Human Glutathione Reductase: Purification of the Crystalline Enzyme from Erythorocytes

Glutathione reductase from human erythrocytes has been purified 40 000‐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 dehydrogenase. 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.