Reduction of 2,6-dichlorophenolindophenol

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 enzyme was also shown to contain FAD. The absorption spectrum is shown in Figure 5. Two moles of FAD were estimated per mole of enzyme protein. The enzyme catalyzed the reduction of 2,6-dichlorophenolindophenol in the presence of phenazine methosulfate. 

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. 

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). 

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