title: Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.
abstract: Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the β-subunit (F41). We therefore replaced this residue with a tyrosine (βF41Y, Hb Mequon). The βF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, βF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the β-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in βF41Y. NO bioavailability was enhanced in βF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the β-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.
authors.forename: Gary G A
authors.initials: GG
authors.lastname: Silkstone
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Silkstone
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.
authors.forename: Michelle
authors.initials: M
authors.lastname: Simons
authors.affiliation: Department of Pure and Applied Biochemistry, Lund University, Box 124, 221 00 Lund, Sweden.
authors.forename: Leif
authors.initials: L
authors.lastname: Bülow
authors.affiliation: Department of Pure and Applied Biochemistry, Lund University, Box 124, 221 00 Lund, Sweden.
authors.forename: Kristian
authors.initials: K
authors.lastname: Kallberg
authors.affiliation: Department of Pure and Applied Biochemistry, Lund University, Box 124, 221 00 Lund, Sweden.
authors.forename: Khuanpiroon
authors.initials: K
authors.lastname: Ratanasopa
authors.affiliation: Department of Neurosciences, University of Parma, Parma, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Ronda
authors.affiliation: Department of Pharmacy, University of Parma, Parma, Italy Institute of Biophysics, National Research Council (CNR), Pisa, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Reeder
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.
authors.forename: Chris E
authors.initials: CE
authors.lastname: Cooper
mesh_terms: D001802:Blood Substitutes; D004579:Electron Transport; D006454:Hemoglobins / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D008055:Lipids; D014443:Tyrosine
keywords: hemoglobin; oxidative stress; reactive oxygen species