N-[3H]Benzoylglycylglycylglycine as a probe for hydroxyl radicals.
N-[4-(3)H]Benzoylglycylglycylglycine ([(3)H]BzG(3)) was tested as a probe for detecting hydroxyl radicals (*OH). Aerated solutions of l-ascorbate generated *OH, which oxidized [(3)H]BzG(3), yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The (3)H(2)O was separated from organic products and remaining [(3)H]BzG(3) on Dowex-1. (3)H(2)O production was much greater with *OH than with other reactive oxygen species (ROS) (e.g., H(2)O(2), superoxide). The slight (3)H(2)O production in the presence of H(2)O(2) or superoxide was blocked by *OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H(2)O(2) or superoxide. This indicates that (3)H(2)O production was caused by *OH and that other ROS only generated any (3)H(2)O by forming traces of *OH. Doses of *OH that caused detectable nonenzymic polysaccharide scission also caused (3)H(2)O production, indicating that [(3)H]BzG(3) 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 (3)H(2)O production, indicating that both processes were due to *OH. Thus, [(3)H]BzG(3) 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.
Miller
Janice G
JG
Edinburgh Cell Wall Group, ICMB, University of Edinburgh, Daniel Rutherford Building, King's Buildings, Edinburgh EH9 3JH, UK.
Fry
Stephen C
SC
D001205:Ascorbic Acid / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000032:analysis*; D009842:Oligopeptides / Q000378:metabolism*; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014867:Water / Q000378:metabolism*
D002614:Chelating Agents; D016166:Free Radical Scavengers; D009842:Oligopeptides; D011134:Polysaccharides; D017382:Reactive Oxygen Species; D014867:Water; C029275:hippuryl-glycyl-glycine; D017665:Hydroxyl Radical; D001205:Ascorbic Acid