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PubMed1983

Inhibition by dithionite and reactivation by iron of the tartrate-resistant acid phosphatase in bone of osteopetrotic (ia) rats.

The staining intensity and inhibitor sensitivity of acid phosphatase activity was determined histochemically in various tissues of normal and ia rat pups by the use of freeze-dried whole body sections. Activity was determined using alpha-naphthylphosphate as substrate and hexazonium pararosaniline as coupler. Sections from ia rats (6 and 24 days old) showed markedly higher enzyme activity in bone than sections from normal littermates. However, there were no differences between ia and normal pups in acid phosphatase activity in soft tissues and developing teeth. Preincubation of sections with 1-100 mM sodium dithionite (an iron-binding agent) caused a dose-related inhibition of enzyme activity in bone of ia and normal pups, but only slight inhibition of activity in soft tissues. Partial restoration of the dithionite-inhibited activity in bone was achieved by subsequent preincubation in 1 mM FeCl2. Addition of 100 mM sodium tartrate to the staining solution of non-preincubated sections caused almost complete inhibition of activity in soft tissues and the developing teeth but no inhibition of the activity in bone that was sensitive to sodium dithionite. These data indicate a) that sodium dithionite can be used as a specific histochemical inhibitor of the tartrate-resistant acid phosphatase and b) that the source of increased acid phosphatase activity in bone from ia rats is mostly from the tartrate-resistant acid phosphatase.

Acid Phosphatase↗
PubMed2005

Sulfur speciation by capillary zone electrophoresis. Determination of dithionite and its decomposition products sulfite, sulfate and thiosulfate in commercial bleaching agents.

In this paper, a capillary zone electrophoretic (CZE) method was developed for the separation of the sulfur species dithionite (S2O4(2-)), sulfite (SO3(2-)), sulfate (SO4(2-)) and thiosulfate (S2O3(2-)). A carrier electrolyte (pH 7.0) containing 1.5 mmol L(-1) pyromellitic (PM) acid, 10 mmol L(-1) Tris(hydroxymethyl)-aminomethane (Tris), 0.5 mmol L(-1) diethylenetriamine (DETA) and 0.1% (v/v) formaldehyde (as stabilizer for S2O4(2-) and SO3(2-)) allowed the determination of the sulfur anions after 9 min CZE separation with indirect UV detection at 214 nm. The addition of 0.1% (v/v) formaldehyde to the sample solution stabilizes dithionite and sulfite as HOCH2SO2- and HOCH2SO3- anions. The procedure was applied for the determination of dithionite and its decomposition products sulfite, sulfate and thiosulfate in commercial formulations of bleaching agents. Dithionite was found to be the major component of the commercial formulations in concentrations between 30.80 and 33.30% (w/w). As anticipated, sulfite, sulfate and thiosulfate were found to be present as decomposition or by-products in the commercial formulations at concentrations of 14.30-14.80, 5.20-5.70 and 0.30-0.40% (w/w), respectively. The results were found to be in good agreement with those of polarographic and spectrophotometric determinations.

Electrophoresis, Capillary↗
PubMed1983

Na-Dithionite Promotes Photosynthetic Sulfide Utilization by the Cyanobacterium Oscillatoria limnetica.

The light- and sulfide-dependent induction process leading to photosystem I-mediated sulfide utilization by Oscillatoria limnetica, for either H(2) evolution or CO(2) photoassimilation, was studied. The identical dependence on pH of the lag length, the inhibition of leucine incorporation and final H(2)S concentration imply that the latter exerts a deleterious effect on nonadapted cells.Na-dithionite (Na(2)S(2)O(4)), Na-sulfite (Na(2)SO(3)), or ethanol cannot serve as photosynthetic electron donors. However, when these compounds were added to the sulfide-containing system, the need for induction was eliminated. At pH 6.9, in the presence of 3.5 millimolar sulfide, these substances (at concentrations of 10 millimolar, 5 millimolar, or 0.4 molar, respectively) completely abolished the delay preceding sulfide-dependent H(2) evolution. It is suggested that all three compounds expose a site capable of directly accepting sulfide electrons.Only dithionite could adapt the cells to sulfide utilization on its own. Sulfite or ethanol acted only in the presence of sulfide. It is implied that this specific activity of dithionite is related to its characteristic low redox potential.Sulfide-dependent H(2) evolution was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but was inhibited by the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, in the presence as well as in the absence of dithionite. In both cases, therefore, the plastoquinone was implied in the electron transport from sulfide.

Journal Article↗
PubMed1984

Chemically coupled spectrophotometric assays based on flow injection analysis: determination of nitrogenase by assays for creatine, ammonia, hydrazine, phosphate, and dithionite.

Micromethods of direct chemical coupling have been developed for several different enzyme reactions, using the principles of flow injection analysis. Samples of 1-25 microliters are injected into a flowing stream of color-forming reagents and the peak of color change is measured after about 1 min. Alternatively, continuous slow infusion of a reacting system (5-100 microliters/min) gives a continuous change of color which can be monitored to derive enzyme reaction rates. These techniques are highly sensitive, requiring a few nanomoles of the substance being detected. Phosphate, ammonia, dithionite, creatine, and hydrazine have been measured. Consumption of reagents is less than 75 ml per hour; typical sample throughout is 30-40 samples per hour by the injection method, and 5 samples per hour by continuous infusion. The procedure has been applied to nitrogenase, continuously monitoring creatine produced from creatine phosphate by creatine kinase which is used to supply a constant level of ATP for nitrogenase. In this way nitrogenase activity can be determined over a wide range of enzyme concentrations. Production of inorganic phosphate directly from ATP, by injection of formaldehyde-quenched samples, was used when coupling to creatine kinase was not possible. Both injection of aliquots and continuous infusion were used for detection of hydrazine during nitrogenase reduction of azide, and the injection method has been used for ammonia assay during dinitrogen reduction. Dithionite oxidation was measured directly from decolorization of iodine, after trapping both dithionite and bisulfite with formaldehyde.

Ammonia↗
PubMed1988

Mechanism of reductive activation of a 5-nitroimidazole by flavoproteins: model studies with dithionite.

The flavoprotein nitroreductases NADPH:cytochrome P-450 reductase and xanthine oxidase catalyzed the cofactor-dependent anaerobic nitro group reduction and covalent binding to protein sulfhydryl groups of the 5-nitroimidazole substrate ronidazole [1-methyl-5-nitroimidazole-2-yl)-methyl carbamate). Studies with variously radiolabeled ronidazole molecules demonstrated that the imidazole ring was intact while greater than 80% of the C-4 3H and 2-carbamoyl group were lost from the covalently bound product. The stoichiometry of cofactor consumption during the enzyme-catalyzed reduction of the substrate could not be determined, so a model nitroreductase system which utilized dithionite as the reductant and agarose-immobilized cysteine as the target for alkylation was developed. Two moles of dithionite was consumed per mole of substrate for maximal reduction of uv absorbance due to the nitro group, for maximal release of C-4 3H, and for maximal covalent binding to agarose-immobilized cysteine. These results indicate that four electrons are required for the reductive activation of the substrate, consistent with formation of a hydroxylamine reactive intermediate. Covalent binding of variously radiolabeled substrate molecules after dithionite reduction exhibited the same labeling pattern as flavoprotein-catalyzed covalent binding, suggesting that covalent binding is mediated by the same species in both chemical and biological systems. The data are consistent with a mechanism where the substrate undergoes four-electron reduction to form a hydroxylamine, which is susceptible to nucleophilic attack at C-4. When water attacks C-4, the 2-carbamoyl group can eliminate to form a Michael-like acceptor which adds thiols at the 2-methylene position.

Animals↗
PubMed1990

Formation and decay of cytochrome c peroxidase compound ES during aerobic reduction with dithionite.

Stopped-flow and rapid scanning studies have clearly demonstrated that mixing of an oxygen-saturated solution of yeast cytochrome c peroxidase with sodium dithionite yields compound ES, indicating generation of H2O2. The formation of compound ES was most pronounced when [Na2S2O4]/[O2] approximately 1, and it reverted to the ferric form while standing. Even in the presence of an excess of dithionite ([Na2S2O4]/[O2] = 3.4) compound ES was formed immediately, but was soon replaced by the ferric form, followed by its final reduction to the ferrous state. The apparent first order rate constant for the decay of compound ES to the ferric form increased linearly with the square root of the dithionite concentration, thus involvement of SO2- in that process being suggested.

Aerobiosis↗
PubMed1985

Calorimetric studies of oxyhemoglobin dissociation. II. Erythrocytic oxygen depletion by sodium dithionite.

Dithionite causes the depletion of dioxygen from suspensions of erythrocytes by reduction of the external dioxygen and not by diffusion into the cell. The molar enthalpy for the reduction shows a small difference with respect to the values found for free hemoglobin; and the normal stoichiometry of 2 moles dithionite/mole dioxygen found there is not observed with erythrocytes. At low hematocrit, the stoichiometry is 2.6:1 and decreases to 1.5:1 at high hematocrit. The change is not due to differences in the hemoglobin saturation or to an inability of dithionite to reduce all dioxygen present at the higher hematocrit. Neither catalase nor peroxidase added to the extracellular volume significantly alters the stoichiometry or the enthalpy of dioxygen reduction by dithionite. Addition of superoxide dismutase, however, restores the normal stoichiometry at high hematocrit and further increases the stoichiometry at low hematocrit. The calorimetrical signal of hydrogen peroxide, clearly seen with free dioxygen, is not present with erythrocytes. In all these cases the total heat evolved is the same.

Animals↗
PubMed1985

Kinetics of dithionite reduction of the heme nonapeptide of cytochrome c.

The kinetics of dithionite reduction of the oxidized heme nonapeptide fragment of horse heart cytochrome c have been measured as a function of ionic strength at pH 7 and pH 9 by the stopped-flow technique. Dithionite concentration dependences indicate that the radical anion monomer, SO2-., is the active reductant. The pH 7 ionic strength dependence suggests that the heme peptide is reacting as a negatively charged molecule (its overall charge is calculated to be -1). Comparison of these results with the known rate of dithionite reduction of cytochrome c indicates that the heme nonapeptide has substantially greater inherent reactivity than cytochrome c, perhaps due to the greater accessibility of the heme.

Animals↗
PubMed1996

Direct 99mTc-labeling of antibodies by sodium dithionite reduction, and role of ascorbate as a stabilizer in cysteine challenge.

A method for the direct 99mTc-labeling of antibodies by dithionite reduction was developed. Among three murine monoclonal IgG1 and one human polyclonal IgG (hIgG) antibodies tested, hIgG was the most quickly reduced by dithionite. These differences may reflect the reactivities of antibody disulfide bonds toward the oxidation products of dithionite. By optimizing reduction conditions to generate enough free sulfhydryl groups, it was possible to radiolabel human IgG and monoclonal antibody 170 with 99mTc with a 90% monomeric antibody efficiency. The process avoided colloid formation. In contrast, about 0.1 sulfhydryl groups per antibody molecule, less than 1% of the possible 36, were detected after treatment with ascorbate (up to 35,000:1 molar ratio) at room temperature for 1 h for the antibodies tested. Sulfhydryl groups generated in antibodies were estimated using a new method: 5-iodoacetamidofluorescein-labeled antibodies quantitated by size exclusion HPLC. Ascorbate was found to prevent antibody aggregate formation in cysteine-challenged samples.

Animals↗
PubMed1987

Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre.

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 X 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 X 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 X 10(5) less than k less than 3 X 10(6) M-1.s-1 and protein conformation change 6 X 10(2) less than k less than 6 X 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.

Adenosine Triphosphate↗
PubMed1989

Kinetics and mechanism of electron transfer from dithionite to microsomal cytochrome b5 and to forms of the protein associated with charged and neutral vesicles.

The kinetics of the dithionite reduction of calf liver microsomal cytochrome b5, both free in solution and bound to dimyristoyl phosphatidylcholine vesicles, are consistent with electron transfer between SO2- and the exposed haem edge of the protein. The vesicle membrane does not hinder the approach of SO2- to the site of electron transfer on the protein. In 0.01 M-Tris/HCl buffer, pH 8.1, ket (25 degrees C), delta H et and delta S et are estimated to be 1.44 x 10(6) M-1.s-1, 7.8 kJ.mol-1 and -92.3 J.K-1.mol-1 respectively. The cytochrome exhibits an acid dissociation, pKa 9.3 +/- 0.3, and the rate of electron transfer from dithionite to the high-pH form is about one-third of that to the neutral-pH form. The effect of ionic strength on the kinetics is consistent with a reaction between like-charged species and is discussed in terms of a number of theoretical models. In systems comprising cytochrome b5 and negatively charged vesicles, the effect of increasing the charge density of mixed dimyristoyl phosphatidylcholine/dicetyl phosphate vesicles and of increasing the concentration of dicetyl phosphate vesicles is to lower the rate of electron transfer from dithionite to the haem moiety of the cytochrome. With vesicles of high charge density, however, the kinetics are complicated by vesicle-induced conformation changes of the cytochrome.

Animals↗
PubMed2002

Dithionite reduction kinetics of the dissimilatory copper-containing nitrite reductase of Alcalegenes xylosoxidans. The SO(2)(.-) radical binds to the substrate binding type 2 copper site before the type 2 copper is reduced.

We report here the first detailed study of the dithionite reduction kinetics of a copper-containing dissimilatory nitrite reductase (NiR). The reduction of the blue type 1 copper (T1Cu) center of NiR preparations that contained both type 1 and type 2 copper atoms, followed biphasic kinetics. In contrast, NiR that was deficient in type 2 copper (T2DNiR), followed monophasic kinetics with a second-order rate constant (T2D)k = 3.06 x 10(6) m(-1) s(-1). In all cases the SO(2)(.-) radical rather than S(2)O(4)(2-) was the effective reductant. The observed kinetics were compatible with a reaction mechanism in which the T1Cu of the fully loaded protein is reduced both directly by dithionite and indirectly by the type 2 Cu (T2Cu) site via intramolecular electron transfer. Reduction kinetics of the T2Cu were consistent with SO(2)(.-) binding first to the T2Cu center and then transferring electrons (112 s(-1)) to reduce it. As SO(2)(.-) is a homologue of NO(2)(-), the NiR substrate, it is not unlikely that it binds to the catalytic T2Cu site. Effects on the catalytic activity of the enzyme using dithionite as a reducing agent are discussed. Reduction of the semireduced T1Cu(I)T2Cu(II) state followed either second-order kinetics with k(2) = 3.33 x 10(7) m(-1) s(-1) or first-order kinetics with 52.6 s(-1) < (T1red)k(1) < 112 s(-1). Values of formation constants of the T1Cu(II)T2Cu(II)-SO(2)(.-) and T1Cu(I)T2Cu(II)-SO(2)(.-) adducts showed that the redox state of T1Cu affected binding of SO(2)(.-) at the catalytic T2Cu center. Analysis of the kinetics required the development of a mathematical protocol that could be applied to a system with two intercommunicating sites but only one of which can be monitored. This novel protocol, reported for the first time, is of general application.

Alcaligenes↗
PubMed1985

Kinetic studies on reduction of cytochromes P-450 and b5 by dithionite.

The kinetics of reduction of cytochromes P-450 and b5 by dithionite had been studied in solution and in microsomal and proteoliposomal membranes by the stopped-flow technique. In all the cases studied the kinetic curves of reduction of cytochrome b5 obey first-order kinetics in relation to cytochrome with the rate constant about 14 s-1 at 10.6 mM dithionite. The kinetic curves of reduction of cytochrome P-450 fit an equation for the sum of two exponentials with the parameters varying from system to system. The simple first-order kinetics of cytochrome P-450 reduction had been observed in the presence of non-ionic detergent Triton N-101. The apparent biphasity of cytochrome P-450 reduction by dithionite should be the result asymmetric distribution of hemoprotein in microsomal and proteoliposomal membranes as well as in cytochrome oligomers in solution.

Animals↗
PubMedDate not supplied

Rapid determination of the transbilayer distribution of NBD-phospholipids in erythrocyte membranes with dithionite.

The assessment of the transverse distribution and mobility of NBD-labelled phospholipid analogues in biological membranes by selective chemical destruction of fluorescent label in the outer monolayer with dithionite has been investigated using resealed erythrocyte ghosts as a model system. The distribution of those analogues can be determined in < 30 s directly in the cell suspension provided the permeation of dithionite across the membrane is suppressed. The results were compared with data on translocation of either NBD- or spin-labelled phospholipid analogues obtained with the technique of back exchange to BSA. It is shown that the passage of dithionite can be mediated by anion-transport systems such as band 3 which is inhibited by DIDS. Appropriate conditions for the applicability of the assay were elucidated also using resealed ghosts having fluorescent NBD-taurine in the intracellular lumen. The application of the assay to measure fast translocation processes, e.g. those mediated by the aminophospholipid translocase, is described.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Colorimetry and constant-potential coulometry determinations of transferrin-bound iron, total iron-binding capacity, and total iron in serum containing iron-dextran, with use of sodium dithionite and alumina columns.

After the parenteral administration of iron-dextran (imferon), the increased total iron concentrations in serum can be determined by atomic absorption spectroscopy and by colorimetric methods involving sodium dithionite, which reductively dissociates iron from the dextran complex. We report that constant-potential coulometry detects only about 55-70% of dextran-bound iron before dithionite reduction and variable amounts after reaction with the reducing agent. In addition, we have developed a procedure for determining transferrin-bound iron, total iron-binding capacity (TIBC), total iron, and dextran-bound iron with the Kodak Ektachem colorimetric system. In determining total serum iron, the sample is first mixed with sodium dithionite, which rapidly dissociates all dextran-bound iron, but does not remove iron from either transferrin or hemoglobin. After the mixture is applied to an Ektachem slide, transferrin-bound iron is released at pH 4 and is detected together with the iron previously bound to dextran. TIBC is determined by mixing serum with ferric citrate in moderate excess and filtering through a small alumina (Al2O3) column, which binds excess free iron and iron-dextran; the iron in the column eluate represents the TIBC. Transferrin-bound iron is determined by applying diluted serum without added ferric citrate to an alumina column and measuring the iron in the column eluate. Dextran-bound iron is equivalent to the difference between total and transferrin-bound iron. Using this method, we found that transferrin iron-binding sites are saturated in vitro by excess iron-dextran less efficiently than by ferric citrate.

Aluminum Oxide↗
PubMed1984

The kinetics of O2 release by human red blood cells in the presence of external sodium dithionite.

Oxygen release by human erythrocytes in the presence of external sodium dithionite was examined by stopped-flow, rapid mixing techniques. The resultant time courses were analyzed quantitatively using a three-dimensional disc model which had been developed previously to describe oxygen uptake (Vandegriff, K. D., and Olson, J. S. (1984) Biophys. J. 45, 825-835). This scheme takes into account diffusion of oxygen through external unstirred solvent layers and intracellular oxygen diffusion and chemical reaction with hemoglobin. Application of this model to deoxygenation time courses required three additional considerations: the reaction of free oxygen with external sodium dithionite, cooperative oxygen binding to intracellular hemoglobin, and the alkaline Bohr effect. The resultant theoretical treatment described accurately both the observed dependence of the deoxygenation rate on dithionite concentration and pH and the exact shapes of the corresponding time courses. Membrane resistance to oxygen diffusion was not required to simulate the observed data as had been suggested previously (Lawson, W. H., Jr., Holland, R. A. B., and Forster, R. E. (1965). J. Appl. Physiol. 20, 912-918). The final, three-dimensional model is general and allows, for the first time, analysis of both oxygen uptake and release kinetics (Vandegriff, K. D., and Olson, J. S. (1984) J. Biol. Chem. 259, 12619-12627).

Dithionite↗
PubMed1981

[Dark and photo-induced changes in absorption and fluorescence spectra of phycobilisomes in the presence of dithionite].

In order to test the possibility of photochemical participation of phycobilin pigments in photosynthesis, the ability of phycobilisomes for reversible photo-induced redox reactions was studied. The photo-induced fast reversible changes in the absorption and fluorescence spectra of phycobilisomes in the presence of dithionite were found. Simultaneously dithionite induced dark changes revealed by the decrease of the absorption and fluorescence yields. However, the dark and photo-induced changes differ in spectral parameters depending on dithionite concentration. The ability of phycobilisomes to photosensitive redox reactions was demonstrated. A possible nature of dark and photo-induced changes in the absorption and fluorescence spectra of phycobilisomes is discussed.

Cyanobacteria↗
PubMed1996

Dithionite-supported hydroxylation of palmitic acid by cytochrome P450BM-3.

The ability of dithionite, an inexpensive reducing agent routinely used to produce the ferrous-carbonyl form of P450, to support P450BM-3-catalyzed hydroxylation of palmitate was studied. The hydroxylation products in the presence of dithionite were 15, 14, and 13-hydroxyhexadecanoate, with relative distributions similar to those observed with NADPH. The hydroxylation reaction was carried out in two separate steps, anaerobic reduction and subsequent oxidation of P450BM-3 by oxygen bubbling. The reduction step was much slower than the oxidation step, thus limiting the overall rate of hydroxylation. Upon addition of dithionite, the reductase domain of P450BM-3 seemed to be reduced before significant reduction of the heme domain occurred. The discovery of new reducing agents for P450-catalyzed reaction raises the possibility of replacing NADPH in specialty chemical hydroxylation catalyzed by P450s, especially catalytically self-sufficient P450s, such as P450BM-3 or recombinant fusion proteins of P450 covalently linked to a reductase.

Bacterial Proteins↗