2.6- Dichlorophenolindophenol, oxidation

Chemical Properties. The most significant chemical property of L-ascorbic acid is its reversible oxidation to dehydro-L-ascorbic acid. Dehydro-L-ascorbic acid has been prepared by uv irradiation and by oxidation with air and charcoal, halogens, ferric chloride, hydrogen peroxide, 2,6-dichlorophenolindophenol, neutral potassium permanganate, selenium oxide, and many other compounds. Dehydro-L-ascorbic acid has been reduced to L-ascorbic acid by hydrogen iodide, hydrogen sulfide, 1,4-dithiothreitol (l,4-dimercapto-2,3-butanediol), and the like (33). 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Although the first purification of bNOS was a monomer, it is now clear that the enzyme in all cases is effective as a dimer. A purified macrophage iNOS was used by Baek and coworkers98 to separate the holoenzyme from the monomers. The subunits do not have NOS activity but do have the ability to oxidize reduced triphosphopyridine nucleotide with either ferricyanide, cytochrome c or dichlorophenolindophenol. When all of the missing factors are present, but not when any is missing, the authors find recombination, as shown in Figure 13. 

Additionally, Wang and Watt have shown that the FeMo protein alone can act as an uptake hvdrogenase(63). Specifically, H2 in the presence of [FeMo] causes the reduction of oxidizing dyes such as methylene blue or dichlorophenolindophenol in the absence of Fe protein. The hydrogen evolution and uptake behavior of nitrogenase proteins forces us to consider the ways in which hydrogen can interact with transition metal sulfur centers. This we discuss in the following section. 

The oxidation of the enamine on El in PDHc by nonlipoic acid acceptors has also been explored for many years. For example, ferricyanide reduction monitored by visible spectroscopy has become a standard test to assay El activity, notwithstanding the attendant problems, including the instability of the thiazolium ring to such conditions. 2,6-Dichlorophenolindophenol (DCPIP) has also been used as an alternative electron acceptor in mechanistic studies of PDHcs75. 

ADR Oxidation (NADH) and reduction [K3Fe(CN)6,2,6-dichlorophenolindophenol] studies 195... 

The aliphatic alcohol oxidase, a FAD-dependent enzyme, catalyzes the oxidation of primary short-chain alcohols to the corresponding aldehydes. Dioxygen can be replaced by synthetic acceptors such as dichlorophenolindophenol or phenazine methosulfate [147l... 

The most satisfactory chemical methods for ascorbic acid analysis are based on the reduction of 2,6-dichlorophenolindophenol or on the formation of a colored dinitrophenylhydrazine derivative. A large number of other reactions, mostly with other oxidizing agents, have been used but have not found general favor (R21). 

The second method of establishing oxidation states employed redox titration using the redox dye dichlorophenolindophenol (DCIP) based on the knowledge that tetrahydropterins reduce DCIP instantaneously while qui-nonoid dihydropterins react slowly and 7,8-dihydropterins do not reduce DCIP at all. As previously mentioned in this chapter, DCIP oxidation of Moeo within several molybdoenzymes was determined to be a two-electron process, suggesting a dihydropterin reduction state that was speculated to be the quinonoid tautomer. The results of stoichiometric additions of DCIP to the molybdenum complexes of reduced pterins showed that no oxidation of... 

The ferric thiocyanate method for peroxide value is based on the oxidation of ferrous to ferric ions, which are determined colorimetrically as ferric thiocyanate. This method is more sensitive and requires a smaller sample (about 0.1 g) than does the iodometric method (Table 5.3). However, the values obtained by the ferric thiocyanate method are higher by a factor of 1.5 to 2 relative to those of the iodometric method. The peroxide values obtained by both methods are of only relative significance. The ferric thiocyanate method is commonly applied to dairy products, which undergo oxidative deterioration at relatively low peroxide values. Other colorimetric methods for peroxide values include the determination of the blue starch-iodine complex in the iodometric method, the red color developed with 1,5-diphenyl-carbohydrazide, the color developed with ferric ions and xylenol orange, ferrous chloride and 2,6-dichlorophenolindophenol, titanium dichloride, and xylenol orange. 

The failure of a generation of investigators to obtain soluble succinic dehydrogenase preparations was caused by the unusually fastidious requirements of this enzyme. Of the ordinary electron acceptors, including methylene blue and dichlorophenolindophenol, none supports the oxidation of succinate. Phenazine methosulfate was found to be reduced by solubilized preparations the enzyme is extracted from acetone powders and was undoubtedly present in many extracts, where it was not detected for lack of a suitable oxidant. A preparation from yeast was reported... 

Characteristically, paracatalytic substrate oxidation and inactivation occur only in the presence of the specific substrate of the enzyme. However, both processes are unspecific with respect to the oxidant. In fact, hexacyanoferrate(III), 2,6-dichlorophenolindophenol, porph3rrindin, tet-ranitromethane, and H2O2 have been successfully employed with various enzymes (see the table). The oxidants differ considerably with respect to the ratio of the rate of oxidation of the carbanion intermediate to the rate of enzyme inactivation, e.g., the inactivation of transaldolase in tiie... 

Of the various oxidation-reduction methods those based upon the reduction of indophenol have been found to be the most satisfactory. 2,6-Dichlorophenolindophenol is a dye that is blue in alkali and pink in acid. It is reduced by ascorbic acid to the colorless leuco form, as shown by following reaction (1). 

Excess substrate inhibited the oxidation of the p-hydroxyphenyl-pyruvate as did also benzoquinone acetic acid. This could be prevented by adding large amounts of ascorbic acid or small amounts of reduced 2,6-dichlorophenolindophenol. The latter was seven hundred times as effective as ascorbic acid with the purified enzyme 214), Zannoni and LaDu speculate that a product formed from p-hydroxyphenylpyruvate is the true inhibiting agent of the reaction. 

Hager et al. 212) carried out a careful investigation of the roles of ascorbic acid, 2,6-dichlorophenolindophenol, and catalase in the formation of homogentisic acid. They concluded that all three are required to prevent inactivation of the enzyme system. The reduced form of the dye, but not the oxidized form, replaced the requirement for ascorbic acid. The dye could be maintained in a reduced state with glutathione. Further study of this phenomena showed that enzyme fraction B of LaDu and Zannoni was inactivated by preincubation with the substrate unless dye was present. 

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