Taming hemoglobin chemistry—a new hemoglobin-based oxygen carrier engineered with both decreased rates of nitric oxide scavenging and lipid oxidation

Abstract

The clinical utility of hemoglobin-based oxygen carriers (HBOC) is limited by adverse heme oxidative chemistry. A variety of tyrosine residues were inserted on the surface of the γ subunit of recombinant fetal hemoglobin to create novel electron transport pathways. This enhanced the ability of the physiological antioxidant ascorbate to reduce ferryl heme and decrease lipid peroxidation. The γL96Y mutation presented the best profile of oxidative protection unaccompanied by loss of protein stability and function. N-terminal deletions were constructed to facilitate the production of recombinant hemoglobin by fermentation and phenylalanine insertions in the heme pocket to decrease the rate of NO dioxygenation. The resultant mutant (αV1del. αL29F, γG1del. γV67F, γL96Y) significantly decreased NO scavenging and lipid peroxidation in vitro. Unlike native hemoglobin or a recombinant control (αV1del, γG1del), this mutation showed no increase in blood pressure immediately following infusion in a rat model of reperfusion injury, suggesting that it was also able to prevent NO scavenging in vivo. Infusion of the mutant also resulted in no meaningful adverse physiological effects apart from diuresis, and no increase in oxidative stress, as measured by urinary isoprostane levels. Following PEGylation via the Euro-PEG-Hb method to increase vascular retention, this novel protein construct was compared with saline in a severe rat reperfusion injury model (45% blood volume removal for 90 minutes followed by reinfusion to twice the volume of shed blood). Blood pressure and survival were followed for 4 h post-reperfusion. While there was no difference in blood pressure, the PEGylated Hb mutant significantly increased survival. Hemoglobin-based oxygen carriers are modified hemoglobin molecules that can be infused as blood substitutes to replace red blood cell transfusions or as oxygen therapeutics to deliver oxygen to damaged tissues not readily accessible by red cells. However, their clinical use has been limited by adverse side effects caused by free radical production and nitric oxide scavenging by extracellular hemoglobin. The researchers used genetic engineering to insert tyrosine residues into fetal human hemoglobin to decrease radical production and phenylalanine residues to decrease nitric oxide scavenging. The resulting novel hemoglobin was tested in rat models to observe the effects on blood pressure and survival rates. The research offers hope for improved treatment for patients in critical need of blood transfusions or with an otherwise compromised oxygen delivery system, such as in sickle cell disease, stroke or sepsis. This summary was written by the author.