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

N-[4-3H]Benzoylglycylglycylglycine ([3H]BzG3) was tested as a probe for detecting hydroxyl radicals (<ce:glyph name="rad" />OH). Aerated solutions of l-ascorbate generated <ce:glyph name="rad" />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 <ce:glyph name="rad" />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 <ce:glyph name="rad" />OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H2O2 or superoxide. This indicates that 3H2O production was caused by <ce:glyph name="rad" />OH and that other ROS only generated any 3H2O by forming traces of <ce:glyph name="rad" />OH. Doses of <ce:glyph name="rad" />OH that caused detectable nonenzymic polysaccharide scission also caused 3H2O production, indicating that [3H]BzG3 is a sensitive <ce:glyph name="rad" />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 <ce:glyph name="rad" />OH. Thus, [3H]BzG3 is a simple, specific, sensitive, and robust probe for detecting <ce:glyph name="rad" />OH production in vitro. It may have applications for in vivo detection of extracellular <ce:glyph name="rad" />OH in arthritic joints and of apoplastic <ce:glyph name="rad" />OH in plant cell walls. [Copyright &y& Elsevier]