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

Research output: Contribution to journalArticle

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 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.

Details

Authors
  • Chris E. Cooper
  • Gary G.A. Silkstone
  • Michelle Simons
  • Badri Rajagopal
  • Natalie Syrett
  • Thoufieq Shaik
  • Svetlana Gretton
  • Elizabeth Welbourn
  • Leif Bülow
  • Nélida Leiva Eriksson
  • Luca Ronda
  • Andrea Mozzarelli
  • Andras Eke
  • Domokos Mathe
  • Brandon J. Reeder
Organisations
External organisations
  • University of Essex
  • University of Parma
  • Semmelweis University
  • CNR Istituto di Biofisica, Pisa
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medicinal Chemistry
  • Hematology

Keywords

  • Blood substitute, Electron transfer, HBOC, Hemoglobin, Oxidative stress, PEGylation
Original languageEnglish
Pages (from-to)106-118
Number of pages13
JournalFree Radical Biology and Medicine
Volume134
Publication statusPublished - 2019
Publication categoryResearch
Peer-reviewedYes