Mechanism of production of hydroxyl radicals in the copper-catalyzed oxidation of dithiothreitol

We have undertaken detailed studies of the mechanisms involved in the production of OH radicals during the copper-catalyzed oxidation of dithiothreitol (DTT), Most Of these studies were conducted in real time, detecting (OH)-O-. based on its reaction with coumarin-3-carboxylic acid to produce the fluorescent derivative 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). Studies of the kinetics of oxidizing DTT show that production of (OH)-O-. occurs in two stages: an initial lag period during which there is little production of 7-OHCCA, and a second reaction phase during which there is more rapid generation of (OH)-O-.. The duration of the initial reaction period depends on the concentrations of both DTT and Cu2+. During this initial stage, oxygen consumption is high, although an increasing concentration of Cu2+ decreases the oxygen consumption. The rate of production of (OH)-O-. during the second phase of the reaction depends on the concentration of Cu2+, but little oxygen is consumed. A mechanism is proposed whereby a Cu2+-DTT complex is formed and catalyzes oxidation of free DTT via formation of an oxygen-containing intermediate, The second phase of the reaction begins after complete oxidation of free DTT and involves production of O-2(.-) and H2O2 followed by generation of (OH)-O-. via reduction of H2O2 by cuprous ion. (C) 1997 by Radiation Research Society.