Dichloroindophenol

A recently characterized class of dehydrogenases are the quinoproteins which contain a pyrroloquinolene quinone prosthetic group and do not require a separate co-factor Electron transfer mediators such as phenazine ethosulphate 2,6-dichloroindophenol and ferricenium ions have been used to recycle the quinoprotein the reduce mediator is detected amperometrically. 

The pH indicator shows the acid or basic properties of sample molecnles. Commonly used for acid indicating are solutions of bromocresol green (20 mg dissolved in 10 ml of ethanol combined with 1 ml of 0.1-molar aqueous NaOH) or bromophenol blue (20 mg dissolved in 10 ml of ethanol, pH-adjusted with 0.1-molar NaOH or 0.2% aqneous citric acid). In the presence of acids, 2,6-dichloroindophenol (40 mg dissolved in 100 ml of ethanol) changes the color from blue to red. The fluorescent dye acridine orange (20 mg dissolved in 100 ml of ethanol) changes pH-dependently the color of its flnorescence from yellow-green to yellow. 

This enzyme [EC 1.3.99.11] catalyzes the reaction of (S)-dihydroorotate with an acceptor substrate to produce orotate and the reduced acceptor. Both iron and zinc ions are needed as cofactors. Acceptor substrates include 2,6-dichloroindophenol, 1,10-phenanthroline, and dioxygen (although dioxygen isn t as effective as the first two). 

This is one vitamin that most laboratories can measure. There are a number of old-fashioned approaches that use 2,6-dichloroindophenol in a titrimetric method such as AOAC 985.33. This works well in some systems but can give rise to false positive results if there are other reducing substances present. It will not detect dehydroascorbic acid (DHA) and so it may well underestimate the actual vitamin C activity if a product contains a significant level of DHA. However, even with for these shortcomings, it is often used as a quick and rough method. In the AOAC there is also a fluorometric method (AOAC 984.26) where ascorbic acid is oxidised to DHA and this is reacted with o-phenylenediamine to give a fluorometric compound which can be detected. This is a robust method that has general applicability. 

Wolthers, Kirsten R., Schimerlik, Michael I. (2002) Neuronal nitric oxide synthase substrate and solvent kinetic isotope effects on the steady-state kinetic parameters for the reduction of 2, 6-dichloroindophenol and cytochrome C3+, Biochemistry 41, 196-204. 

Cytochrome 62 is stereospecific for l(- -)-lactate. It also oxidizes other a-hydroxymonocarboxylic acids at slow rates 80, 96). As electron acceptors ferricyanide, methylene blue, 2,6-dichloroindophenol, 1,2-naphthoquinone 4-sulfonate, and cytochrome c have been used. This wide acceptor specificity is characteristic of a number of flavoproteins, which are generally capable of reducing quinoid structures and ferric compounds 97). However, as will be seen below, cytochrome c is considered to be the physiological electron acceptor for the yeast L-lactate dehydrogenase. 

D-Lactate cytochrome c reductase can oxidize D-2-hydroxymonocar-boxylic acids, but only D-lactate and D-2-hydroxybutyrate are oxidized at appreciable rates. The enzyme exhibits a similar high specificity for electron acceptors. It reacts with cytochrome c and phenazine methosul-fate as electron acceptors, but not with ferricyanide, methylene blue, 2,6-dichloroindophenol, and menadione 308, 312, 313). With D-lactate as substrate and at Fmax with respect to acceptor, phenazine methosulfate is reduced at 30° eight times as fast as cytochrome c 308). The values at 30° and pH 7.5 are D-lactate, 0.29 mM n-2-hydroxybutyrate,... 

The determination of ascorbic acid in foods is based, in part, on its ability to be oxidized or to act as a reducing agent. The most common method for determination of vitamin C in foods is the visual titration of the reduced form with 2,6-dichloroindophenol (DCIP) (4-7). Variations in this procedure include the use of a potentiometric titration (6), or a photometric adaptation (S) to reduce the diflSculty of visually determining the endpoint in a colored extract. The major criticisms of this technique are that only the reduced vitamin, and not the total vitamin C content of the food, is measured, and that there can be interference from other reducing agents, such as sulfhydryl compounds, reductones, and reduced metals (Fe, Sn, Cu), often present in foods. The DCIP assay can be modified to minimize the effects of the interfering basic substances, but the measurement is still only of the reduced form. Egberg et al. (9) adapted the photometric DCIP assay to an automated procedure for continuous analysis of vitamin C in food extracts. 

Oxidation of 2,6-dichloroindophenol (sodium salt) can be carried out on Ti02 with unit quantum efficiency. This particular reaction has been suggested as a method for testing for photocatalytic activity of semiconductor powders . ... 

It was discovered in 1958 that anaerobically grown yeast contains a form of lactate dehydrogenase which is different from the d- and L-lac-tate cytochrome c reductases of aerobic yeast (306, 319). The enzyme has been partially purified (320, 321), and shown to contain flavin (320-322). Gel filtration studies have suggested a molecular weight of about 100,000 (320, 321). Preparations of the enzyme oxidize several d-2-hydroxyacids to the respective keto acids in a reversible manner (320). For the forward reaction, ferricyanide, 2,6-dichloroindophenol, menadione, and methylene blue have been used as electron acceptors, and for the reverse reaction leucomethyl viologen and FMNHa are effective electron donors (320). A number of L-2-hydroxyacids and 2-keto acids have been shown to be competitive inhibitors. Oxalate, cyanide, o-phenanthro-line, and EDTA are also potent inhibitors (320, 321, 323, 324). The inhibition by metal chelators develops slowly and is reversed by addition of Zn, Co, Mn +, or Fe + (320, 323-326). Substrates prevent the inhibition by chelators at concentrations considerably lower than their respective Km values (327). It has been suggested that EDTA inactivation involves the removal of a metal, most probably Zn +, from the substrate binding site of the enzyme (325, 326, 328, 329). However, others have... 

Measurements of 2,6-dichloroindophenol (DCPIP) photoreduction were carried out as previously described, with a Hitachi Model 100-60 Spectrophotometer modified for cross-illumination (12). 

The properties of spinach chloroplasts adsorbed onto diethylaminoethyl-cellulose have been reported and the immobilized chloroplasts used for the continuous photoreduction of 2,6-dichloroindophenol as an indicator of photosystem II activity in the chloroplasts. The formation of ATP from ADP and orthophosphate via the cyclic photophosphorylation system has been achieved using chloroplasts copolymerized with polyvinylalcohol onto an electrode. Citrobacter freundii cells immobilized by co-casting with collagen have been used in a flow type sensor for cephalosporins. ... 

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