Structural requirements for the ferrihemoglobin-forming activity of glutathione S-conjugates of 4-dimethylaminophenol

4-Dimethylaminophenol (DMAP) is a suitable cyanide antidote that rapidly forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. Deleterious methemoglobinemia, because of the catalytic cycling, is prevented by side reactions of oxidized DMAP with thiols, particularly with glutathione (GSH). In human red cells, both in vitro and in vivo, the formation of a transient bis-glutathione and a stable tris-glutathione adduct was observed. To investigate the reactivity of GSH adducts of DMAP, we synthesized various thioethers by oxidizing DMAP with PbO2 in 0.1 M sulfuric acid followed by reaction with GSH. The following compounds were isolated and characterized by 1H-NMR spectroscopy and determination of the pK values: 4-dimethylamino-2-(glutathione-S-yl)-phenol (2-GS-DMAP), 4-dimethylamino-3-(glutathione-S-yl)-phenol (3-GS-DMAP), 4-dimethylamino-2,5-bis(glutathione-S-yl)-phenol (2,5-bis GS-DMAP), 4-dimethylamino-2,6-bis(glutathione-S-yl)-phenol (2,6-bis GS-DMAP), and 4-dimethylamino-2,3,6-tris(glutathione-S-yl)-phenol (2,3,6-tris GS-DMAP). Ferrihemoglobin-forming activity was investigated with oxyhemoglobin, alkylated with N-ethylmaleimide (Hb-NES) to prevent binding of oxidized compounds to the protein SH groups. DMAP, 2,6-bis-GS-DMAP, and 2-GS-DMAP (0.1 mM each) completely oxidized Hb-NES (0.6 mM) in a decreasing order of activity (pH 7.4, 37 degrees C, air); the other derivatives were quite inactive. The same thioether reactivity was observed during autoxidation. Ferrihemoglobin formation by the reactive thioethers was greatly enhanced when the oxygen tension was increased from 2 to 100%. In contrast, variation of the oxygen tension had only marginal effects on the activity of DMAP.(ABSTRACT TRUNCATED AT 250 WORDS)