An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase.
1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.
D000818:Animals; D002846:Chromatography, Affinity; D011756:Diphosphates; D005942:Gluconates; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008099:Liver / Q000201:enzymology; D009249:NADP; D010734:Phosphogluconate Dehydrogenase / Q000032:analysis*; D011732:Pyridoxal Phosphate; D012756:Sheep
D011756:Diphosphates; D005942:Gluconates; D008024:Ligands; D009249:NADP; D011732:Pyridoxal Phosphate; D010734:Phosphogluconate Dehydrogenase