Studies on the electron transfer system. XXV. The isolation and properties of a lipoflavoprotein with diaphorase activity from beef heart mitochondria.

The electron transport particle prepared from beef heart mitochondria contains two flavoprotein enzymes in approximately equal amounts: (a) the succinic dehydrogenase and (a) the reduced diphosphopyridine nucleotide dehydrogenase (1). When the electron transport particle is exposed to 9 per cent ethanol and pH 5 at 38’, the DPNH dehydrogenase is liberated from the particle almost quantitatively, and the flavoprotein thus solubilized has been isolated by de Bernard in a highly purified state (2). The de Bernard flavoprotein catalyzes the oxidation of DPNH by both cytochrome c and ferricyanide. The functional flavin group of the de Bernard enzyme is not flavin adenine dinucleotide. Some years previously Mahler et al. (3) had isolated from pig heart muscle a soluble flavoprotein enzyme which corresponds closely in properties with those of the de Bernard enzyme. The method of extraction of the Mahler flavoprotein from the particles was the same as for the de Bernard flavoprotein. In 1939 Straub (4) isolated from a particulate suspension originating from pig heart muscle a soluble flavoprotein with DPNH dehydrogenase activity. Unlike the de Bernard and Mahler enzymes the Straub flavoprotein shows no cytochrome c reductase activity but is active with methylene blue as electron acceptor. Furthermore the prosthetic group of the enzyme is flavin adenine dinucleotide. The relationship between the Straub enzyme on the one hand and the Mahler and de Bernard enzymes on the other has been the subject of lively discussion and speculation in recent years. Three observations induced us to probe more deeply into the DPNH dehydrogenase problem. First, particles from which the de Bernard flavoprotein has been isolated contain acid-extractable flavin exclusively in the form of flavin adenine dinuclcotide, whereas the prosthetic group of the de Bernard enzyme is not flavin dinucleotide. Second, the DPNH-ferricyanide activity of the electron transport particle is much higher per unit of acidextractable flavin than that of the de Bernard enzyme. Third, the de Bernard enzyme can be extracted from particles which show no DPNH-cytochrome c reductase activity. Thus the