Interactions of immobilized and free triazine dyes with glucose-6-phosphate dehydrogenase from yeast.
Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) prepared from baker's yeast binds to immobilized Cibacron Blue F3G-A and Procion Red HE-3B. In this paper the two dyes are compared with respect to their use in the purification of this enzyme. Cibacron Blue chromatography was found useful at an early stage of purification for the removal of contaminating hexokinase, phosphoglucose isomerase and phosphoglucomutase. With Procion Red HE-3B Sepharose the NADP dependent enzymes phosphogluconate dehydrogenase and glutathione reductase are separable from glucose-6-phosphate dehydrogenase. Unlike Cibacron Blue gel chromatography, the enzyme can be specifically eluted from Procion Red HE-3B Sepharose by a NADP gradient. Other monochlorotriazine dyes like Xirone Brillant Red BHD, 4BHD, 6BHD and GHD and the dichlorotriazine dye Procion Brown MX-5BR immobilized to Sepharose have only little binding affinity to glucose-6-phosphate dehydrogenase. The binding behaviour of different immobilized triazine dyes for pre-purified and purified glucose-6-phosphate dehydrogenase is compared. In addition, the influence of the free dyes on the activity of glucose-6-phosphate dehydrogenase is studied. It is demonstrated that the results of kinetic and binding studies with the purified enzyme are not uncritically applicable for the selection of a dye as ligand for affinity chromatography during enzyme preparation.
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014227:Triazines / Q000378:metabolism*
D014227:Triazines; C011535:procion brilliant red H 3B; D009249:NADP; C012842:Cibacron Blue F 3GA; D005954:Glucosephosphate Dehydrogenase