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Adem, S and Ciftci, M (2012) Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step. Protein Expr. Purif. 81:1-4

The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.

PubMed Online version:10.1016/j.pep.2011.08.031

Animals; Buffers; Chromatography, Affinity/methods; Electrophoresis, Polyacrylamide Gel; Glucosephosphate Dehydrogenase/chemistry; Glucosephosphate Dehydrogenase/isolation & purification; Glucosephosphate Dehydrogenase/metabolism; Glutathione Reductase/chemistry; Glutathione Reductase/isolation & purification; Glutathione Reductase/metabolism; Kidney/chemistry; Kidney/enzymology; Molecular Weight; Phosphogluconate Dehydrogenase/chemistry; Phosphogluconate Dehydrogenase/isolation & purification; Phosphogluconate Dehydrogenase/metabolism; Rats; Rats, Sprague-Dawley; Sepharose/analogs & derivatives; Sepharose/chemistry