Application Note: Using BioTek's Synergy HT Reader To Measure Reactive Oxygen Species (ROS) Generation In Stimulated Cells

By Paul Held Ph. D, BioTek Instruments, Inc., Kheng Newick, Graduate Student, University of Vermont

Reactive Oxygen Species (ROS) is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. Reactive oxygen species were originally thought to be only released by phagocytic cells as part of their role in host defense. Recent work has demonstrated that ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signaling cascades.

While the specific role that ROS plays is poorly understood, their physical nature makes them ideal candidates for being second messengers with cells. These molecules are small in size and diffuse only a short distance before they dissipate. In addition, there are several controlled mechanisms by which they are generated that work in conjunction with multiple means of their degradation.

While several enzymes are recognized as being able to produce ROS moieties, NADPH oxidase is the most significant. NADPH oxidase activity is controlled by a complex regulatory system that involves the G-protein Rac. In resting cells a membrane embedded heterodimer of two polypeptides (p22-phox and gp91-phox), which also contains two heme groups as well as a FAD group, enables the transfer of electrons from cytosolic NADPH across the membrane to molecular oxygen without NADPH oxidase activity . It is believed that the charge compensation occurs when gp91-phox polypeptide also acts as a proton pump. Upon stimulation, a number of polypeptides (p47- phox, p67-phox and p40phox) translocates to the inner face of the plasma membrane to form a fully active enzyme complex that contains NADPH oxidase activity Part of the active complex is the G-protein Rac, which upon stimulation dissociates from GDI, binds GTP and associates with the membrane . The assembled complex then catalyzes the formation of hydrogen peroxide (H2O2) from oxygen and hydrogen ions.