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PubMed2006

Dependence of turbidity oscillations of self-oscillating polymers on the selective recognition of the crown ether receptors contained in the polymer chain.

The turbidity oscillations of self-oscillating polymers in the Belousov-Zhabotinsky (BZ) reaction system depending on the crown ether receptors contained in the polymer network have been studied. The three monomers are copolymerized, namely, N-isopropylacrylamide, the metal catalyst monomer for the BZ reaction, and the crown ether receptor monomer, to prepare the self-oscillating polymers used in this study. The turbidity oscillations are characterized by monitoring the transmittance of the polymer solution in the BZ reaction system at a specific wavelength of 570 nm. The oscillations are varied by crown ether receptors used in the polymerization process, i.e., BCAm(6) or BCAm(5), for the selective recognition of specific cations between potassium and sodium ions in the solution. The selective recognition of the BCAm receptors in the polymer chain for the two ions has brought out a variation in the turbidity oscillations by a change in the hydrophilicity of the polymer chain. The oscillations of the polymer solution composed of the BCAm(5) receptor are more influenced by sodium ion, while the polymer solution of BCAm(6) receptor is affected by potassium ion. However, the oscillation patterns of the redox changes obtained by these solution systems look much alike despite the differences in the polymer chain by crown ether receptors and cations of bromate used for the BZ reaction.

Crown Ethers↗
PubMed2006

Combined propofol sedation with sub-Tenon's lidocaine/mercaine infusion for strabismus surgery in adults.

AIMS: To evaluate the safety and efficacy of propofol sedation combined with sub-Tenon's anaesthesia for strabismus surgery in adults. METHODS: Thirty-two consecutive patients aged 31-85 years underwent strabismus surgery under general (n=16) or local (n=16) anaesthesia. In the local anaesthesia (study) group, sedation was induced with a loading dose of midazolam, fentanyl, and propofol, followed by continuous infusion of propofol, 3-6 mg/k/h to deep sedation. A nasal tube was inserted to prevent airway obstruction. Sub-Tenon's anaesthesia included infusion of a 3-4 ml mixture (50 : 50) of lidocaine 2%/mercaine 0.5%. General anaesthesia consisted of premedication with midazolam, followed by fentanyl, esmeron-bromate, propofol, and tracheal intubation. Duration of surgery and anaesthesia, intraoperative oculocardiac reflex and arrhythmias, time to discharge, postoperative pain, nausea and vomiting, and patient and surgeon satisfaction were evaluated. RESULTS: The local anaesthesia group had a significantly shorter operative and anaesthesia time, fewer episodes of oculocardiac reflex or arrythmia/bradycardia requiring treatment, fewer early or late episodes of nausea and vomiting, and less pain. The patients and surgeon in this group reported higher satisfaction. CONCLUSION: Propofol sedation with local sub-Tenon's injection of lidocaine/mercaine is recommended for the induction and maintenance of anaesthesia during unilateral or bilateral strabismus surgery in adults. The method is quick and effective, without systemic or ocular side effects.

Adult↗
PubMed2001

HPLC analysis of complete BZ systems. Evolution of the chemical composition in cerium and ferroin catalysed batch oscillators: experiments and model calculations.

In the last few years many new reaction routes and intermediates have been discovered in the mechanism of the Belousov-Zhabotinsky (BZ) reaction with the aid of high performance liquid chromatography (HPLC). These previous HPLC studies, however, were limited to the Ce(4+)-organic substrate (malonic or bromomalonic acid) systems only. Very recently some measurements were made on a cerium catalysed full BZ system but only in its induction period. The present work follows the evolution of the main chemical components in a cerium and in a ferroin catalysed full BZ system from the start until the end of the oscillatory regime in a batch reactor. While recording the potential oscillations of a bromide selective electrode we measured from time to time the concentration of the following components: malonic and bromomalonic acids and bromate as main components; malonyl malonate, ethanetetracarboxylic and bromoethenetricarboxylic acids which are recombination products of organic free radicals; oxidized intermediates: tartronic, oxalic (OA) and mesoxalic (MOA) acids, and brominated products: dibromoacetic and tribromoacetic acids. Recombination products are generated in the intervals when the autocatalytic reaction is "switched off". In the course of the autocatalytic periods, however, the organic radicals react with the inorganic bromine dioxide radical mainly which leads to the formation of MOA and OA. Due to a very fast Ce(4+)-MOA reaction, MOA can be detected in the ferroin catalysed BZ system only. Our model calculations deal exclusively with the cerium catalysed system. The suggested new Marburg-Budapest-Missoula (MBM) model includes both negative feedback loops (bromous acid-bromide ion Oregonator type and bromine dioxide-organic free radicals Radicalator type feedback) and the recently discovered radical-radical recombination reactions. Comparison of the experimental data with the model calculations shows a good qualitative agreement but some open problems still remain. To overcome these problems oxygen atom transfer and other redox reactions are proposed.

Journal Article↗
PubMed2002

Examples of the role of analytical chemistry in environmental risk management research.

Analytical chemistry is an important tier of environmental protection and has been traditionally linked to compliance and/or exposure monitoring activities for environmental contaminants. The adoption of the risk management paradigm has led to special challenges for analytical chemistry applied to environmental risk analysis. Namely, methods developed for regulated contaminants may not be appropriate and/or applicable to risk management scenarios. This paper contains examples of analytical chemistry applied to risk management challenges broken down by the analytical approach and analyte for some selected work in our laboratory. Specific techniques discussed include stable association complex electrospray mass spectrometry (cESI-MS), gas chromatography-mass spectrometry (GC-MS), split-flow thin cell (SPLITT) fractionation and matrix-assisted laser desorption time of flight mass spectrometry (MALDI-ToF-MS). Specific analytes include haloacetic acids (HAA9), perchlorate, bromate, triazine degradation products, metal-contaminated colloids and Cryptosporidium parvum oocysts.

Acetates↗
PubMed2002

Advances in the determination of inorganic ions in potable waters by ion chromatography.

Ion chromatography (IC) is now a well-established methodology for the analysis of ionic species. The technique is applicable to the determination of a wide range of solutes in many sample types, although the determination of inorganic ions in potable waters continues to be the most widely used application of ion chromatography. Many standardization and regulatory bodies, such as the American Society for Testing and Materials (ASTM), International Organization for Standardization (ISO), and US Environmental Protection Agency (EPA), have approved methods of analysis based upon IC, most of which have been published within the last decade. Recent developments in the field of IC, such as the use of higher capacity columns, larger loop injections, more complex sample preparation and detection schemes, have been incorporated into these new approved methods. These advances allow the determination of environmentally significant contaminants, such as common inorganic anions, bromate, perchlorate and chromate, at trace levels in potable waters using ion chromatography.

Chromatography, Ion Exchange↗
PubMed2005

Responsive brushes and gels as components of soft nanotechnology.

Progress in the development of generic molecular devices based on responsive polymers is discussed. Characterisation of specially synthesised polyelectrolyte gels, "grafted from" brushes and triblock copolymers is reported. A Landolt pH-oscillator, based on bromate/ sulfite/ferrocyanide, with a room temperature period of 20 min and a range of 3.1 < pH < 7.0, has been used to drive periodic oscillations in volume in a pH responsive hydrogel. The gel is coupled to the reaction and changes volume by a factor of at least 6. A continuously stirred, constant volume, tank reactor was set-up on an optical microscope and the reaction pH and gel size monitored. The cyclic force generation of this system has been measured directly in a modified JKR experiment. The responsive nature of polyelectrolyte brushes, grown by surface initiated ATRP, have been characterised by scanning force microscopy, neutron reflectometry and single molecule force measurements. Triblock copolymers, based on hydrophobic end-blocks and either polyacid or polybase mid-block, have been used to produce polymer gels where the deformation of the molecules can be followed directly by SAXS and a correlation between molecular shape change and macroscopic deformation has been established. The three systems studied allow both the macroscopic and a molecular response to be investigated independently for the crosslinked gels and the brushes. The triblock copolymers demonstrate that the individual response of the polyelectrolyte molecules scale-up to give the macroscopic response of the system in an oscillating chemical reaction.

Journal Article↗
PubMed2003

New enzyme belonging to the family of molybdenum-free nitrate reductases.

A novel molybdenum-free nitrate reductase was isolated from the obligate chemolithoautotrophic and facultative anaerobic, (halo)alkaliphilic sulphur-oxidizing bacterium Thioalkalivibrio nitratireducens strain ALEN 2. The enzyme was found to contain vanadium and haem c as cofactors. Its native molecular mass was determined as 195 kDa, and the enzyme consists of four identical subunits with apparent molecular masses of 57 kDa. Apart from nitrate, the enzyme can utilize nitrite, chlorate, bromate, selenate and sulphite as electron acceptors. Moreover, it also has a haloperoxidase activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Transient complex oscillations in a closed chemical system with coupled autocatalysis.

In this study, hydroquinone was introduced to the classic Belousov-Zhabotinsky (BZ) reaction to build up coupled autocatalytic feedbacks. Various complex dynamical behaviors including successive period-adding bifurcations, irregular oscillations, and frequency modulations were observed in the coupled reaction system. Not only the complexity of oscillations but also the time period during which complex oscillations persist were found to depend greatly on the initial concentration of hydroquinone, which was expected to manifest the coupling strength in the studied system. Dependence of the observed transient complex oscillations on concentrations of ferroin, sulfuric acid, bromate, and malonic acid was also characterized systematically. Numerical simulations with a modified BZ model via incorporating reactions involving hydroquinone and products of hydroquinone qualitatively reproduced the influence of hydroquinone seen in experiments.

Journal Article↗
PubMed1995

The vacuolar ATPase: sulfite stabilization and the mechanism of nitrate inactivation.

Using vacuolar membranes from Neurospora crassa, we observed that sulfite prevented the loss of vacuolar ATPase activity that otherwise occurred during 36 h at room temperature. Sulfite neither activated nor changed the kinetic behavior of the enzyme. Further, in the presence of sulfite, the vacuolar ATPase was not inhibited by nitrate. We tested the hypothesis that sulfite acts as a reducing agent to stabilize the enzyme, while nitrate acts as an oxidizing agent, inhibiting the enzyme by promoting the formation of disulfide bonds. All reducing agents tested, dithionite, selenite, thiophosphate, dithiothreitol and glutathione, prevented the loss of ATPase activity. On the other hand, all oxidizing agents tested, bromate, iodate, arsenite, perchlorate, and hydrogen peroxide, were potent inhibitors of ATPase activity. The inhibitory effect of the oxidizing agents was specific for the vacuolar ATPase. The mitochondrial ATPase, assayed under identical conditions, was not inhibited by any of the oxidizing agents. Analysis of proteins with two-dimensional gel electrophoresis indicated that nitrate can promote the formation of disufide bonds between proteins in the vacuolar membrane. These data suggest a mechanism to explain why nitrate specifically inhibits vacuolar ATPases, and they support the proposal by Feng and Forgac (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13244-13230) that oxidation and reduction of critical cysteine residues may regulate the activity of vacuolar ATPases in vivo.

Adenosine Triphosphatases↗
PubMedDate not supplied

Application of cDNA microarray technology to in vitro toxicology and the selection of genes for a real-time RT-PCR-based screen for oxidative stress in Hep-G2 cells.

Large-scale analysis of gene expression using cDNA microarrays promises the rapid detection of the mode of toxicity for drugs and other chemicals. cDNA microarrays were used to examine chemically induced alterations of gene expression in HepG2 cells exposed to a diverse group of toxicants at an equitoxic exposure concentration. The treatments were ouabain (43 microM), lauryl sulfate (260 microM), dimethylsulfoxide (1.28 M), cycloheximide (62.5 microM), tolbutamide (12.8 mM), sodium fluoride (3 mM), diethyl maleate (1.25 mM), buthionine sulfoximine (30 mM), potassium bromate (2.5 mM), sodium selenite (30 microM), alloxan (130 mM), adriamycin (40 microM), hydrogen peroxide (4 mM), and heat stress (45 degrees C x 30 minutes). Patterns of gene expression were correlated with morphologic and biochemical indicators of toxicity. Gene expression responses were characteristically different for each treatment. Patterns of expression were consistent with cell cycle arrest, DNA damage, diminished protein synthesis, and oxidative stress. Based upon these results, we concluded that gene expression changes provide a useful indicator of oxidative stress, as assessed by the GSH:GSSG ratio. Under the conditions of this cell culture test system, oxidative stress upregulated 5 genes, HMOX1, p21(waf1/cip1), GCLM, GR, TXNR1 while downregulating CYP1A1 and TOPO2A. Primers and probes for these genes were incorporated into the design of a 7-gene plate for RT-PCR. The plate design permitted statistical analysis and allowed clear discrimination between chemicals inducing oxidative vs nonoxidative stress. A simple oxidative stress score (0-1), based on the responses by the 7 genes (including p-value) on the RT-PCR plate, was correlated with the GSH:GSSG ratio using linear regression and ranking (Pearson product) procedures. These analyses yielded correlation coefficients of 0.74 and 0.87, respectively, for the treatments tested (when 1 outlier was excluded), indicating a good correlation between the biochemical and transcriptional measures of oxidative stress. We conclude that it is essential to measure the mechanism of interest directly in the test system being used when assessing gene expression as a tool for toxicology. Tables 1-15, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Dose-Response Relationship, Drug↗
PubMedDate not supplied

Induction of transitional cell hyperplasia in the urinary bladder and aberrant crypt foci in the colon of rats treated with individual and a mixture of drinking water disinfection by-products.

Cancer of the urinary bladder and colon are significant human health concerns. Epidemiological studies have suggested a correlation between these cancers and the chronic consumption of chlorinated surface water containing disinfection by-products (DBPs). The present study was designed to determine if exposure to DBPs would cause preneoplastic or neoplastic lesions in the urinary bladder and colon of rats, and what effect a mixture of DBPs would have on these lesions. Male and female Eker rats were treated via drinking water with low and high concentrations of potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, or bromodichloromethane individually or in a mixture for 10 months. The urinary bladders and colons were examined for the presence of preneoplastic lesions. Cell proliferation in the urothelium was examined using immunohistochemical staining for bromodeoxyuridine. Aberrant crypt foci (ACF), as well as the number of individual crypts in each ACF, were identified and counted microscopically after staining with 0.2% methylene blue. Colon crypt cell proliferation and mitotic index were determined using immunohistochemical staining for proliferating cell nuclear antigen. Labeling indexes for the urinary bladder and colon were calculated based on the percentage of positively labeled cells. Treatment with the high dose of MX caused transitional epithelial hyperplasia and cell proliferation in the rat urinary bladder, and this effect was diminished in the high dose mixture animals. Treatment with 4 individual DBPs, as well as a mixture of them, caused the development of ACF, the putative preneoplastic lesion of colon cancer.

Animals↗
PubMed1997

Increased sensitivity of scid heterozygous mice to ionizing radiation.

In the present study, acute effects of ionizing radiation on animal survival, bone marrow cells and fibroblast cell lines of scid homozygous, scid heterozygous and wild-type mice with the same C.B-17 genetic background were examined. The sensitivities to ultraviolet light (UV) and various chemicals, bleomycin, mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanosulphonate, 5-fluorouracil, 6-mercaptopurine, 4-nitroquinoline 1-oxide and potassium bromate) were also investigated. In addition, micronucleus testing of whole-body irradiated mice was performed. Scid heterozygous mice were found to be less sensitive than the homozygotes but more sensitive to ionizing radiation than wild-type mice, not only in vivo but also for bone marrow cells in vitro, suggesting partial dominance under both conditions. In contrast, there were no differences in sensitivity to UV light and various chemicals, as compared with wild-type and scid heterozygous cell lines, either in vitro or in the micronucleus test.

Animals↗
PubMed1998

Experimental study of oxidative DNA damage.

Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical compounds have been studied in animal experiments mainly in rats and mice, and generally with measurement of 8-oxodG with HPLC-EC. A large number of well-known carcinogens induce 8-oxodG formation in liver and/or kidneys. Moreover several animal studies have shown a close relationship between induction of dative DNA damage and tumour formation. In principle the level of oxidative DNA damage in an organ or cell may be studied by measurement of modified bases in extracted DNA by immunohistochemical visualisation, and from assays of strand breakage before and after treatment with repair enzymes. However, this level is a balance between the rates of damage and repair. Until the repair rates and capacity can be adequately assessed the rate of damage can only be estimated from the urinary excretion of repair products albeit only as an average of the entire body. A number of model compounds have been used to induce oxidative DNA damage in experimental animals. The hepatocarcinogen 2-nitropropane induces up to 10-fold increases in 8-oxodG levels in rat liver DNA. The level of 8-oxodG is also increased in kidneys and bone marrow but not in the testis. By means of 2-nitropropane we have shown correspondence between the increases in 8-oxodG in target organs and the urinary excretion of 8-oxodG and between 8-oxodG formation and the comet assay in bone marrow as well potent preventive effects of extracts of Brussels sprouts. Others have shown similar effects of green tea extracts and its components. Drawbacks of the use of 2-nitropropane as a model for oxidative DNA damage relate particularly to formation of 8-aminoguanine derivatives that may interfere with HPLC-EC assays and have unknown consequences. Other model compounds for induction of oxidative DNA damage, such as ferric nitriloacetate, iron dextran, potassium bromate and paraquat, are less potent and/or more organ specific. Inflammation and activation of an inflammatory response by phorbol esters or E. coli lipopolysaccharide (LPS) induce oxidative DNA damage in many target cells and enhance benzene-induced DNA damage in mouse bone marrow. Experimental studies provide powerful tools to investigate agents inducing and preventing oxidative damage to DNA and its role in carcinogenesis. So far, most animal experiments have concerned 8-oxodG and determination of additional damaged bases should be employed. An ideal animal model for prevention of oxidative DNA damage has yet to he developed.

Animals↗
PubMed2000

A multiple-purpose design approach to the evaluation of risks from mixtures of disinfection by-products.

Drinking water disinfection has effectively eliminated much of the morbidity and mortality associated with waterborne infectious diseases in the United States. Various disinfection processes, however, produce certain types and amounts of disinfection by-products (DBPs), including trihalomethanes (THM), haloacetic acids, haloacetonitriles, and bromate, among others. Human health risks from the ubiquitous exposure to complex mixtures of DBPs are of concern because existing epidemiologic and toxicologic studies suggest the existence of systemic or carcinogenic effects. Researchers from several organizations have developed a multiple-purpose design approach to this problem that combines efficient laboratory experimental designs with statistical models to provide data on critical research issues (e.g., estimation of human health risk from low-level DBP exposures, evaluation of additivity assumptions as useful for risk characterization, estimation of health risks from different drinking water treatment options). A series of THM experiments have been designed to study embryonic development, mortality and cancer in Japanese medaka (Oryzias latipes) and liver and kidney endpoints in female CD-1 mice. The studies are to provide dose-response data for specific mixtures of the 4 THMs, for the single chemicals, and for binary combinations. The dose-levels and mixing ratios for these experiments were selected to be useful for development and refinement of three different statistical methods: testing for departures from dose-additivity; development of an interactions-based hazard index; and use of proportional-response addition as a risk characterization method. Preliminary results suggest that dose-additivity is a reasonable risk assessment assumption for DBPs. The future of mixtures research will depend on such collaborative efforts that maximize the use of resources and focus on issues of high relevance to the risk assessment of human health.

Alanine Transaminase↗
PubMed2003

Photocatalytic degradation of two selected textile dye derivatives, eosine yellowish and p-rosaniline, in aqueous suspensions of titanium dioxide.

Thephotocatalytic degradation of two selected textile dye derivatives, eosine yellowish (1) and p-rosaniline (2) has been investigated in aqueous suspensions of titanium dioxide under a variety of conditions. The degradation was studied by monitoring the change in substrate concentration employing UV spectroscopic technique and decrease in Total Organic Carbon (TOC) content as a function of irradiation time under a variety of conditions. The degradation of the dye was studied under different conditions such as pH, catalyst concentration, substrate concentration, different types of TiO2 and in the presence of electron acceptor such as hydrogen peroxide (H2O2), potassium bromate (KBrO3), and ammonium persulphate (NH4)2S2O8) besides molecular oxygen. The degradation rates were found to be strongly influenced by all the above parameters. The photocatalyst Degussa P25 was found to be more efficient as compared with other photocatalyst in the case of p-rosaniline, whereas UV100 was found to be better for the degradation of eosine yellowish. The xanthene dye (1) was found to degrade faster as compared to the triphenylmethane dye (2). The degradation products were analyzed by GC/MS technique and plausible mechanism for the formation of products have been proposed.

Catalysis↗
PubMed2005

Heterogeneous photocatalytic degradation of picloram, dicamba, and floumeturon in aqueous suspensions of titanium dioxide.

Heterogeneous photocatalytic degradation of three-selected herbicide derivatives: (1) picloram (4-Amino-3,5,6-trichloropyridine-2-carboxylic acid, (2) dicamba (2-Methoxy-3,6-dichlorobenzoic acid, and (3) floumeturon (N,N-Dimethyl-N-[3-(trifluoromethyl)phenyl]-urea) has been investigated in aqueous suspensions of titanium dioxide under a variety of conditions. The degradation was studied by monitoring the change in substrate concentration employing UV spectroscopic technique and decrease in total organic carbon (TOC) content as a function of irradiation time under a variety of conditions. The degradation of the herbicide was studied under different conditions such as pH, catalyst concentration, substrate concentration, different types of TiO2, and in the presence of electron acceptors such as hydrogen peroxide (H2O2), potassium bromate (KBrO3), and ammonium persulphate (NH4)2S2O8 besides molecular oxygen. The degradation rates were found to be strongly influenced by all the above parameters. The photocatalyst Degussa P25 was found to be more efficient as compared with other photocatalysts in the case of dicamba (2) and floumeturon (3), whereas Hombikat UV100 was found to be better for the degradation of picloram (1). The herbicide picloram (1) was found to degrade faster as compared to dicamba (2) and floumeturon (3). The degradation products were analyzed by gas chromatography-mass spectrometry (GC/MS) technique, and plausible mechanisms for the formation of products have been proposed.

Dicamba↗
PubMed1992

Purification and properties of trimethylamine N-oxide reductase from aerobic photosynthetic bacterium Roseobacter denitrificans.

Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90,000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172,000. The midpoint redox potential of heme c was +200 mV at pH 7.5. Absorption maxima appeared at 418,524, and 554 nm in the reduced state and 410 nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase.

Bacteria↗
PubMed1986

Purification and properties of nitrate reductase from Mitsuokella multiacidus.

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).

Bacteroides↗