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.
An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase
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P Griffiths, A Houlton, M J Adams, M J Harvey, P D G Dean; An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase. Biochem J 1 March 1977; 161 (3): 561–568. doi: https://doi.org/10.1042/bj1610561
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