Redox active cysteine residues including βCys93 are part of hemoglobin's “oxidation hotspot”. Irreversible oxidation of βCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.
Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2) and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA) could easily induce complete pDNA cleavage while fetal hemoglobin (HbF) was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals.
Developing a functional blood substitute as an alternative to donated blood for clinical use is believed to relieve present and future blood shortages, and to reduce the risks of infection and blood type mismatching. Hemoglobin vesicle (HbV) encapsulates a purified and concentrated human-derived Hb solution in a phospholipid vesicle (liposome). The in vivo safety and efficacy of HbV as a transfusion alternative have been clarified. Auto-oxidation of ferrous Hb in HbV gradually increases the level of ferric methemoglobin (metHb) and impairs the oxygen transport capabilities. The extension of the functional half-life of HbV has recently been proposed using an electron mediator, methylene blue (MB), which acts as a shuttle between red blood cells (RBC) and HbV. MB transfers electron energies of NAD(P)H, produced by RBC glycolysis, to metHb in HbV. Work presented here focuses on screening of 15 potential electron mediators, with appropriate redox potential and water solubility, for electron transfer from RBC to HbV. The results are assessed with regard to the chemical properties of the candidates. The compounds examined in this study were dimethyl methylene blue (DMB), methylene green, azure A, azure B, azure C, toluidine blue (TDB), thionin acetate, phenazine methosulfate, brilliant cresyl blue, cresyl violet, gallocyanine, toluylene blue, indigo carmine, indigotetrasulfonate, and MB. Six candidates were found to be unsuitable because of their insufficient diffusion across membranes, or overly high or nonexistent reactivity with relevant biomolecules. However, 9 displayed favorable metHb reduction. Among the suitable candidates, phenothiazines DMB and TDB exhibited effectiveness like MB did. In comparison to MB, they showed faster reduction by electron-donating NAD(P)H, coupled with showing a lower rate of reoxidation in the presence of molecular oxygen. Ascertaining the best electron mediator can provide a pathway for extending the lifetime and efficiency of potential blood substitutes.
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