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PubMed1985

Differential investigation of the capacity of succinate oxidation in human skeletal muscle.

Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6-dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder.

2,6-Dichloroindophenol↗
PubMed1993

An enzymatic assay of inorganic phosphate in serum using nucleoside phosphorylase and xanthine oxidase.

We have developed a new enzymatic assay for the determination of inorganic phosphate (Pi) in serum, using nucleoside phosphorylase (NP) and xanthine oxidase (XOD). Pi and inosine react to form hypoxanthine and ribose-1-phosphate. The hypoxanthine is oxidized to xanthine, which is further oxidized to uric acid. In these two reactions 2,6-dichlorophenol-indophenol (DCIP) is reduced to a colourless compound and the decrease in colour is measured spectrophotometrically at 600 nm. The assay is automated with an RA-XT analyser. The precision of the automated assay is acceptable (C.V. < 3.5%) and results are accurate and linear across a range of values from 0.2-2.5 mmol/l. The assay correlates well with molybdate methods carried out on SMAC III and RA-XT analysers (r values 0.99 and 0.98, respectively), and seems to be less prone to non-specific sample interference than the usual RA-XT method. The enzymatic assay described seems to be suitable for the routine determination of serum Pi.

Humans↗
PubMed1992

Thermoluminescence evidence for light-induced oxidation of tyrosine and histidine residues in manganese-depleted photosystem II particles.

In the thermoluminescence (TL) glow curve of photosystem II, particles depleted of manganese, a tyrosine modifier, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) abolishes the TL band appearing around -55 degrees C (TL-55). Addition of a histidine modifier, diethylpyrocarbonate results in the disappearance of the band peaking around -30 degrees C (TL-30). NBD treatment also abolishes the EPR signal IIfast of oxidized tyrosine donor, Yz, and inhibits the electron transport from diphenylcarbazide to 2,6-dichlorophenol-indophenol. It is concluded that the TL-55 and TL-30 bands can be assigned to oxidized tyrosine (Yz+) and histidine (His+) residues, respectively, which participate in electron transfer from manganese to the reaction center of chlorophyll, P680+.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

High-specific binding of Fe(II) at the Mn-binding site in Mn-depleted PSII membranes from spinach.

The interaction of Fe(II) and Fe(III) with the 'high-affinity Mn-binding site' in Mn-depleted photosystem II (PSII) was investigated using diphenilcarbazide (DPC)/2,6-dichlorophenol-indophenol (DCIP) inhibition assay. Fe(III) was ineffective in the inhibition of DPC-DCIP reaction while Fe(II) decreased the rate of DCIP photoreduction supported by DPC in the same concentration range as Mn(II). The effectivity of the interaction of Fe(II) with the high affinity Mn-binding site depends on different anions in the same manner as for Mn(II) and coincides with hierarchy observed for the stimulation of O2 evolution. The Fe(II) binding is accompanied by its oxidation. By using reductants it was shown that the high affinity site contains a redox-active component and the reduction of this component totally prevents the binding of Fe(II).

2,6-Dichloroindophenol↗
PubMed1986

Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane.

NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.

Animals↗
PubMed1988

Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate.

Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.

Animals↗
PubMed1988

Some pitfalls and considerations of plasma ammonia estimation.

Several approaches to the estimation of plasma ammonia have been tested and compared: indophenol, Nessler, iodometric techniques were studied as well as enzymatic spectrophotometric and radiometric methods. Their lower safe limits of estimation were determined and it was found that most of them were not viable for plasma estimations because of its very low ammonia levels. The need for a concentration/purification step and the lack of repetability in this phase at very low concentrations, made very unreliable the utilization of all methods studied except for the enzymatic radiometric method tested that was barely usable for plasma ammonia estimations.

Ammonia↗
PubMed1995

Mutation to glutamine of histidine 373, the catalytic base of flavocytochrome b2 (L-lactate dehydrogenase).

Flavocytochrome b2 catalyzes the two-electron oxidation of L-lactate. Reducing equivalents are transferred first to FMN then to heme b2 in the same subunit, finally to cytochrome c or a non-physiological acceptor. The enzyme's three-dimensional structure, when analyzed in the light of existing mechanistic knowledge, suggested that His 373 is the active site base which initiates the substrate chemical transformation by abstracting the lactate alpha-proton. We report here the properties of a mutant enzyme with glutamine substituted histidine at position 373. The mutated enzyme preparations show a 10(4)-fold decrease in catalytic activity. We find that most of this residual activity can be eliminated by treatments with: 1) fluoropyruvate, an affinity label for His 373; and 2) 2- hydroxy-3-butynoate, a suicide reagent which normally forms an adduct with FMN but in this case leaves the bulk of the prosthetic group intact. Furthermore, although spectral titrations do not detect any binding of oxalate, this reagent inhibits the mutant enzyme with the same kinetic behaviour as for the wild-type enzyme. We conclude that the enzyme preparations contain about 1 in 10(4) molecules of wild-type flavocytochrome b2; this is probably due to codon misreading during biosynthesis. Thus the H373Q enzyme displays at most 10(5)-fold less activity than the wild-type enzyme. We report values for the spectrally determined binding constants of sulfite, pyruvate and D-lactate for the mutant enzyme. Finally, we show that 2,6-dichlorophenol indophenol, which is a 10-fold more sensitive routine electron acceptor than ferricyanide, accepts electrons only from heme b2 and not from the flavin.

2,6-Dichloroindophenol↗
PubMed1992

Pressure modulation of cytochrome-to-cytochrome electron-transfer. Models and enzyme reactions.

The kinetics of electron-transfer involved in reactions of reduction of 2,6-dichlorophenol indophenol and Fe(CN)3-(6) by L-ascorbic acid and reduction of ferric cytochrome c by both L-ascorbic acid and reduced hydroxylamine oxidoreductase were studied as a function of three parameters: ionic strength, pressure (1-2000 bar) and temperature (4-20 degrees C) using the high-pressure stopped-flow method. From measurements, the thermodynamic parameters of activation volume (delta V++), and, when possible, activation enthalpy and entropy (delta H++ and delta S++) have been calculated. We found, for these four systems, that the pressure has revealed solvation effects involved in electron-transfer. For the reduction of ferric cytochrome c by reduced hydroxylamine oxidoreductase (a cytochrome-to-cytochrome electron-transfer), we have not obtained evidence for a conformational change.

2,6-Dichloroindophenol↗
PubMed1989

Evidence for the presence of a new NAD+-dependent formate dehydrogenase in Pseudomonas sp. 101 cells grown on a molybdenum-containing medium.

The facultatively methylotrophic bacterium Pseudomonas sp. 101, grown on methanol in presence of molybdate, contains a new formate dehydrogenase (N-FDH) catalyzing NAD+-dependent oxidation of formate. The activity of this N-FDH could also be measured in presence of artificial electron acceptors, ferricyanide and 2,6-dichlorophenol indophenol. This new enzyme is absent in cells grown on a methanol-containing medium with tungstate, where only another two, previously described formate dehydrogenases, which are active only with NAD+ or only with artificial acceptors, respectively, were determined. The N-FDH was partially purified by a combination of ion-exchange and gel-filtration chromatography, and was shown to differ in its properties from the known NAD+-dependent counterpart.

Aldehyde Oxidoreductases↗
PubMed1993

High-performance liquid chromatographic determination of plasma ascorbic acid in relationship to health care.

We have developed a simple reversed-phase high-performance liquid chromatographic method for determining plasma ascorbic acid level and studied the relationship between its plasma concentration and fruit and vegetable intake and plasma dopamine-beta-hydroxylase activity. The samples were pretreated by precipitating the proteins and injected onto the column. Elution with a methanol gradient in sodium phosphate buffer was carried out by monitoring the absorbance at 265 nm, and the peak corresponding to ascorbic acid was well separated from other peaks of reagents used for pretreatment and from plasma endogenous components. The proposed method correlated well with the conventional dichlorophenol-indophenol method. Mean levels of ascorbic acid in normal human plasma were 0.86 +/- 0.36 mg/dl for males (twenty subjects, 19-28 years old) and 1.01 +/- 0.30 mg/dl for females (twenty subjects, 19-21 years old). There was good correlation between plasma ascorbic acid levels and dopamine-beta-hydroxylase levels, reflecting activities of daily living, but no correlation was found between these levels and dietary consumption of vegetables or fruits.

2,6-Dichloroindophenol↗
PubMed1991

Spectrophotometric determination of cimetidine in the presence of its acid-induced degradation products.

Cimetidine has been determined in the presence of its acid-induced degradation products using a second derivative (D2-) spectrophotometric method (method I) or a colorimetric method (method II). The former is based on D2-value measurement at 216 nm, whilst the latter depends on charge-transfer complexation with dichlorophenol-indophenol. The two methods are proved to be stability indicating, since plots of log C% versus time were linear. The application to cimetidine determination in tablets and ampoules gave good results.

Cimetidine↗
PubMed1991

The reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells: free radical formation and NAD(P)H: quinone-acceptor oxidoreductase (DT-diaphorase) activity.

The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ.

Antineoplastic Agents↗
PubMed1992

2,6-Dichlorophenolindophenol is a competitive inhibitor for xanthine oxidase and is therefore not usable as an electron acceptor in the fluorometric assay.

Xanthine oxidase has been recognized as an important source of oxygen free radicals in ischemia-reperfusion injury. In order to study this enzyme in biological tissues, the conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a very sensitive fluorometric assay. Xanthine oxidase is typically assayed in the presence of pterin only, while an electron acceptor which replaces NAD+ is used to determine the combined xanthine dehydrogenase plus xanthine oxidase activity. 2,6-Dichlorophenol-indophenol has been used as an electron acceptor in this assay. However, it was found in this study that it acts as an effective competitive inhibitor for xanthine oxidase. We concluded that methylene blue is the electron acceptor of choice in the fluorometric assays for xanthine oxidase.

2,6-Dichloroindophenol↗
PubMed1994

Monitoring the activity of glucose oxidase during the cultivation of Aspergillus niger using novel amperometric sensor with 1, 1'-dimethylferricinium as a mediator.

1, 1'-dimethylferricinium (DMF+), a deep blue, and stable mediator, was prepared from a water-soluble 1, 1'-dimethylferrocene(DMF):2-hydroxypropyl- beta-cyclodextrin complex via enzymatic oxidation using immobilised bilirubin oxidase. This mediator was superior to other soluble ferrocenes, notably carboxyferrocene, in terms of both solubility (110 mM vs 0.5 mM) and oxidation potential (150 mV vs 300 mV against Ag/AgCl). Although the cyclic voltammogram of DMF+ was electrochemically equivalent to DMF, the use of the former resulted in a significantly lower background current (< 10 nA vs 30 nA). Because of its higher solubility, concentrated stock solutions of DMF+ can be prepared and supplied to the electrode. This is of particular importance when the signal is severely limited by the rate at which the working electrode can oxidase DMF to DMF+. A linear response of current versus units of glucose oxidase (GOD) was obtained up to 0.5 unit/ml. The detection limit was estimated to be 0.03 unit/ml and the response time was 2.5 min or less. The amperometric system was used successfully to follow the GOD activity during the growth of Aspergillus niger a well-known GOD producer. The results obtained correlated well with a standard absorbance-based assay using dichlorophenol-indophenol (DCPIP). The KM of GOD for the glucose in the lysate was measured as 38 mM. A reduced response and higher KM (48 mM) of the cell homogenate, compared to the lysate, illustrated the requirement for the DMF+ and glucose to diffuse across the cell membrane to interact with GOD in whole cells.

Aspergillus niger↗
PubMed2004

Stabilization and characterization of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae.

Histidine-tagged homocitrate synthase from Saccharomyces cerevisiae was purified to about 98% using a Ni-NTA resin and stabilized using a combination of 100 mM guanidine hydrochloride, 100 mM alpha-cyclodextrin, and 600 mM ammonium sulfate. The enzyme was assayed using dichlorophenol indophenol (DCPIP) as an oxidant to oxidize the CoASH produced in the reaction. A stoichiometry of 1:1 was obtained between DCPIP and CoASH. Kinetic parameters for the stable enzyme at pH 7.5 are: Km (AcCoA), 24 microM: Km (alpha-kg), 1.3 mM; and kcat, 37 min(-1). The enzyme, in the absence of reactants, self-associates, as suggested by size exclusion chromatography. Fluorescence and circular dichroic spectra suggested a partially exposed tryptophan residue and a mixed (alpha/beta) secondary structure for the enzyme. Fluorescence quenching studies with KI, CsCl, and acrylamide suggest that the microenvironment around the single tryptophan residue of the enzyme has some positive charge.

2,6-Dichloroindophenol↗
PubMed2004

Electrochemical investigation of the dynamics of Mycobacterium smegmatis cells' transformation to dormant, nonculturable form.

Dynamics of transformation of Mycobacterium smegmatis cells by cultivation under nonoptimal conditions (partial starvation) to dormant, nonculturable form has been studied. For this aim, an electrochemical method was developed to detect both viable and 'viable but nonculturable' (VBNC) cells. The current produced by bacteria placed at the electrode surface was measured in the presence of 2,6-dichlorophenol indophenol (DCIP) at the applied potential of 350 mV. It has been established that electrochemical activity changes parallel with the growth of biomass. The transition of M. smegmatis to a dormant, nonculturable state goes along with the decrease of the detection current up to 20% of the maximum level. This means that nonculturable cells have rather high rest metabolic activity. The course of the CFU values has a complicated character during bacterial growth. The placement of the bacterial culture on the solid medium appears to cause a new stress that stops proliferation and stimulates aggregation. Both processes distort CFU measurement results. The quantitative estimation of the viable but nonculturable cells by counting colonies, measuring optical density and current produced by bacteria has been discussed.

2,6-Dichloroindophenol↗
PubMed2006

Escherichia coli-catalyzed bioelectrochemical oxidation of acetate in the presence of mediators.

Bioelectrocatalytic oxidation of acetate was investigated under anaerobic conditions by using Escherichia coli K-12 (IFO 3301) cells cultured on aerobic media containing poly-peptone, glucose or acetate as the sole carbon source. It was found that all E. coli cells cultured on the three media work as good catalysts of the electrochemical oxidation of acetate as well as glucose with Fe(CN)6(3-), 2,3-dimethoxy-5-methyl-1,4-benzo-quinone (Q0), 2,6-dichloro-indophenol, or 2-methyl-1,4-naphthoquinone as artificial electron acceptors (mediators). Acetate-grown E. coli cells exhibited the highest relative activity of the acetate oxidation against the glucose oxidation. On the other hand, all the artificial electron acceptors used work as inhibitors for the catalytic oxidation of acetate at increased concentrations. The inhibition phenomenon can be interpreted in terms of competitive substrate inhibition as a whole. Apparent values of Michaelis constant, catalytic constant, and inhibition constant were evaluated by amperometric methods. Q0 is an effective artificial mediator as evidenced by a large reaction rate constant between the cell and Q0 at least at low concentrations (<50 microM). However, Fe(CN)6(3-) is a promising mediator in biosensor applications because the inhibition constant is very large and it works as an electron acceptor even under aerobic conditions.

2,6-Dichloroindophenol↗