Abstract
6-Phosphogluconate dehydrogenase from human erythrocytes has been purified 5000-fold from haemolysates with an overall yield of 6%. Bulk fractionations followed by gradient elutions were carried out on DEAE-Sephadex and CM-Sephadex, and gel-filtration on Sephadex G-200. Ammonium sulphate fractionations were used after the CM-Sephadex and Sephadex G-200 steps. The final preparation had a specific activity of 10 units/mg, and was homogeneous on ultracentrifugation. Although other proteins were absent, electrophoresis indicated the presence of isoenzymes. The pruification procedure was completed in 3 weeks, and gave 50 mg enzyme from 35 l blood. The procedure allows the concurrent isolation of glucose-6-phosphate dehydrogenase. The dependence of the enzyme activity on ionic stregth, pH and NADPH was investigated. Under cellular conditions the total potential activity is the same for the two hexosemonophosphate dehydrogenases. The flow through the pathway would be controlled by the level of NADPH.
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