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 erythrocytes. It is suspected that variants 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 HPLC we were able to obtain elution profiles for the standards BSA and Alcohol Dehydrogenase. We were also able to obtain profiles for varying concentrations of the enzyme G6PD. Leading edge elution times were determined by integration in Excel? Our results indicate that lowering the enzyme concentration through serial dilution encourages the dissociation of the G6PD dimer. Size exclusion studies at increased NaCl concentrations are in progress to determine if increased salt concentrations will encourage dimer dissociation at higher enzyme concentrations. We expect to determine the monomer-dimer equilibrium concentration, calculate KD, and repeat the experiment with a variant to gain some insight as to how mutations alter enzymatic activity.
