Critical residues in D-amino acid oxidase. A pulse-radiolysis and inactivation study.

The enzyme D-amino acid oxidase and its apoenzyme have been irradiated at pH 5.5--10 under conditions designed to assess the inactivating effect of OH radicals and the selective free radicals Br2- and (SCN)2-. Near neutral pH, removal of the coenzyme FAD from the enzyme results in greater inactivation by selective free-radical attack. From pulse-radiolysis spectra, this increase is associated with attack on tyrosine and tryptophan residues in the protein. A large increase in inactivation of both the haloenzyme and apoenzyme by selective free-radical attack is seen with increasing alkalinity. This is consistent with attack on tyrosine being of major importance.     

Pulse radiolysis of lactate dehydrogenase.

Pulse radiolysis has been used to investigate the rates and transient spectra for the reactions of free radicals with beef heart lactate dehydrogenase at pH 7. Analysis of the results leads to second-order rate-constants for eaq-, .OH, .I, .Br2-, .I2- and .(CNS)2- which are, respectively, 24, 21, 10, 0.55, 0.43 and 0.15 in units of 10(10) M-1 s-1 with uncertainties of +/- 20 per cent. Those for .I and .I2- are similar to the corresponding rate-constants for the related enzyme alcohol dehydrogenase. The spectra of the transient species produced by .OH, .Br2- and .(CNS)2- all showed evidence for reactions with tyrosine and tryptophan residues, and in general terms the magnitudes of the rate-constants appeared to increase with the oxidizing abilities of the radicals. The implication of the results for understanding the mechanism of deactivation by free radicals is discussed.     

Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in peroxynitrite-treated erythrocyte membranes.

The treatment of erythrocyte membranes with peroxynitrite (ONOO-), a cytotoxic species formed in vivo by the almost completely diffusion controlled reaction of nitric oxide (NO*) and the superoxide anion (O2*-), led to the loss of the EPR signal of the nitroxide radical 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). The decrease in the TEMPO EPR signal was peroxynitrite concentration dependent in the studied peroxynitrite concentration range (100-1000 microM). The absence of such a phenomenon in the control membranes (not treated with peroxynitrite) and in a buffer treated with peroxynitrite indicates that the effect must be caused by nitroxide radicals reacting with the products of peroxynitrite reactions with membrane components. To find out which membrane components are responsible for the decrease in EPR signal, this effect was studied in simple model systems (protein and lipid suspensions). The same phenomenon was observed in both lipid and protein systems treated with peroxynitrite, but in protein solutions the effect was greater and occurred for lower peroxynitrite concentrations. A clear effect of the loss of the EPR signal was observed for both erythrocyte membranes and bovine serum albumin (BSA) solution for a peroxynitrite concentration of 100 microM, while in the case of linolenic acid suspension, a significant difference between control and peroxynitrite-treated samples was achieved for a peroxynitrite concentration of 1000 microM. A comparison of the results obtained for the lipid and protein systems suggests that the reaction of nitroxide radicals with protein derived species plays the main role in the observed decrease in the TEMPO EPR signal in peroxynitrite treated erythrocyte membranes.     

The oxidation of ferrocytochrome c by Br2-, (SCN)2-, N3 and OH radicals studied by pulsed-electron and gamma-ray radiolysis

The reactions of ferrocytochrome c with Br2-, (SCN)2-, N3 and OH radicals were followed by measuring the change in the optical spectra of cytochrome c on gamma-irradiation as well as the rate of change of absorbance upon pulse irradiation. Ferrocytochrome c is oxidized to ferricytochrome c by Br2-, (SCN)2- or N3 radical with an efficiency of about 100% through a second-order process in which no intermediates were observed. The rate constants in neutral solutions at I = 0.073 are 9.7 . 10(8) M-1 . s-1, 7.9 . 10(8) M-1, 1.3 . 10(9) M-1 . s-1 for the oxidation by Br2-, (SCN)2- and N3 radicals, respectively. The rate constants do not vary appreciably in alkaline solutions (pH 8.9). The ionic strength dependence was observed for the rate constants of the oxidation by br2- and (SCN)2-. Those rate constants estimated on the assumption that the radicals react only with the amino acid residues with the characteristic steric correction factors were less than one-tenth of the observed ones. These results suggest that the partially exposed region of the heme is the probable site of electron transfer from ferrocytochrome c to the radical. Hydroxyl radicals also oxidize ferrocytochrome c with a high rate constant (k greater than 1 . 10(10) M-1 . s-1), but with a very small efficiency (5%).     

Investigation of radical-based damage of RNase A in aqueous solution and lipid vesicles

The gamma-irradiation of bovine pancreatic ribonuclease A (RNase A) in aqueous solution were investigated at different doses by vibrational spectroscopy as well as enzymatic assay, electrophoresis, and HPLC analysis. Both functional and structural changes of the protein were caused by attack of H(*) atoms and (*)OH radicals. In particular, Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and amino acidic residues that are preferential sites of the radical attack (i.e., tyrosine and methionine). After partial structural changes by the initial radical attack, the internal sulfur-containing amino acid residues were rendered susceptible to transformation. By using the biomimetic model of dioleoyl phosphatidyl choline vesicle suspensions containing RNase A, the damage to methione residues could be connected to a parallel alteration of membrane unsaturated lipids. In fact, thiyl radical species formed from protein degradation can diffuse into the lipid bilayer and cause isomerization of the naturally occurring cis double bonds. As a consequence, trans unsaturated fatty acids are formed in vesicles and can be considered to be markers of this protein damage.     

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.     

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.     

Radical-Induced Damage to Proteins: E.S.R. Spin-Trapping Studies

The reactions of hydroxyl radicals generated from Fe¹¹/H₂O₂ and Cu¹¹/H₂O₂ redox couples with a variety of proteins (BSA, histones, cytochrome c, lysozyme and protamine) have been investigated by e.s.r. spin trapping. The signals obtained, which are generally anisotropic in nature, characterize the formation of partially-immobilized spin-adducts resulting from attack of the HO- radicals on the protein and subsequent reaction of the protein-derived radicals with the spin trap. Similar spin adducts are observed on incubation of two haem-proteins (haemoglobin and myoglobin) with H₂O₂ in the absence of added metal ions implying a reaction at the haem centre followed by internal electron transfer reactions.

Two strategies have been employed to obtain information about the site(s) of radical damage in these proteins. The first involves the use of a variety of spin traps and in particular DMPO: with this particular trap the broad spectra from largely immobilized radicals show characteristic a(β-H) values which enable carbon-, oxygen- and sulphur-centred radicals to be distinguished. The second involves the use of enzymatic cleavage of first-formed adducts to release smaller nitroxides, with isotropic spectra, which allow the recognition of β-proton splittings and hence information about the sites of radical damage to be obtained. These results, which allows backbone and side-chain attack to be distinguished, are in agreement with random attack of the HO^. radical on the protein and are in accord with studies carried out on model peptides. In contrast the use of less reactive attacking radicals [N₃·, ·CH(CH₃)OH] and oxidising agents (Ce⁴⁺) provides evidence for selective attack on these proteins at particular residues.     

Intra- and intermolecular oxidation of oxymyoglobin and oxyhemoglobin induced by hydroxyl and carbonate radicals

The mechanism of the reactions of myoglobin and hemoglobin with *OH and CO3*- in the presence of oxygen was studied using pulse and gamma-radiolysis. Unlike *NO2, which adds to the porphyrin iron, *OH and CO3*- form globin radicals. These secondary radicals oxidize the Fe(II) center through both intra- and intermolecular processes. The intermolecular pathway was further demonstrated when BSA radicals derived from *OH or CO3*- oxidized oxyhemoglobin and oxymyoglobin to their respective ferric states. The oxidation yields obtained by pulse radiolysis were lower compared to gamma-radiolysis, where the contribution of radical-radical reactions is negligible. Full oxidation yields by *OH-derived globin radicals could be achieved only at relatively high concentrations of the heme protein mainly via an intermolecular pathway. It is suggested that CO3*- reaction with the protein yields Tyr and/or Trp-derived phenoxyl radicals, which solely oxidize the porphyrin iron under gamma-radiolysis conditions. The *OH particularly adds to aromatic residues, which can undergo elimination of H2O forming the phenoxyl radical, and/or react rapidly with O2 yielding peroxyl radicals. The peroxyl radical can oxidize a neighboring porphyrin iron and/or give rise to superoxide, which neither oxidize nor reduce the porphyrin iron. The potential physiological implications of this chemistry are that hemoglobin and myoglobin, being present at relatively high concentrations, can detoxify highly oxidizing radicals yielding the respective ferric states, which are not toxic.     

Reactivity of ubiquinones and ubiquinols with free radicals

The reactivity of quinones 1-4 and of the corresponding quinols 5-8 towards carbon- and oxygen-centred radicals were studied. All quinones bearing at least one nuclear position free, readily react with alkyl and phenyl radicals to afford the alkylated quinones 12-24; however, quinones 1 and 3 reacted with 2-cyano-2-propyl radical to yield products (the mono- and di-ethers 9-11) derived from the attack on the carbonylic oxygen. The reactions carried out on quinones with the benzoyloxy radical led to no reaction products and in the case of Q₁₀, the isoprenic chain also remained unchanged. Quinols 5-8 reacted only with oxygencentred radicals (benzoyloxy and 2-cyano-2-propylperoxy radicals) to give the corresponding quinones. The isoprenic chain of Q₁₀ did not undergo attack even with peroxy radicals. Carbon-centred radicals resulted unable to abstract hydrogen from the studied quinols.     

Free-radical reactions induced by OH-radical attack on cytosine-related compounds: a study by a method combining ESR, spin trapping and HPLC.

Free-radical reactions induced by OH-radical attack on cytosine-related compounds were investigated by a method combining ESR, spin trapping with 2-methyl-2-nitrosopropane and high-performance liquid chromatography (HPLC). Cytidine, 2'-deoxycytidine, cytidine 3'-monophosphate, cytidine 5'-monophosphate, 2'-deoxycytidine 5'-monophosphate and their derivatives, of which 5,6-protons at the base moiety were replaced by deuterons, and polycytidylic acid (poly(C] were employed as samples. OH radicals were generated by X-irradiating an N2O-saturated aqueous solution. Five spin adducts were separated by HPLC. Examination of them by ESR spectroscopy and UV photospectrometry showed that spin adducts assigned to C5 and C6 radicals due to OH addition to the 5,6 double-bond, a deaminated form of the spin adduct derived from a C5 radical due to the cyclization reaction between C5' of the sugar and C6 of the base, and a spin adduct assigned to the C4' radical due to H abstraction by OH radicals were produced. From these results the sites of OH-radical attack and the subsequent radical reactions in cytosine-related compounds were clarified.     

Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in ozone-treated erythrocyte membranes

In ozone-treated erythrocyte membrane suspension a slow decrease occurs in the EPR signal of 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). Because of the absence of such a phenomenon in control membranes and ozonized buffer, this effect must be caused by reaction of nitroxide radicals with products of ozone reactions with membrane components. To find out which components are responsible for the decrease in EPR signal we studied this effect in simple model systems. The same phenomenon was observed both in lipid and protein systems treated by ozone. For unsaturated fatty acids, the correlation between the rate of decrease in EPR signal and the number of double bonds in the lipid molecule was very strong. This suggests that the observed decrease in the nitroxide radical TEMPO EPR signal in ozone-treated erythrocyte membranes is a complex process, but probably the most important reaction is recombination of nitroxide radicals with organic free radicals produced both in the process of lipid peroxidation and ozonolysis of double bonds.     

Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in ozone-treated erythrocyte membranes

In ozone-treated erythrocyte membrane suspension a slow decrease occurs in the EPR signal of 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). Because of the absence of such a phenomenon in control membranes and ozonized buffer, this effect must be caused by reaction of nitroxide radicals with products of ozone reactions with membrane components. To find out which components are responsible for the decrease in EPR signal we studied this effect in simple model systems. The same phenomenon was observed both in lipid and protein systems treated by ozone. For unsaturated fatty acids, the correlation between the rate of decrease in EPR signal and the number of double bonds in the lipid molecule was very strong. This suggests that the observed decrease in the nitroxide radical TEMPO EPR signal in ozone-treated erythrocyte membranes is a complex process, but probably the most important reaction is recombination of nitroxide radicals with organic free radicals produced both in the process of lipid peroxidation and ozonolysis of double bonds.     

Hypochlorous acid damages erythrocyte membrane proteins and alters lipid bilayer structure and fluidity

Treatment of human erythrocyte membranes with active forms of chlorine (hypochlorous acid and chloramine T) resulted in a concentration-dependent inhibition of the membrane Na(+), K(+)- and Mg(2+)-ATPases. Membrane protein thiol group oxidation was consistent with inactivation of enzymes and preceded oxidation of tryptophan residues and chloramine formation. Erythrocyte exposure to hypochlorous acid led to complex changes of cell membrane rigidity and cell morphological transformations: cell swelling, echinocyte formation, and haemolysis. The inhibition of ion pump ATPases of human erythrocyte membranes may be due to direct oxidation of essential residues of enzyme (thiol groups) and structural rearrangement of the membrane.     

Hot spot kinetics of the sonolysis of aqueous acetate solutions

Water and acetate solutions were irradiated under argon by 300 kHz ultrasonic waves. Oxygen was found to be generated besides the products H2 and H2O2, already known. In the presence of acetate the O2 yield decreased rapidly while that of H2O2 decreased more slowly. Succinic acid was found as a product of the attack of OH radicals on acetate. Appreciable amounts of glyoxylic and glycolic acid and smaller amounts of formaldehyde and carbon dioxide were also detected. They resulted from the reaction of sonolytically generated oxygen with CH2CO2- radicals, produced upon attack of OH on acetate. Methane was a minor product of sonolysis. At acetate concentrations above 0.4 mol dm-3 CO2 and CO became the predominant products of sonolysis. This is explained by a second kind of action of ultrasound on dissolved acetate, i.e. by a thermal decomposition. This decomposition is possibly facilitated by radical attack on acetate. The results are discussed in terms of a 'structured hot spot' model, in which three regions for the occurrence of chemical reactions are postulated: a hot gaseous nucleus, an interfacial region with radial gradient in temperature and local radical density; and the bulk solution at ambient temperature.     

Effect of reactive oxygen species on the erythrocyte calcium-pump function in protein-energy malnutrition

The presence of detectagle amounts of non-heme iron in erythrocyte ghost membranes have been postulated to lead to the initiation of membrane lipid peroxidation and the attendant perturbation of membrane functions. We have investigated the presence of non-heme iron and endogenous products of lipid peroxidation in erythrocyte membranes of normal and kwashiorkor (KWA) subjects and assessed the susceptibility of the membranes to exogenously generated reactive oxygen species. The modulation of the basal and calmodulin-stimulated calcium-pumping activity of these membranes by reactive oxygen species was also assessed. The results show the presence of significant amounts of non-heme iron and endogenous free radical reaction products in the red cell membranes of KWA subjects compared with that of normal children. Estimation of the extent of lipid peroxidation in the presence of exogenously generated reactive oxygen species further revealed that erythrocyte ghost membranes of KWA subjects are more susceptible to oxidative stress than those of normal individuals. Although both the basal and calmodulin-stimulated activities of the membrane-bound Ca²⁺-pump enzyme in normal and KWA subjects were inhibited by oxygen-free radicals, the erythrocyte enzyme in KWA subjects showed higher susceptibility to inhibition by oxygen free radicals than that of normal individuals. We propose that the reduced erythrocyte calcium-pump function in KWA is not unconnected with excessive generation of reactive oxygen species.Abbreviations PMSF phenylmethylsulfonylfuloride - TLCK N--p-tosyl-l-lysine chloromethylketone - EGTA ethyleneglycol-bis (B-aminoethylether) N,N-tetraacetic acid - EDTA ethylene diamine tetraacetic acid - ATP Adenosine 5-triphosphate - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - Tris-HCl Tris (hydroxymethyl) aminomethane-hydrochloride - SDS Sodium dodecyl sulphate - TBAR thiobarbituric acid-reactive products TBA, thiobarbituric acid - TCA trichloroacetic acid     

A method to evaluate the antioxidant system for radicals in erythrocyte membranes

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.     

Zinc release from irradiated yeast alcohol dehydrogenase

The release of Zn2+ from gamma-irradiated yeast alcohol dehydrogenase has been measured using atomic absorption spectrometry. Radiolysis is accompanied by release of Zn2+ at a rate which is dependent on the nature of the free radicals available for reaction. Hydroxyl radicals and hydrogen atoms readily cause zinc release with G values of 0.13 and 0.11 (/100 eV) respectively, whereas hydrated electrons are considered not to contribute to the demetallization process. The radiolytically generated radical anions I2-., (SCN)2-. and Br2-. enhance the rate of zinc release. Evidence is presented that the enzyme is demetallized as a result of free radical reactions at cysteine and histidine residues.     

Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants

Cupric ions (Cu2+) and ferric ions (Fe3+) added to hydrogen peroxide generate hydroxyl radicals (OH) capable of degrading deoxyribose with the formation of thiobarbituric acid-reactive products. This damage can be inhibited by catalase, OH radical scavengers and specific metal ion chelators. All proteins tested nonspecifically inhibited copper-dependent damage but have little effect on the iron-dependent reaction. Copper ions appear to bind to the proteins which prevents formation of OH radicals in free solution. However, OH radicals are still generated at a site-specific location on the protein molecule. Protein damage is detected as fluorescent changes in amino acid residues.     

Antioxidant properties of melatonin: a pulse radiolysis study

Various one-electron oxidants such as OH*, tert-BuO*, CCl3OO*, Br2*- and N3*, generated pulse radiolytically in aqueous solutions at pH 7, were scavenged by melatonin to form two main absorption bands with lambda(max) = 335 nm and 500 nm. The assignment of the spectra and determination of extinction coefficients of the transients have been reported. Rate constants for the formation of these species ranged from 0.6-12.5x10(9) dm3 mol(-1) s(-1). These transients decayed by second order, as observed in the case of Br2*- and N3* radical reactions. Both the NO2* and NO* radicals react with the substrate with k = 0.37x10(7) and 3x10(7) dm3 mol(-1) s(-1), respectively. At pH approximately 2.5, the protonated form of the transient is formed due to the reaction of Br2*- radical with melatonin, pKa ( MelH* <=> Mel* + H+) = 4.7+/-0.1. Reduction potential of the couple (Mel*/MelH), determined both by cyclic voltammetric and pulse-radiolytic techniques, gave a value E(1)7 = 0.95+/-0.02 V vs. NHE. Repair of guanosine radical and regeneration of melatonin radicals by ascorbate and urate ions at pH 7 have been reported. Reactions of the reducing radicals e(aq)- and H* atoms with melatonin have been shown to occur at near diffusion rates.