Tetramethyl-p-phenylenediamine Research Articles

Catalase activity of chloroperoxidase and its interaction with peroxidase activity.

The catalatic activity of chloroperoxidase (CPO) was demonstrated to exhibit saturation kinetics under steady-state conditions, which were not observed with catalase under comparable conditions. Results were obtained using reaction mixtures of CPO and H2O2 at pH 6.2, rapid spectral scan and single wavelength measurements, and transient- and steady-state reaction conditions. The observed rectangular hyperbolae (measurement of rates of disappearance of H2O2 and appearance of O2) could be fit quantitatively to [equation: see text] where v is rate of O2 evolution, [CPO]0 is total enzyme concentration, B1 = (9 +/- 1) x 10(2) s-1, and B2 = (3.3 +/- 0.4) x 10(-3) M. The results indicated formation of a complex of compound I (CPO-I) and H2O2, which dissociated to native CPO, O2, and H2O with a rate constant of (9 +/- 1) x 10(2) s-1. The determination of the peroxidatic activity of CPO was performed using demethylation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) under steady-state conditions. Attempts to determine Michaelis-Menten constants for the substrates TMPD and H2O2 gave rise to apparently anomalous data. Our data showed that the modified ping-pong mechanism established for horseradish peroxidase is applicable to the peroxidatic reaction catalyzed by chloroperoxidase. Both peroxidatic and catalatic reactions occurred in the reaction system containing H2O2, a reducing substrate, and CPO. A combined reaction mechanism was proposed for CPO-catalyzed reactions in which the modified ping-pong mechanism was applicable for the peroxidatic reactions and the formation of a CPO-I-H2O2 complex occurred for the catalatic reaction.

An alternative cytochrome oxidase of Paracoccus denitrificans functions as a proton pump

Deletion of the genes encoding subunit I of cytochrome aa 3 in P. denitrificans yields mutants that lack cytochrome aa 3 but which are still capable of oxidizing ubiquinol by O 2 via the cytochrome bc 1 complex, as revealed by myxothiazol-sensitive respiration on succinate. An alternative terminal oxidase must be responsible for the myxothiazol-sensitive activity. The H +/e − ratio of proton translocation by mutant cells approached 3.0 for oxidation of succinate by O 2, while it was near 2.0 either with ferricyanide as electron acceptor (engaging the bc 1 complex only), or with O 2 as acceptor in the presence of myxothiazol (engaging the ubiquinol oxidase). However, proton translocation coupled to oxidation of N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) plus ascorbate amounted to much less than 1.0 H +/e −. This may be due to interference by another TMPD-oxidising activity that is not engaged during electron flux to oxygen via the cytochrome bc 1 complex. We conclude that Paracoccus can express an alternative cytochrome b-containing terminal oxidase, which translocates protons at least when operating in conjunction with the cytochrome bc 1 complex.

Mechanism of maltose uptake and glucose excretion in Lactobacillus sanfrancisco

Lactobacillus sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells of L. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog alpha-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system.

The use of ultramicroelectrodes for the determination of diffusion coefficients

A combination of chronoamperometric and voltammetric responses at microdisk and conventional size disk electrodes respectively, was used to determine the diffusion coefficient of an electroactive species and the ultramicroelectrode diameter simultaneously. The method does not require knowledge of the bulk concentration and requires only the number of electrons participating in the electrode reaction and a value for the area of the conventional size disk electrode. Subsequent determination of the concentration of electroactive species is also possible. This approach is demonstrated by evaluating the diffusion coefficient of N,N,N′,N′-tetramethyl-p-phenylendiamine (TMPD) in pure and binary mixtures of aprotic solvents in perchlorate media. Parallel determinations were carried out from potentiodynamic I—E curves obtained for ultramicroelectrodes of different radii and corrected for ideal steady state conditions. The diffusion coefficients thus determined were in good agreement with those determined using other electrochemical techniques.

Muscle mitochondrial ATP production in progressive supranuclear palsy.

Six patients with progressive supranuclear palsy (PSP) and 12 age-matched disease-free subjects participated in this study designed to compare rates of ATP production by intact mitochondria from biopsied skeletal muscle. When pyruvate and malate were used as metabolic substrates, rates of ATP production were 0.184 +/- 0.025 mumol/min/U of citrate synthase (CS) activity (a mitochondrial marker) in control subjects and 0.131 +/- 0.051 mumol/min/U of CS in PSP patients. In the presence of succinate, rates of ATP formation were 0.137 +/- 0.02 mumol/min/U of CS in controls and 0.109 +/- 0.04 mumol/min/U of CS in patients. With N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ascorbate, rates were 0.034 +/- 0.008 mumol/min/U of CS in controls and 0.022 +/- 0.01 mumol/min/U of CS in PSP subjects. Differences between the control and PSP populations reached statistical significance with pyruvate/malate and TMPD/ascorbate. No differences in either muscle histopathology or histochemistry were found between patient and control subjects. Results of this study suggest that oxidative phosphorylation defects occur in muscle mitochondria from patients with PSP.

2-D Measurement of Fuel Vapor Concentration in Diesel Spray via Exciplex-Based Fluorescence Technique.

To measure the fuel vapor concentration in an unsteady evaporating spray, the exciplex-forming method, which produces spectrally separated fluorescence from the liquid and vapor phase, was applied in this study. Two experiments were conducted to investigate the qualitative and quantitative applicability of the technique in a high-temperature and high-pressure atmosphere during the fuel injection period. In one experiment, we examined the thermal decomposition of TMPD (NNN' N'-tetramethyl-P-phenylene-diamine) dopant in a high-temperature and high-pressure atmosphere during a period of time shorter than 10 ms. In the other, we calibrated the relationship between fluorescence intensity and vapor concentration of TMPD at different temperatures. Then, the distributions of fuel vapor concentration in diesel sprays were measured.

Magnetic Properties of CT Complexes between 2,2″,5,5″-Tetrakis(dimethylamino)terphenyls and TCNQF4

Abstract 2,2″,5,5″-Tetrakis(dimethylamino)-p- and m-terphenyls (1 and 2), in which two N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) moieties are linked through p- and m-phenylene units, respectively, were synthesized, and their CT complexes with TCNQF4 were prepared. In the complexes, 1 and 2 possess two S = 1/2 spins per molecule. The intramolecular interaction between the spins of the two TMPD moieties was found to be antiferromagnetic (2J/k = −42 K) in 1, and ferromagnetic (2J/k = 240 K) in 2.

Excited-state photophysics of donor-appended cobalt(II) porphyrins from picosecond transient absorption spectroscopy

Picosecond absorption spectra, obtained with 4-ps pump and probe pulses, are reported for the Co(II) complex of octaethylporphyrin (OEP) and of octaalkylporphyrins with single meso substituents: phenyl, N,N,N[prime],N[prime]-tetramethyl-p-phenylenediamine (TMPD), dimethylaniline-N,N,N[prime],N[prime]-tetramethyl-p-phenylenedia mine (DMA-TMPD), or a phenyl-TMPD. The donor substituents were attached in order to to investigate the possibility of transferring charge to the putative Co[sup (I)]P[sup +] species transiently formed in the excited-state deactivation of cobalt(II) porphyrins, thereby increasing the lifetime of a Co(I) phototransient. The substituents shifted the porphyrin ground-state absorption bands only slightly, and the redox potentials of the constituent parts of the constructs were essentially unaffected by the substitution. For all of the cobalt(II) porphyrins, photoexcitation produced transient bleaching of the ground-state Soret band and an induced absorption at ca. 410 nm. Ground-state recovery was complete within 50 ps in all cases. The lifetime of the phototransient was determined to be 12 [+-] 4 ps for OEP, as well as all the meso-substituted octaalkylporphyrins. 35 refs., 10 figs., 1 tab.

Purification and partial characterization of thiosulphate dehydrogenase from Thiobacillus acidophilus

SUMMARY: Thiosulphate dehydrogenase (EC 1.8.2.2; thiosulphate:acceptor oxidoreductase) was purified to apparent homogeneity from Thiobacillus acidophilus by a combination of ammonium sulphate precipitation, hydrophobic interaction chromatography, anion-exchange chromatography and gel filtration. The enzyme catalysed the oxidation of thiosulphate (S2O2- 3) to tetrathionate (S4O2- 6) with potassium ferricyanide as an artificial electron acceptor. The molecular mass of the native enzyme, as determined by gel filtration, was 102 ± 4.2 kDa. The enzyme contained two different subunits with a molecular mass of 24 ± 0.9 and 20 ± 1.0 kDa (SDS-PAGE), respectively. Both subunits contained c 553-type haem with absorption bands at 553, 524 and 416 nm. A 77 K spectrum of purified thiosulphate dehydrogenase revealed that the absorption at 553 nm is due to different haem groups. A cytochrome content of 5.3 mole c-type haem per mole of native enzyme was calculated. The pH optimum of the purified enzyme was 3. Apart from ferricyanide, Wurster's blue (the free radical of tetramethyl p-phenylenediamine) and horse heart cytochrome c could also serve as electron acceptors, though less effectively than ferricyanide. At pH 7.0, the K m for thiosulphate was 0.54 mM. The K m could not be determined at the pH optimum due to the chemical reactivity of thiosulphate at low pH values. Sulphite was a potent inhibitor of enzyme activity.

Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells.

Microfluorimetric and patch-clamp techniques have been combined to determine the relationship between changes in mitochondrial metabolism, the activity of KATP channels and changes in intracellular free calcium concentration ([Ca2+]i) in isolated pancreatic beta-cells in response to glucose, ketoisocaproic acid (KIC) and the electron donor couple tetramethyl p-phenylenediamine (TMPD) and ascorbate. Exposure of cells to 20 mM glucose raised NAD(P)H autofluorescence after a delay of 28 +/- 1 s (mean +/- S.E.M., n = 30). The mitochondrial inner membrane potential, delta psi m (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+]i rose after 129 +/- 13 s (n = 5). The amplitudes of the metabolic changes were graded appropriately with glucose concentration over the range 2.5-20 mM. All variables responded to KIC with shorter latencies: NAD(P)H autofluorescence rose after a delay of 20 +/- 3 s (n = 5) and rhodamine 123 changed after 21 +/- 3 s (n = 6). The electron donor couple, TMPD with ascorbate, rapidly hyperpolarized delta psi m and raised [Ca2+]i. When [Ca2+]i was raised by sustained exposure to 20 mM glucose, TMPD had no further effect. TMPD also decreased whole-cell KATP currents and depolarized the cell membrane, measured with the perforated patch configuration. These data are consistent with a central role for mitochondrial oxidative phosphorylation in coupling changes in glucose concentration with the secretion of insulin.

Efficiency of insecticidal crystal protein production in a Bacillus thuringiensis mutant with derepressed expression of the terminal oxidase aa 3 during sporulation

A Bacillus thuringiensis respiratory mutant (AB1 strain) that shows premature sporulation and insecticidal crystal protein (ICP) production was isolated. The mutant strain harbours the cryIC and cryID insecticidal genes and could be important for the production of ICP highly toxic to Spodoptera sp. The mutant was selected by its increased capacity to oxidize. N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). In this strain, cytochrome aa3 expression is not repressed during the sporulation phase, in contrast with the wild-type strain. The growth, spore production, dissolved O2, O2 consumption, CO2 evolution rate and ICP production were recorded as a function of time. The AB1 mutant strain has a similar growth yield to the wild-type strain, but begins sporulation at least 4 h earlier. The AB1 strain consumes 74.5% more O2 than the wild-type strain, during the fermentation process. The mutation on strain AB1 has an important positive effect on ICP production. This procedure shows that ICP production could be increased during fermentation by increasing the respiration capacity of Bacillus thuringiensis.

Rhizobium phaseoli cytochrome c-deficient mutant induces empty nodules on Phaseolus vulgaris L.

A Rhizobium phaseoli cytochrome mutant, unable to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), was isolated after Mu-dl (Kan lac) mutagenesis of the wild-type strain CE-3. Mutant strain CFN4202 had sixfold less haem-c but similar levels of b type, o and aa3 cytochromes than the wild-type strain. CFN4202 strain also showed reduced NADH- and TMPD-oxidase activity than the wild-type strain. Succinate-oxidase activities were very similar. Western blot experiments, using antiserum against bovine c1 and c cytochromes, revealed that both proteins were present in CFN4202 membranes, suggesting a defect of haem binding to cytochrome c. Nodules formed by this strain in Phaseolus vulgaris did not contain bacteroids. These data suggest that the cytochrome c-aa3 chain or some other respiratory chain, containing c-type cytochromes in R. phaseoli, is essential for bacterial division during the early steps of the symbiotic interaction with the legume-host.

Rates and efficiencies of contact-ion-pair formation in photolyzed mixtures of TMPD with halogenated compounds in nonpolar solvents

Time variation of microwave dielectric absorption has been examined for photolyzed solutions of N,N,N,N'-tetramethyl-p-phenylenediamine (TMPD) admixed with halogen-containing compounds AX (AX=CCl 4 , C 2 H 5 I, C 2 H 5 Br, C 2 H 5 Cl, C 6 H 5 I, C 6 H 5 Br, C 6 H 5 Cl, C 6 H 2 F, and C 6 F 6 ) in nonpolar solvents (mostly benzene). In solutions with CCl 4 , C 2 H 5 I, C 6 H 5 I, C 6 H 5 Br, and C 6 H 5 Cl, the dielectric absorption signals show first-order growths with appreciable intensities, indicating formation of a contact ion pair (CIP) TMPD + X - (X denotes a halogen atom) by reactions of these compounds with the excited triplet state of TMPD

Isolation and Characterization of a Mo 6+ -Reducing Bacterium

A Mo -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo (10 mM), the bacterium reduced Mo to form molybdenum blue. Approximately 27% of Mo added to the medium was reduced after 28 h of cultivation. The reduction of Mo with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo reduction. NADH and N,N,N',N' -tetramethyl-p-phenylenediamine served as electron donors for Mo reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo reduction. Both ferric and stannous ions strongly enhanced the activity of Mo reduction by NADH.

Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrificans

Light inhibited each step of the denitrification process in whole cells of Rhodobacter sphaeroides forma sp. denitrificans. This inhibition by light was prevented in the presence of exogenous electron donors like N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) plus ascorbate or in the presence of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone). Addition of myxothiazol restored the inhibition by light in uncoupled cells. Measurements of light-induced absorbance changes under these conditions showed that this inhibition is due, for the steps of reduction of nitrite to dinitrogen, to the photooxidation of cytochromes c1 plus c2 and not due to the photoinduced membrane potential. Moreover, the presence of oxygen inhibited almost all of the reduction of nitrate and nitrous oxide but only 70% of the reduction of nitrite to nitrous oxide. These inhibitions were overcome in the presence of TMPD plus ascorbate. This implies that the inhibition in presence of oxygen was due to a diversion of the reducing power from the denitrifying chain to the respiratory chain. It was concluded from this series of experiments that the reduction of nitrate to nitrite is inhibited when the ubiquinone pool is partly oxidized and that nitrite and nitrous oxide reductions are inhibited when cytochromes c1 plus c2 are oxidized by photosynthesis or respiration.

Exciplex Method for Remote Probing of Fuel Droplet Temperature. Effects of Ambient Conditions on Fluorescence.

An exciplex-based fluorescence method was employed for remote, nonintrusive, instantaneous thermometry of a fuel droplet evaporating or burning in a stream of gaseous mixture. The fluorescence emission spectra from a droplet doped with naphthalene and N, N, N', N'-tetramethyl-p-phenylenediamine(TMPD)were measured with an optical multichannel analyzer. The effects of several parameters including the composition of the ambient gas and the concentrations of the dopants on the fluorescence emission spectra were studied. The results showed that the oxygen in the ambient gas was a major quencher for the fluorescence and that the fluorescence emission spectra varied with the concentration of naphthalene and TMPD. The velocity of the ambient gas had no appreciable effect on the fluorescence emission spectra. It was suggested that the temperature dependence of the ratio of fluorescence intensities at two different wavelengths for a droplet burning in air was the same as that for a droplet evaporating in nitrogen.

Effects of p-phenylenediamines and adriamycin on primary culture of rat skeletal muscle cells

In vitro toxicity of p-phenylenediamine derivatives and adriamycin were examined using cultured rat myofibers prepared by the selective plating method, with slight modification. When the myofibers were cultured for 2 days with 100 μM N, N, N′, N′-tetramethyl p-phenylenediamine, atrophy and/or swelling of the cells were observed, and the toxic effect was reduced as the number of N-methyl groups in the phenylenediamine molecule was decreased. Adriamycin, at concentrations of greater than 0.25 μM, caused cell injury. Decline of cellular creatine phosphokinase activities generally preceded the apparent morphological changes. The primary culture of rat myofibers responded sensitively to the agents which were myotoxic in vivo.

Cytochromes c‐dependent aerobic respiration of Paracoccus denitrificans

AbstractTerminal parts of the respiratory chain of Paracoccus denitrificans containing cytochromes c have been investigated through the use of NNN′Nprime;‐tetramethyl‐p‐phenylenediamine (TMPD) as an electron donor. The cells have been shown to possess two major membrane bound TMPD oxidases characterized by their different sensitivities to cyanide. The more sensitive one, identified as cytochrome aa3, appears to be produced under oxygen‐rich growth conditions, whereas a less sensitive and as yet uncharacterized enzyme accompanied with only moderate amounts of cytochrome aa3 functions when oxygen becomes limiting. The soluble periplasmic cytochrome cd1 does not contribute significantly to the total oxygen consumption rate, although its maximal catalytic capacity may be high. The fact thap the activity of alternative TMPD oxidase is not proportional to cytochrome o content is discussed in relation to possible occurrence of two distinct cytochrome o‐type terminal oxidases.

Solvent effects on the kinetics of heterogeneous electron transfer processes. The TMPD/TMPD ·+ redox couple

Solvent effects on the heterogeneous electron exchange between N, N, N′, N′-tetramethyl- p-phenylendiamine (TMPD) and its monocation radical (TMPD ·+) have been studied by chronocoulometry at a platinum electrode in perchlorate solutions in a wide range of aprotic and hydrogen-bonded solvents. Formal potentials, diffusion coefficients, anodic transfer coefficients and formal rate constants have been evaluated. The variation of the formal potential with the solvent can be interpreted by determining the solvent—solvent—solute interactions using Taft theory. Correlations between predicted and experimental formal potentials for the Taft model were an improvement over those obtained using the Gutmann donor number approach. A weak correlation between the anodic transfer coefficient and the Taft β parameter was also found. The variations in the rate constants were analyzed on the basis of current electron-transfer models using a statistical method to separate the effects of the longitudinal relaxation time τ L from those of the solvent permittivity parameter γ. It has been shown that the dynamics of solvent relaxation affect the heterogeneous electron-transfer rate in this case, but the rate constants depend on τ −0.53 instead of τ −1, as theory predicts. The degree of reaction adiabaticity and the relative sizes of the inner- and outer-sphere components of the Gibbs energy of activation were considered.

CIDEP study on photooxidation of N,N,N′,N′-tetramethyl- p-phenylenediamine by maleic anhydride. Enhanced S—T −1 mixing by radical-ion pair system

The photooxidation process of N,N,N′,N′-tetramethyl- p-phenylenediamine (TMPD) by maleic anhydride was studied by a time-resolved ESR method. The CIDEP spectra assigned to the anion radical of maleic anhydride revealed that the reaction takes place in the excited triplet state of TMPD. The analysis of the intensity ratio of the hyperfine lines leads to the conclusion that the S—T −1 mixing of the radical pair mechanism remarkably contributes to the total emissive spin polarization especially in the low concentration region of maleic anhydride. This S—T −1 mixing is probably enhanced by the Coulombic interaction in a radical-ion pair.