Glucose 6-phosphate dehydrogenase (G6PD) was purified from buffalo (Bubalus bubalis) erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: hemolysate preparation and 2',5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the two consecutive procedures, the enzyme, having a specific activity of 69.7 EU/mg proteins, was purified 650-fold with a yield of 31%. Optimal pH, stable pH, optimal temperature, molecular weight, and K-m and V-max values for NADP(+) and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition K-i values and the type of inhibition were determined by means of Lineweaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADPH, and NADH. (C) 2003 Elsevier Science (USA). All rights reserved.