Cross-Linked Hemoglobin-Superoxide Dismutase-Catalase Scavenges Oxygen-Derived Free Radicals and Prevents Methemoglobin Formation and Iron Release

Abstract

In this study, we prepared PolyHb-SOD-catalase (intermolecularly cross-linked hemoglobin, superoxide dismutase (SOD), and catalase). We found that PolyHb-SOD-catalase is effective in scavenging oxygen-derived free radicals. In the xanthine/xanthine oxidase system, the initial rate of cytochrome c reduction was 2.13 ± 0.26 nmoles cyt. c/min for PolyHb alone. PolyHb-SOD-catalase reduced this to 0.56 ± 0.08 nmoles cyt. c/min because of its ability to eliminate superoxide (O₂). Addition of PolyHb to 200 μM of hydrogen peroxide (H₂O₂), changed the H₂O₂ level slightly to 192 ± 0.4 μM. Addition of PolyHb-SOD-catalase, on the other hand, lower the level to 41 ± 0.3 μM. Results also show that both effects were dependent on the concentration of SOD-catalase cross-linked with hemoglobin. Oxidative challenge with H₂O₂ resulted in minimal changes in the absorbance spectra of PolyHb-SOD-catalase. With PolyHb, there were spectral changes reflecting the formation of methemoglobin and heme degradation. Furthermore, the amount of iron released, after incubation with 250 μM H₂O₂, was 6.8 ± 1.8 μg/dl for PolyHb-SOD-catalase and 76.6 ± 1.0 μg/dl for PolyHb. These results show that cross-linked SOD-catalase prevents oxidative reactions involving the hemoglobin component of PolyHb-SOD-catalase.