Studies of Glucose-6-Phosphate Dehydrogenase. Lauren Chew & John Crawley

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes cellular biochemical oxidation-reduction reactions. Glucose-6-phosphate undergoes oxidation to 6-phosphogluconolactone while nicotinamide adenine dinucleotide phosphate (NADP+) is reduced to NADPH. This redox reaction is the primary step in the Pentose phosphate pathway. By way of this reaction, G6PD maintains sufficient cellular levels of NADPH, which is required for biosynthetic processes and protection against oxidative damage in red blood cells. It is suspected that variants (G6PD mutations) alter enzymatic activity in one of two ways. One, the variants mapped at the dimer interface affect the cohesive strength of the dimer. Two, the variants remotely affect the G6PD active sites thereby influencing the reaction rate. It follows that the association boundary warrants investigation. Using size-exclusion chromatography theory in conjunction with High Performance Liquid Chromatography (HPLC), we were able to obtain large zone elution profiles for Alcohol Dehydrogenase and Beta-Amylase. Leading edge centroid elution times were determined by integration using Simpson's Rule. Our results show that Alcohol Dehydrogenase had a greater elution time than Beta-Amylase. Size-exclusion studies are in progress to obtain elution times for different concentrations of G6PD. We expect to determine the monomer-dimer equilibrium concentration, calculate Keq, and repeat the experiment with a variant to gain some insight as to how mutations alter enzymatic activity.