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.