Preparation Of D-Glucose-6-Phosphate Dehydrogenase And Its Substrates Prior To Reactivity With Ozone

Kristin Linzmeyer, Carroll College, Helena, MT

Abstract

Ozone is becoming more important in the area of disinfection of water and food. In order to determine how much ozone is needed to inactivate microorganisms that contaminate water and food supplies, the mechanisms of ozone toxicity must be understood. In a project in which ozone was used to oxidize an enzyme model, which in some experiments was encapsulated in liposomes, the activity before and after ozonation of the enzyme was recorded to determine how the enzyme affected the enzyme's rate of reaction. The enzyme, D-glucose 6-phosphate dehydrogenase (DG6P dehydrogenase), was studied to determine at what concentration it needed to be, as well as its substrates, NAD+ and D-glucose 6-phosphate. In addition to these parameters, any interfering substances needed to be discovered before any experiments with ozone commenced. One such substance, Triton xlOO, is a detergent that is used to break open the liposome membrane; the other, L-cysteine, is the amino acid that is most reactive with ozone, and thus it is used to terminate the ozone's oxidative attack on the enzyme. These experiments were accomplished with enzyme assays. The conclusions reached were that neither Triton xlOO nor L-cysteine were inhibitive toward DG6P dehydrogenase. Furthermore, the concentration of NAD+ could be as low as 4.5 mM and that of DG6P equalling 3.0 mM so that neither one of these reagents was the limiting factor in the reaction. The enzyme, DG6P dehydrogenase, had a sufficient concentration at 3.0 mM. The enzyme could now be used in experiments involving ozone.