A superoxide dismutase–human hemoglobin fusion protein showing enhanced antioxidative properties

Abstract

Much of the toxicity of Hb has been linked to its redox activity; Hb may generate reactive oxygen species, such as the superoxide anion. Superoxide is intrinsically toxic, and superoxide dismutase (SOD) provides important cellular protection. However, if the Hb molecule is located outside the red blood cell, the normal protection systems involving SOD and catalase are no longer closely associated with it, exposing Hb and its cellular surroundings to oxidative damage. In order to produce less toxic Hb molecules, we have explored gene fusion to obtain homogeneous SOD–Hb conjugates. The chimeric protein was generated by coexpressing the human Hb α‐chain/manganese SOD gene together with the β‐chain gene in Escherichia coli. We show that the engineered SOD–Hb fusion protein retains the oxygen‐binding capacity and, moreover, decreases cytotoxic ferrylHb (HbFe4+) formation when challenged with superoxide radicals. The SOD–Hb fusion protein also exhibits a 44% lower autoxidation rate and higher thermal stability than Hb alone.

The Redox Activity of Hemoglobins: From Physiologic Functions to Pathologic Mechanisms

“…Subsequently, HBOCs were produced that include superoxide dismutase and catalase covalently linked to Hb through chemical modifications to decrease ROS formation, resulting from Hb autooxidation. Efforts are also under way to produce such protein complexes through chimeric ''fusion'' proteins, with Hb and superoxide dismutase expressed as a single polypeptide chain (112). Such products have been shown to decrease hydroxyl radical production and to scavenge free radicals in rat models of ischemia=reperfusion injury (69,213) and prevent ferryl Hb formation in vitro when challenged with hydrogen peroxide (70,112).…”

“…Efforts are also under way to produce such protein complexes through chimeric ''fusion'' proteins, with Hb and superoxide dismutase expressed as a single polypeptide chain (112). Such products have been shown to decrease hydroxyl radical production and to scavenge free radicals in rat models of ischemia=reperfusion injury (69,213) and prevent ferryl Hb formation in vitro when challenged with hydrogen peroxide (70,112). The one drawback with these Hbs linked with antioxidant proteins is that the reaction between lipid hydroperoxides and Hb, shown to be a significant pathogenic mechanism after hemolytic events, is relatively unaffected by the presence of superoxide dismutase and catalase.…”

Enhancement of Solubility and Specific Activity of a Cu/Zn Superoxide Dismutase by Co-expression with a Copper Chaperone in Escherichia coli

“…SOD activity was measured according to the inhibition of nitroblue tetrazolium (NBT) reduction by superoxide radicals generating from NADH/phenazine methosulfate (PMS) reaction at aerobic and non-acidic pH conditions as previously described (24). The absorbance at 560 nm was monitored during 5 min as an index of NBT reduction using a UV-visible spectrophotometer and calculated the enzyme inhibition (%) to define the half maximal inhibitory concentration values (IC50) and specific enzymatic activity.…”

Angiopep-2-Mediated Delivery of Human Manganese Superoxide Dismutase in Brain Endothelial Cells and its Protective Effect Against Oxidative Stress

“…Superoxide dismutase (SOD) activity was measured according to the inhibition of nitroblue tetrazolium (NBT) reduction by superoxide radicals generating from NADH/ phenazine methosulfate (PMS) reaction at aerobic and nonacidic pH conditions as described previously (Grey et al 2009). The absorbance at 560 nm was monitored during 5 min as an index of NBT reduction using a UV-Vis spectrophotometer, then calculated the enzyme inhibition (%) to define IC 50 values and specific enzymatic activity.…”