Diaphorase

The diaphorases are a ubiquitous class of flavin-bound enzymes that catalyze the reduction of various dyes which act as hydrogen acceptors from the reduced form of di-and tri-phosphopyridine nucleotides, i.e., NADH, NADPH. The first such enzyme to be purified was that from heart muscle ( Straub 1939). Almost twenty years later heart diaphorase was shown to be identical to lipoyl dehydrogenase ( Massey 1958, 1963). Other diaphorases have been described and purified from various bacteria, plants and mammalian organs. Diaphorase activity of a partially purified extract of Clostridium kluyveri cells, originally described as a source of NADH and NADPH oxidase ( Ciotti and Kaplan 1957), was observed in this laboratory and its use applied to the coupled, colorimetric determinations of dehydrogenases and ethanol. (Teller 1958). These methods, based on the decolorization of 2,6-dichlorophenolindophenol were improved by the substitution of a tetrazolium dye which becomes chromogenic on reduction. ( Brower and Woodbridge 1970; Nachlas et al. 1960).\n\nNAD(P)H: (guinone-acceptor) oxidoreductase (EC 1.6.99.2)\n\nApplication:\nSuitable for use as a calibrator or control serum. \n\nStandard: Diaphorase-03\n\nSpecific Activity: (same/more than) 20U/mg protein (25 degrees C, pH 7.5)\n\nUnit Definition:\nOne unit is equivalent to a decrease in absorbance of 1.0 per a minute at 600nm with 2,6-dichlorophenolindophenol and NADH at 25 degrees C, pH 7.5. \n\nStorage and Stability:\nMay be stored at 4 degrees C. For long-term storage, aliquot and store at 4 degrees C. Do not freeze. Aliquots are stable for at least 6 months. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Further dilutions can be made in assay buffer.

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