Hemoglobin-based oxygen carriers. Structural alterations that affect free radical formation
Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that the chemical modifications used to generate HBOCs of specific types alter their ability to generate or interact with free radicals. Three HBOCs were studied: Hb-FMDA and HbBv-FMDA, produced by the reaction of human or bovine oxyHb with the fumaryl-diaspirin, mono-(3,5-dibromosalicyl) fumarate; and Hb-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. Exposure of oxyHbA to H2O2 causes generation of free radicals capable of cleaving dimethylsulfoxide to produce formaldehyde (HCHO). Relative to the reaction rate for HbA (630 ± 130 M/min) the rates of HCHO formation were roughly 70% for Hb-DBBF, 50% for Hb-FMDA, and 16% for HbBv-FMDA. Exposure to H2O2 also caused spectral changes (indicative of oxidative modifications of hemoglobin) at varied rates for the HBOCs analyzed. Although these rates were not directly correlated with the rate of free radical formation, addition of mannitol or thiourea (hydroxyl radical scavengers) slowed both the rate of spectral changes and of formaldehyde generation. The relative ability of the ferric derivatives of the HBOCs to participate in free radical reactions was monitored by assays of non-enzymatic NADPH oxidation and aniline hydroxylation (reactions mediated by reactive oxygen species). HbBv-FMDA showed significantly slower rates than the other HBOCs in both assays: in 2 minutes, up to 37% of oxidized Hb-DBBF was reduced; in contrast, in 20 minutes 30% of oxidized Hb-FMDA and only 2.5% of oxidized HbBv-FMDA were reduced. Paraminophenol, generated within 5 minutes as a result of Hb-mediated hydroxylation of aniline, was 13.5, 10, and 1 μM for Hb-DBBF, Hb-FMDA, and HbBv-FMDA, respectively. The observed differences between HBOCs in these assays indicate differences in their ability to generate free radicals as well as differences in their reactivity with reactive oxygen species.
