PURIFICATION AND PROPERTIES OF GLUTATHIONE REDUCTASE OF HUMAN ERYTHROCYTES

1. 1. Human erythrocyte glutathione reductase (NAD(P)H: oxidized glutathione oxidoreductase, EC 1.6.4.2) was purified 47 000-fold by column chromatography. The enzyme contained FAD as the prosthetic group. From the flavin content a minimum mol. wt. of 56 600 was calculated. 2. 2. The mol. wt. was determined by gel filtration with Sephadex G-200 and was found to be 115 000 ± 4000. Blue dextran-2000 was not usable to determine the void volume of the column as it interacted with glutathione reductase. 3. 3. The Km value for FAD binding to be apoenzyme was temperature dependent. The Arrhenius plot of the rate of recombination was nonlinear. Restoration of full activity occurred at least through several steps. 4. 4. Preincubation of the apoenzyme with FMN retarded the restoration of the activity with FAD. Full restoration of the activity did not guarantee that the original enzyme conformation had been acquired. © 1969.