N-[3H]Benzoylglycylglycylglycine as a probe for hydroxyl radicals

Abstract

N-[4-3H]Benzoylglycylglycylglycine ([3H]BzG3) was tested as a probe for detecting hydroxyl radicals (OH). Aerated solutions of l-ascorbate generated OH, which oxidized [3H]BzG3, yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The 3H2O was separated from organic products and remaining [3H]BzG3 on Dowex-1. 3H2O production was much greater with OH than with other reactive oxygen species (ROS) (e.g., H2O2, superoxide). The slight 3H2O production in the presence of H2O2 or superoxide was blocked by OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H2O2 or superoxide. This indicates that 3H2O production was caused by OH and that other ROS only generated any 3H2O by forming traces of OH. Doses of OH that caused detectable nonenzymic polysaccharide scission also caused 3H2O production, indicating that [3H]BzG3 is a sensitive OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent 3H2O production, indicating that both processes were due to OH. Thus, [3H]BzG3 is a simple, specific, sensitive, and robust probe for detecting OH production in vitro. It may have applications for in vivo detection of extracellular OH in arthritic joints and of apoplastic OH in plant cell walls.