Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.
1. A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification. 2. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx. 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73%. 3. A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out. NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents. A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution. 4. Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B. The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction. 5. Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B.
D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification*; D007934:Leuconostoc / Q000201:enzymology*; D008024:Ligands; D009243:NAD; D009249:NADP; D011189:Potassium Chloride; D014227:Triazines
D004396:Coloring Agents; D008024:Ligands; D014227:Triazines; D009243:NAD; D009249:NADP; D011189:Potassium Chloride; D005954:Glucosephosphate Dehydrogenase