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PubMed1998

Dithionite reduced carbon monoxide complex of cytochrome P450cam is a monomer.

Sedimentation experiments on cytochrome P450cam (CYP101) has been performed to compare the molecular mass of the protein in the oxidized state and as carbon monoxide complex. The oxidized protein in the absence of beta-mercaptoethanol is a dimer with a molecular mass of 92 kDa. Addition of mercaptoethanol avoids completely the dimerization. Dithionite reduced P450cam in the presence of carbon monoxide has been found to be a monomeric protein.

Camphor 5-Monooxygenase↗
PubMed1975

Indirect sulphonation of mucopolysaccharides and glycogen in tissue sections with the use of bisulphite or dithionite after periodic acid oxidation.

Tissue mucopolysaccharides and glycogen can be indirectly sulphated after being oxidized by periodic acid and treated with sodium bisulphite or dithionite in aqueous solution. The sulphated sites are darkly stained by Roluidine Blue and realted dyes at pH less than 1.0. The background is very pale or colourless. The stained sections resist extraction with 1% hydrochloric acid for 48 hr, but can be extracted by 5% ammonium hydroxide in ethanol in 1 hr. Other oxidizing agents cannot be substituted for periodic acid.

Dithionite↗
PubMed1984

Kinetic studies on the reduction of iron(III)deuteroporphyrin--human serum albumin complex with dithionite ion.

The effects of human serum albumin (HSA) on the rate of dithionite reduction of iron(III)deuteroporphyrin (iron(III)Dp) have been investigated in order to further characterize the porphyrin binding site and the changes manifested in this site under various conditions. These studies were performed under pseudo-first-order conditions, and in the presence of carbon monoxide as a "trapping agent" for the reduced iron(II)porphyrin. The rate of reduction of the free iron(III)Dp in phosphate buffer at pH 7.4 follows second-order kinetics with a rate constant (4.2 X 10(9) M-1 s-1) suggestive of a diffusion-controlled process. A six-orders of magnitude decrease in the rate of reduction was observed with iron(III)Dp was complexed with HSA. This result is consistent with HSA-bound porphyrin being less accessible to the aqueous environment. Additional studies demonstrated that both pH and anions induce various alterations in the complex that are reflected in the rate of reduction of iron(III)porphyrin.

Binding Sites↗
PubMed1986

The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan.

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

P450BM-3: reduction by NADPH and sodium dithionite.

Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids. The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system. Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da. The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components. FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration. The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents. The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues. Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide. Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group. The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed. The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.

Anaerobiosis↗
PubMed1982

Binding of metyrapone to dithionite-reduced cytochrome P-450 from rats treated with xenobiotics.

The in vitro binding of metyrapone to dithionite-reduced cytochrome P-450 in hepatic microsomes from rats treated in vivo with thirteen different xenobiotics was studied spectrophotometrically. The proportion of cytochrome P-450 that bound metyrapone increased 1.8-fold to about 78% following treatment with phenobarbitone (PB) and PB-type inducers (trans-stilbene oxide, 2,2',4,4'-tetrachloro-, 2,2',4,5,5'-pentabromo- and 2,2',4,4',5,5'-hexachlorobiphenyl) but remained unaltered following treatment with 3-methylcholanthrene (MC) and MC-type induces (benzo[a]pyrene, beta-naphthoflavone and 3,3',4,4'-tetrabromobiphenyl). The simultaneous induction of the PB-inducible and MC-inducible forms of cytochrome P-450 by administering Aroclor 1254 or by coadministering PB with MC increased the proportion of cytochrome P-450 that bound metyrapone to 74 and 78% respectively. PB treatment increased whereas MC treatment decrease the binding affinity for metyrapone by approximately 20-fold. Treatment with isosafrole or metyrapone itself failed to stimulate metyrapone binding. In contrast, pregnenolone-16 alpha-carbonitrile was indistinguishable from PB in its ability to increase the binding capacity and binding affinity for metyrapone. Our results indicate that metyrapone binding is not specific for cytochrome P-450b, the major PB-inducible hemoprotein, as has been proposed [V. Luu-The, J. Cumps and P. Dumont, Biochem. biophys. Res. Commun. 93, 776 (1980)].

Animals↗
PubMed1993

Site-specific metal-induced damage of mitomycin C-crosslinked DNA fragments in the presence of sodium dithionite.

Purified singly mitomycin C-crosslinked DNA duplex fragments were treated with Cu(II) in the presence of sodium dithionite. Cleavage products were analyzed through single-nucleotide resolving denaturing polyacrylamide gel electrophoresis. In addition to the previously described non-specific cleavage, which may be attributed to the production of hydroxyl radical, a novel site-specific cleavage in the immediate vicinity of the MC-crosslink was observed. Possible mechanisms of this reaction are discussed.

Base Sequence↗
PubMed1986

Calorimetric studies of oxyhemoglobin ligand dissociation. III. Evidence for a hemoglobin-catalase-superoxide dismutase integrated system in the oxygen depletion by dithionite.

Calorimetric studies of the effect of superoxide dismutase and/or catalase on the reduction of dioxygen into water by dithionite in oxyhemoglobin have been carried out and the results compared with those in red cell hemolysates. In the absence of the enzymes the stoichiometry (moles dithionite/mole dioxygen) is less than the value of 2:1 which was found previously in red cell hemolysates [Forlani et al., J. Inorg. Biochem. 20, 147-155 (1984)]. In the presence of either superoxide dismutase or catalase alone the stoichiometry increases but is still less than 2:1. In the presence of both enzymes the stoichiometry and the shape of the thermogram is that previously observed for hemolysates, suggesting the presence of a hemoglobin-catalase-superoxide dismutase integrated system. The absence of a calorimetric signal for hydrogen peroxide in the reduction of oxyhemoglobin in the presence of superoxide dismutase suggests a wider biological role of superoxide dismutase than previously thought.

Catalase↗
PubMed1993

X-ray absorption spectroscopic studies of a transient intermediate in the reaction of cyanide metmyoglobin with dithionite by using rapid freezing.

The reaction of cyanide metmyoglobin (Mb+CN-) with dithionite produces a transient intermediate, supposed to be cyanide-ligated ferrous myoglobin. The Fe K-edge X-ray absorption spectrum of the intermediate has been measured by using rapid freezing and compared with those of Mb+CN- and deoxymyoglobin (deoxyMb). The shapes of the XANES (X-ray Absorption Near Edge Structure) spectra of Mb+CN- and the intermediate are very similar, including the intensity ratios of the peak C1 to D. This indicates that CN- remains bound with a linear Fe-C-N configuration in the intermediate. The absorption edge of the intermediate is shifted to 1.2 eV lower energy than that of Mb+CN-, reflecting a valence change in the heme iron. The EXAFS (Extended X-ray Absorption Fine Structure) spectrum of the intermediate closely resembles that of Mb+CN- but significantly differs from that of deoxyMb. Analysis shows that the average iron-nearest neighbor atom distance is 1.99 +/- 0.01 A for both Mb+CN- and the intermediate and 2.05 +/- 0.01 A for deoxyMb. These results imply that the local structure around the heme iron of Mb+CN- does not change upon reduction until the cyanide ligand is released.

Animals↗
PubMed2004

Reduction of nitrogenase Fe protein from Azotobacter vinelandii by dithionite: quantitative and qualitative effects of nucleotides, temperature, pH and reaction buffer.

Oxidized Fe protein from Azotobacter vinelandii (Av2(0)) was reduced by dithionite (DT) in the absence and presence of nucleotides, over the temperature range 10-40 degrees C, over the pH range 7-8, and in various buffers--inorganic phosphate, TES, HEPES, and Tris. The reduction of each species of Fe protein--Av2(0), Av2(0)(MgATP)2, and Av2(0)(MgADP)2--was resolved into at least three exponential phases, with relative amplitudes of each phase varying over the range of experimental conditions, suggesting a dynamic population shift of kinetically distinct species. The rapid phase of Av2(0) reduction predominated at low temperature and pH, and in Tris buffer; rapid Av2(0)(MgATP)2 reduction was favored at high temperature and pH, and in phosphate buffer; and Av2(0)(MgADP)2 reduction was favored under more physiologically relevant conditions of 20 degrees C, pH 7.5, and in phosphate buffer. The rates of reduction of Fe protein species did not change with buffer, but temperature and pH do have an effect on the rates. With the appropriate constants, an empirically derived equation estimates the rate of Fe protein reduction at any temperature and pH within the limits 10-40 degrees C and pH 7-8, for a given species of Fe protein, and a given phase of the reaction. At 23.0 degrees C and pH 7.4, the rate of the dominant phase of Av2(0) reduction is 1.9 x 10(8) M(-1) s(-1). Under the same conditions, the rates of the two dominant phases of Av2(0)(MgATP)2 reduction are 1.2 x 10(6) and 1.5 x10 (5) M(-1) s(-1); and the rate of the dominant phase of Av2(0)(MgADP)2 reduction is 3.5 x 10(6) in M(-1) s(-1). Thermodynamic activation parameters for each phase of reduction were calculated. No breaks in the Arrhenius plots for any Fe protein species were observed.

Adenine Nucleotides↗
PubMed1979

Reduction of methemerythrin-anion adducts by dithionite ion.

The reduction by dithionite ion (in excess) of methemerythrin-anion adducts, Hr+X-, to deoxyhemerythrin, Hr degree, has been examined at 25 degrees and pH 6.3 and 8.2. The results accord with the scheme: S2O42- in equilibrium 2SO2- rapid Hr+X- in equilibrium Hr++X- k-1, k1 Hr++SO2- leads to PRODUCT k2 with X- = Br-, HCO2-, CNO-, and F-, k2[SO2-] greater than k1[X-], and the pseudo first-order rate constant, kobs (= k-1), is independent of [X-] and [S2O42-]. Only with X- = NCS- is k2[SO2-] approximately k1[X-] and kobs = a[S2O42-]1/2 (b[NCS-] + [S2OR2-]1/2)-1. Values at pH 6.3 of k-1 (sec-1) and k1 (M-1 sec-1), obtained by anation and anion displacement reactions, are 2.3 x 10(-3), 1.6 x 10(-2) (Br-); 1.5 x 10(-3), 1.2 x 10(-2) (HCO2-); 1.3 x 10(-4), 0.52 (CNO-) and approximately 2 x 10(-4), 3.3 x 10(-3) (CN-, pH 7.0). Values of k-1 from reduction and displacement methods are in good agreement with each other. The value of k2 (1.6 x 10(5) M-1 sec-1, pH 6.3) in somewhat smaller than that for reduction of the met form of hemoproteins. There is only a small effect of pH on rates. Direct reduction of Hr+CN- does not occur, in contrast with Mb+CN-.

Animals↗
PubMed1986

Hydrogen evolution from dithionite and H2 photoproduction by hydrogenase incorporated into various hydrophobic matrices.

The effects of surfactants, lipids and amphiphilic viologen mediators on H2 production from dithionite as well as on Ru(bpy) sensitized H2 photoproduction by hydrogenase from Thiocapsa roseopersicina was studied. Three systems which differed as to the nature of the hydrophobic matrix around the hydrogenase were tested. An enhanced hydrogenase activity was observed in the presence of surfactants, in the 1-6 mM concentration range. Hydrogenase showed a selectivity for the amphiphilic viologens, 2C7-diCl was the most efficient electron mediator in both reactions. H2 photoproduction seemed not to be feasible in the detergent-hydrogenase system because of intensive foaming. Hydrogenase incorporated into liposomes catalyzed H2 photoevolution efficiently but the rate was decreasing in time, though reversibly. Using intact bacterial cells instead of purified hydrogenase yielded stable H2 photoevolution for at least 12 hours. This system offers several advantages for potential practical applications.

Cetrimonium↗
PubMed1976

Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite.

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2--with k greater than 10(8)M-1-S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2-- at 23 degrees C. Further reductions by SO2-- occurred in three slower phases with rate constants in the range 10(4) -10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-- of the Fe-protein alone (k greater than 10(8)M-1-S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 X 1O(2)s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.

Adenosine Triphosphate↗
PubMed1980

Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells.

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.

Animals↗
PubMed1983

Reactions of cytochrome c oxidase with sodium dithionite.

The reduction of cytochrome c oxidase (EC 1.9.3.1) by dithionite was investigated by stopped-flow spectrophotometry and flow-flash techniques in the presence of CO. Of the two haem groups present in the enzyme, that associated with cytochrome alpha is the first reduced. The second-order rate constants for reduction of a number of redox proteins (cytochrome c, stellacyanin and azurin) by the S2O4(2-) and SO2.- anions are reported, and the values are compared with those determined for cytochrome c oxidase. These results are discussed in terms of the accessibility and charge distribution of the electron-entry site of cytochrome c oxidase.

Dithionite↗
PubMed1988

The vanadium- and molybdenum-containing nitrogenases of Azotobacter chroococcum. Comparison of mid-point potentials and kinetics of reduction by sodium dithionite of the iron proteins with bound magnesium adenosine 5'-diphosphate.

The mid-point potentials of the Fe protein components (Ac2 and Ac2* respectively) of the Mo nitrogenase and V nitrogenase from Azotobacter chroococcum were determined in the presence of MgADP to be -450 mV (NHE) [Ac2(MgADP)2-Ac2*ox.(MgADP)2 couple] and -463 mV (NHE) [Ac2* (MgADP)2-Ac2*ox.(ADP)2 couple] at 23 degrees C at pH 7.2. These values are consistent with a flavodoxin characterized by Deistung & Thorneley [(1986) Biochem. J. 239, 69-75] with Em = -522 mV (NHE) being an effective electron donor to both the Mo nitrogenase and the V nitrogenase in vivo. Ac2*ox.(MgADP)2 and Ac2*ox.(MgADP)2 were reduced by SO2.- (formed by the predissociation of dithionite ion, S2O4(2-)) at similar rates, k = 4.7 X 10(6) +/- 0.5 X 10(6) M-1.s-1 and 3.2 X 10(6) +/- 0.2 X 10(6) M-1.s-1 respectively, indicating structural homology at the electron-transfer site associated with the [4Fe-4S] centre in these proteins.

Adenosine Diphosphate↗
PubMed1995

Bathophenanthrolene disulfonic acid and sodium dithionite effectively remove surface-bound iron from Caco-2 cell monolayers.

Iron uptake by Caco-2 cell monolayers is commonly assessed by incubating the cells under radiolabeled iron solutions, removing the radiolabeled solution, rinsing to stop uptake and measuring the radioactivity retained by the cells. It is therefore essential to differentiate between iron that is nonspecifically bound to the cell surface from that which has been taken up by the cell. We report here on a method for removal of surface-bound iron from Caco-2 cell monolayers. We used a 140 mmol/L NaCl, 10 mmol/L PIPES, pH 6.7 solution containing 5.0 mmol/L sodium dithionite (Na2S2O4) and 5.0 mmol/L bathophenanthroline disulfonic acid to reduce, remove and chelate iron bound to the cell surface. We validated our method by demonstrating the removal of 97% of an insoluble iron complex from the apical surface of Caco-2 cell monolayers. Our data indicate that the removal solution does not damage the apical membrane and thereby does not have access to intracellular iron; thus only surface bound iron is removed. The remaining cell-associated iron represents that which has been transported into the cell. We present data on the uptake and nonspecific binding of iron from iron complexes of both ferrous and ferric forms, and show that iron removal treatment resulted in uptake measurements that agree more closely with accepted principles of iron uptake by intestinal epithelium. The iron removal method used in this study should provide investigators with a valuable tool for accurately determining iron uptake by epithelial cells in culture.

Biological Transport↗
PubMed1985

Oxygen binding to dithionite-reduced chloroperoxidase.

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.

Chloride Peroxidase↗