purification and characterization of glucose 6-phosphate dehydrogenase from rainbow trout (oncorhynchus mykiss) erythrocytes

Abstract

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP⁺ oxidoreductase, EC 1.1.1.49; G6PD) from rainbow trout (Oncorhynchus mykiss) erythrocytes was purified, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure consisted of three steps: haemolysate preparation, ammonium sulphate precipitation, and 2’,5’-ADP Sepharose 4B affinity gel chromatography, which took one working day. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 14.51 EU/mg proteins, was purified with a yield of 70.40% and 1 271.19-fold. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis was carried out. SDS polyacrylamide gel electrophoresis showed a single band for the enzyme. Optimal pH, stable pH, optimal temperature, molecular weight, and K_M and V_max values for NADP⁺ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, the effect of NADPH on the enzyme was investigated and K_i value and the type of inhibition were determined by means of Lineweaver-Burk graph obtained for NADPH.