Enhancement by nitrite of peroxide-induced degradation of uric acid and 3-N-ribosyluric acid

The oxidation of uric acid and 3-N-ribosyluric acid by hydrogen peroxide and methemoglobin was stimulated by the addition of sodium nitrite, which alone has no effect on the urates. The urates were not oxidized by either hydrogen peroxide alone or hydrogen peroxide and sodium nitrite unless methemoglobin was present. t-Butyl hydroperoxide also oxidized the urates in the presence of methemoglobin, but the reaction was not stimulated by sodium nitrite. The addition of either sodium azide or potassium cyanide reduced the rate of the reaction with either hydrogen peroxide or t-butyl hydroperoxide both in the presence and absence of sodium nitrite. Possible explanations for the stimulation by nitrite of peroxide-induced degradation of urates are presented.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chicken ceruloplasmin. Evidence in support of a trinuclear cluster involving type 2 and 3 copper centers

Ceruloplasmin was isolated to purity from chicken plasma by a single-step chromatography on amino-ethyl-derivatized Sepharose. Molecular mass, as estimated by nonreducing sodium dodecyl sulfate-electrophoresis, was approximately 140 kDa, slightly higher than that found for ceruloplasmins from other sources. Specific activity as p-phenylenediamine oxidase was five times higher than that reported for mammalian ceruloplasmins. The copper content was estimated to be 5.01 +/- 0.35 atoms per protein molecule, 50% of which was EPR-detectable. The EPR spectrum was completely devoid of any signal typical of the type 2 copper as seen in the other blue multicopper oxidases and in ceruloplasmin from mammalian species. Anaerobic reduction of chicken ceruloplasmin resulted in the disappearance of the 330 nm optical band typical of type 3 copper, which was followed by the appearance of an EPR signal typical of type 2 copper. Subsequently, the type 1 copper and finally the newly formed type 2 copper were reduced. The original optical and EPR spectra were recovered within few minutes upon exposure of reduced ceruloplasmin to air. It is concluded that in oxidized chicken ceruloplasmin type 2 copper interacts with the diamagnetic pair responsible for the 330 nm absorption in such a way as to become EPR-undetectable and that the interaction is relieved by reduction of the pair. Whether this interaction is intrinsically weaker in other blue oxidases and ceruloplasmins studied or is lost with standard preparation procedures remains to be established.     

Combined effect of gamma-irradiation and sodium nitrite on the oxidative sensitivity of rat hemoglobin

gamma-Irradiation has been defined to increase in the rats blood the methemoglobin level providing for shortening the initiation phase and accelerates the autocatalytic phase initiation, reduces the period of half transforming hemoglobin into methemoglobin and increases the velocity of its oxidation. Alongside with the latter there is observed a violation of methemoglobin concentration growth dependence on the animals irradiation dose (in the range of 0.16-0.50 Gr). The hemoglobin oxygenation reaction kinetics with the initial level of hemoglobin unexceeding 3% has been determined as having a biexponential character. The reaction kinetics parameters don't depend on ionizing radiation and number of sodium nitrite oxidized subunits formed in the process of reaction in the case if their composition unexceeds 50% of the total level.     

Distinction of the two type I coppers in bovine ceruloplasmin

Redox potentials of the two type I copper ions, ‘blue copper ions’, of bovine ceruloplasmin (ferroxidase, iron(II): oxygen oxidoreductase, EC 1.16.3.1) were determined to be 370 and 390 mV (vs. NHE). These two type I copper ions were clearly differentiated during the anaerobic reduction process of oxidized ceruloplasmin and the reoxidation process of completely reduced ceruloplasmin by using absorption, circular dichroic and electron paramagnetic resonance spectroscopies. One of the blue copper ions is reduced faster and reoxidized very slowly, and is assumed to be located away from the active site of ceruloplasmin. On the other hand, the other blue copper ion, which is reduced more slowly and reoxidized rapidly, is supposed to interact with other types of coppers, such as type II (non-blue) and type III (EPR undetectable) coppers. The active site of ceruloplasmin is considered to be comprised of one type I, one type II and a pair of type III copper ions.     

The conformational states of cytochrome oxidase in the mitochondrion.

THE Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized" oxidase. With increasing activity the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.     

Partial reduction of aquomethemoglobin on a sephadex G-25 column as detected by magnetic circular dichroism spectroscopy and revised extinction coefficients for aquomethemoglobin

In a typical preparation of aquomethemoglobin, oxyhemoglobin is oxidized with potassium ferricyanide, and the resultant mixture of methemoglobin and potassium ferro- and ferricyanides is separated on a Sephadex G-25 column. We find that about 1% of the heme is reduced on the column and is eluted with the methemoglobin. Magnetic circular dichroism spectra show that the reduced species is oxyhemoglobin. Magnetic circular dichroism is more sensitive than is absorption spectroscopy to small amounts of oxyhemoglobin in such solutions; we can detect its presence at the 0.1% level. A redetermination of the extinction coefficients for methemoglobin gives a value of 0.80 for the absorbance ratio A⁵⁷⁰/A⁶³⁰ at pH 6. This value lies clearly outside the currently accepted range of 0.83 to 0.87.     

Hexose monophosphate shunt-stimulated reduction of methemoglobin by divicine

Reduced divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, reduces methemoglobin efficiently in intact erythrocytes and in hemolysates. Oxidized divicine produces the same effect when glucose or an NADPH-generating system is added to intact erythrocytes or to hemolysates. Although NADPH, NADH, and GSH have no direct methemoglobin-reducing activity in vitro, they convert oxidized divicine to the reduced hydroquinone species, which is responsible for the electron transfer to methemoglobin. Reduction of methemoglobin is optimally observed under nitrogen since, in the presence of oxygen, reduced divicine undergoes autoxidation. Several lines of evidence rule out the reduction of methemoglobin by divicine through an enzyme-catalyzed process, although it is certainly sustained by the hexose monophosphate shunt activity of erythrocytes through the generation of both NADPH and GSH. Thus, the strong enhancing effect that glucose produces on the divicine-dependent methemoglobin reduction within intact normal erythrocytes is completely absent in erythrocytes from glucose-6-phosphate dehydrogenase-deficient subjects. This distinctive behavior might account for the enhanced methemoglobin levels that are found both in vitro in glucose-6-phosphate dehydrogenase-deficient erythrocytes exposed to divicine and in vivo as a typical feature of the acute hemolytic crisis of favic patients.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

A comparative study on the influence of cysteamine and metyrapone on mixed-function oxygenase activities in variously pretreated liver microsomes from rats and mice.

It has been found that metyrapone can inhibit both type I and type II mixed-function oxygenase reactions, while cysteamine inhibits only type I activity in this mammalian system. Following pretreatment with phenobarbital and 3-methylcholanthrene the half-maximal inhibiting concentrations for the O-demethylation of paranitranisol are increased for cysteamine and decreased for metyrapone. Both cysteamine and metyrapone give type II binding spectra with oxidized cytochrome P-450. The negative and positive peaks are at 393 and 426 nm respectively for metyrapone, and 410 and 434 nm for cysteamine. Cysteamine showed no binding comparable to that of metyrapone for reduced cytochrome P-450. Metyrapone showed little or no inhibition of the NADH cytochrome-c reductase (EC 1.6.1.1) or NADPH (EC 1.6.2.3) cytochrome-c reductase while cysteamine had a more or less strong inhibiting effect depending on the pretreatment of animals. Neither the binding to P-450 heme nor the inhibition of NADH and NADPH cytochrome-c reductase correlates well with cysteamine inhibition of total activity. It is therefore suggested that cysteamine reacts with an intermediate electron carrier of non-heme iron or glycoprotein character thus inhibiting mixed-function oxygenase activity.     

Singlet oxygen formation during hemoprotein catalyzed lipid peroxide decomposition

The singlet oxygen trap diphenylfuran was rapidly oxidized to cis dibenzoylethylene during the decomposition of linoleic acid hydroperoxide catalyzed by ceric ions, methemoglobin or hematin. This conversion was enhanced in a deuterated medium and inhibited by other singlet oxygen quenchers or traps. The chemiluminescence accompanying the decomposition of the linoleic acid hydroperoxide was also markedly enhanced in a deuterated medium and inhibited by other singlet oxygen quenchers or traps. Antioxidants markedly inhibited these reactions. It is concluded that singlet oxygen is formed in substantial quantities during the metal catalyzed decomposition of linoleic acid hydroperoxide.     

Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion

The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.     

Bactericidal effect of Fe2+, ceruloplasmin, and phosphate.

Fe2+, when combined with ceruloplasmin or phosphate, was bactericidal to Escherichia coli at pH 5.0, and when Fe2+, ceruloplasmin, and phosphate were combined, a bactericidal effect was observed under conditions, i.e., short incubation period, in which Fe2+ plus ceruloplasmin and Fe2+ plus phosphate were ineffective. Bactericidal activity increased with the ceruloplasmin or phosphate concentration to a maximum and then decreased as their concentration was further increased. Fe2+ was oxidized in the presence of ceruloplasmin, phosphate, or, in particular, a combination of the two. A bactericidal effect was observed when there was only a partial loss of Fe2+, with more extensive oxidation resulting in a loss of bactericidal activity. The bactericidal effect of Fe2+ plus ceruloplasmin and/or phosphate was unaffected by catalase or superoxide dismutase and was not associated with iodination. Fe-EDTA was also bactericidal at an Fe2+: EDTA molar ratio of 1:0.5, where Fe2+ was partially oxidized. However, in contrast to Fe2+ plus ceruloplasmin and/or phosphate, bactericidal activity was inhibited by catalase and was associated with iodination. Combinations of Fe2+ and Fe3+ were not bactericidal under the conditions employed. A requirement for Fe2+ plus either a product of Fe2+ oxidation or an iron ceruloplasmin and/or phosphate chelate for bactericidal activity is proposed.     

Inhibition by thiols of copper(II)-induced oxidation of oxyhemoglobin.

The ability of thiols, 2-imidazolethiones and uric acid to protect bovine oxyhemoglobin from copper(II)-induced oxidation to methemoglobin was investigated. The oxidation of oxyhemoglobin by Cu(II) proceeded in two phases: (1) an initial rapid reaction (less than 30 s) followed by (2) a slower reaction that carried it to completion. Thiols, including N-acetyl-L-cysteine, DL-dithiothreitol, reduced glutathione, DL-homocysteine, 2-mercaptoethanol and 2- and 3-mercaptopropionic acid, whose sulfhydryl groups were slowly oxidized by Cu(II) (with the exception of 2-mercaptopropionic acid), protected oxyhemoglobin in both phases of the reaction. Other thiols, including L-cysteine, cysteamine, and D-penicillamine, whose sulfhydryl groups were readily oxidized by Cu(II), protected hemoglobin initially, but within 2-4 min, the rate of methemoglobin formation was the same as Cu(II)-treated oxyhemoglobin. 2-Mercaptoimidazole and 1-methyl-2-mercaptoimidazole, which complex Cu(II) and inhibit Cu(II)-catalyzed oxidation of ascorbic acid, also protected hemoglobin in the initial phase, but not in the second phase. Uric acid, L-ergothioneine, and thiourea did not protect oxyhemoglobin in either the fast or slow phase. Cu(II) may have a coordination site involved in the oxidation of hemoglobin that is not blocked by the 2-imidazolethiones, uric acid, or the oxidized thiols. It is concluded that certain thiols that complex Cu(II) and are not rapidly oxidized will protect oxyhemoglobin from Cu(II)-induced oxidation, but the thiols are no longer effective once they are oxidized.     

Metabolic effects of hemoglobin gene expression in plants

Hemoglobin (Hb) genes are ubiquitous in plants. Several classes have been identified and are expressed during infection by nitrogen-fixing symbionts, as a result of tissue hypoxia, during seed germination, and in developing (e.g. meristematic) tissues. The induction of the Hb gene by hypoxia is linked to a decrease in ATP levels and is mediated by Ca(2+). Numerous investigations have led to the conclusion that the main function of hypoxically-induced Hb is to metabolize nitric oxide (NO) formed as a by-product of nitrate/nitrite reduction. In this function, Hb serves as a part of an NO dioxygenase system, using traces of oxygen to convert NO to nitrate. It operates in conjunction with a methemoglobin reductase protein, which reduces the oxidized form of Hb (methemoglobin) formed in the course of the NO dioxygenase reaction. The complete reaction serves to maintain the cellular energy and redox state. Plant hemoglobins may also function to modulate effects of plant hormones that employ NO as a downstream signal transduction component.     

Structure and function of cytochrome-c oxidase

Recent works on the structure and the function of cytochrome-c oxidase are reviewed. The subunit composition of the mitochondrial enzyme depends on the species and is comprised of between 5 and 13 subunits. It is reduced to 1 to 3 subunits in prokaryotes. The complete amino acid composition has been derived from protein sequencing. Gene sequences are partially known in several eukaryote species. Metal centers are only located in subunits I and II. The mitochondrial cytochrome-c oxidase is Y-shaped; the arms of the Y cross the inner membrane, the stalk protrudes into the intermembrane space. The bacterial enzyme has a simpler, elongated shape. A number of data have been accumulated on the subunit topology and on their location within the protein. All available spectrometric techniques have been used to investigate the environment of the metal centers as well as their interactions. From the literature, attention must be paid to what may be considered or not as an active form. The steady improvement of the instrumentation has yielded evidence for different kinds of heterogeneities which could reflect the in vivo situation. The 'pulsed' and 'resting' conformers have been well characterized. The 'oxygenated' form has been identified as a peroxide derivative of the fully oxidized cytochrome-c oxidase. The mammalian enzyme has been isolated in fully active monomeric form which does not preclude the initially suggested dimeric behavior in situ. The role of the lipids is still largely investigated, mainly through reconstitution experiments. Kinetic studies of electron transfer between cytochrome c and cytochrome-c oxidase lead to a single catalytic site model to account for the multiphasic kinetics. Results related to the low temperature investigation of the intermediate steps in the reaction between oxygen and cytochrome-c oxidase received a sound confirmation by the resolution of compound A at room temperature. It is also pointed out that the so-called mixed valence state might not be a transient state in the catalytic reduction of oxygen. The functioning of cytochrome-c oxidase as a proton pump has been supported by a number of experimental results. Subunit III would be involved in this process. The redox link to the proton pump has been suggested to be at the Fea-CuA site. The molecular mechanism responsible for the proton pumping is still unknown.     

The interaction between nitrogen oxides and hemoglobin and endothelium-derived relaxing factor

Among nitrogen oxides, NO and NO₂ are free radicals and show a variety of biological effects. NO₂ is a strongly oxidizing toxicant, although NO, not oxidizing as NO₂, is toxic in that it interacts with hemoglobin to form nitrosyl-and methemoglobin. Nitrosylhemoglobin shows a characteristic electron spin resonance (ESR) signal due to an odd electron localized on the nitrogen atom of NO and reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase in red cells. NO was found to inhibit the reductase activity. Part of NO inhaled in the body is oxidized by oxygen to NO₂, which easily dissolves in water and converts to nitrite. The nitrite oxyhemoglobin autocatalytically after a lag. The mechanism of the oxidation, particularly the involvement of superoxide, was controversial. The stoichiometry of the reaction has now been established using nitrate ion electrode and a methemoglobin free radical was detected by ESR during the oxidation. Complete inhibition of the autocatalysis by aniline or aminopyrine suggests that the radical catalyzes conversion of nitrite to NO₂, which oxidizes oxyhemoglobin. Recently NO was shown to be one of endothelium- derived relaxing factors and the relaxation induced by the factor was inhibited by hemoglobin and potentiated by superoxide dismutase.     

Modification of redox properties of cells by ortho-benzoquinones

Ortho-benzoquinones have been studied for their effect on the processes of methemoglobin formation in the rabbit erythrocytes and reduction of potassium ferricyanide by the HeLa cells. It is established that the incubation of cells in the presence of lipophilic quinone OBQ-1 results in the formation of the intracellular methemoglobin in erythrocytes and the intensification of the ferricyanide reduction by HeLa cells. OBQ-2 differing in the presence of the polar sulphogroup does not react with the intracellular oxyhemoglobin and exerts no effect on the ferricyanide reduction. It is supposed that OBQ-1 may change the ratio of the oxidized and reduced metabolites in a cell, thus inducing in it a state of the oxidative stress. A conclusion is drawn hat ortho-benzoquinones are able to modify the redox properties of cells, the efficiency of modification depending on the chemical structure of the compounds.     

Cyanosis by methemoglobinemia in tadpoles of Cochranella granulosa (Anura: Centrolenidae)

Tadpoles inhabit generally well oxygenated rivers and streams, nevertheless they were found in areas with limited oxygen availability inside the rivers. To assess this feature, I examined factors that influence centrolenid tadpole behaviour using Cochranella granulosa. The tadpoles were reared in well-oxygenated and hypoxic environments and their development, survivorship and growth were compared. The tadpoles in oxygenated water acquired a pale color, while tadpoles in hypoxic water grew faster and were bright red and more active. In the oxygenated water, the ammonium, which had its origin in the tadpoles' urine and feces, was oxidized to nitrate. In contrast, in the hypoxic treatment, the nitrogen compounds remained mainly as ammonium. Presumably, the nitrate in oxygenated water was secondarily reduced to nitrite inside the long intestine coils, because all symptoms in the tadpoles point to methemoglobinemia, which can occur when the nitrite passes through the intestine wall into the bloodstream, transforming the hemoglobin into methemoglobin. This could be checked by a blood test where the percentage of methemoglobin was 2.3% in the blood of tadpoles reared in hypoxic condition, while there was a 19.3% level of methemoglobin in the blood of tadpoles reared in oxygenated water. Together with the elevated content of methemoglobin, the growth of the tadpoles was delayed in oxygenated water, which had high nitrate content. The study about quantitative food-uptake showed that the tadpoles benefit more from the food in hypoxic water, although they spent there more energy moving around than the tadpoles living in oxygenated but nitrate-charged water.