A comparative study of dihydrolipoyl dehydrogenase and diaphorase.

The purification and some of the properties of a flavoprotein component of a-ketoglutaric dehydrogenase complex have been described in a previous communication (1). In several of its properties, this flavoprotein shows a remarkable resemblance to the flavoprotein first isolated by Straub (2) and later studied by Savage (3). They have the same prosthetic group (flavin adenine dinucleotide) and are both highly fluorescent. They catalyze the reduction of 2,6dichlorophenolindophenol by reduced diphosphopyridine nucleotide at comparable rates. They have the same electrophoretic mobility at pH 7.2 and at pH 6.5. Finally, the spectrum of the flavoprotein component of arketoglutaric dehydrogenase complex and the spectrum obtained after its reduction with excess DPNH are very similar to the spectra obtained under similar conditions by Savage. While the above work on the components and reaction sequence of the ac-ketoglutaric dehydrogenase complex was under investigation and partially completed in our laboratory, the physiological role of diaphorase was being investigated by Massey. He reported that diaphorase prepared by a new and more gentle procedure had dihydrolipoyl dehydrogenase activity (4). He later showed that cu-ketoglutaric dehydrogenase complex can be obtained in a resolved form consisting of a flavoprotein and a colorless component, and that the flavoprotein can be replaced by diaphorase in reconstituting the full activity of the complex. From these results, he proposed that the true role of the classic diaphorase is that of a component of the LYketoglutaric dehydrogenase complex (5). In this paper we wish to compare further the spectral and enzymatic properties of the flavoprotein isolated by our method (1) from a-ketoglutaric dehydrogenase complex and the flavoprotein isolated by Straub. These enzymes have previously been referred to in terms of their original assay, dihydrolipoyl dehydrogenase for the former and diaphorase for the latter. Since both preparations catalyse both reactions, the first will be referred to as cr-ketoglutaric dehydrogenase flavoprotein and the second as Straub flavoprotein in this paper.