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.
Highlights
There is a need for a sustainable, long-lasting, artificial blood substitute due to increased demand, the risk from new blood-borne diseases, difficulty in matching rare blood types, and religious aversion to blood products [1]
E. coli BL21 (DE3) cells transformed with pETDuet plasmid containing genes for the adult or fetal wild-type Hb were grown in 2 l Erlenmeyer flasks containing 1.4 l growth medium at 37◦C, and agitated at 180 rpm until an OD600 ∼2 was achieved
We did not observe any statistical differences in the yields between recombinant adult Hb (rHbA) and recombinant fetal Hb (rHbF) (Table 1)
Summary
There is a need for a sustainable, long-lasting, artificial blood substitute due to increased demand, the risk from new blood-borne diseases, difficulty in matching rare blood types, and religious aversion to blood products [1]. There is a growing realization that at a lower dose these products can facilitate oxygen delivery to compromised tissue even in the presence of adequate numbers of red blood cells In these cases, the products are acting not as providers of oxygen, but as facilitators of oxygen transferred from the red cells to the hypoxic tissue. Hb is purified from animal erythrocytes (including human), removing or reducing the stroma content; this Hb is treated to increase its size and vascular retention rate by cross-linking, polymerization, conjugation, or encapsulation [4] These approaches may increase protein stability, they have the potential to increase product heterogeneity and to alter reactivity [5]
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