Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol

Abstract

The influences of buffers and iron chelators on the rate of autoxidation of Fe2+ were examined in the pH range 6.0-7.4. The catalysis by Fe2+ and Fe3+ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe2+, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30 degrees C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe3+ over Fe2+, Fe2+ autoxidizes rapidly in the pH range 6.0-7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe2+ stabilize both Fe2+ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe3+ in non-chelating buffers or in the presence of strong chelators of Fe2+, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe2+ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe2+ and Fe3+. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe2+ or both dithiothreitol and iron.