The catalytic properties of glutathione reductase from human erythrocytes have been studied over a range of buffer conditions and substrate concentrations. This study provides optimal conditions for determining the basic kinetic parameters of the enzyme.The catalytic behaviour of glutathione reductase is consistent with spatially separated binding sites for its substrates.In certain assays anomalies were observed which are correlated with an inactivation of the enzyme by NADPH. Concurrent sedimentation experiments showed that NADPH promoted aggregation of the enzyme. Both inactivation and aggregation could be connected with oxidation of thiols at the active site.The relation of the properties of glutathione reductase to cellular Conditions is discussed.Glutathione reductase contains FAD and a disulphide at its active site [l]. The catalytic reaction requires reduction of the site by NADPH, producing a semiquinone of FAD, a sulphur radical and a thiol P I : GSSGGlutathione reductase has attracted some attention as a representative example of flavin enzymes, and various attempts have been made to determine the order of attachment of substrates and release of its products. Following reduction of the active site by NADPH, NADP may be released before or after the catalytic steps involving glutathione. The relation between reaction rates and substrate concentrations suggests early release of NADP [l-41. In contrast, the competitive inhibition shown by NADP towards NADPH implies release of NADP after reduced glutathione [5 -81. Mannervik has combined these alternatives in a branched mechanism, whereby the reaction sequence depends on the relative substrate concentrations [9,10].Much of the work on glutathione reductase has been carried out on the enzyme from yeast [1,7-121.

Glutathione reductase from human erythrocytes has been purified 40000-fold in 10 steps with an overall yield of 36 %.The procedure included bulk separations on DEAE-Sephadex and CM-Sephadex, gel-filtration on Sephadex G-200, and salt fractionation with ammonium sulphate. The purified enzyme was crystallised from aqueous solution.The final preparation had a specific activity of 240 units/mg. The purification procedure was completed in 4 weeks, and gave 40 mg enzyme from 35 1 blood. The schedule for glutathione reductase allowed the concurrent isolation of glucose-6-phosphate dehydrogenase and 6-phosphogluconate deh ydrogenase.In solutions of thiols, the glutathione reductase preparation was homogeneous, as indicated by gel-filtration, ultracentrifugation and electrophoresis. In the absence of thiols, the enzyme showed a tendency to form aggregates. Some of the physical properties of the enzyme are discussed in relation to previous difficulties encountered in its isolation.Glutathione reductase catalyses the reaction between the oxidised form of glutathione and NADPH [1,21:The NADPH for the above reaction is provided by the initial steps of the hexosemonophosphate pathway involving glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These two dehydrogenases and glutathione reductase therefore provide a sequence of reactions whereby NADPH is produced and used to maintain glutathione in the reduced state. The reduced glutathione contributes to the viability of the erythrocyte by stabilising other thiols, including those in the cell membrane, in haemoglobin and in the cellular enzymes [3,4]. Although haemolytic anaemias have been associated with glutathione reductase deficiency, a partial depression of activity of the red cell enzyme is common, and is connected with the incomplete saturation of the apoenzyme with its prosthetic group, FAD [5]. This condition could arise from an inadequate intake of Enzyme. Glutathione reductase (EC 1.6.4.2).riboflavin [6]. There is interest in the mechanism of action of glutathione reductase as a representative example of flavin enzymes. The catalytic process is thought to proceed through single electron transfer mediated by the semiquinone form of FAD [7]. However, the sequence of steps in the catalytic mechanism is not clear.Despite the interesting features of the catalytic process and the apparent importance of glutathione reductase to the stability of the red cell, there is limited information on this enzyme relating to its cellular concentration, specific activity and its physical and catalytic properties. This lack of information arises from the difficulty in obtaining sufficient material from erythrocytes for subsequent study of the physicochemical properties of the enzyme. Thus the previous attempts at isolation of glutathione reductase from erythrocytes have only approached purity by a sacrifice of most of the enzyme [8 -101. The previous yields of purified material ranged from 0.3 [lo] to 1.3 mg Previous work in this laboratory has been carried out on th...

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