Abstract
Abstract 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 (s20,w = 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 (Km) for methemoglobin and cytochrome b5 were 3.1 x 10-4 m and 7.1 x 10-6 m, respectively. Rapid reduction of methemoglobin was observed in the presence of a small amount of cytochrome b5, which can be explained by the enzymatic reduction of cytochrome b5 and subsequent nonenzymatic reduction of methemoglobin by the reduced cytochrome.
Highlights
Dercral enzymepreparationswhich cat,alyzethe reduction of methemoglobinhave been isolated from human erythrocytes (l-6)
Human erythrocytes were obtained from the central laboratory of the Kanazama Red Cross Blood Transfusion Service
All ot.her chemicals Jvere obtaiiied from the usual commercial sources. h calcium phosphate gel column was prepared according to the method of Price and Greenfield (10)
Summary
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. Various hemoproteins and 2,6-dichlorophenolindophenol served as electron acceptors for the enzymatic reaction. 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. Scott and McGraw (3) hare isolated a NADH-dehydrogenase which has methemoglobin reductase activity and is lacking in red cells of patients with hereditary methemoglobinemia (7). It was shown that this ;“\‘hDI-I-dehydrogenase is the major pyridine nucleotide dehydrogenase in human red cells (8). We describe the purification and propeities of t,he major pyridine nucleotide dehydrogenase from human eryth-. Rocytes with pa,rticular reference to t,he reduction of methemoglobin
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