Purification of reduced pyridine nucleotide dehydrogenase from human erythrocytes and methemoglobin reduction by the enzyme.

Reduced pyridine nucleotide dehydrogenase was purified 44,000-fold from normal human erythrocytes by procedures including ammonium sulfate fractionation, calcium phosphate gel chromatography, Sephadex G-100 gel filtration, and isoelectric focusing. The most purified enzyme preparation showed a single homogeneous peak (s~~,~ = 2.77 S) upon ultracentrifugation and was nearly homogeneous on acrylamide gel disc electrophoresis. Absorption spectrum of the enzyme, fluorimetry of flavin, and effect of inhibitors showed that the enzyme contained no flavin nucleotides and no heme as a coenzyme. Various hemoproteins and 2,6-dichlorophenolindophenol served as electron acceptors for the enzymatic reaction. Among the hemoproteins, cytochrome b5 was the most effective electron acceptor. Assuming a molecular weight of 28,000, the enzyme has a molecular activity of 6.5 for methemoglobin and 4,550 for cytochrome b5 with NADH as an electron donor. The Michaelis constants (K,) for methemoglobin and cytochrome b5 were 3.1 X 10M4 M and 7.1 X 10-e M, respectively. Rapid reduction of methemoglobin was observed in the presence of a small amount of cytochrome bs, which can be explained by the enzymatic reduction of cytochrome bS and subsequent nonenzymatic reduction of methemoglobin by the reduced cytochrome.