Purification of human erythrocyte glucose 6-phosphate dehydrogenase and glutathione reductase enzymes using 2 ′,5 ′-ADP Sepharose 4B affinity column material in single chromatographic step

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The enzymes of glucose 6-phosphate dehydrogenase and glutathione reductase were purified from human erythrocytes in one chromatographic step consisting of the use of the commercially available resin 2 ′,5 ′-ADP Sepharose 4B by using different washing buffers. Ammonium sulfate (30–70%) precipitation was performed on the hemolysate before applying to the affinity column. Using this procedure, G6PG, having the specific activity of 22.9 EU/mg proteins, was purified with a yield of 43% and 9150-fold; GR, having the specific activity of 20.7 EU/mg proteins, was purified with a yield of 26% and 8600-fold. The purity of the enzymes was checked on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and each purified enzyme showed a single band on the gel. This procedure has advantages of preventing of enzyme denaturation, short experimental duration, and use of less chemical materials for purification of the enzymes.