Chemically modified human or bovine hemoglobins (Hb) have been developed as oxygen-carrying therapeutics and are currently under clinical evaluation. Oxidative processes, which are in many cases enhanced when modifications are introduced that lower the oxygen affinity, can limit the safety of these proteins. We have carried out a systematic evaluation of two modified human Hbs (O-R-polyHbA0 and DBBF-Hb) and one bovine Hb (polyHbBv). We have both measured the oxidative products present in the Hb preparations and followed the oxidative reactions during 37 °C incubations. Autoxidation, the primary oxidative reaction which initiates the oxidative cascade, is highly correlated with P50 (R = 0.987; p < 0.002). However, when the results for the other oxidative processes are compared, two different classes of oxidative reactions are identified. The formation of oxyferrylHb, like the rate of autoxidation, increases for all modified Hbs. However, the subsequent reactions, which lead to heme damage and eventually heme degradation, are enhanced for the modified human Hbs but are actually suppressed for bovine-modified Hbs. The rhombic heme measured by electron paramagnetic resonance, which is the initial step that causes irreversible damage to the heme, is found to be a reliable measure of the stability of ferrylHb and has the tendency to produce degradation products, DBBF-Hb, a Hb-based oxygen carrier (HBOC) for which toxic side effects have been well documented, has the highest level of rhombic heme (41-fold greater than for HbA0), even though its rate of autoxidation is relatively low. These findings establish the importance of these secondary oxidative reactions over autoxidation in evaluating the toxicity of HBOCs.
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