Purification and characterization of diaphorases from some Drosophila species

Diaphorase-1 and diaphorase-2 were isolated from two Drosophila species, D. virilis and D. melanogaster, and purified by gel filtration, affinity chromatography, immunoaffinity chromatography, and ion-exchange chromatography. The molecular weights of both enzymes were the same in each species. The molecular weight of diaphorase-1 was the same under both denaturating and nondenaturating conditions, close to 60,000, indicating a monomeric structure. Sodium dodecyl sulfate (SDS) electrophoresis of the purified diaphorase-2 revealed the presence of a single protein band of 55,000 Da, while the molecular weight of the native enzyme was found to be 67,000. The two diaphorases were further characterized by their pH optima, isoelectric points, and kinetic parameters, and antibodies were raised in rabbits against the purified enzymes from D. virilis. The antibodies showed no cross-reactions but recognized the corresponding diaphorases in D. melanogaster and D. novamexicana as well as D. virilis. The data obtained confirmed the hypothesis of an independent genetic control of diaphorase-1 and diaphorase-2 in Drosophila.