The iron-catalyzed oxidation of dithiothreitol is a biphasic process: Hydrogen peroxide is involved in the initiation of a free radical chain of reactions

Dithiothreitol (DTT) is the most common agent used to reduce disulfide bonds in proteins. In the presence of transition metals and O-2, however, DTT can induce oxidative damage in biomolecules. By means of polarographic measurements, it was established that the DTT oxidation catalyzed by Fe3+ is a biphasic process, characterized by a lag phase of several minutes during which O-2 is consumed at a slow rate, by a mechanism involving the DTT-dependent reduction of Fe3+ to Fe2+ and the conversion of O-2 to H2O2. Some lines of evidence indicate that the reduction of Fe3+ is the rate-limiting step: (i) The replacement of Fe3+ with Fe2+ leads to a decrease in the length of the lag phase. (ii) The rate of Fe2+ formation by DTT is the same as the initial rate of O-2 uptake. (iii) The rate of sulfhydryl oxidation under anaerobic conditions is very slow. EDTA stimulates the iron-catalyzed oxidation of DTT probably by accelerating Fe3+ reduction. The lag phase is followed by a rapid uptake of O-2 involving both O-2(.-)-dependent and O-2(.-)-independent free radical reactions, which can proceed, albeit more slowly, in the absence of H2O2 (i.e., in the presence of catalase). Glutamine synthetase is inactivated faster when added during the phase of rapid O-2 uptake than when added before the beginning of the reaction. In both cases, catalase protects the enzyme, suggesting that only reactive species generated by H2O2 decomposition are able to induce the inactivation. (C) 1996 Academic Press, Inc.