Purification of glucose-6-phosphate dehydrogenase from red blood cells and from human leukocytes: Description of a new method of purification by electric elution of the enzyme with NADP +

Abstract

The authors have highly purified glucose-6-phosphate dehydrogenase from erythrocytes, and leukocytes from a patient with chronic myeloid leukemia. The stages of purification include a chromatography on DEAE-Sephadex and an elution of glucose-6-phosphate dehydrogenase from a CM-Sephadex column by its own coenzyme NADP +. This method permits an overall yield of 55%, and 80 to 90% for the stage of elective elution of a stable enzymatic extract, whose specific activity is 170 units/mg of proteins. This extract is homogeneous from an immunological point of view. Sodium dodecylsulphate acrylamide gel electrophoresis shows the persistence of traces of impurities which could be completely eliminated by a chromatography on hydroxyapatite gel. The mechanism of the fixation and elution of glucose-6-phosphate dehydrogenase on CM-Sephadex is discussed. The utilization of leukemia leukocytes permits the purification of a quantity of enzymatic material sufficient for physicochemical and structural studies from a single donor. Since such a tissue is always less affected by glucose-6-phosphate dehydrogenase deficiency than erythrocytes, it alone can permit the study of certain unstable variants.