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At least 19 recordsLinked to original sources
PubMed1992

Aqueous humor hydrogen peroxide analysis with dichlorophenol-indophenol.

Hydrogen peroxide is now reported to be a normal aqueous humor component present, in the low microM concentration range, in the animal species which have been studied. This finding was established with the exclusive use of the dichlorophenol-indophenol method of analysis. In this procedure, aqueous humor is added to a blue, oxidized dichlorophenol-indophenol solution. The 605 nm absorbance of this solution immediately decreases in response to the reducing action of ascorbate present in the sample. The extent of reoxidation of the solution upon the addition of peroxidase, as measured by the increase in its 605 nm absorbance, can be quantitatively related to the concentration of H2O2 in the sample. A close examination of this method revealed that reduced dichlorophenol-indophenol spontaneously reoxidizes at a rate of 0.03 nmol min-1 microM-1, with generation of H2O2. H2O2 generation was unequivocally established by analysis of the temporal dependency of the absorbance increase produced by peroxidase in the absence of added H2O2 and by the sensitivity of this phenomenon to catalase. This spontaneous production of H2O2, on the other hand, cannot be attributed to ascorbate auto-oxidation because added ascorbate quantitatively reacts with dichlorophenol-indophenol, provided that an excess of the latter is maintained. This method then has an enormous potential to overestimate H2O2 in any sample. On the other hand, the response of the assay system to a given level of H2O2 depends on the level of reduction previously produced by ascorbate. This results in an artifactual positive correlation between ascorbate and H2O2 levels in samples containing variable amounts of ascorbate. In spite of these serious limitations the method can still be useful to measure H2O2 if appropriate precautions are taken. When using it for the analysis of rabbit aqueous humor H2O2 without correcting for the H2O2 generated during the assay and ignoring differences in the level of ascorbate in the samples, we obtained an average value of 25.3 microM H2O2, which coincides with that reported in the literature for the rabbit, but is obviously incorrect. When analysing aqueous humor there was the additional variable of the aqueous humor itself inhibiting the rate of dichlorophenol-indophenol auto-oxidation and so the final, corrected figure for H2O2 concentration in the aqueous humor became uncertain, since the auto-oxidation of the substrate could not be properly subtracted.(ABSTRACT TRUNCATED AT 400 WORDS)

2,6-Dichloroindophenol↗
PubMed1977

Mitochondrial membrane-linked reactions in carcinogenesis: change in steroselective uncoupling of oxidative phosphorylation by aliphatic dicarbonyls and in the Arrhenius plot of NADH-indophenol reductase.

The previously observed alterations in the energy transducing system of rat liver mitochondria during 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) carcinogenesis were investigated using aliphatic dicarbonyl compounds as molecular probes and the effect of temperature on the membrane-linked NADH-indophenol reductase. The vicinal diketone, diacetyl, uncouples oxidative phosphorylation in normal rat liver mitochondria while the higher diketones, acetylacetone and acetonylacetone, are increasingly less effective in that order; diacetyl totally abolishes respiratory control with substrates the oxidation of which involves the NADH leads to CoQ segment, but only partially with succinate which bypasses this segment. Diacetyl, likewise, uncouples oxidative phosphorylation in liver mitochondria from rats fed 3'-Me-DAB, but the mitochondria are most resistant to this uncoupling (in terms of the P/O ratio) at the time period when the respiratory control index (determined in the absence of diacetyl) is at the dye-induced minmum. This time period is at 3 to 4 weeks of dye administration, representing the cumulative dose for tumorigenesis threshold. At this threshold period of feeding 3'-Me-DAB, discontinuities in the Arrhenius plot of the mitochondrial membrane-localized NADH-indophenol reductase appear, with a return toward the control state (no break) at 8 weeks, only to reappear in the plot of the enzyme from tumor mitochondria, suggesting sequential membrane phase transitions in the mitochondria during azo dye carcinogenesis.

2,6-Dichloroindophenol↗
PubMed1988

Reduction of exogenous quinones and 2,6-dichlorophenol indophenol in cytochrome b-deficient yeast mitochondria: a differential effect on center i and center o of the cytochrome b-c1 complex.

The reduction of duroquinone (DQ), 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), and dichlorophenol indophenol (DCIP) by succinate and NADH was investigated in yeast mitochondria which have no spectrally detectable cytochrome b. Succinate reduces DB in the cytochrome b-deficient mitochondria at rates comparable to that observed in wild-type mitochondria, suggesting that succinate:ubiquinone oxidoreductase is unaffected by the lack of cytochrome b. In the mutant mitochondria, succinate does not reduce DQ or DCIP at significant rates; however, NADH reduces both DQ and DCIP at rates similar to that of the wild-type mitochondria in a myxothiazol, but not antimycin, sensitive reaction. The Ki for myxothiazol in this reaction is close to that for electron transfer through the cytochrome b-c1 complex. In addition, myxothiazol does not inhibit NADH:ubiquinone oxidoreductase. These results confirm our previous suggestion that the cytochrome b-c1 complex is involved in electron transfer from the primary dehydrogenases to DQ and DCIP and suggest that cytochrome b is not the binding site for myxothiazol.

2,6-Dichloroindophenol↗
PubMed1979

[Activation, by various aldoses, of dichlorophenol-indophenol reduction by endogenous constituents of a preparation of glucose dehydrogenase from Pseudomonas fluorescens].

Dichlorophenol-indophenol is reduced neither by D-glucose nor by the endogenous components of a particulate purified glucose-dehydrogenase from Pseudomonas fluorescens, when these two classes of compounds acts individually. In contrast, the dye is quickly reduced by the endogenous components when the reaction occurs in the presence of glucose, without a direct participation of glucose in the reduction. In this effect D-glucose can be replaced by D-mannose, D-galactose or D-xylose, but not by D-fructose.

2,6-Dichloroindophenol↗
PubMedDate not supplied

Aids for in vitro Mycobacterium lepraemurium investigations: estimation of oxidation-reduction potentials and pO2 with 2, 6 dichlorophenol indophenol.

The partial success in cultivating a "host-dependent" microbe provided the incentive to develop methods which may aid the growth of the organism. The oxidation-reduction potential (ORP) of NC-5, an aerobic, cysteine containing medium which supports the limited in vitro growth of Mycobacterium lepraemurium, is measurable with the redox dye, 2, 6 dichlorophenol indophenol. Both cysteine and autoclaved glucose can be used to adjust to ORP. Glucose autoclaved in phosphate buffer but not in aqueous solutions reduced the dye. The dye was also reduced in glucose solutions by atmospheres containing 10% and 1%-2% pO2. With exposure to 20% pO2 the reoxidation of the dye was slow but complete. Thus, the dye in glucose solutions provides a general method for estimating pO2 above the level of anaerobiosis. Proper adjustment and monitoring of the ORP and pO2 may enhance growth.

2,6-Dichloroindophenol↗
PubMed1979

[The influence of infusions and amino-acids on the determination of ammonia by means of indophenol-reaction (author's transl)].

It will analyse and discuss the influence of fructose-, glucose-, sorbitol- and argininmalat-infusions, of ascorbit acid and vitamin-B-complex as well as of 13 different amino-acids on the determination of ammonia by means of indophenol-reaction. With this the frequent of liver-cirrhosis i.v. administer substances trouble the determination of ammonia just as little (exception: arginin-malat), how amino-acids in physiological and little pathological range.

Amino Acids↗
PubMed1990

Nonenzymatic NADPH-dependent reduction of 2,6-dichlorophenol-indophenol.

The reduction of 2,6-dichloroindophenol (DCIP) by direct interaction with NADPH was studied. The results indicate that reduction proceeds via a direct electron transfer from NADPH to DCIP, with no oxygen consumption, and a rate constant of k = 4.69 M-1.s-1. The reduced DCIP can rapidly transfer its electrons to potassium ferricyanide (K3Fe(CN)6) or ferricytochrome c, but not to nitro blue tetrazolium. Superoxide dismutase inhibits DCIP reduction in an oxygen-dependent manner by favoring the reoxidation of the reduced DCIP. We therefore conclude DCIP is not suitable for detecting O2- when the nucleotides NADH or NADPH are present.

2,6-Dichloroindophenol↗
PubMed1992

Correction for creatine interference with the direct indophenol measurement of NH3 in steady-state nitrogenase assays.

Creatine was identified as a major source of interference with the direct phenol/hypochlorite colorimetric determination of ammonia in nitrogenase reaction mixtures. A method is described for removing other compounds which inhibit color development and for compensating for the interference produced by creatine. This method avoids time-consuming microdiffusion and also routinely makes available the efficiency of ATP hydrolysis coupled to substrate reduction (ATP/2e ratio) with N2 as a reducible substrate. Using this method we determined values for this ratio at 30 degrees C of 4.87 +/- 0.03 during the reduction of protons to H2 and 7.16 +/- 0.14 during the reduction of N2 by the vanadium-containing nitrogenase of Azotobacter chroococcum.

Adenosine Triphosphate↗
PubMed1976

Photometric determination of nitrogen. Wet incineration followed by formation of indophenol blue with salicylate/hypochlorite.

Ammonia in wet incineration residues has been analyzed photometrically by a modified Berthelot reaction using a water-free digestion mixture. Phenol is substituted by salicylate in the dye-producing reaction. The method can be adapted for any biological material with a minimum content of 0.04 mug N/sample. Nonprotein nitrogen may be determined in the supernatant of TCA precipitates.

Humans↗
PubMed1976

Steady-state kinetics of high molecular weight (type-I) NADH dehydrogenase.

(1) Studies of the steady-state kinetics of the NADH dehydrogenase activity of Complex I (NADH: Q oxidoreductase) revealed that the reaction mechanism with the one-electron acceptor ferricyanide or the two-electron acceptor 2,6-dichloro-indophenol is ping pong bi bi, with double substrate inhibition. NADH inhibits the reaction of the reduced form of the flavoprotein with the acceptors, and the acceptors prevent NADH from reacting with the oxidized form. This implies that both NADH and acceptors react with the same site on NADH dehydrogenase. (2) The velocity at infinite NADH and acceptor concentrations (corrected for the double substrate inhibition) is much larger with ferricyanide than with the indophenol. It is concluded that the latter binds to the reduced enzyme. Thus, with ferricyanide the rate constant measured refers to the dissociation of bound NAD+ from the reduced enzyme (k2) and with the indophenol to the rate constant of oxidation of reduced enzyme by bound acceptor (k4). The latter value is not an estimate for the situation in vivo, where ubiquinone is the acceptor. (3) The rate constant of the dissociation of bound NAD+ from the reduced enzyme (k2) increases with pH. It is suggested that an ionizing group on the enzyme is involved in the dissociation. (4) After extraction of ubiquinone from Complex I with pentane curve relating activity at infinite ferricyanide concentration to NADH concentration changes from hyperbolic to sigmoidal. The hyperbolic curve is restored by reincorporating ubiquinone. It is concluded that ubiquinone is an effector for NADH dehydrogenase.

2,6-Dichloroindophenol↗