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

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 ? subunit (?F41Y) 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 ? and ? subunits of Hb to determine if this effect of ?F41Y 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 (?T84Y, ?L91Y and ?F85Y) 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??M), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (?T84Y) 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 ?T84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, ?T84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. ?T84Y 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.

SUBMITTER: Cooper CE 

PROVIDER: S-EPMC6597946 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature