Enzymatic activation of hydrazine derivatives. A spin-trapping study

The oxidative metabolism of hydralazine, isoniazid, iproniazid, and phenylhydrazine has been studied using spin-trapping techniques. The oxidation of these hydrazine derivatives, catalyzed by horseradish peroxidase and prostaglandin synthetase, produces reactive free radical intermediates. Enzymatic activation of hydralazine produce the nitrogen-centered hydralazyl radical (RNHNH); phenylhydrazine formed only the phenyl radical. Iproniazid, on the other hand, formed both the isopropyl radical and a hydroperoxy radical. The formation of the hydroperoxy radical was not inhibited by superoxide dismutase. The horseradish peroxidase-catalyzed oxidation of isoniazid produced two different carbon-centered radicals. The identity of these radicals is not clear; however, they may arise from an acyl (RCO) radical and an alkyl (R) radical.     

Concerted Action Of Reduced Glutathione And Superoxide Dismutase In Preventing Redox Cycling Of Dihydroxypyrimidines, And Their Role In Antioxidant Defence

Dialuric Acid, the reduced form of the β-cell toxin alloxan, and the related fava bean derivatives divicine and isouramil, autoxidize rapidly in neutral solution by a radical mechanism. GSH promotes redox cycling of each compound, with concomitant GSH oxidation and H₂O₂ production. With superoxide dismutase present, there is a lag period in which little oxidation occurs, followed by rapid oxidation. GSH extends this lag and decreases the subsequent rate of oxidation, so that with superoxide dismutase and a sufficient excess of GSH. coupled oxidation of GSH and each pyrimidine is almost completely suppressed. This mechanism may be a means whereby GSH in combination with superoxide dismutase protects against the cytotoxic effects of these reactive pyrimidines. Superoxide dismutase may also protect cells against oxida-tive stress in other situations where GSH acts as a radical scavenger. and we propose that the concerted action of GSH and superoxide dismutase constitutes an important antioxidant defence     

Concerted Action Of Reduced Glutathione And Superoxide Dismutase In Preventing Redox Cycling Of Dihydroxypyrimidines,And Their Role In Antioxidant Defence

Dialuric Acid, the reduced form of the β-cell toxin alloxan, and the related fava bean derivatives divicine and isouramil, autoxidize rapidly in neutral solution by a radical mechanism. GSH promotes redox cycling of each compound, with concomitant GSH oxidation and H₂O₂ production. With superoxide dismutase present, there is a lag period in which little oxidation occurs, followed by rapid oxidation. GSH extends this lag and decreases the subsequent rate of oxidation, so that with superoxide dismutase and a sufficient excess of GSH. coupled oxidation of GSH and each pyrimidine is almost completely suppressed. This mechanism may be a means whereby GSH in combination with superoxide dismutase protects against the cytotoxic effects of these reactive pyrimidines. Superoxide dismutase may also protect cells against oxida-tive stress in other situations where GSH acts as a radical scavenger. and we propose that the concerted action of GSH and superoxide dismutase constitutes an important antioxidant defence     

The catalytic effect of the carcinogen "4-nitroquinoline-N-oxide" on the oxidation of vitamin C.

The carcinogen 4-nitroquinoline-N-oxide was found to mediate the reaction between ascorbate and oxygen. The oxidation of ascorbate was initiated by the production of the nitro radical anion which reacted with oxygen to produce the oxygen superoxide radical anion, peroxide and hydroxyl radical. The production of partially reduced oxygen intermediates resulted in additional reactions with ascorbate. The consumption of oxygen could be either completely blocked by reacting the nitro radical with ferricytochrome c or partially blocked by the combined effects of superoxide dismutase and catalase. The consumption of oxygen could be enhanced by reducing the hydroxyl radicals with dimethylsulfoxide.     

The reaction between indole 3-acetic acid and horseradish peroxidase

Three distinct phases of the reaction between indole 3-acetic acid (IAA) and horse-radish peroxidase (isoenzymes B and C) were observed. When 100 μm IAA was added to an aerobic solution of the 7μm enzyme at pH 5.0 the oxidation of IAA occurred after a lag time of several seconds, during which the enzyme was partially converted into peroxide Compound II. At a time when the lag time was over the conversion of the enzyme into a green hemoprotein, called P-670 suddenly occurred at a considerable speed. The oxidation of IAA was almost over at the end of the second phase. The last phase was the restoration of the free enzyme from the remaining Compound II.Ascorbate and cytochrome c peroxidase elongated the lag phase of IAA oxidation. From these inhibition experiments it was suggested that a peroxide form of IAA would react with peroxidase to form its peroxide compounds as does hydrogen peroxide and cause the oxidation of IAA. A reaction path that the enzyme is directly reduced by IAA might be involved as an initiation step but appeared to play no essential role in the oxidation of IAA at steady state.Contrary to the cases with dihydroxyfumarate and NADH, Superoxide dismutase did not inhibit the aerobic oxidation of IAA by peroxidase. IAA peroxide radical instead of superoxide anion radical was suggested to be an intermediate in the oxidation of IAA.On the basis of stoichiometric relation of reactions between IAA and peroxidase peroxide compounds a tentative scheme of P-670 formation during the oxidation of IAA was presented.     

Co-ordinate regulation of low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase and synthase gene expression in HepG2 cells.

We have examined the effects of O2-derived free radicals on oxymyoglobin, the myocardial intracellular protein involved in the storage and transport of O2. The oxyradicals generated by the xanthine/xanthine oxidase system decreased the concentration of oxymyoglobin. Based on the decreases in absorbance peaks at 581 nm and 415 nm it is estimated that out of a 10 nmol decrease in oxymyoglobin, 5 nmol appears to be oxidized to ferrimyoglobin (deoxygenation), while haem was removed from the other 5 nmol of haem protein. These processes were inhibited by both catalase alone and superoxide dismutase in combination with catalase, but not by either superoxide dismutase alone or deferoxamine. These results suggest that among H2O2, OH. and O2.-, only H2O2 causes the removal of haem and the oxidation of oxymyoglobin. Furthermore, the oxyradicals also released 3 microM free iron from oxymyoglobin, which is at least 5-fold less than the 15 nmol loss of oxymyoglobin. The loss of oxymyoglobin also preceded the release of free iron. These results indicate that oxymyoglobin oxidation and haem removal occur before the removal of free iron. Thus myoglobin appears to be highly susceptible to free radical attack, and this may represent yet another mechanism of free radical-mediated cellular injury.     

Mechanism of action of D-serine dehydratase. Identification of a transient intermediate.

Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2 degrees C show that during the steady-state course of D-serine conversion the absorption maximum of the Schiff base of the cofactor pyridoxal 5'-phosphate (pyridoxal-P) is shifted from 415 to 442 nm. Furthermore, the progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320 to 500 nm. A point by point construction of successive spectra from these stopped-flow traces at various time intervals after the start of reaction resulted in a series of consecutive spectra exhibiting two isobestic points at 353 and 419 nm. The half-time of the absorbance changes occurring at 330 and 455 nm was found to be 6.5 ms, suggesting the observation of a single, enzyme-bound intermediate. The spectral data with substrate and inhibitors provide evidence that the intermediate is the Schiff base of alpha-aminoacrylate and pyridoxal-P. The proposed assignment is strongly supported by experiments of apodehydratase with transient-state analogues which exhibit a similar absorbance shift on binding to apoenzyme. Moreover, these results suggest that the phosphate group of the substrate--pyridoxal-P complex serves as the main anchoring point during catalysis. A reaction mechanism of the D-serine dehydratase is presented.     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.     

Dual effects of superoxide dismutase on the autoxidation of 1,4-naphthohydroquinone

The autoxidation of 1,4-naphthohydroquinone, in a phosphate, EDTA buffer at pH 7.4, exhibits an autocatalysis whose lag phase becomes more pronounced in the presence of either the Cu,Zn- or the Mn-containing superoxide dismutases. In contrast, the autoxidation of a second aliquot of the hydroquinone, added after complete oxidation of the first, is linear and is accelerated by superoxide dismutase. Catalase or inactive superoxide dismutase were without effect in either situation. These results are explicable in terms of a free radical chain reaction which is initially propagated by O2- and then, as the quinone accumulates, by univalent reduction of the quinone by the hydroquinone. Reduction of the quinone by O2- diminishes the overall rate of oxidation. It is not necessary to postulate catalysis by superoxide dismutase of the reduction of the semiquinone by O2-.     

Concentration of ultraviolet-B radiation absorbing compounds in leaves of a range of fynbos species

Leaves from 38 species representing 17 families were collected from contrasting elevations (100 to 824 m above sea level) in sclerophyllous mountain fynbos vegetation near Hermanus, South Africa, and the leaf percentage dry mass (PDM), specific leaf mass (SLM) and ultraviolet-B (UV-B, 280 to 320 nm) absorbance properties determined. Leaf PDM, SLM and UV-B absorbance were generally high compared to mesophyllous plants. Leaves collected at high elevation exhibited higher absorbances per unit dry mass at both 280 and 320 nm than those from the same species at low elevation. No differences in absorbance per unit leaf area were observed. UV-B absorbance properties differed between families, with high values obtained for the Apiaceae, Asteraceae, Cyperaceae, Ericaceae, Penaeaceae and Proteaceae, but lower values for the Anacardiaceae, Fabaceae and Geraniaceae. A positive correlation was found between absorbance at 280 nm per unit leaf area and SLM. It was concluded that most fynbos species, on account of their highly sclerophyllous leaves and large accumulation of UV-B absorbing compounds (flavonoids and related phenolics) may be well protected against future increases in UV-B radiation.     

Inhibition of lipoxygenase-dependent lipid peroxidation by quercetin: Mechanism of antioxidative function

Quercetin inhibited soybean lipoxygenase-1-dependent linoleic acid peroxidation. Two to three μM quercetin was required for 50% inhibition. During the inhibition, quercetin was oxidized. The oxidation was observed as an absorbance decrease at about 380 nm and an absorbance increase at about 335 nm. Inhibition of linoleic acid peroxidation by quercetin seems to be due to reduction by the reagent of the linoleic acid radical formed as an intermediate during lipoxygenation. Quercetin oxidation was suppressed by ascorbate under conditions when ascorbate did not affect lipoxygenase-dependent linoleic acid peroxidation. The results suggest that ascorbate can reduce the quercetin oxidized by the linoleic acid radical back to quercetin. Based on the results, the significance of a redox reaction between oxidized quercetin and ascorbate is discussed.     

Mechanism of NADH-sensitized formation of DNA breaks during irradiation with near UV light]

NADH-photosensitized in vitro formation of single-stranded breaks in plasmid DNA pBR322 depends on both the concentration of the sensitizer and the influence of near-UV radiation (320-400 nm). Scavengers and inhibitors of different activated oxygen species (sodium azide, sodium benzoate, catalase and superoxide dismutase) prevent the formation of breaks in full or partly. The data obtained show that hydroxyl radical (.OH) and singlet oxygen (1O2) are directly involved in the induction of breaks. In this process hydrogen peroxide (H2O2) plays the role of an intermediate in the reaction of .OH formation from superoxide anion-radical (O2-.) which is the first NAD.H-photogenerated product.     

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. I. Evidence for superoxide anion involvement in the oxidation of NADPH2.

1. The oxidation of NADPH2 by leucocyte granules, as measured at acid pH in the presence of Mn-2+, was found to be inhibited by superoxide dismutase. 2. Omission of Mn-2+ markedly lowered the oxidase activity at acid pH, which was still inhibited by superoxide dismutase. 3. At alkaline pH the oxidase activity was lower than at acid pH. 4. During oxidation of NADPH2 by leucocyte granules, reduction of cytochrome c occurred which was partially inhibited by superoxide dismutase. 5. It was concluded that NADPH2 oxidation occurs through an enzymatic reaction and a nonenzymatic chain reaction. Superoxide anion (O-minus-2 and NADPH- free radical would be involved in the chain reaction. The differential sensitivity of NADPH2 oxidation to superoxide dismutase in different experimental conditions (see above 1, 2 and 3) was explained on the basis of changes in the properties of the chain reaction.     

Cyanide catalyzes the oxidation of alpha-hydroxyaldehydes and related compounds: monitored as the reduction of dioxygen, cytochrome c, and nitroblue tetrazolium

Cyanide catalyzed the oxidation of α-hydroxycarbonyls and of related compounds. In the cases of glyceraldehyde 3-phosphate and of dihydroxyacetone phosphate the tautomeric enediol was the obligatory intermediate which reacted with cyanide yielding the active reductant. Cytochrome c, nitroblue tetrazolium, and dioxygen were all reduced by this reductant. In the case of dioxygen the product was the superoxide radical which could then secondarily reduce cytochrome c or nitroblue tetrazolium. In air-equilibrated reaction mixtures, at 25 °C, approximately 35% of cytochrome c reduction and 95% of nitroblue tetrazolium reduction was mediated by superoxide, as judged from susceptibilities to inhibition by superoxide dismutase. Since the oxidations observed were univalent, carbon-centered radicals appear to be necessary intermediates, and their secondary reactions generated a multiplicity of products, seen as smears on thin-layer chromatograms. Free cyanide must be regenerated during these secondary reactions, since cyanide functioned catalytically in the overall process. A partial mechanism has been proposed in explanation of these observations.     

Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation

Hematoxylin, a natural dye commonly used as a histological stain, generates superoxide upon oxidation to its quinonoid product, hematein. The parameters affecting this reaction were assessed in developing a new and versatile assay for superoxide dismutase. The autoxidation of hematoxylin to hematein was accompanied by an increase in absorbance between 400 and 670 nm. The autoxidation rate was proportional to hematoxylin concentration and increased with pH above 6.55. Trace metals accelerated the autoxidation and this effect was eliminated by EDTA. Superoxide dismutase inhibited the autoxidation 90-95% below pH 7.8, but above pH 8.1 the rate was augmented by superoxide dismutase. The rate inhibition at low pH was proportional to the superoxide dismutase concentration up to 70% inhibition. The rate acceleration at high pH was proportional to superoxide dismutase concentration up to approximately 200% acceleration. The autoxidation rate was not significantly affected by ethanol, cyanide, azide, hydrogen peroxide, or catalase. However, the reaction was inhibited by the reducing agents NADH, reduced glutathione, ascorbate, and dithiothreitol, and by undialyzed extracts of Escherichia coli B. When cell extracts were dialyzed prior to assay, the degree of inhibition observed was proportional to the concentration of superoxide dismutase in the extract. These observations form the basis for negative and positive assays of superoxide dismutase which are inexpensive and simple to perform. The negative assay has the added advantage of being applicable at physiological pH.     

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.     

Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion (MPP+): reductive activation by NADPH cytochrome P-450 reductase

Formation of free radical intermediates from 1--methyl-4-phenylpyridinium ion(MPP+) has been studied using spin-trapping techniques. Incubation of MPP+ with purified NADPH cytochrome P-450 reductase and NADPH under anaerobic conditions failed to produce any detectable radical intermediates. However, in the presence of air and a spin-trap, a significant stimulation of superoxide and hydroxyl radicals was detected. Formation of these toxic radicals from MPP+ was inhibited by superoxide dismutase, catalase, and ethanol. Under identical conditions, however, considerably less of these radicals were formed with MPP+ in comparison to paraquat, a lung toxin containing two pyridinium moieties.     

UV-visible spectral identification of the solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid

Solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid were investigated by spectroscopy. The spectral data showed the formation of a stable organomanganese intermediate, which was responsible for the rise in the absorbance at 420 nm. This result enables unambiguous interpretation of the absorbance change at 420 nm, as the intermediate permanganate ions could be isolated on the solid supports.     

Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system.

Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosine and bityrosine in aqueous solution at pH 7.4 resulted in light emission in the visible region. Electrolysis of tyrosine emitted light which peaked at 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H(2)O(2)-tyrosine system the oxidation-reduction of tyrosine emitted light with two prominent peaks, 490 and 530 nm, and was not quenched by SOD. The phenoxyl neutral radical of the tyrosine in HRP-H(2)O(2)-tyrosine system was detected by electron spin resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; the spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H(2)O(2)-tyrosine reaction system. Changes in absorption spectra of HRP and chemiluminescence intensities during HRP-catalyzed oxidation of tyrosine suggest that for photon emission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in this study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(*+)) and the bityrosine cation radical (BT(*+))     

Redox intermediates in the catalase cycle of catalase-peroxidases from Synechocystis PCC 6803, Burkholderia pseudomallei, and Mycobacterium tuberculosis

Monofunctional catalases (EC 1.11.1.6) and catalase-peroxidases (KatGs, EC 1.11.1.7) have neither sequence nor structural homology, but both catalyze the dismutation of hydrogen peroxide (2H2O2 --> 2H2O + O2). In monofunctional catalases, the catalatic mechanism is well-characterized with conventional compound I [oxoiron(IV) porphyrin pi-cation radical intermediate] being responsible for hydrogen peroxide oxidation. The reaction pathway in KatGs is not as clearly defined, and a comprehensive rapid kinetic and spectral analysis of the reactions of KatGs from three different sources (Synechocystis PCC 6803, Burkholderia pseudomallei, and Mycobacterium tuberculosis) with peroxoacetic acid and hydrogen peroxide has focused on the pathway. Independent of KatG, but dependent on pH, two low-spin forms dominated in the catalase cycle with absorbance maxima at 415, 545, and 580 nm at low pH and 418 and 520 nm at high pH. By contrast, oxidation of KatGs with peroxoacetic acid resulted in intermediates with different spectral features that also differed among the three KatGs. Following the rate of H2O2 degradation by stopped-flow allowed the linking of reaction intermediate species with substrate availability to confirm which species were actually present during the catalase cycle. Possible reaction intermediates involved in H2O2 dismutation by KatG are discussed.