Abstract
Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (βF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: βF41Y; βK66Y; βF71Y; βT84Y; βF85Y; and βL96Y. As with βF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (βT84Y, and βF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in βT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 μM ascorbate over a time period of 60 min increasing from 10% in βF41Y to over 50% in βT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.
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References
Cooper CE, Silaghi-Dumitrescu R, Rukengwa M et al (2008) Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC). Biochim Biophys Acta 1784:1415–1420
Dunne J, Caron A, Menu P et al (2006) Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo. Biochem J 399:513–524
Vandegriff KD, Malavalli A, Minn C et al (2006) Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates. Biochem J 399:463–471
Reeder BJ, Grey M, Silaghi-Dumitrescu RL et al (2008) Tyrosine residues as redox cofactors in human hemoglobin: Implications for engineering non toxic blood substitutes. J Biol Chem 283:30780–30787
Reeder BJ, Svistunenko DA, Cooper CE et al (2012) Engineering tyrosine-based electron flow pathways in proteins: the case of aplysia myoglobin. J Am Chem Soc 134:7741–7749
Silkstone GG, Silkstone RS, Wilson MT et al (2016) Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design. Biochem J 473:3371–3383
Silkstone RS, Silkstone G, Baath JA et al (2016) The betaLys66Tyr variant of human hemoglobin as a component of a blood substitute. Adv Exp Med Biol 876:455–460
Acknowledgments
We acknowledge financial support from BBSRC (BB/L004232/1).
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Cooper, Reeder and Silkstone have patents relating to the introduction of tyrosine residues into recombinant hemoglobin as a component of a blood substitute.
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Silkstone, G.G.A., Simons, M., Rajagopal, B.S., Shaik, T., Reeder, B.J., Cooper, C.E. (2018). Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes. In: Thews, O., LaManna, J., Harrison, D. (eds) Oxygen Transport to Tissue XL. Advances in Experimental Medicine and Biology, vol 1072. Springer, Cham. https://doi.org/10.1007/978-3-319-91287-5_35
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DOI: https://doi.org/10.1007/978-3-319-91287-5_35
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