]) was oxidized in vitro using concentrations of hypochlorite (50–500 μM) which might be achieved by activated neutrophils in vivo, high molecular weight species were observed on SDS polyacrylamide gels. The reaction was concentration-, temperature- and time-dependent. The high molecular weight apoprotein complexes were resistant to heating in SDS and DTT, suggesting covalent, but non-disulfide bond, cross-linking. Negligible amounts of lower molecular weight degradation produts were formed. Bityrosine formation, measured by fluorescence and HPLC analysis, was found to increase with the amount of hypochlorite added. However, the molar concentration of bityrosine could not account for cross-linking, even if it was assumed that every bityrosine was intermolecular. Hypochlorite-oxidized Lp(a) and LDL were both effective as ligands for loading mouse peritoneal macrophages in vitro. We conclude that hypochlorite produced in inflammatory reactions might be important in the generation of atherogenic forms of lipoproteins.

Oxidative damage to lysozyme by the hydroxyl radical: comparative effects of scavengers

The hydroxyl radical (OH .) is a highly-damaging reactive oxygen species, given its high reactivity and the consequent generation of secondary free radicals. This study was aimed at determining the qualitative and quantitative aspects of OH . scavenging by pentoxifylline (Ptx, a methylxanthine), uric acid and thymine on the OH ./mol lysozyme; moreover, SDS-PAGE showed a loss of native lysozyme (14.4 kDa), the presence of dimer and trimer aggregates and characteristic fragmentation. Tryptophan fluorescence was lost before aggregation became detectable in terms of bityrosine formation. Increasing concentrations of OH . scavengers gave increasing protection of lysozyme activity. Although all three compounds scavenge OH . with high rate constants, their effects were different: uric acid and Ptx prevented aggregation and preserved enzyme activity, whereas thymine preserved activity but did not prevent aggregation. These differences appear to be related to the formation of reducing secondary radicals, underlining the importance of this mechanism in the effects of scavengers.

The effect of diabetes and dietary ascorbate supplementation on the oxidative modification of rat lens beta L crystallin.

The level of characteristic markers of protein oxidative modification (tryptophan oxidation and sulfhydryl group loss as well as carbonyl and bityrosine formation) and glycation (AGEP formation) have been measured in beta L crystallin purified from the lenses of control, diabetic, and ascorbate-supplemented diabetic animals. These markers were also determined following the application of an in vitro graded oxidative insult. Prior to the application of stress, diabetic lens crystallins, in comparison with control, exhibited a higher content of bityrosine and AGEPs, a lower level of nonoxidized tryptophan, and a loss of sulfhydryl groups. After exposure to the oxidative insult there was a stress-proportional increase of the parameters in all beta L crystallins, irrespective of their source. The effects were most pronounced in the diabetic, in which the already-elevated indicators of oxidative damage were further increased. Dietary supplementation of the diabetic group with ascorbate had a marked effect in preventing beta L crystallin modification in vivo, alleviating the loss of sulfhydryl groups and the oxidation of tryptophan, partially preventing the formation of AGEP and carbonyl groups, but not affecting the formation of bityrosine. Supplementation also inhibited the increase in susceptibility of diabetic beta L crystallin to in vitro oxidative stress, preventing sulfhydryl group loss as well as carbonyl and AGEP group formation. The results are discussed in relation to the proposal that diabetes renders lens crystallins more susceptible to oxidative stress and that this may be a causative factor in cataractogenesis. The possible role of ascorbate in the inhibition, or attenuation, of cataractogenesis is examined.

The superoxide radical reacts with tyrosine-derived phenoxyl radicals by addition rather than by electron transfer

Radiolytically generated azide radicals have been used for the formation of tyrosyl radical, TyrO˙ from tyrosine. The TyrO˙ radicals combine (2k= 4.5 × 108 dm3 mol–1 S–1, determined by pulse radiolysis) yielding bityrosine in a > 90% yield. Bityrosine formation is not suppressed in the presence of oxygen [k(TyrO˙+ O2) < 1 × 103 dm3 mol–1 S–1].When TyrO˙ and O2˙– radicals are generated side by side in a 1:1.2 ratio, bityrosine formation is strongly suppressed and (2S,3aR,7aS)- and (2S,3aS,7aR)-3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acids 10 become the major final products. Their hydroperoxidic precursor is only short-lived (t½= 4.2 h at room temperature and pH 8). Upon its decay H2O2 is released. Product 10 is believed to be formed by the addition of O2˙– to the ortho- and para-position of the phenoxyl radical, followed by protonation, ring closure and hydrolysis.Based on material balance considerations an electron transfer from O2˙– to TyrO˙, although thermodynamically feasible, must play a minor role (⩽10%). The rate constant k(O2˙–+ TyrO˙) has been determined by pulse radiolysis to be 1.5 × 109 dm3 mol–1 S–1.

Oxidative damage to bovine serum albumin induced by hydroxyl radical generating systems of xanthine oxidase + EDTA−Fe 3+ and ascorbate + EDTA−Fe 3+

Oxidative damage to bovine serum albumin (BSA) was induced by hydroxyl radical (HO •) generating systems of xanthine oxidase (XO) + EDTA−Fe 3+ and ascorbate + EDTA−Fe 3+. Formation of bityrosine and loss of tryptophan were observed in the ascorbate + EDTA−Fe 3+ system and carbonyl formation was induced by both systems. Mannitol and ethanol very strongly inhibited the carbonyl and/or bityrosine formation, indicating that the oxidative damage to BSA was due to HO •. The sulfhydryl (SH) groups of BSA were very sensitive to the XO + EDTA−Fe 3+ but not to the ascorbate + EDTA−Fe 3+ system. Catalase but not hydroxyl radical scavengers or superoxide dismutase strongly inhibited the loss of SH groups, indicating that H 2O 2 is involved in their oxidation. Fragmentation of BSA was observed during exposure to the XO + EDTA−Fe 3+ and ascorbate + EDTA−Fe 3+ systems and the products presented a broad band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Little formation of amine groups was observed in these systems, indicating that little peptide bond cleavage occurred. BSA exposed to the ascorbate + EDTA−Fe 3+ system was more readily degraded by trypsin than that exposed to the XO + EDTA−Fe 3+ system. Elastase degraded BSA exposed to the ascorbate + EDTA−Fe 3+ system but not to the XO + EDTA−Fe 3+ system.