Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) is the first enzyme of the oxidative pentose phosphate cycle and catalyses the conversion of glucose-6-phosphate (G6P) to 6-phosphoglucono-δ-lactone and transfers one electron to NADP+ producing one NADPH. Conversion of G6P to 6-phosphoglucono-δ-lactone is proportional to the production of NADPH. The increase in NADPH concentration results in an increase in absorbance at 340 nm. To assay G6PDH activity, therefore, production of NADPH is determined by measuring increase in absorbance at 340 nm spectrophotometrically. This increase rate is then converted to unit of activity and specific activity of G6PDH. In this procedure, a generalized method is given for bacterial G6PDH assays emphasizing on a cyanobacterium Synechocystis sp. PCC6803 (Schaeffer and Stanier, 1978; Karakaya et al., 2008, 2012) and a heterotrophic bacterium E.coli (Hylemon and Phibbs, 1972; Barnel et al., 1990).