Determination of NADH2-ferricyanide oxidoreductase (cytochrome b5 reductase, diaphorase) activity of human erythrocytes by an analysis of the time-dependence of NADH2 oxidation.
The time-dependence of the reaction of human erythrocyte diaphorase activity has been studied by the use of NADH2 and ferricyanide as substrates. Reaction was found to be first-order with respect to NADH2 concentration, and zero-order with respect to ferricyanide concentration. These findings indicate that human erythrocyte diaphorase has a Km value for NADH2 by far higher than, and for ferricyanide by far lower than, the concentration of the substrates used, i.e. 0.1 and 0.2 mmol/l, respectively. The diaphorase activity determination method, described in the present communication, has been used in 19 healthy adults and children. Diaphorase activity was found to be 7.29 +/- 3.69 1 SD mumol NADH2 oxidized/ml packed cells per min, at 25 degrees C, and pH 7.00.
Papandreou
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Hematology Laboratory, Pendeli General Children's Hospital, Athens, Greece.
Rakitzis
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D000328:Adult; D002648:Child; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction
D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase; D008058:Dihydrolipoamide Dehydrogenase