Expression, Purification, and Characterization of Mammalian and Earthworm Hemoglobins

The frequent shortages, risks of disease transmission, and storage issues associated with donated blood illustrate a significant demand for a red blood cell (RBC) substitute. Such a substitute should be able to effectively transport oxygen throughout the body with minimal side effects. Several hemoglobin-based oxygen carriers (HBOCs) have been developed and clinically tested, but they have all caused severe side effects. The problems associated with these HBOCs may all be attributed to removing hemoglobin from the RBC. Therefore, this work focuses on the use of the extracellular hemoglobin of the earthworm Lumbricus terrestris (LtEc) as a new class of HBOC. Since earthworms lack RBCs, their hemoglobin is freely dissolved in the bloodstream and has already adapted to solve many of the challenges facing modern synthetic HBOCs. It has a lower rate of oxidation, avoids harmful side reactions with nitric oxide (NO), and it is extremely stable. We have developed a novel purification technique to highly purify large amounts of LtEc at costs that are comparable to donated blood. The LtEc product also transports oxygen similarly to human blood. Transfusion of LtEc into hamsters does not elicit the harmful side effects observed with other HBOCs and preliminary studies have not revealed any immune or allergic reactions in vivo. Therefore, this work shows that LtEc might be an effective and safe oxygen carrier that warrants further study and suggests the need for a paradigm shift in the HBOC field from cellular to extracellular hemoglobins.