Comparison of the oxidative reactivity of recombinant fetal and adult human hemoglobin: implications for the design of hemoglobin-based oxygen carriers

Abstract

Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been engineered to replace or augment the oxygen carrying capacity of erythrocytes. However, clinical results have generally been disappointing, in part due to the intrinsic oxidative toxicity of Hb. The most common HBOC starting material is adult human or bovine Hb. However, it has been suggested that fetal Hb may offer advantages due to decreased oxidative reactivity. Large-scale manufacturing of HBOC will likely and ultimately require recombinant sources of human proteins. We, therefore, directly compared the functional properties and oxidative reactivity of recombinant fetal (rHbF) and recombinant adult (rHbA) Hb. rHbA and rHbF produced similar yields of purified functional protein. No differences were seen in the two proteins in: autoxidation rate; the rate of hydrogen peroxide reaction; NO scavenging dioxygenase activity; and the NO producing nitrite reductase activity. The rHbF protein was: less damaged by low levels of hydrogen peroxide; less damaging when added to human umbilical vein endothelial cells (HUVEC) in the ferric form; and had a slower rate of intrinsic heme loss. The rHbA protein was: more readily reducible by plasma antioxidants such as ascorbate in both the reactive ferryl and ferric states; less readily damaged by lipid peroxides; and less damaging to phosphatidylcholine liposomes. In conclusion in terms of oxidative reactivity, there are advantages and disadvantages to the use of rHbA or rHbF as the basis for an effective HBOC.

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin

“…All recombinant proteins successfully bound and released oxygen, and the cysteine mutations had no particular influence on the oxygen binding property of HbF ( Table 1 ). These data are largely in agreement with a recent study comparing HbA and HbF [30] .…”

Allogeneic cord blood transfusions prevent fetal haemoglobin depletion in preterm neonates. Results of the CB‐TrIP study

“…[27][28][29] This protective mechanism neutralises peroxides and removes radicals through a peroxide radical self-termination reaction that generates more stable molecules. 29 This function is critical for the progressive adaptation to the oxygen-rich post-natal environment, when enzymatic antioxidant systems are still immature. Notably, fetal Hb has higher pseudo-peroxidase activity than the adult form, with significantly faster reconversion of the reactive ferryl-heme.…”

“…Notably, fetal Hb has higher pseudo-peroxidase activity than the adult form, with significantly faster reconversion of the reactive ferryl-heme. 28,29 Moreover, it more efficiently maintains the tetrameric integrity of haemoglobin, thus preventing the release of toxic free heme groups. 27 Finally, fetal haemoglobin has greater ability to generate unbound nitric oxide via the oxidative denitrosylation, explaining its beneficial effect in preventing vasocclusive crises in sickle cell disease.…”

Artificial Oxygen Carriers—Past, Present, and Future—a Review of the Most Innovative and Clinically Relevant Concepts

“…In contrast, disposability of bovine blood is nearly unlimited. Instead of using Escherichia coli or Saccharomyces cerevisiae, there are now efforts to obtain recombinant Hgb (Varnado et al, 2013) from plants, e.g., from Nicotiana benthamiana (Eriksson and Bülow, 2017), and furthermore researchers are focusing more and more an fetal Hgb, which is more stable than adult Hgb (Ratanasopa et al, 2016;Simons et al, 2018). Free, unprocessed mammal Hgb that is not encased with any type of membrane is associated with typical problems (Table 3) (Cardenas et al, 2017).…”