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 KM and Vmax 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 Ki value and the type of inhibition were determined by means of Lineweaver-Burk graph obtained for NADPH.

Keywords

• keywords: purification
• characterization
• glucose 6-phosphate dehydrogenase
• oncorhynchus mykiss
• erytrocyte