Oxidative Hemolysis and Precipitation of Hemoglobin

In a preceding report (1) it was shown that those redox compounds that cause Heinz body anemias in vivo produce a sequence of changes in vitro in solutions of crystalline hemoglobin culminating in the precipitation of hemoglobin as spherical granules. These granules appear to be identical with Heinz bodies. The sequence of changes produced in hemoglobin by these compounds includes: 1) the formation of methemoglobin, 2) an increase in electrophoretic and chromatographic mobility, 3) the appearance of irreversible hemochromes such as sulfhemoglobin and 4) frank precipitation. It was found that similar changes in hemoglobin were produced rapidly by "simple" oxidants such as ferricyanide and slowly by prolonged incubation of hemoglobin alone under oxygen. Compounds that cause Heinz body anemias apparently are converted under oxygen into redox intermediates or free radicals which catalyze the oxidative breakdown of hemoglobin, a process which proceeds spontaneously only slowly in vitro and presumably still more slowly during cellular aging in vivo. There are many reasons to suspect the involvement of sulfhydryl (thiol) groups in the processes described above. Such groups are readily oxidized under physiologic conditions and are involved in maintaining the integrity of the red cell (2) and the physiologic activity of hemoglobin (3). The studies of Beutler and his co-workers (4, 5) have established an association between intracellular reduced glutathione (GSH) levels and the susceptibility in vitro and in vivo of red cells to injury by oxidant drugs. Based on studies of "choleglobin" formation in the presence of ascorbic acid, Mills (6) has advanced the hypothesis that GSH protects hemoglobin from oxidative