Hemoglobin 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 scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the alpha (Y42) subunit of hemoglobin, but absent in the homologous position in the beta subunit (F41). We therefore replaced this residue with a tyrosine (bF41Y, Hb Mequon). The bF41Y 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) hemoglobin to liposomes. However, bF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally heme damage in the beta subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid (HPODE) was 5-fold slower in bF41Y. Nitric oxide bioavailability was enhanced in bF41Y 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 beta subunit, but unchanged in the alpha subunit. We conclude that the addition of a redox-active tyrosine mutation in hemoglobin 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 an HBOC product.
- oxidative stress
- reactive oxygen species
- ©2016 The Author(s)
This is an Accepted Manuscript; not the final Version of Record. You are encouraged to use the final Version of Record that, when published, will replace this manuscript and be freely available under a Creative Commons licence.