Straub’s diaphorase

A significant advance in understanding of the mechanism of the dihydrolipoic dehydrogenase reaction resulted from the discovery by Massey (1958) that the classic flavoprotein first isolated by Straub (1939), and widely known as Straub s diaphorase, behaves as a powerful dihydrolipoic dehydrogenase. Diaphorase activity was measured with ferricyanide as electron acceptor, Eq. (27), and dihydrolipoic dehydrogenase activity by... 

Searls and Sanadi (1959, 1960a) and Massey et al. (1960) observed an increase in absorbancy in the region between 500 and 600 mju (maximum at 530 m/i), concomitant with a decrease at 455 mju, on reduction of pig heart dihydrolipoic dehydrogenase with DPNH and with dihydrolipoic acid.A similar effect was noted previously by Savage (1957) on reduction of Straub s diaphorase with DPNH. These results recalled similar observations by Beinert (1957) with other flavoproteins which were attributed to the formation of a flavin semiquinone. Massey ei al. (1960) have made a detailed study of the stoichiometry of formation of the 530-mju band and the kinetics of its formation and disappearance under a variety of conditions, and attributed it to a flavin semiquinone which is an obligatory intermediate in the catalytic cycle of the enzyme. Addition of p-chloro-mercuriphenyl sulfonate to the partially reduced flavoprotein resulted in disappearance of the red color and further reduction of the flavin. This observation was interpreted as indicating that the flavin semiquinone is stabilized by interaction with a protein sulfhydryl group. 

Diaphorase and Cytochrome Reductase. Enzymes have been isolated from animal sources that have many of the properties of the various yeast enzymes. The first, and simplest, was liberated from particulate structures by Straub, who employed dilute ethanol and ammonium sulfate at 43 C. The enzyme could then be purified and was named a diaphorase. Diaphorase was coined to identify a widespread group of enzymes that transfer electrons from DPNH to dyes. Many of the purified flavoproteins have been found to oxidize pyridine nucleotides, and almost all of the flavoproteins can reduce dyes. Straub s diaphorase reduces methylene blue but not cytochrome c. Slight modification of the isolation procedure was found by workers at the Enzyme Institute of the University of Wisconsin to yield a cytochrome reductase. The relation between these preparations is not known, but it is possible that one is derived from the other. Cytochrome reductase contains 4 atoms of iron for each flavin, whereas Straub s preparation contains little iron. Both proteins have molecular weights around 75,000, and contain 1 equivalent of FAD. 

Both the oxidation-reduction potential and the fluorescence of flavin nucleotides are modified profoundly by attachment of the nucleotide to various proteins. Flavin enzymes have been reported to have oxidation-reduction potentials at pH 7 ranging from —0.4 to 0.187. The combination to proteins also results in shifts of the absorption maxima. The 450 m u band is found at 451 mju in Straub s diaphorase and at 455 m/t in Haas yellow enzyme, while the 375 m/t band appears at 359 m/t and 377 m/t in these preparations. Most flavin enzymes do not fluoresce, and it is assumed that the quenching of fluorescence implies binding of the flavin to the enzyme through N-3. Straub s diaphorase, unlike most other flavoproteins, does fluoresce. This may be evidence that this diaphorase is a partially degraded cytochrome reductase. 

The low molecular weight form of mitochondrial NADH dehydrogenase was first isolated from pig heart muscle by Edelhoch et al. (60) and Mahler and his associates 57 in 1952. The mitochondrial origin of the enzyme was demonstrated by de Bernard 81. These and similar preparations reported subsequently by Mackler 63, Kumar et al. 63, and Pharo et al. 64 were isolated by extracting the source material (heart muscle or various submitochondrial preparations) with 9-11% ethanol at pH 4.8-5.S and 43°-45°, a procedure originally devised for isolation of the Straub diaphorase (lipoyl dehydrogenase) 85. Two other preparations of basically similar composition and catalytic proper-... 

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