Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP(+) oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep liver by a simple and rapid method. The purification process consisted of two steps: preparation of the homogenate, and 2, 5'-adenosine diphosphate (ADP) Sepharose 4B affinity chromatography. Through the use of these two consecutive steps, the enzyme was purified with a yield of 35.6% and 1,920 fold, having the specific activity of 11.2 enzyme units (EU/mg protein). A K-M of 0.176 mM and a V-max of 0.0179 EU/ml were obtained for G6-P, and 0.0194 mM and 0.0223 EU/ml for NADP(+). Enzymatic activity was measured spectrophotometrically according to Beutler's method at 340 nm and optimal pH and assay temperature were determined. By means of a Lineweaver-Burk plot, the inhibitor constant for NADPH was determined to be K-i, 4.707 +/- 0.49 muM and it was shown to inhibit the enzyme in a non-competitive manner. The purification of enzyme was monitored by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE showed a single band at similar to55 kDa for the enzyme and gel filtration chromatography indicated it to be a dimer.