Improved biotinylation of glucose-6-phosphate dehydrogenase using active-site-blocking agents.
Characteristics of the biotinylation of glucose-6-phosphate dehydrogenase are presented. The enzyme is inactivated in the presence of N-hydroxysuccinimido biotin but can be protected by an appropriate concentration of NADPH used as an active-site blocker. A Ki of 1.6 +/- 1.0 microM calculated for NADPH for this protection shows it to be an active-site phenomena. Enzyme inactivation is irreversible with consistent kinetic results requiring the presence of 10 mM EDTA. An improved methodology was developed for biotinylation allowing 100% protection of the enzyme with loading factors up to 30.8 mol of biotin per enzyme.
D001665:Binding Sites / Q000187:drug effects; D001710:Biotin / Q000031:analogs & derivatives; D055598:Chemical Phenomena; D002621:Chemistry; D005954:Glucosephosphate Dehydrogenase; D007700:Kinetics; D009249:NADP; D013388:Succinimides
D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; D009249:NADP; D001710:Biotin; D005954:Glucosephosphate Dehydrogenase