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.
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.
Erat
Mustafa
M
Biotechnology Application and Research Center, Atatürk University, Erzurum, Turkey. eratm@atauni.edu.tr
D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D005980:Glutathione Reductase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012685:Sepharose / Q000737:chemistry*
D012685:Sepharose; D005954:Glucosephosphate Dehydrogenase; D005980:Glutathione Reductase