Light generation with Fenton's reagent its relationship to granulocyte chemiluminescence

A simple chemical system consisting of FeSO₄ and H₂O₂ (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H₂O₂ was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH^.) and singlet oxygen (¹Δ_gO₂) quenchers suppressed chemiluminescence of the FeSO₄ + tryptophan + H₂O₂ system; and, deuterium oxide (²H₂O) enhanced chemiluminescence of both FeSO₄ reactions. These observations suggest that a radical chain reaction involving both OH^. and ¹Δ_gO₂ is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H₂O₂. Since iron and H₂O₂ are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.     

Chemically shifted singlet oxygen spectrum.

The estimated light emission spectrum was determined for a singlet oxygen (1O2)-producing system, NaOCl + H2O2, alone and in the presence of tryptophan and bovine serum albumin. Tryptophan and bovine serum albumin caused a decrease in the red emission of 1O2 and an increase in the amount of shorter wavelength light. This effect was due to chemiluminescence rather than fluorescence. Arachidonic acid caused a similar spectral shift, while guanosine demonstrated a late chemiluminescent reaction of predominantly short wavelength light in the presence of 1O2.     

Cytochrome c-catalyzed oxidation of organic molecules by hydrogen peroxide.

Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.     

Luminol peroxidation catalyzed by human isoferritins

The role of ferritin in catalyzing the oxidation of luminol with the production of chemiluminescence was investigated. The effect of pH was compared to its effect on K3Fe(CN)6-catalyzed oxidation and different pH optima were recorded for the two catalysts. The ferrous iron chelator, bipyridyl, enhanced the production of chemiluminescence catalyzed by FeSO4 and ferritin but had little effect on the K3Fe(CN)6-catalyzed reaction. Desferal reduced the level of chemiluminescence in the presence of FeSO4 and ferritin but was a much more effective inhibitor of chemiluminescence catalyzed by K3Fe(CN)6. The hydroxyl radical scavenger, mannitol, had little effect upon light production whereas superoxide dismutase inhibited light production. The addition of antihuman spleen ferritin completely inhibited activity. The catalytic activity of both H and L rich ferritins was affected by iron content. Activity increased until the Fe/protein ratio reached 0.04 micrograms Fe/micrograms protein and then decreased with increasing iron content. Thus activity is controlled by the iron content of the molecule and influenced by its subunit composition as is the uptake of iron into ferritin. These findings suggest that ferroxidation by ferritin is associated with the ability to generate radicals of the nitrogenous base luminol with the production of chemiluminescence. Although activity is greatest at alkaline pH there is significant activity at pH 7.4. Ferritin therefore may be able to generate free radical reactions in vivo with the acidic isoferritin being most active.     

Anti-oxidant activity of spermine and spermidine re-evaluated with oxidizing systems involving iron and copper ions

This study was designed to investigate the direction of redox reactions of spermine and spermidine in the presence of iron and copper. The redox activity of spermine and spermidine was assessed using a variety of methods, including their ability to: (1) reduce Fe(3+) to Fe(2+) ions; (2) protect deoxyribose from oxidation by Fe(2+)-ethylene diaminetetraacetic acid, Fe(3+)-ethylene diaminetetraacetic acid systems with and without H(2)O(2); (3) protect DNA from damage caused by Cu(2+)-H(2)O(2), and Fe(2+)-H(2)O(2) with and without ascorbic acid; (4) inhibit H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence; (5) scavenge diphenyl-picryl-hydrazyl radical. Spermine and spermidine at concentration 1mM reduced 1.8+/-0.3 and 2.5+/-0.1 nmol of Fe(3+) ions during 20 min incubation. Both polyamines enhanced deoxyribose oxidation. The highest enhancement of 7.6-fold in deoxyribose degradation was found for combination of spermine with Fe(3+)-ethylene diaminetetraacetic acid. An 10mM spermine and spermidine decreased CuSO(4)-H(2)O(2)-ascorbic acid- and FeSO(4)-H(2)O(2)-ascorbic-induced DNA damage by 73+/-6, 69+/-4% and 90+/-5, 53+/-4%, respectively. They did not protect DNA from CuSO(4)-H(2)O(2) and FeSO(4)-H(2)O(2). Spermine apparently increased the CuSO(4)-H(2)O(2)-dependent injury to DNA. Polyamines attenuated H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence. Total light emission from specimens containing 10mM spermine or spermidine was attenuated by 85.3+/-1.5 and 87+/-3.6%. During 20 min incubation 1mM spermine or spermidine decomposed 8.1+/-1.4 and 9.2+/-1.8% of diphenyl-picryl-hydrazyl radical. These results demonstrate that polyamines of well known anti-oxidant properties may act as pro-oxidants and enhance oxidative damage to DNA components in the presence of free iron ions and H(2)O(2).     

Protein damage and degradation by oxygen radicals. II. Modification of amino acids

Exposure of proteins to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical (.OH + O2-) causes gross structural modification. Such modified proteins can undergo spontaneous fragmentation or can exhibit substantial increases in proteolytic susceptibility. In the present study, with the representative protein bovine serum albumin (BSA), we report that alterations to primary structure underlie such gross structural modifications. All amino acids in BSA were susceptible to modification by both .OH and .OH + O2- +O2), although tryptophan, tyrosine, histidine, and cysteine were particularly sensitive. At a radical/BSA molar ratio (nmol of radicals/nmol of BSA) of 10, we observed an average 9-10% destruction of amino acids; whereas at a ratio of 100, the average loss was 45%. Decreasing tryptophan fluorescence provided a useful index of amino acid loss and exhibited a clear dose dependence with .OH or with .OH + O2- (+O2). Linear production of the biphenol bityrosine was observed with .OH treatment. In contrast, .OH + O2- (+O2) induced only a limited bityrosine production rate which reached an early plateau. Studies with various chemical scavengers (t-butyl alcohol, isopropyl alcohol, mannitol, urate) and gasses (N2O, N2, O2, air) revealed that .OH is the primary radical responsible for all amino acid modifications, but that O2- and O2 can further transform the products of .OH reactions. Thus, O2-/O2 can potentiate .OH-dependent destruction of many amino acids (e.g. tryptophan) while inhibiting production of bityrosine by reacting with tyrosyl (phenoxyl) radicals. No amino acid loss or bityrosine production occurred with exposure to O2- (+O2) alone. Amino acid modifications caused both by .OH alone and by .OH + O2- (+O2) progressively affected the overall electrical charge of BSA. In a pH range of 3.7-6.2, some 16 new isoelectric focusing bands were induced by .OH, and some eight new bands were induced by .OH + O2- (+O2). The alterations to primary structure observed provide the key to an understanding of the link between oxidative modification and increased proteolytic susceptibility.     

Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis.

Defined radical species generated radiolytically were allowed to attack proteins in solution. The hydroxyl radical (OH.) in the presence of O2 degraded bovine serum albumin (BSA) to specific fragments detectable by SDS/polyacrylamide-gel electrophoresis; fragmentation was not obvious when the products were analysed by h.p.l.c. In the absence of O2 the OH. cross-linked the protein with bonds stable to SDS and reducing conditions. The superoxide (O2-.) and hydroperoxyl (HO2.) radicals were virtually inactive in these respects, as were several other peroxyl radicals. Fragmentation and cross-linking could also be observed when a mixture of biosynthetically labelled cellular proteins was used as substrate. Carbonyl and amino groups were generated during the reaction of OH. with BSA in the presence of O2. Changes in fluorescence during OH. attack in the absence of O2 revealed both loss of tryptophan and changes in conformation during OH. attack in the presence of O2. Increased susceptibility to enzymic proteolysis was observed when BSA was attacked by most radical systems, with the sole exception of O2-.. The transition-metal cations Cu2+ and Fe3+, in the presence of H2O2, could also fragment BSA. The reactions were inhibited by EDTA, or by desferal and diethylenetriaminepenta-acetic acid ('DETAPAC') respectively. The increased susceptibility to enzymic hydrolysis of radical-damaged proteins may have biological significance.     

Oxidative damage to fibronectin. II. The effect of H2O2 and the hydroxyl radical.

The effect of H2O2 and the hydroxyl radical (.OH) on fibronectin was investigated. .OH was generated in three ways: (i) by radiolysis with 60Co under N2O, or by the Fenton system using either (ii) equimolar Fe(2+)-EDTA and H2O2 or (iii) H2O2 and catalytic amounts of Fe(2+)-EDTA recycled with ascorbate. Each system had a different effect. H2O2 alone caused no changes, even at an 800-fold molar excess. Radiolytic .OH caused a rapid loss of tryptophan fluorescence, an increase in bityrosine fluorescence, and extensive crosslinking. The Fenton system using Fe-EDTA, H2O2, and ascorbate caused a loss in tryptophan fluorescence, a smaller increase in bityrosine than was seen with radiolytic .OH, and a threefold increase in carbonyl groups. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis fragmentation of fibronectin was seen. In contrast, when .OH was generated with equimolar Fe-EDTA and H2O2, the only change was a small increase in bityrosine fluorescence at the highest dose of oxidant. None of the systems used affected cysteine. All the changes except the loss of tryptophan by radiolytic .OH were completely inhibited with mannitol. The differences seen with radiolytic .OH and the Fe-EDTA, H2O2, ascorbate system were not solely due to O2 in the latter system since similar results were obtained under N2. The differences between radiolytic .OH and the Fenton systems could be partly due to the components of the latter systems reacting with .OH and thus competing with fibronectin. Our results demonstrate that the extent and type of fibronectin damage by .OH is dependent on the mode of radical generation.     

Problems concerning the biochemical action of superoxide dismutase (erythrocuprein).

The decay of the tetraperoxochromate- (V) complex (CrO83theta) was examined to study the substrate specificity of erythrocuprein (super-oxide dismutase). The decay of CrO83theta proved rather complex in aqueous solutions. Apart from the two known oxygen species O2theta and singlet oxygen (1 deltagO2), H2O2 and probably OH radicals were formed. No unequivocal evidence for the appearance of superoxide was obtained. The possible electron transfer from Cr5 to Fe3 (cytochrome c) was also discussed. In Tris buffer, pH 7.8, there were absolutely no signs of superoxide or OH radical formation. In fact, pulse radiolysis measurements employing a homogeneous OH source demonstrated that the Tris and OH radicals react with each other. One mol of H2O2 was generated from 1 mol of CrO83theta in Tris buffer. By contrast, only 0.5 mol H2O2 could be determined when the CrO83theta decay was carried out in 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, pH 7.8. The phenomenon of reducing oxidized cytochrome c could not fully be assigned to a superoxide-mediated reduction, since erythrocuprein was unable to inhibit this cytochrome c reduction efficiently. The energetic oxygen species (1deltag O2, OH etc.) appearing during the CrO83theta decay gave rise to a clearly detectable chemiluminescence. In this system, erythrocuprein was very active regardless of which buffer was used. Even in the absence of a chemiluminescent mediating agent, which might have interferred with the enzyme, erythrocuprein proved capable of inhibiting the CrO83theta-induced chemiluminescence in a rather specific way. No such specificity was seen in the presence of low molecular weight Cu-chelates including Cu(Tyr)2, Cu(Lys)2 and Cu(His)2. The ability to suppress chemiluminescence was approximately 3 orders of magnitude less pronounced than that of the native enzyme. It is presumed that erythrocuprein reacts with oxygen species other than the superoxide radical.     

Chelated iron-catalyzed OH. formation from paraquat radicals and H2O2: mechanism of formate oxidation

Traces of iron, when complexed with either EDTA or diethylenetriaminepentaacetic acid (DTPA), catalyze an OH.-producing reaction between H2O2 and paraquat radical (PQ+.): H2O2 + PQ+.----PQ++ + OH. + OH-.[1]. Kinetic studies show that oxidation of formate induced by this reaction occurs by a Fenton-type mechanism, analagous to that assumed in the metal-catalyzed Haber-Weiss reaction, in which the rate determining step is H2O2 + Fe2+ (chelator)----Fe3+(chelator) + OH. + OH-,[7]; with k7 = 7 X 10(3) M-1 s-1 for EDTA and 8 X 10(2) M-1 s-1 for DTPA at pH 7.4. PQ+. rapidly reduces both Fe3+ (EDTA) and Fe3+ (DTPA), and hence allows both agents to catalyze [1] with comparable efficiency, in contrast to the much lower efficiency reported for the latter as a catalyst for the Haber-Weiss reaction. The catalytic properties of these chelating agents is attributed to their lowering of E0 (Fe3+/Fe2+) by 0.65 V, thus making [7] thermodynamically possible at pH 7. Approximately 2.5% of the OH. produced is consumed by internal or "cage" reactions, which decompose the chelator and produce CO2; however, the majority (97%) diffuses into the bulk solution and participates in competitive reactions with OH. scavengers.     

Interaction of tert-butyl hydroperoxide with hypochlorite induces peroxyl radicals. A chemiluminescence study

The interaction of hypochlorite (HOCl/OCl-) with tert-butyl hydroperoxide ((CH3)3COOH) was investigated by chemiluminescence. It was shown that the addition of HOCl/OCl- to (CH3)3COOH induces a fast chemiluminescent flash. The intensity of this flash increases with the increase in both HOCl/OCl- and (CH3)3COOH concentration. The chemiluminescence is quenched in a concentration-dependent manner in the presence of free radical spin traps N-tert-butyl nitrone and alpha-(4-pyridyl-1-oxyl)-N-tert-butyl nitrone. This fact proves that free radicals take part in the interaction of HOCl/OCl- and (CH3)3COOH. Hypochlorite yielded a very similar chemiluminescence spectrum in its reaction with (CH3)3COOH as Ce4+. It differed considerably from the spectrum in the system H2O2 and HOCl/OCl-. It is well known that the interaction of Ce4+ and (CH3)3COOH produces peroxyl radicals. These results confirm the hyothesis that the interaction of HOCl/OCl- and (CH3)3COOH is mediated by peroxyl radicals. Thus, organic hydroperoxides always present in unsaturated lipids can induce lipid peroxidation processes in the reaction with HOCl/OCl-.     

Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (相似文献   

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway.

The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diode-array spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO-. Mb/H2O2 produced a short-living light emission that was suppressed by the addition of BH4. Simultaneous addition of POBN, BH4 and Mb/H2O2 produced the same six-line EPR spectrum, with a signal intensity depending on BH4 concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO- (302 nm) as well as substantial modifications of the initial BH4 spectrum with both oxidant systems. These data demonstrated that BH4 oxidation, either by ONOO- or by Mb/H2O2, occurred with the production of activated species and by radical pathways.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase. A requiem for H2O4.

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O2- during the first few minutes of the reaction. H2O2 decreases this accumulation of O2- presumably because of the Haber-Weiss reaction (H2O2+O2- leads to OH- +OH+O2) and very small amounts of superoxide dismutase eliminate it. This accumulation of O2- was demonstrated in terms of a burst of reduction of cytochrome c, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H2O4-, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

The effects of alpha-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.     

The hydroxylation of the salicylate anion by a Fenton reaction and T-radiolysis: a consideration of the respective mechanisms

The yield of 2,3- and 2,5-dihydroxybenzoates (dHB's) from the reaction of .OH radicals with salicylate (SA) ions has been measured as a function of pH and in the presence of oxidants. Under steady-state radiolysis conditions, the production of these products occurs via the reactions .OH + SA----HO-SA. (radical adduct) HO-SA. H+.OH+----2-carboxyphenoxyl radical (SA.) + H2O HO-SA. + SA.----2,3-/2,5-dHB + SA The addition of the oxidants O2, Fe3+ edta, or Fe(CN)63- increases the relative yield of 2,5-dHB/2,3-dHB from about 0.2 to 1. A model to account for this effect is presented. Steady-state radiolyses of 3- and 4-hydroxybenzoate give dihydroxybenzoate products consistent with the phenol group being an ortho-para director in the electrophilic attack of the hydroxyl radical on the aromatic ring. A comparison of product distributions from the reaction of ferrous edta with hydrogen peroxide using salicylate as a scavenger strongly suggests that the same hydroxyl radical adducts are formed as in the radiation experiments.     

Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein

Proteolytic degradation of oxidatively damaged [3H] bovine serum albumin [( 3H]BSA) was studied during incubation with cell-free erythrocyte extracts and a wide variety (14) of purified proteases. [3H]BSA was pretreated by exposure (60Co radiation) to the hydroxyl radical (.OH), the superoxide anion radical (O2-), or the combination of .OH + O2- + oxygen. Treated (and untreated) samples were dialyzed and then incubated with erythrocyte extract or proteases for measurements of proteolytic susceptibility (release of acid-soluble counts). Both .OH and .OH + O2- + caused severalfold increases in proteolytic susceptibility (with extract and proteases), but O2- alone had no effect. Proteolytic susceptibility reached a maximum at 15 nmol of .OH/nmol of BSA and declined thereafter. In contrast, proteolytic susceptibility was still increasing at an .OH + O2-/BSA molar ratio of 100 (50% .OH + 50% O2-). Degradation in erythrocyte extracts was conducted by a novel ATP- and Ca2+-independent pathway, with maximal activity at pH 7.8. Inhibitor profiles indicate that this pathway may involve metalloproteases and serine proteases. Comparisons of proteolytic susceptibility with multiple modifications to BSA primary, secondary, and tertiary structure revealed a high correlation (r = 0.98) with denaturation/increased hydrophobicity by low concentrations of .OH. Covalent aggregation reactions (BSA cross-linking) may explain the declining proteolytic susceptibility observed at .OH/BSA molar ratios greater than 20. Protein denaturation may also have caused the increased proteolytic susceptibility induced by .OH + O2- + O2, but no simple correlation could be obtained. Results with .OH + O2- + O2 appear to have been complicated by direct BSA fragmentation reactions involving (.OH-induced) protein radicals and oxygen. These data indicate a direct and quantitative relationship between protein damage by oxygen radicals and increased proteolytic susceptibility. Oxidative denaturation may exemplify a simple, yet effective inherent mechanism for intracellular proteolysis.     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Generation of *OH initiated by interaction of Fe2+ and Cu+ with dioxygen; comparison with the Fenton chemistry

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.     

Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure

Proteins which have been exposed to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical and oxygen (.OH + O2- + O2) exhibit altered primary structure and increased proteolytic susceptibility. The present work reveals that alterations to primary structure result in gross distortions of secondary and tertiary structure. Denaturation/increased hydrophobicity of bovine serum albumin (BSA) by .OH, or by .OH + O2- + O2 was maximal at a radical/BSA molar ratio of 24 (all .OH or 50% .OH + 50% O2-). BSA exposed to .OH also underwent progressive covalent cross-linking to form dimers, trimers, and tetramers, partially due to the formation of intermolecular bityrosine. In contrast, .OH + O2- + O2 caused spontaneous BSA fragmentation. Fragmentation of BSA produced new carbonyl groups with no apparent increase in free amino groups. Fragmentation may involve reaction of (.OH-induced) alpha-carbon radicals with O2 to form peroxyl radicals which decompose to fragment the polypeptide chain at the alpha-carbon, rather than at peptide bonds. BSA fragments induced by .OH + O2- + O2 exhibited molecular weights of 7,000-60,000 following electrophoresis under denaturing conditions, but could be visualized as hydrophobic aggregates in nondenaturing gels (confirmed with [3H]BSA following treatment with urea or acid). Combinations of various chemical radical scavengers (mannitol, urate, t-butyl alcohol, isopropyl alcohol) and gases (N2O, O2, N2) revealed that .OH is the primary species responsible for alteration of BSA secondary and tertiary structure. Oxygen, and O2- serve only to modify the outcome of .OH reaction. Furthermore, direct studies of O2- + O2 (in the absence of .OH) revealed no measurable changes in BSA structure. The process of denaturation/increased hydrophobicity was found to precede either covalent cross-linking (by .OH) or fragmentation (by .OH + O2- + O2). Denaturation was half-maximal at a radical/BSA molar ratio of 9.6, whereas half-maximal aggregation or fragmentation occurred at a ratio of 19.4. Denaturation/hydrophobicity may hold important clues for the mechanism(s) by which oxygen radicals can increase proteolytic susceptibility.