| 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). Source: Clostridium kluyveri Form: Supplied as a dialyzed, lyophilized powder Activity: 30units per mg dry weight Unit Definition: 1 unit equals a decrease in absorbance of 1.0 per minute at 25C, pH 7.5 with 2,6-dichlorophenolinodophenol as the chromogen. Molecular Weight: 24kD Composition: The enzyme contains one molecule of flavin mononucleotide per molecule. The amino acid composition has been determined. Optimum pH: 8.5. At pH 7.4 and 9.4 the activity is reduced 50%. Both NADH and NADPH share this optimum. Constants: Km for NADH was reported to be 9x10e-5 and for NADPH it was much lower. Inhibitors: N-ethylmaleimide inactivates diaphorase at concentrations of under 5mM. Activators: Excess flavin mononucleotide (FMN) appears to stimulate the reaction with dichlorophenol-indophenol slightly. Specificity: Either NADH or NADPH may be used as reductants. However, no exchange of hydrogen between the coenzymes is catalyzed. Neither oxygen nor cytochrome C is reduced by Cl. kluyveri diaphorase. Stabilizers: NADH, NADPH and FMN protect the enzyme against denaturation by urea and guanidine. Quality Control: SDS-PAGE Storage and Stability: Lyophilized and reconstituted products are stable for 6 months after receipt at -20C. Reconstitute with sterile buffer. Aliquot to avoid repeated freezing and thawing. Store at -20C. Liquid is light sensitive. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. |
