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Engineering tyrosine residues into hemoglobin enhances heme reduction, decreases oxidative stress and increases vascular retention of a hemoglobin based blood substitute

Cooper, Chris E.; Silkstone, Gary G.A.; Simons, Michelle; Rajagopal, Badri; Syrett, Natalie; Shaik, Thoufieq; Gretton, Svetlana; Welbourn, Elizabeth; Bülow, Leif LU and Eriksson, Nélida Leiva LU , et al. (2019) In Free Radical Biology and Medicine 134. p.106-118
Abstract

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every... (More)

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product.

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published
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keywords
Blood substitute, Electron transfer, HBOC, Hemoglobin, Oxidative stress, PEGylation
in
Free Radical Biology and Medicine
volume
134
pages
13 pages
publisher
Elsevier
external identifiers
  • scopus:85059619029
ISSN
0891-5849
DOI
10.1016/j.freeradbiomed.2018.12.030
language
English
LU publication?
yes
id
76a6eccb-8598-4bb9-985d-80cdb7eb57a1
date added to LUP
2019-01-17 11:52:14
date last changed
2019-02-20 11:43:21
@article{76a6eccb-8598-4bb9-985d-80cdb7eb57a1,
  abstract     = {<p>Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (&lt; 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product.</p>},
  author       = {Cooper, Chris E. and Silkstone, Gary G.A. and Simons, Michelle and Rajagopal, Badri and Syrett, Natalie and Shaik, Thoufieq and Gretton, Svetlana and Welbourn, Elizabeth and Bülow, Leif and Eriksson, Nélida Leiva and Ronda, Luca and Mozzarelli, Andrea and Eke, Andras and Mathe, Domokos and Reeder, Brandon J.},
  issn         = {0891-5849},
  keyword      = {Blood substitute,Electron transfer,HBOC,Hemoglobin,Oxidative stress,PEGylation},
  language     = {eng},
  pages        = {106--118},
  publisher    = {Elsevier},
  series       = {Free Radical Biology and Medicine},
  title        = {Engineering tyrosine residues into hemoglobin enhances heme reduction, decreases oxidative stress and increases vascular retention of a hemoglobin based blood substitute},
  url          = {http://dx.doi.org/10.1016/j.freeradbiomed.2018.12.030},
  volume       = {134},
  year         = {2019},
}