Towards the Development of Hemerythrin-Based Blood Substitutes

Abstract

Hemerythrin is proposed as an alternative to hemoglobin-based blood substitutes. In contrast to hemoglobin, hemerythrin exhibits negligible reactivity towards oxidative and nitrosative stress agents (peroxide, nitric oxide, nitrite). Protocols for attachment of polyethylene glycol and glutaraldehyde cross-linking of Hr are described. These derivatizations appear to have favorable effects on O(2) affinity and autoxidation rates for use in blood substitutes. Based on lessons learned from hemoglobin-based blood substitutes, these derivatizations should also help limit extravasation and antigenicity of a hemerythrin-based blood substitute.

Towards hemerythrin-based blood substitutes: Comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells

“…Native bovine hemoglobin (Hb) (Antonini and Brunori 1971) and recombinant hemerythrin (Hr) (Mot et al 2010) were obtained as previously described and manipulated in phosphate buffer saline (PBS) unless otherwise mentioned. Hemoglobin and hemerythrin concentrations in text are given per heme rather than per tetramer.…”

“…Hemoglobin and hemerythrin concentrations in text are given per heme rather than per tetramer. Protocols for obtaining glutaraldehyde-polymerized-Hr (poly-Hr), polyethylene glycol-derivatized Hr (PEG-Hr) and glutaraldehydederivatized Hb were previously described in detail (Deac et al 2009;Mot et al 2010). PBS, RPMI-1640, Hank's media, FCS, glutamine, penicillin-streptomycin solution, Histopaque 1077 and Trypan blue solution were acquired from Sigma.…”

“…Hr was shown to avoid most of the stress-related sidereactions cited above for hemoglobin, showing much slower, if any, reactions with hydrogen peroxide, nitric oxide and nitrite (Kryatov et al 2005;Mot et al 2010). Furthermore, we described protocols for chemical modification of Hr with polyethylene glycol (PEG) and glutaraldehyde, pointing out that the effects of these chemical modifications on O 2 affinity, autoxidation rate and molecular weight of Hr appear to be favourable for blood substitute applications.…”

Protein-Based Blood Substitutes: Recent Attempts at Controlling Pro-Oxidant Reactivity with and Beyond Hemoglobin

“…Hemerythrin (Hr) was shown to avoid reactivity towards hydrogen peroxide, nitric oxide and nitrite. Another advantage is the higher molecular weight than Hb (108 kDa vs 64 kDa), which should lead to lower levels of extravasation or elimination through the kidney [42,43]. Until now, glutaraldehyde-polymerized hemerythrin and polyethylene glycol-derivatized hemerythrin were successfully obtained.…”

“…Until now, glutaraldehyde-polymerized hemerythrin and polyethylene glycol-derivatized hemerythrin were successfully obtained. The effects of these chemical modifications on molecular weight, autooxidation rate and oxygen affinity appear to be favorable for blood substitute applications [42,43,44,45]. …”