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PubMed1993

Structure/activity relationships in porphobilinogen oxygenase and horseradish peroxidase. An analysis using synthetic hemins.

The apo-enzymes of porphobilinogen oxygenase and horseradish peroxidase were reconstituted with hemin IX, deuterohemin IX, 2,4-diacetyldeuterohemin IX, 2-vinyl-4-deuterohemin IX and hemin I. The apoproteins did not reconstitute with the dimethyl or diethyl esters of hemin IX. The native enzymes and the synthetic hemoproteins showed similar oxygenase activities toward porphobilinogen in the presence of dithionite and oxygen. They also showed peroxidase activity in the presence of H2O2, which was affected by the side-chain substitution pattern of the hemes. Oxygenase activities, however, were not affected by the heme structure. Iron chelators completely inhibited the oxygenase, but not the peroxidase activities. The EPR spectra of the native and synthetic porphobilinogen oxygenase showed that dithionite reduction produced a rapid disappearance of the high-spin heme-iron signal at g = 6.0. It reappeared 1 min later but the enzyme retained its catalytic activity. The changes in the EPR spectra could be correlated with the biphasic kinetics of the oxygenase reaction which was very fast during the first minute and then decreased to a half-value rate. The oxygenase reaction was inhibited by addition of superoxide dismutase during the fast rate phase, but not during the slower phase. These results could be explained by the formation of a superoxide anion during the first minute of the oxygenase reaction, after which a protein-stabilized radical (g = 2.0) is generated (very likely a tyrosyl radical). The latter then oxidizes the substrate porphobilinogen and facilitates its reaction with O2 to give oxopyrrolenines.

Animals↗

PubMed1994

Pre-steady-state MgATP-dependent proton production and electron transfer by nitrogenase from Azotobacter vinelandii.

MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 degrees C is 14 +/- 4 s-1. No proton production with a rate constant comparable to the observed rate constant of electron transfer (kobs approximately 100 s-1) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +/- 0.3 mol H+produced/mol MoFe protein with oxidized Fe protein, and 1.1 +/- 0.1 mol H+produced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electrons(transferred)/H-uptake was calculated to be 1.2 +/- 0.2. After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP-dependent proton production (kobs approximately 35 s-1). The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein; with dithionite-reduced Fe protein a value of 3.1 +/- 0.4 mol H+produced/mol MoFe protein was found (or 0.5 +/- 0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite-reduced Fe protein with MgADP resulted in the production of 0.17 +/- 0.05 mol H+/mol Fe protein. All reported absorbance changes were absent when the experiments were performed in a buffered solution. The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s-1, which is followed by H+ production (kobs approximately 14 s-1). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP-induced proton production decreases.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗

PubMed1995

A seven-iron ferredoxin from the thermoacidophilic archaeon Desulfurolobus ambivalens.

A seven-iron ferredoxin was isolated from aerobically grown cells of the hyperthermoacidophilic archaeon Desulfurolobus ambivalens (DSM 3772). The protein is monomeric, with an apparent molecular mass of 15 kDa and contains 7 iron atoms/molecule. The N-terminal sequence shows a large similarity (70% identity) with that of the ferredoxin isolated from the archaeon Sulfolobus acidocaldarius. The EPR characteristics in both the native (oxidized) and dithionite-reduced states of this protein allowed an unequivocal identification of a [3Fe-4S]1+/0 center, with a reduction potential of -270 +/- 20 mV, at pH 7.5. The protein also contains a [4Fe-4S]2+/1+ center with a very low reduction potential (Eo = -540 mV, pH 7.0), which yields a rhombic EPR spectrum upon reduction with sodium dithionite at high pH. The reduction potentials of both centers are slightly pH dependent between pH 6 and 9. The [3Fe-4S] ferredoxin center is able to accept electrons from pyruvate oxidase and NADH oxidase isolated from D. ambivalens. This ferredoxin is present in large amounts (at least 130 mg/kg wet cells), which allowed the unequivocal observation of oxidized [3Fe-4S] clusters in intact D. ambivalens cells.

Amino Acid Sequence↗

PubMed1997

Alkene monooxygenase from Nocardia corallina B-276 is a member of the class of dinuclear iron proteins capable of stereospecific epoxygenation reactions.

Nocardia corallina B-276 possesses a constitutive multi-component alkene monooxygenase which catalyses the epoxidation of terminal and sub-terminal alkenes. The epoxygenase component of this system has been purified with an overall yield of 35%. The electron paramagnetic resonance spectrum of the oxidised protein has a weak signal at g = 4.3, which we ascribe to rhombic iron and a free radical signal at g(ave) = 2.01. Upon partial reduction with dithionite using methyl viologen as a mediator, a signal at g(ave) = 1.9 appeared. Upon further reduction with excess dithionite a signal at g = 15 appeared with the concomitant disappearance of the g(ave) = 1.9 signal. These results indicate that the epoxygenase contains a bridged dinuclear iron centre similar to that found in a variety of proteins involved in oxygen transport and activation as well as desaturation of fatty acids. Analysis of the products of the reaction indicates that AMO is capable of stereospecific epoxidation of alkenes producing the R-enantiomer in high yield, a reaction catalysed by very few oxygenase enzymes. Whole cells gave lower enantiomeric excess values for the epoxide and a stereospecific epoxidase enzyme has been proposed to account for this difference. Although alkene monooxygenase was not inhibited by ethyne, a potent inhibitor of soluble methane monooxygenase with which alkene monooxygenase shares many common features, it was weakly inhibited by propyne with an apparent Km value of 340 microM. The mechanistic implications of these physicochemical features of the enzyme are discussed.

Alkenes↗

PubMed1998

Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster.

FNR is a global regulator that controls transcription of genes whose functions facilitate adaptation to growth under O2 limiting conditions. It has long been appreciated that the activity of FNR must be regulated by O2 availability, since FNR dependent gene expression is observed in vivo only under anaerobic conditions, while similar levels of this protein are present in both aerobic and anaerobic grown cells. Recent progress in this field has shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster and that this [4Fe-4S]2+ cluster is sufficiently unstable toward O2 to make it suitable as an O2 sensor. The presence of the [4Fe-4S] cluster increases dimerization of FNR which is correlated with an increase in site-specific DNA binding of FNR, a property expected of transcription factors of the FNR/CRP family. According to Mössbauer spectroscopy on purified FNR and cells containing overexpressed FNR, the [4Fe-4S]2+ cluster of FNR is converted by O2 to a [2Fe-2S]2+ in high yield. The [2Fe-2S]2+ cluster can be reconverted to the [4Fe-4S]2+ cluster on reduction with dithionite in vitro raising the possibility that the [2Fe-2S]2+ cluster is a biologically inactive intermediate which may be more readily available for reconstitution into the [4Fe-4S]2+ form than the Fe-free apoform. The ability to observe, by Mössbauer spectroscopy, the Fe-S clusters of FNR in cells containing high levels of FNR should be of value in further unraveling how FNR functions in vivo. Attempts to reduce the [4Fe-4S]2+ cluster of FNR with dithionite indicated that the redox potential of the +1/+2 couple is < or = -650 mV and that the [4Fe-4S]+ cluster form is, therefore, not likely to occur in vivo.

Aerobiosis↗

PubMed1992

Intracellular pH changes induced by hypoxia and anoxia in isolated sheep heart Purkinje fibres.

The effects were studied of hypoxia on intracellular ion activities in sheep heart Purkinje fibres. The intracellular pH (pHi), surface pH (pHs), intracellular potassium activity (aki), and intracellular sodium activity (aNai) of the cells were recorded using liquid ion exchanger-filled microelectrodes. Various methods of inhibiting oxidative phosphorylation were compared for their effect on pHi. These methods were the use of hypoxia, anoxia or NaCN (2 mM). Hypoxia was produced by degassing solutions under reduced pressure then bubbling with 100% nitrogen gas. Anoxia was produced in a similar manner but with the addition of the reducing agent sodium dithionite (0.5 mM) to remove all traces of oxygen from the solutions. Anoxia caused the most marked changes. Concentration of sodium dithionite between 0.1 and 1 mM produced similar maximum rates of acidification. High concentrations (5 or 20 mM) could produce larger intracellular acidifications apparently unrelated to anoxia. The effects of hypoxia and NaCN were similar. Inhibition of Na(+)-H+ exchange with amiloride (1 mM) had little effect on the pH changes produced by hypoxia. Periods of hypoxia exceeding 1 h still resulted in rapid, readily reversible changes in pHi. Hypoxia caused a rise in aNai, the effect being larger in anoxic conditions. The intracellular K+ activity decreased in hypoxia with further decreases in anoxic conditions. The intracellular ion changes produced during hypoxia are discussed in terms of the production of lactic acid by the cells and changes in the ATP supply to the Na(+)-K+ pump.

Adenosine Triphosphate↗

PubMed1978

Absence of strand breaks in deoxyribonucleic acid treated with metronidazole.

The deoxyribonucleic acid (DNA)-degrading potential of metronidazole was evaluated in vitro by three techniques: determination of melting curve, measurement of viscosity, and centrifugation in neutral or alkaline sucrose gradients. Studies were performed on calf thymus DNA and on (3)H-labeled or unlabeled pneumococcal and T7 phage DNA after treatment with metronidazole alone or metronidazole reduced by sodium dithionite in the presence of DNA. This latter process is known to elicit covalent binding of metronidazole to DNA. Reduced or unreduced metronidazole had no effect on the melting properties, viscosity, or sedimentation velocity of the nucleic acids studied. Sodium dithionite alone, however, caused a 25% decrease in the intrinsic viscosity of pneumococcal DNA, and decreased the sedimentation velocity of pneumococcal and T7 phage DNA in both neutral and alkaline sucrose gradients. These data suggest that degradation of DNA is not important in the interaction of metronidazole with nucleic acids, an interaction assumed relevant to the cytotoxic, radiosensitizing, and mutagenic activities of this compound.

Centrifugation, Density Gradient↗

PubMed1982

Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa.

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.

Azides↗

PubMed1983

Characterization of a new type of dissimilatory sulfite reductase present in Thermodesulfobacterium commune.

A new type of dissimilatory bisulfite reductase, desulfofuscidin, was isolated from the nonsporeforming thermophilic sulfate-reducing microorganism Thermodesulfobacterium commune. The molecular weight of the enzyme was estimated at 167,000 by sedimentation equilibrium, and the protein was pure by both disc electrophoresis and ultracentrifugation. The bisulfite reductase was a tetramer and had two types of subunits with an alpha(2)beta(2) structure and an individual molecular weight of 47,000. The enzyme exhibited absorption maxima at 576, 389, and 279 nm, with a weak band at 693 nm. Upon the addition of dithionite, the absorption maxima at 576 and 693 nm were weakened, and a new band appeared at 605 nm. The protein reacted with CO in the presence of dithionite to give a complex with absorption peaks at 593, 548, and 395 nm. The extinction coefficients of the purified enzyme at 576, 389, and 279 nm were 89,000, 310,000, and 663,000 M(-1) cm(-1), respectively. Siroheme was detected as the prosthetic group. The protein contains 20 to 21 nonheme iron atoms and 16 to 17 acid-labile sulfur groups per molecule. The data suggest the presence of four sirohemes and probably four (4Fe-4S) centers per molecule by comparison with desulfoviridin, the dissimilatory sulfite reductase from Desulfovibrio species. The protein contains 36 cysteine residues and is high in acidic and aromatic amino acids. The N-terminal amino acids of the alpha and beta subunits were threonine and serine, respectively. With reduced methyl viologen as electron donor, the major product of sulfite reduction was trithionate, and the pH optimum for activity was 6.0. The enzyme was stable to 70 degrees C and denatured rapidly above this temperature. The dependence of T. commune bisulfite reductase activity on temperature was linear between 35 and 65 degrees C, and the Q(10) values observed were above 3. The presence of this new type of dissimilatory bisulfite reductase in T. commune is discussed in terms of taxonomic significance.

Amino Acid Sequence↗

PubMed1991

Menaquinol-nitrate oxidoreductase of Bacillus halodenitrificans.

When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membrane-bound nitrate reductase (NR) that was solubilized by 2% Triton X-100 but not by 1% cholate or deoxycholate. Purification on columns of DE-52, hydroxylapatite, and Sephacryl S-300 yielded reduced methyl viologen NR (MVH-NR) with specific activities of 20 to 35 U/mg of protein that was stable when stored in 40% sucrose at -20 degrees C for 6 weeks. 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropone-1-sulfonat e (CHAPSO) and dodecyl-beta-D-maltoside stimulated enzyme activity three- to fourfold. Membrane extractions yielded purified NR that separated after electrophoresis into a 145-kDa alpha subunit, a 58-kDa beta subunit, and a 23-kDa gamma subunit. The electronic spectrum of dithionite-reduced, purified NR displayed peaks at 424.6, 527, and 557 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. Analyses revealed a molybdenum-heme-non-heme iron ratio of 1:1:8 for the NR and the presence of molybdopterin. Electron paramagnetic resonance (EPR) signals characteristic of iron-sulfur centers were detected at low temperature. EPR also revealed a minor signal centered in the g = 2 region of the spectra. Upon reduction with dithionite, the enzyme displayed signals at g = 2.064, 2.026, 1.906, and 1.888, indicative of the presence of low-potential iron-sulfur centers, which resolve most probably as two [4Fe-4S]+1 clusters. With menadiol as the substrate for nitrate reduction, the Km for nitrate was 50-fold less than that seen when MVH was the electron donor. The cytochrome b557-containing enzyme from B. halodenitrificans is characterized as a menaquinol-nitrate:oxidoreductase.

Bacillus↗

PubMed1995

Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1.

Although reductive dehalogenation by anaerobic microorganisms offers great potential for the degradation of halocarbons, little is known about the biochemical mechanisms involved. It has previously been demonstrated that the dehalogenase activity involved in 3-chlorobenzoate dehalogenation by Desulfomonile tiedjei DCB-1 is present in the membrane fraction of the cell extracts. We report herein the purification of a 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of D. tiedjei DCB-1. The dehalogenase activity was monitored by the conversion of 3-chlorobenzoate to benzoate with reduced methyl viologen as a reducing agent. The membrane fraction of the cell extracts was obtained by ultracentrifugation, and the membrane proteins were solubilized with either the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) or Triton X-100 in the presence of glycerol. The solubilized dehalogenase was purified by ammonium sulfate fractionation and a combination of anion exchange, hydroxyapatite, and hydrophobic interaction chromatographies. This procedure yielded about 7% of the total dehalogenase activity with a 120-fold increase in specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified dehalogenase consisted of two subunits with molecular weights of 64,000 and 37,000. The enzyme converted 3-chlorobenzoate to benzoate at its highest specific activity in 10 mM potassium phosphate buffer (pH 7.2) at 38 degrees C. The enzyme was yellow and probably a heme protein. The enzyme had an adsorbance peak at 408 nm. The dithionite-reduced enzyme displayed absorbance peaks at 416, 522, and 550 nm. The dithionite-reduced enzyme was able to complex with carbon monoxide. The nature of the heme chromophore is currently unknown.

Benzoates↗

PubMed1967

Determination of iron in urine with special reference to the desferrioxamine test.

A simple and rapid method for the determination of ferrioxamine iron in urine, in which the colour reagent is added directly to centrifuged urine after reduction with dithionite, has been critically evaluated and compared with a method of iron determination after wet ashing. The coefficient of variation of both methods was less than 5%. A very high correlation was found between the values obtained with the two methods, but the simple method gave consistently lower values, averaging about 85% of those by the wet ashing method. Iron present in the urine sediment or bound in a soluble form which is not split off by dithionite will not be measured with the simple method. Despite its lower values the simple method gives uniform and reliable results, and is considered suitable for the performance of the desferrioxamine test.

Deferoxamine↗

PubMed1999

A flavoprotein mechanism appears to prevent an oxygen-dependent inhibition of cGMP-associated nitric oxide-elicited relaxation of bovine coronary arteries.

The redox state of the heme of soluble guanylate cyclase (sGC) may regulate the sensitivity of vascular tissue to nitric oxide (NO). In this study, diphenyliodonium (DPI) is used as an inhibitor of flavoprotein oxidoreductases to examine their potential role in the expression of NO-elicited cGMP-associated arterial relaxation and sGC stimulation. The relaxation of endothelium-removed bovine coronary arteries (BCAs) precontracted with 30 mmol/L KCl to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or to NO is markedly suppressed by 10 micromol/L DPI under an atmosphere of 21% O(2) (5% CO(2)). In contrast, DPI has minimal effects on the relaxation to SNAP under 95% N(2) (5% CO(2)). If BCAs are treated with DPI under 21% O(2) and then exposed to the hemoprotein reductant sodium dithionite (1 mmol/L) under N(2), there is a partial reversal of the inhibitory effects of DPI compared with BCAs that were not treated with dithionite. DPI did not inhibit relaxation elicited by 8-bromo-cGMP or forskolin. Increases in tissue cGMP levels stimulated by SNAP were eliminated by pretreatment of BCAs with DPI under 21% O(2) but not under N(2). Activation of sGC by SNAP in BCA homogenate was also eliminated when vessels were pretreated with 10 micromol/L DPI under 21% O(2), but DPI did not have an inhibitory effect when directly added to the assay of sGC activity. These observations are consistent with a flavoprotein-dependent oxidoreductase functioning to prevent the expression of a novel O(2)-dependent process from oxidizing the heme on sGC and inhibiting NO-elicited cGMP-mediated BCA relaxation.

Animals↗

PubMed1989

A new microphotometric method for measurement of cytochrome P-450 in sections of liver.

We developed a new microphotometric method for measuring the amounts of cytochrome P-450 (P-450) in fresh frozen sections of liver. Four serial frozen sections cut from the liver were separately incubated in 50 mM Tris-HCl buffer (pH 8.0) alone, in buffer containing sodium dithionite, in buffer saturated with carbon monoxide (CO), and in buffer saturated with CO and containing sodium dithionite. The difference between absorbance at 450 nm and that at 490 nm was measured in these sections with a simple microphotometer system. This method yielded precise amounts of P-450 in sections by measuring the true extinction of P-450 and by minimizing the effect of contaminating hemoproteins. Livers of adult rats contained large amounts of P-450, which was greater in perivenular hepatocytes than in periportal hepatocytes. In livers of newborn rats, however, small amounts of the enzyme were distributed evenly throughout the lobule.

Aging↗

PubMed1986

Kinetics of oxygen uptake and release by red blood cells of chicken and duck.

The specific conductance (G) for O2 transfer by red blood cells (RBCs) of chicken and muscovy duck was measured using the experimental (stopped-flow) and analytical techniques (RBC model) previously applied to human RBC (Yamaguchi, Nguyen Phu, Scheid & Piiper, 1985). Avian RBCs behaved similarly to human RBCs: G values were of similar magnitude; G for O2 uptake decreased with time and increasing O2 saturation; G for O2 release at high levels of dithionite decreased slightly with decreasing O2 saturation; G for O2 release was higher than G for O2 uptake. The deoxygenation kinetics of oxyhaemoglobin in solution was similar for both avian species. The G measured for O2 release at high dithionite concentration, considered to represent a good approximation to intra-erythrocyte O2 diffusion conductance, averaged (in mmol min-1 Torr-1 ml-1 RBC) 0.33 for chicken and 0.25 for duck (at 41 degrees C, pH of the suspension = 7.5, O2 saturation range 0.4-0.8). These species differences can be explained by differences in cell size, the RBC volume averaging 104 micron3 in the chicken and 155 micron3 in the duck. Compared with human RBCs, the G estimates for avian RBCs are somewhat smaller than would be predicted from size differences, which can be explained by the discoid shape of mammalian RBCs which constitutes an advantage compared with the ovoid avian RBC.

Animals↗

PubMed1982

A new device for the determination of microsomal cytochrome P-450 in renal tissue preparations from various species contaminated with mitochondria and hemoglobin.

Using a spectrophotometer connected to a microcomputer, the carbon monoxide difference spectrum of renal microsomal dithionite-reduced cytochrome P-450 was measured avoiding the effects of the contaminating mitochondrial cytochromes and hemoglobin by subtracting the CO difference spectrum of a succinate-treated microsomal suspension from that of a dithionite-treated one. By this method, we quantitatively determined the microsomal cytochrome P-450 in kidneys from various species. The absorption peak of the renal microsomal cytochrome P-450 was about 452 nm in rats and about 450 nm in mice, hamsters, rabbits, guinea pigs, dogs, and pigs. Compared with the renal cytochrome P-450 contents in rats, the contents on a g tissue basis were greater in pigs, dogs, and mice and were about the same in hamsters and rabbits. The renal cytochrome P-450 contents in guinea pigs were less than those in rats.

Animals↗

PubMedDate not supplied

Determination of redox-sensitive phosphorus in field runoff without sediment preconcentration.

Reduction-induced phosphorus (P) release from particles transported by field runoff has been poorly studied for want of a method that could be used for large surveys. To rectify this shortcoming, we modified the bicarbonate-dithionite (BD) extraction step of a sediment P speciation scheme for analyzing redox-sensitive P in runoff without sample preconcentration. The extraction comprised the addition of bicarbonate (pH buffer) and dithionite (reducing agent) into a runoff sample, 15 min of gentle shaking, filtration, and sample digestion. The samples were greatly reduced (Eh < -200 mV), and Fe and P were solubilized, but Al solubility was not increased. Phosphorus release from rock phosphates (calcium phosphates) was greater in the BD extraction than in water or bicarbonate solution, although no more than 0.2% of the total P was released. For runoff from a very fine Typic Cryaquept, the particulate phosphorus (PP) versus BD-PP relationship was linear up to a PP concentration of about 1.0 mg L(-1), but over the whole PP range studied (up to 2.6 mg L(-1)) somewhat better described by an exponential equation (BD-PP = 0.297 x PP(0.766); r2 = 0.91, n = 79). The minimum detectable value given by the method was relatively low, 0.023 mg L(-1), but reproducibility varied, with the coefficient of variation for 10 samples analyzed with 5 replicates ranging from 1.8 to 28.5%. Considering the variable reproducibility of the results and the lack of suitable reference material, the method needs further refinement and testing if it is to be used for quantitative determination of redox-sensitive P in runoff.

Agriculture↗

PubMed1999

Interaction of peroxynitrite and hydrogen peroxide with dinitrosyl iron complexes containing thiol ligands in vitro.

The interaction of peroxynitrite with thiolate dinitrosyl iron complexes (DNIC) has been examined and compared with the interaction with H2O2. Peroxynitrite oxidized DNIC containing various thiolate ligands--cysteine, glutathione, and bovine serum albumin. Analysis of the oxidation suggested a two-electron reaction and gave third-order rate constants of (9.3 +/- 0.5).109 M-2.sec-1 for DNIC with BSA, (4.0 +/- 0.3).108 M-2.sec-1 for DNIC with cysteine, and (1. 8 +/- 0.3).107 M-2.sec-1 for DNIC with glutathione at 20 degrees C and pH 7.6. Peroxynitrite was more reactive towards DNIC than towards sulfhydryls. Addition of sodium dithionite after the reaction led to significant restoration of the EPR signal of DNIC with cysteine. The reaction of glutathione DNIC with H2O2 was about 600 times slower than with ONOO- and not reversed by sodium dithionite. Thus peroxynitrite, in contrast to hydrogen peroxide, changes the pool of nitrosocompounds which can be responsible for interconversion, storage, and transportation of nitric oxide in vivo.

Cysteine↗