Isolation and Purification of Human Glucose-6-Phosphate Dehydrogenase from E. coli. Marci Raney and Cindy Kopp

Abstract

Glucose 6-phosphate dehydrogenase, an enzyme in red blood cells, converts glucose 6-phosphate to 6-phosphogluconolactone and reduces NADP to NADPH in the pentose phosphate pathway. G6PD deficiency affects approximately 400 million people worldwide and is the most common human enzyme deficiency. A deficiency and/or specific mutation in G6PD may lead to the destruction of red blood cells through a process called hemolytic anemia. Upon exposure to oxidative agents the cell membrane becomes distorted and eventually ruptures. Massive destruction of cells in this manner may lead to death. To date, most of the biochemical facts discovered have been the result of experiments completed with intact red blood cells. Currently, over 400 variants of this enzyme are known. Once the structure of these variants is better understood, it may be possible to modify or silence the structural flaws to eliminate the pathologies associated with certain variants. Several isolation and purification experiments were performed to purify significant quantities of the enzyme from E. coli for further structural analysis. The greatest success in extracting the enzyme from E. coli cells was accomplished with sonication, the administration of high frequency sound waves. The enzyme was overexpressed in cells after subjection to high temperatures for brief periods of time during incubation. G6PD was successfully further purified with the use of an affinity matrix called Affi-gel Blue, low concentrated salt solutions, and ammonium sulfate precipitation.