Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

Kinetics of methyl viologen reduction by hydrogen catalyzed by hydrogenase from Desulfovibrio vulgaris

The reduction of methyl viologen by hydrogen with hydrogenase was studied kinetically. The initial rate of the reduction was expressed as

where k′, K₂, and K₃ are constants and [S′] is the concentration of methyl viologen.According to this equation, a sequential mechanism was proposed. By combining the mechanism of hydrogen production from the reduced methyl viologen, a reaction mechanism for the reduction and oxidation of methyl viologen was proposed.     

Purification and properties of hydrogenase from Megasphaera elsdenii

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.     

Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase

Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor. The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed. The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined. The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V. The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential. Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide. No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.     

Effect of enzymic assay conditions on sulfite reduction catalysed by desulfoviridin from Desulfovibrio gigas.

The type and the amount of end products resulting from sulfite reduction catalysed by a single partially purified desulfoviridin preparation from Desulfovibrio gigas were shown to depend upon the enzymic assay conditions employed. Both manometric and spectrophotometric assays were used, with reduced methyl viologen serving as the electron donor in each system. Trithionate, thiosulfate, tetrathionate and sulfide were identified as possible end products. In the manometric assays, sulfide production was favoured by high reduced methyl viologen concentrations, low sulfite concentrations and a pH value of 7.0 as opposed to 6.0. In the spectrophotometric assays, results approaching the stoichiometric conversion of sulfite to sulfide were obtained only at high initial reduced methyl viologen concentrations.     

A circadian rhythm in the rate of light-induced electron flow in three leguminous species

Three legume species, Pisum sativum L., Glycine max (L.), and Phaseolus vulgaris L., were grown in light-dark cycles and then maintained in constant dim light. During the constant conditions, chloroplasts were isolated throughout the day and assayed for various light-reaction activities. Similar results were found for all three species. The rate of whole-chain, light-induced electron flow (H₂O to methyl viologen) was rhythmic over a 24-hour period provided an uncoupler of photophosphorylation was present. Chloroplasts varied in their response to uncouplers on a 24-hour basis and the per cent stimulation of electron flow was rhythmic. Neither PSII activity (H₂O to DCPIP or light-induced pH changes in the presence of K₃Fe(CN)₆), PSI activity (DCPIPH₂ ascorbate to methyl viologen) or the rate of oxidation of hydroquinone (TMQH₂ to methyl viologen) could be identified as a rate-limiting step for the rate of electron flow. The capability to photophosphorylate as measured by a photosynthetic control assay was also constant with time. A rhythm in oxygen evolution was also observed with leaf mesophyll cell suspensions isolated from Pisum. The possible involvement of dynamic changes in the composition or configuration of the thylakoid membrane is discussed.     

Ammonia assimilation and glutamate formation in the anaerobe Selenomonas ruminantium.

Selenomonas ruminantium was found to possess two pathways for NH4+ assimilation that resulted in net glutamate synthesis. One pathway fixed NH4+ through the action of an NADPH-linked glutamate dehydrogenase (GDH). Maximal GDH activity required KCl (about 0.48 M), but a variety of monovalent salts could replace KCl. Complete substrate saturation of the enzyme by NH4+ did not occur, and apparent Km values of 6.7 and 23 mM were estimated. Also, an NADH-linked GDH activity was observed but was not stimulated by KCl. Cells grown in media containing non-growth-rate-limiting concentrations of NH4+ had the highest levels of GDH activity. The second pathway fixed NH4+ into the amide of glutamine by an ATP-dependent glutamine synthetase (GS). The GS did not display gamma-glutamyl transferase activity, and no evidence for an adenylylation/deadenylylation control mechanism was detected. GS activity was highest in cells grown under nitrogen limitation. Net glutamate synthesis from glutamine was effected by glutamate synthase activity (GOGAT). The GOGAT activity was reductant dependent, and maximal activity occurred with dithionite-reduced methyl viologen as the source of electrons, although NADPH or NADH could partially replace this artificial donor system. Flavin adenine dinucleotide, flavin mononucleotide, or ferredoxin could not replace methyl viologen. GOGAT activity was maximal in cells grown with NH4+ as sole nitrogen source and decreased in media containing Casamino Acids.     

Heparin inhibition of ferredoxin-NADP reductase in chloroplast thylakoid membranes

Heparin, an anionic polysaccharide, inhibited the ferredoxin-catalyzed reduction of NADP in spinach chloroplast thylakoid membranes. Under the same conditions of assay, heparin did not interfere markedly with photoreduction of methyl viologen, anthraquinone sulfonate, or ferredoxin. A kinetic analysis of the heparin-induced interference with NADP photoreduction showed partial competitive inhibition. Heparin also interfered with NADPH oxidation by membrane-bound ferredoxin-NADP reductase (with dichlorophenol-indophenol as the acceptor) by a mechanism that involves partial competitive inhibition. This reaction was sensitive to the presence of salts; increasing ionic strength increases the heparin Ki for inhibition of NADPH oxidation. These results show that heparin binds to ferredoxin-NADP reductase, and in doing so interferes with binding to the reductase by both ferredoxin and NADP(H). Since heparin is redox inactive and does not interfere with the photophosphorylation reaction, it is a useful inhibitor of thylakoid membrane reactions which require the catalytic activity of ferredoxin-NADP reductase.     

The effects of photooxidative stress on photosystem I measured in vivo in Chlamydomonas

The effects of different photooxidative stresses on the function of photosystem I were measured in vivo in Chlamydomonas reinhardtii. Pholooxidative stresses included strong light, light combined with chilling to 0 °C, and light combined with several concentrations of methyl viologen. Photosystem I function was measured in vivo using the absorbance change at 820 nm associated with P₇₀₀ oxidation. Photosystem II function was measured in vivo using chlorophyll fluorescence. Strong light or light combined with chilling caused inhibition of photosystem II function earlier than inhibition of photosystem I function. When photosystem I was inhibited, however, it did not recover. Light combined with 5 mmol m^?3 methyl viologen caused inhibition of photosystem I function earlier than inhibition of photosystem II. If the methyl viologen concentration was reduced to 1 mmol m^?3, the damage to PSI was accelerated by addition of 90 mmol m^?3 chloramphenicol. This effect of chloroamphenicol suggests a role for chloroplast-encoded proteins in protecting photosystem I against photooxidative damage caused by methyl viologen.     

A hydrogen biosensor made of clay,poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode

A hydrogen gas (H(2)) biosensor was developed in which hydrogenase (H(2)ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H(2)ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay-PBV-H(2)ase modified electrode. The clay-PBV-H(2)ase electrode catalyzed the oxidation of H(2) to protons (H(+)) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38+/-2 kJ/mol at pH 7. The catalytic current of the clay-PBV-H(2)ase electrode increased linearly when exposed to increasing concentrations of H(2) gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H(2) by the clay-PBV-H(2)ase electrode was from 7 to 10. Electron transfer process in the clay-PBV-H(2)ase electrode is discussed.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Membrane association of the carbon monoxide oxidation system in Rhodopseudomonas gelatinosa.

A comparison of the distribution of CO oxidation activity between soluble and particulate protein fractions obtained after disruption of CO-grown Rhodopseudomonas gelatinosa 1 by French pressure cell breakage and osmotic lysis of spheroplasts suggested that, in situ, the enzyme complex was associated with the cell membrane. An improved, strictly anaerobic method is given for spectrophotometric measurement of CO oxidation activity based on the carbon monoxide:methyl viologen oxidoreductase reaction.     

Supramolecular formation of antibodies with viologen dimers: utilization for amplification of methyl viologen detection signals in surface plasmon resonance sensor

Monoclonal antibodies for 1-(carboxypentyl)-1'-methyl-4,4'-bipyridinium dichloride have been prepared. The complex formation of one of the antibodies, 10D5, with viologen dimer has been studied by a biosensor technique based on surface plasmon resonance. The dissociation constants of the complex between antibody 10D5 and methyl viologen or viologen dimer are found to be (2.0 +/- 0.2) x 10 (-7) and (1.5 +/- 0.5) x 10 (-7) M, respectively. Enhancement of response signal intensities in SPR is observed by the addition of the antibody solution to the viologen dimer-antibody complex indicating the formation of linear supramolecules between the antibody and viologen dimer. Amplification of methyl viologen sensing processes is realized by the inhibition of the complex formation between antibodies and viologen dimer-antibody complexes by methyl viologen and signal enhancement due to the supramolecular formation of the antibody and viologen dimer. The sensitivity in this system is found to be 2 orders larger than that obtained in the simple addition of methyl viologen to the antibody immobilized to the surface of the sensor chip.     

Kinetic parameters for hydrogen evolution by the NAD-linked hydrogenase of Alcaligenes eutrophus

The hydrogen-evolving reaction of the purified soluble NAD-linked hydrogenase of Alcaligenes eutrophus was used to determine kinetic parameters of the enzyme. The H₂-evolving activity with methyl viologen as electron mediator was 20-fold as compared to that with NADH. In the assay with dithionite-reduced methyl viologen (K _m 0.7 mM) the hydrogenase was most active at a redox potential of –560 mV and exhibited a pH optimum of 7.0. The K _m for protons, the second substrate for H₂ evolution, was 6.2 nM. With electrochemically reduced methyl viologen the pH optimum was shifted to pH 6.0. Double-reciprocal plots of reaction rates versus proton concentrations intercepted at the ordinate for different methyl viologen concentrations. At different pH values such an intercept was also observed with the dye as the varied substrate. The kinetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H₂ is released. Hydrogenase coupled to thylakoid membranes resulted in a constant H₂ evolution rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Effect of oxidative stress inductors on the photosynthetic apparatus in cyanobacterium Synechocystis sp. PCC 6803 Prq20 mutant resistant to methyl viologen

The damaging effect of oxidative stress inductors: methyl viologen, benzyl viologen, cumene hydroperoxide, H2O2, menadion, and high irradiance on the photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC 6803 in cells of the wild type strain and the methyl viologen-resistant Prq20 mutant with the disrupted function of the regulatory gene prqR has been investigated by measuring the delayed fluorescence of chlorophyll a and the rate of CO2dependent -O2 gas exchange. It has been shown that the damage to the photosynthetic apparatus in the Prq20 mutant as compared with the wild type was less in the presence of methyl viologen and benzyl viologen. Reasons for the enhanced resistance of the photosynthetic apparatus in the mutant Prq20 to methyl viologen and benzyl viologen are discussed.     

Properties of Stable Hydrogenase from the Purple Sulfur Bacterium Lamprobacter modestohalophilus

Some properties of a hydrogenase from the recently isolated phototrophic sulfur bacterium Lamprobacter modestohalophilus strain Syvash and its resistance to a number of inactivating factors have been investigated. The enzyme consists of two subunits, 64 and 30 kD; pI = 4.5. The optimal pH was 8.5-9.5 for hydrogen uptake and 4.0 for H2 evolution. Hydrogenase preparations were resistant to the effects of O2, CO, and temperature, revealing high stability under storage. A considerable inactivation of the enzyme was observed at temperatures above 80 degrees C; the temperature optimum of methyl viologen reduction by H2 was 85 degrees C. Inhibitory effects of Ni2+, Cd2+, and Mg2+ on the hydrogenase activity were shown to be reversible and competitive with respect to methyl viologen in the hydrogen oxidation reaction.     

Metabolism of carbon monoxide by Rhodopseudomonas gelatinosa: cell growth and properties of the oxidation system.

Rhodopseudomonas gelatinosa 1 grew as an anaerobic facultative methylotroph with carbon monoxide as the sole carbon and energy source. Carbon from CO was assimilated into cell material via the ribulose 1,5-bisphosphate carboxylase cycle. The CO oxidation system in R. gelatinosa was induced during growth with the gas substrate. Light-grown cells did not oxidize CO. Surprisingly, when strain 1 cells grown in the dark with CO were transferred to growth with both CO and light, they continued to use CO and then photometabolized after the CO gas flow was stopped. This change in the energy-yielding substrate resulted in a diauxic growth response. The use of CO in preference to light energy forms the basis of a system in the cells that controls photosynthetic differentiation. CO oxidation was assayed as CO-methyl viologen oxidoreductase. Methyl viologen reduction only occurred with CO; the dye was not reduced with other C1 compounds. In vitro methyl viologen was reduced best at 24 degrees C and at pH values above 8.5. Whole cells exhibited a Km of 12.5 microM for CO and a Vmax of 3,800 nmol of CO oxidized per mg of protein per min. This was a low-potential oxidation reaction that readily reduced the viologen dye triquat (1,1'-trimethylene-2,2'-dipyridilium dibromide) (E degrees' = -548 mV).     

Antigenic similarities between ferredoxin-dependent nitrite reductase and glutamate synthase fromChlamydomonas reinhardtii

Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green algaChlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferrodoxin-binding domains.     

A novel cis-element that is responsive to oxidative stress regulates three antioxidant defense genes in rice

All organisms have defense systems against oxidative stress that include multiple genes of antioxidant defense. These genes are induced by reactive oxygen species under condition of oxidative stress. In this study, we found that a 28-bp motif is conserved on the promoter regions of three antioxidant defense genes in rice (Oryza sativa): cytosolic superoxide dismutase (sodCc1), cytosolic thioredoxin (trxh), and glutaredoxin (grx). We demonstrated that the 28-bp sequence acts as a cis-element responsive to oxidative stress by transient expression assay and designated it as CORE (coordinate regulatory element for antioxidant defense). The CORE was activated by methyl viologen treatment and induced a 3.1-fold increase in expression of the reporter gene, but it did not respond to hydrogen peroxide. The expressions of the sodCc1, trxh, and grx genes were coordinately induced by methyl viologen, suggesting that multiple genes involved in antioxidant defense are controlled by a common regulatory mechanism via CORE. Application of the mitogen-activated protein kinase kinase inhibitor caused the constitutive induction of the sodCc1, trxh, and grx genes and the activation of CORE without methyl viologen treatment. These results indicate that a mitogen-activated protein kinase cascade is involved in the gene regulation mediated by CORE.