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PubMed1981

Kinetic distinction between cytochromes a and a3 in cytochrome c oxidase. Rapid scanning stopped flow study of anaerobic reduction by a neutral and a negatively charged donor.

Anaerobic reduction of cytochrome c oxidase by 5,10-dihydro-5-methylphenazine (reduced PMS) and by sodium dithionite were studied by rapid scanning stopped flow spectrophotometry. In both cases the decay of the Soret band of the oxidized oxidase is not uniform. With reduced PMS, the reduction involves two molecules of reductant (4 electrons)/oxidase molecule. The first stage of the reduction exhibits an isosbestic point in the Soret region at 437 nm. This shifts to 428 nm in later stages of the reaction. The reduction of the oxidase by sodium dithionite is also complete and apparently involves SO2 radical. In this case the spectra show an isosbestic point at approximately 420 nm which shifts to 432 nm as the reaction proceeds. For each of the reductants the reaction is best described by three phases: the first is a second order reaction between the oxidase and the reductant, followed by two first order processes which appear to describe the intramolecular electron redistribution within the oxidase molecule. The results agree with the assignment of the Soret band of the oxidase molecule to cytochrome a3 with an absorption maximum near 410 nm and to cytochrome a which has its maximum absorption hear 430 nm. If these assignments are correct, the present data show that reduced PMS, an uncharged molecule, reacts more rapidly with cytochrome a than it does with cytochrome a3, while the negatively charged radical anion, SO2, appears to have more direct access to cytochrome a3.

Anaerobiosis↗
PubMed1978

Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states.

NADPH-cytochrome P-450 reductase was isolated from liver microsomes of phenobarbital-induced rats. The enzyme exhibits an apparent minimal molecular weight of 76,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 molecule each of FMN and FAD. Trypsin treatment of the reductase yields an enzyme with an apparent minimal molecular weight of 69,000 which retains the ability to reduce cytochrome c but has no activity toward cytochrome P-450. Various spectrophotometric titrations were performed to examine the electron-accepting properties of the purified NADPH-cytochrome P-450 reductase and, in particular, to determine the oxidation state of the stable semiquinone form produced by air oxidation of NADPH-reduced enzyme. Titration of the air-stable semiquinone form of the reductase with ferricyanide indicated that 1 mol/2 mol of flavin was required for complete oxidation. Furthermore, a spectrum corresponding to that of the air-stable semiquinone form was produced by the addition of approximately 0.5 mol of reductant/2 mol of flavin when the oxidized enzyme was titrate with NADPH or dithionite under anaerobic conditions. The spectral changes which accompanied the overall reduction of oxidized enzyme to the reduced form with dithionite produced four sets of isosbestic points, and the spectrophotometric titration curve consisted of four approximately equal phases. In the titration with NADPH, no significant further reduction was observed after the addition of approximately 1.5 mol/2 mol of flavin. However, the enzyme was fully reduced by NADPH when an NAPH-generating system was used to prevent the accumulation of NADP. Our results establish that the air-stable semiquinone form is a 1-electron-reduced form, rather than a half-reduced (2-electron-reduced) form as maintained by others and are in agreement with earlier studies (Iyanagi, T., Makino, N., and Mason, H.S. (1974) Biochemistry 13, 1701-1710) with the purified trypsin-solubilized reductase. Accordingly, the air-stable species represents a form of the NADPH-cytochrome P-450 reductase in which one of the two flavins exists in the semiquinone state and the other in the oxidized state.

Animals↗
PubMed1978

The reaction of ferrous leghemoglobin with hydrogen peroxide to form leghemoglobin(IV).

Ferrous leghemoglobin reacts with hydrogen peroxide to form the stable product, leghemoglobin(IV). The reaction follows second order kinetics (k = 2.24 X 10(4) M-1 S-1 at 20 degrees C) and may be regarded as a single-step, two-electron oxidation. Ferric leghemoglobin is not an intermediate. The oxidation state of leghemoglobin(IV) is established by reductive titration with dithionite; 2 eq of dithionite are required to convert 1 mol of leghemoglobin(IV) to ferrous leghemoglobin. An outstanding property of leghemoglobin(IV) is its stability, little change is noted after 12 h at 25 degrees C. Leghemoglobin(IV) differs from the higher oxidation states of other hemoglobins and myoglobins in that it does not react with hydrogen peroxide to form the oxygenated protein.

Dithionite↗
PubMed1981

Ferredoxin and ferredoxin reductase activities in bovine thyroid. Possible relationship to iodotyrosine deiodinase.

NADPH-ferredoxin reductase and ferredoxin activities have been identified in bovine thyroid particulate fractions (mainly mitochondria) after ultrasonication and DEAE-cellulose chromatography. The proteins were identified by their ability to reconstitute NADPH-cytochrome c reductase activity when used in combination. NADPH ferredoxin reductase and ferredoxin also catalyzed NADPH-dependent deiodination of L-diiodotyrosine; bovine adrenodoxin and adrenodoxin reductase could partially replace the thyroidal components in NADPH-dependent deiodination of L-diiodotyrosine. Both these reconstitutive activities were substantially inhibited by the iron chelators, alpha, alpha'-dipyridyl and o-phenanthroline. Deiodination by the NADPH-ferredoxin reductase-ferredoxin system was inhibited by the addition of a previously characterized dithionite-responsive flavoprotein iodotyrosine deiodinase (preparation F), isolated and purified from bovine thyroid particulate fractions after solubilization with steapsin. Ferredoxin reductase alone showed dithionite-responsive deiodinase activity and elution profiles of this activity on gel filtration before and after steapsin treatment suggest that preparation F may be a form of ferredoxin reductase modified by steapsin.

Animals↗
PubMed1978

Measurement of sickling by controlled temperature increase.

Previous experiments to study the rate of red cell sickling have employed rapid mixing apparatus for SS cells with dithionite and have shown that the half-time of sickling is quite rapid, on the order of seconds. An alternative approach is to slow down the rate by taking advantage of the negative temperature coefficient of the process. We developed a method in which deoxygenation of a cell suspension is carried out at 0 degrees C. A linear temperature gradient to 37 degrees C is applied, and a gradual increase in the percentage of sickled cells is observed. At a heating rate of 1.5 degrees C/min the temperature at which half of the cells became sickled was 19 degrees C for SS cells treated with dithionite, 22 degrees C for SC cells, 28 degrees C for AS cells, 22 degrees C for cyanate-treated SS cells, and 23 degrees C for SS cells in the presence of 0.1 M butylurea. Thus this method promises to be useful for the study of sickling rates and the screening of potential anti-sickling agents.

Anemia, Sickle Cell↗
PubMed1980

Interference of imferon in colorimetric assays for iron.

Imferon (Merrell-National), an iron-dextran complex, is widely used in patients with iron deficiency. It is present in the circulation in appreciable amounts for two to three weeks after administration and interferes with all tested colorimetric iron assays, both with and without deproteinization. The amount of the plasma Imferon iron interference depends primarily on the choice of reductant. With dithionite it is essentially 100%. In the presence of ascorbic acid and hydroxylamine, the interference depends also on assay conditions, especially temperature, but also incubation time and pH. The minimum interference in a homogeneous assay was about 3%. The relative amount of interference from hemoglobin iron under the various assay conditions is different from that of Imferon iron. In the presence of a reducing agent, the dextran-iron complex decomposes--instantaneously with dithionite, and gradually with sulfite, ascorbic acid, and hydroxylamine. The freed iron becomes dialyzable, can react with bathophenanthroline, and elutes on a Sephadex G-50 or G-15 column in the same fractions as an ammonium ferrous sulfate.

Ascorbic Acid↗
PubMed1980

A survey of reducing agents for the synthesis of tetraphenylarsonium oxotechnetiumbis(ethanedithiolate) from [99Tc] pertechnetate in aqueous solution.

We have studied the effectiveness of various reducing agents in the production of the well-characterized complex [99TcO(SCH2CH2S)2]- from pertechnetate in aqueous solution. The reductants tested included sodium dithionite, hypophosphorous acid, formamidine sulfinic acid, dithiothreitol, hydrazine, and hydroxylamine. Of these, only sodium dithionite in the pH range 11--13 was found to give quantitative yields of the required technetium complex.

Arsenicals↗
PubMed1995

Genetic functions of isolated maize mitochondria under model changes of redox conditions.

The template functions of maize mitochondrial genome have been studied under model conditions of redox potential created by the addition of potassium ferricyanide as an oxidising agent and sodium dithionite as a reducing agent. The addition of both potassium ferricyanide and sodium dithionite to the isolated mitochondria causes the changes in the activity of the mitochondria RNA and DNA synthesis. The data obtained indicate the possibility of redox regulation of genetic functions in isolated mitochondria.

Alkaloids↗
PubMed1977

Evidence that puriified liver microsomal cytochrome P-450 is a one-electron acceptor.

Two forms of highly purified liver microsomal cytochrome P-450, P-450LM2, and P-450LM4, have been titrated with standardized solutions of sodium dithionite under anaerobic conditions. Only 1 electron was consumed per hemin molecule, and reoxidation of the reduced heme is accompanied by the transfer of 1 electron to oxidizing agents such as cytochrome c, cytochrome b5, or potassium ferricyanide. The present results are in disagreement with earlier dithionite titrations and reoxidation experiments which indicate that liver microsomal cytochrome P-450 is a 2-electron acceptor, but are in accord with previous potentimetric titrations and product yield data which indicate that this hemeprotein is a 1-electron acceptor. The cause for the discrepancy between the present and previous results is not fully understood.

Animals↗
PubMedDate not supplied

[Irreversible changes in cytochrome P-420 differential spectrum amplitude in microsomal membranes. New properties of Cu(II)-EDTA complex].

The influence of EDTA on the CO-differential spectra of cytochrome P-420 formed in the rat liver microsomes under the effect of 1 mM CuCl2 in the presence of dithionite has been studied. It was shown that the level of revealed cytochrome P-420 was diminished two-fold following 10 mM EDTA addition if dithionite concentration initially added to the medium was about 2 mM. Cytochrome b5 had no changes. Cytochrome P-420, formed under the effect of desoxycholate, was insensitive to EDTA, but its amount decreased after addition of CuCl2. It is supposed to proceed under chelatation of heme Fe(II).

Animals↗
PubMedDate not supplied

Ferricytochrome c encapsulated in silica nanoparticles: structural stability and functional properties.

Using a modified sol-gel technique, we have succeeded in encapsulating ferric cytochrome c in silica nanoparticles obtained from hydrolysis and polycondensation of tetramethylorthosilicate. Particles dimensions have been determined with dynamic light scattering; this technique yields an hydrodynamic radius of about 100 nm, each nanoparticle containing about 10(2)-10(3) proteins. If stored in the cold at low ionic strength, nanoparticles are stable for more than one week, even if a slow radius increase with time is observed. CD measurements show that encapsulated proteins exhibit substantially increased stability against guanidinium hydrochloride induced denaturation. Reduction kinetics of encapsulated ferric cytochrome c by sodium dithionite, measured with standard stopped flow techniques, are slower by a factor of ten with respect to those measured in solution. Analogous experiments with myoglobin suggest that this slowing down is due to the diffusion time of dithionite within the silica matrix. Indeed, if a smaller ligand like CO is used, the intrinsic kinetic properties of encapsulated proteins are found to be unaltered even in the millisecond time range. The reported data show that our nanoparticles are extremely useful both for basic research, to study the stability and functions of encapsulated proteins, and for their potential biotechnological applications.

Animals↗
PubMed2003

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation.

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.

Bioreactors↗
PubMed1999

Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.

Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids. The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.

Allosteric Regulation↗
PubMed2002

High-level expression of human liver monoamine oxidase A in Pichia pastoris: comparison with the enzyme expressed in Saccharomyces cerevisiae.

The high-level expression, purification, and characterization of recombinant membrane-bound human liver monoamine oxidase A (MAO-A) in Pichia pastoris is described. Two liters of fermentation culture produces 1170 units (660 mg) of MAO-A. The enzyme is purified in a 35% yield, is homogeneous on denaturing gel electrophoresis, and exhibits a single species (60,512 +/- 6 Da) on electrospray mass spectrometry. It contains 1 mol of 8alpha-S-cysteinyl FAD/mole of enzyme and exhibits >95% functionality. In contrast, the Saccharomyces cerevisiae-expressed enzyme is partially processed by C-terminal serine removal as demonstrated by mass spectra. The amino termini of both P. pastoris- and S. cerevisiae-expressed MAO-A are acetylated on the N-terminal methionine. The steady-state kinetic properties of P. pastoris-expressed MAO-A are similar to those of S. cerevisiae-expressed MAO-A using the following substrates: phenethylamine, p-CF(3)-benzylamine, dopamine, serotonin, and kynuramine. Reductive titrations demonstrate that the recombinant enzyme is reduced by 1 mol of substrate or dithionite as expected for the two electron equivalents required for flavin reduction. Absorption and EPR spectra show no radical species in the resting enzyme while the anionic flavin radical is formed in 50% yield during the reductive titration with dithionite. These data demonstrate significant advantages in the heterologous expression of human MAO-A in P. pastoris compared with the published S. cerevisiae system in higher expression level (329 mg/L) and in a higher level of homogeneity of the isolated enzyme.

Cloning, Molecular↗
PubMed1991

Transformation of trichloroethylene by sulfate-reducing cultures enriched from a contaminated subsurface soil.

Trichloroethylene (TCE) was reductively dechlorinated to cis-1,2-dichloroethylene (cDCE) by sulfate-reducing cultures enriched from a contaminated subsurface soil. The highest observed transformation rate of TCE was 213 mumol l-1 per day at 35 degrees C. The predominant biotransformation product was cDCE. However, further dechlorination of cDCE was not observed in most of the cultures. Methane production was insignificant and active sulfate reduction was achieved by maintaining excess sulfate. A comparison of sodium sulfide and sodium dithionite for their effect on the transformation of TCE revealed that the latter is a better reducing agent. The extent of TCE transformation in 25 days was ca. 20% higher in the dithionite-amended cultures. A decrease in the rate and extent of TCE transformation was observed with an increase in the concentration of bromoethanesulfonate up to 50 mM.

Bacteria↗
PubMed1987

Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes. Reinvestigation of assay conditions.

Assay conditions in determining total cytochrome P-450 in four laboratories were compared. Although the determination was derived from the original Omura and Sato method in each laboratory, the four standard protocols differed slightly, resulting in considerable differences in the results. Since the cytochrome P-450 content is usually expressed per mg protein, the protein assay conditions were evaluated as well. Furthermore, we compared the cytochrome P-450 values obtained by the CO- and the dithionite (DT)-difference methods. The effect of a number of variables in the assay was investigated. The influence of the storage temperature of the microsomes was ascertained as well as effects of the gassing time with CO and the time between addition of dithionite, CO-gassing and the recording of the difference spectra. After evaluating these variables a standard operation procedure was established. Using this procedure the interlaboratory coefficient of variation for total cytochrome P-450 was 4.8%, a value which was comparable to the intralaboratory coefficients of variation. The final results also show that the millimolar extinction coefficient for the DT-difference method is higher than for the CO-difference method.

Animals↗
PubMed1986

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans.

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

Ammonia↗
PubMed1981

Orientation of complex III in the yeast mitochondrial membrane: labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate.

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [125I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochrome b revealed that the two core proteins and cytochrome b were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochrome c1 and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromes b and c1 was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15-30% of ;the total dithionite-reducible b and 100% of the cytochrome c1: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochrome b but insignificant amounts of cytochrome c1. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.

Diazonium Compounds↗