ISOLATION AND PURIFICATION OF HUMAN ERYTHROCYTE GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM SMALL AMOUNTS OF BLOOD

1. 1. A simple procedure for the purification of glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) from 500 ml of human blood is described. 2. 2. The procedure consists of three main steps: DEAE-Sephadex chromatography; recycling gel filtration on Sephadex G-100; CM-Sephadex chromatography with specific elution by substrate. 3. 3. The enzyme, obtained with an overall yield of about 60%, has a specific activity of about 190 units/mg in diluted state, and it is shown to be homogeneous by electrophoresis on cellulose acetate gel and by N-terminal amino acid determination. 4. 4. The procedure is particularly suited for structural studies of rare glucose-6-phosphate dehydrogenase variants, but it can be scaled-up for the study of common glucose-6-phosphate dehydrogenase variants associated with low activity. © 1969.