Diaphorase from Clostridium kluyveri, or Lipoyl dehydrogenase, has been used in a study to assess the protein-protein interactions in assembly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic metabolism. Lipoyl dehydrogenase has also been used in a study to investigate the redox regulation of tyrosine nitration and 3-nitrotyrosine reduction by antioxidants.
Methylene blue (MB)-containing polyacrylamide nanoparticle platforms (NPs) were tested in solution with diaphorase from Clostridium kluyveri and the cofactor NADH. this test done done to check whether the encapsulation of MB in NPs could prevent the reduction of MB, and thus protect its photodynamic effectiveness. The enzyme from Sigma has been used along with aldehyde dehydrogenase to construct a biosensor for acetaldehyde by immobilization. It has also been used in the reoxidation of NADP to NADPH. This reaction simultaneously catalyzed the conversion of resazurin into the highly fluorescent resorufin, and also allowed the detection of minute amounts of NAADP. NAADP was initially converted to NADP and NADPH by other enzymes.
Diaphorase from Clostridium kluyveri has been used in lactate dehydrogenase assay in gill cells and impregnated on the strip for formate dehydrogenase assay.
Diaphorase catalyzes the oxidation of either β-NADH or β-NADPH in the presence of an electron acceptor such as methylene blue or 2,6-dichlorophenolindophenol. Diaphorases which are specific for either β-NADH or β-NADPH are known. The pig heart enzyme of Straub seems to have native diaphorase (β-NADH specific) as well as lipoic and lipoamide dehydrogenase activities. It is reported to be a single protein. However, Massey reports that diaphorase is probably a denatured lipoamide dehydrogenase. Pre-incubation of the pig heart preparation with Cu2+ reduces the lipoamide dehydrogenase activity and proportionately increases the β-NADH diaphorase activity. In our laboratory, we have demonstrated this copper effect to some degree on the pig heart enzyme, but no appreciable effect was observed on the Clostridium kluyveri or torula yeast preparations. The lipoamide dehydrogenase:diaphorase ratio is a measure of the denaturation.
