Three Different Purification Protocols in Purification of G6PD from Sheep Brain Cortex
 Cihangir ŞENGEZER*, Nuriye Nuray ULUSU*^,o
 * Hacettepe University, Faculty of Medicine, Department of Biochemistry Ankara, Turkey

^oCorresponding Author

Summary

The aim of this study was to determine the most efficient purification protocol for purification of glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) from sheep brain cortex. G6PD was purified by 2', 5'-ADP-Sepharose 4B affinity and DEAE Sepharose Fast Flow ion exchange chromatography columns in different orders. In all purification protocols, 105,000 x g supernatants were used after a homogenization step. In the first purification protocol, the supernatant was loaded onto an affinity column after an ultracentrifugation step, followed by ion exchange chromatography. In the second purification protocol, the supernatant was dialyzed after ultracentrifugation, then loaded onto an ion exchange column. The enzyme was eluted by linear KCl gradient. G6PD activity containing fractions were collected and dialyzed, then loaded onto an affinity chromatography column. The third protocol was similar to the first, except for addition of a dialysis step after ultracentrifugation. In the first protocol, the yield and the specific activity were found to be 68.33% and 51.25 U/mg protein, respectively. In the second purification protocol, G6PD was obtained with a yield of 24.89% and had a specific activity of 3.63 U/mg protein. In the third, the enzyme was obtained with a yield of 27.08% and had a specific activity of 7.8 U/mg protein. In this study, we compared three different protocols for purification of G6PD from sheep brain cortex.
 
Key Words :
Glucose-6-phosphate dehydrogenase, purification, brain cortex.