The identity of diaphorase and lipoyl dehydrogenase

Abstract

A method of purification of diaphorase not involving any heat treatments is described. The product is shown to be identical with the enzyme obtained by previous methods of purification. Diaphorase has been found to be a potent lipoyl dehydrogenase; the identity of lipoyl dehydrogenase and diaphorase has been demonstrated by the constant ratio of the two activities during purification, by inhibition studies and by the direct demonstration that reduced diaphorase is rapidly reoxidized by lipoic acid and derivatives. The reaction rates of DPNH oxidation and DPN + reduction by the enzyme employing a variety of lipoic acid derivatives have been detrermined. The turnover numbers at 25° vary from 1000 with DL-lipoic acid to about 80,000 with DL-lipoamide. The redox potential of the lipoic acid couple and its variation with pH is also reported.