Oxidative processes, which are in many cases enhanced when modifications are introduced that lower the oxygen affinity, can limit the safety of these HBOC proteins. We have carried out a systematic evaluation of two modified human Hbs (O-R-PolyHbAo and DBBF-Hb) and one bovine Hb (PolyHbBv). We have measured both the oxidative products present in the Hb preparations and followed the oxidative reactions during 37oC incubations. Autoxidation, the primary oxidative reaction which initiates the oxidative cascade is highly correlated with P50 (R = 0.987; p< 0.002). However, when comparing the results for the other oxidative processes, 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 actually suppressed for bovine modified Hbs. The rhombic heme (distorted geometry) 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 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 HbAo), 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. This work was recently published in Biochemistry 41, 7407-7415, 2002. An extension of these studies; we developed a more reliable rapid mixing method to detect heme degradation products in several chemically modified Hb solutions. We recently described a stopped-flow fluorescence method for the detection of fluorescent heme degradation products in Hb solutions challenged with high concentration of H2O2 and reported the rate constants for these processes (Anal. Biochem. 308:186-188, 2002). We have also recently reported that DBBF-Hb, and analogue of Baxter's DCLHb, under mild oxidative stress can cause endothelial cells to die apoptotically and neurotically. In this study we clearly documented a role for ferryl-DBBF-Hb as the causative agent of these changes (Blood 98:3315-3323, 2001)
