Glucose 6-phosphate dehydrogenase of human erythrocytes. I. Purification and characterization of normal (B+) enzyme.

Human erythrocyte glucose 6-phosphate dehydrogenase (o-glucose 6-phosphate :nicotinamide adenine dinucleotide phosphate oxidoreductase, EC 1.1.1.49) was purified by column chromatography with diethylaminoethyl cellulose, calcium phosphate gel, carboxymethyl cellulose, diethylaminoethyl Sephadex, and carboxymethyl Sephadex. A homogeneous preparation was obtained in over-all yield of about 50%. The specific activity (750) and the yield were significantly greater than those so far reported for the enzyme. The sedimentation patterns of analytical ultracentrifugation and interference pattern of sedimentation equilibrium indicated a homogeneous preparation. The sedimentation constant (s%J was 10.0 S at a protein concentration of 0.3 %. The molecular weight was estimated as 240,000. The molecular weight was about 43,000 in 4 N guanidineHCl, indicating that the enzyme consisted of six similar size subunits. Removal of nicotinamide adenine dinucleotide phosphate from the enzyme caused dissociation into inactive (or weakly active) subunits with a molecular weight of about one-half of the native protein. The enzyme was partially inactivated by dilution without dissociating into subnuits. Optimal pH and Michaelis constants for the primary substrates were determined. Analogues of n-glucose 6-phosphate accepted as substrates were 2-deoxy-D-glucose 6-phosphate and D-galactose 6-phosphate. Nicotinamide adenine dinucleotide was a weak substrate. Amino acid composition of the enzyme was determined.