Poly(ethylene glycol) Surface-Conjugated Apohemoglobin as a Synthetic Heme Scavenger

Abstract

Apohemoglobin (apoHb) contains vacant hydrophobic heme-binding pockets that can bind to a variety of hydrophobic molecules. Thus, apoHb is a promising protein for drug delivery, bioimaging, and heme scavenging. Unfortunately, apoHb has a short half-life and precipitates at physiological temperature. In this study, apoHb was surface-conjugated with poly(ethylene glycol) (PEG) to improve the therapeutic potential of apoHb. The scalable PEGylation process had >95% protein yield with ∼10 to 12 PEGs attached to each apoHb αβ dimer. The resulting PEG-apoHb had an average molecular weight of ∼80 to 90 kDa and a hydrodynamic diameter of 11 nm. PEG-apoHb maintained high heme-binding affinity and 30−40% of the hemebinding activity. Moreover, heme-bound and heme-free PEG-apoHb bound to haptoglobin, enabling PEG-apoHb to potentially target CD163+ macrophages and monocytes. Finally, PEG-apoHb was stable at physiological temperature with minimal precipitation. In summary, the in vitro results shown demonstrate that PEG-apoHb could be an effective in vivo heme scavenger during states of hemolysis.

Tangential flow filtration facilitated fractionation and PEGylation of low and high‐molecular weight polymerized hemoglobins and their biophysical properties

“…PEGylation was performed as previously described (Pires et al, 2020). Briefly, the PolybHb fraction was thawed at 4°C and diluted to approximately 5 mg/ml in PBS.…”

Purification and analysis of a protein cocktail capable of scavenging cell‐free hemoglobin, heme, and iron

“…As natural mechanisms to remove Hb are limited, cell‐free Hb elicits oxidative damage to proteins, lipids, nucleic acids, and other macromolecules 3 . Free heme is involved in various vascular pathologies and, due to its hydrophobicity, avidly binds to lipid surfaces or plasma proteins, mediating their oxidation 2,4 . Lastly, iron also causes oxidative damage through generation of reactive oxygen species 5–7 .…”

Enhanced Photodynamic Therapy Using the Apohemoglobin-Haptoglobin Complex as a Carrier of Aluminum Phthalocyanine

“…A promising new candidate for transport and delivery of PS molecules is apohemoglobin (apoHb), the apoprotein obtained via removal of the heme groups from hemoglobin (Hb). , The vacant heme-binding pocket of apoHb facilitates binding of hydrophobic molecules, creating an aqueous drug delivery vehicle for hydrophobic molecules such as PS . Therefore, binding of PS to the heme-binding pocket of apoHb should ensure that PS remains in a monomeric state, similar to the role of apoHb in maintaining heme in a nonaggregated form.…”

“…However, targeting CD163+ macrophages relies on Hp binding in vivo , and apoHb is unstable at physiological temperature . Fortunately, apoHb is stabilized when bound to Hp, enabling potential biomedical applications .…”

“…16,17 The vacant heme-binding pocket of apoHb facilitates binding of hydrophobic molecules, creating an aqueous drug delivery vehicle for hydrophobic molecules such as PS. 18 Therefore, binding of PS to the heme-binding pocket of apoHb should ensure that PS remains in a monomeric state, similar to the role of apoHb in maintaining heme in a nonaggregated form. Moreover, the apoprotein clears via the same pathway as cell-free Hb in which CD163+ macrophages and monocytes uptake the protein after haptoglobin (Hp) binding.…”