The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.
1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.
D000345:Affinity Labels; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D010734:Phosphogluconate Dehydrogenase / Q000302:isolation & purification*; D011189:Potassium Chloride; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013696:Temperature; D014227:Triazines
D000345:Affinity Labels; D004396:Coloring Agents; D014227:Triazines; D011189:Potassium Chloride; D010734:Phosphogluconate Dehydrogenase