Polymerization of human hemoglobin using the crosslinker 1,11‐bis(maleimido)triethylene glycol for use as an oxygen carrier

Abstract

Vasoconstriction and systemic hypertension are the main side effects associated with transfusion of current commercial polymerized hemoglobins (PolyHbs). It is hypothesized that the presence of free tetrameric hemoglobin (Hb) in the PolyHb solution is the root cause of these side effects. Therefore, increasing the size of PolyHbs and reducing the amount of free Hb in solution should dually reduce the extent of vasoconstriction and systemic hypertension. However, all current commercial PolyHb preparations have a small fraction of free Hb in solution. Hence, there is an urgent need to develop novel chemical strategies to synthesize large PolyHb molecules with a higher degree of polymerization without free Hb in solution. In this study, a Hb-based oxygen carrier was synthesized by polymerizing human Hb using a dimaleimide poly(ethylene glycol) derivative (1,11-bis(maleimido)triethylene glycol). The resultant PolyHb has a weight-averaged molecular weight of 1.49 ± 0.62 MDa, O₂ affinity (P₅₀) of 2.75 ± 0.55 mm Hg, and Hill coefficient (n) of 0.97 ± 0.07. Light scattering analysis of the PolyHb dispersion confirmed the absence of free Hb monomers, dimers, and tetramers in solution. This work is significant, as it should enable future engineering of nonvasoactive PolyHbs with potential applications in transfusion medicine.

Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters

“…The high O 2 -affinity variant may be advantageous as a transfusion-related medication. Another possible reason of the side-effect by Hb derivatives is overoxygenation [ 33 – 36 ]. The small Hb injected into the bloodstream facilitates the diffusive O 2 transport to the blood vessel wall, initiating overoxygenation of the surrounding tissues.…”

“…These physiological responses engender vasoconstriction. The high O 2 -affinity (low P 50 ) avoids early O 2 offloading on the arterial vessels and may be beneficial for O 2 release in the capillaries [ 34 – 36 ].…”

Facile Method of Constructing Polyproteins for Single-Molecule Force Spectroscopy Studies

“…Maleimide–thiol coupling-based bioconjugation methods have been used extensively in biochemistry because of the rapid and specific reaction of maleimide with sulfhydryl groups under mild experimental conditions. , Here we employ this maleimide–thiol coupling chemistry to develop an efficient method of constructing polyproteins for single-molecule AFM studies. The basic experimental design is schematically depicted in Figure.…”

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

“…The residues mediating the distinctive self-association remain unknown. P. palpebrosus Hb lacks the only surface Cys residue known in humans (␤93Cys) (98), and ␤49Cys, which causes polymerization through intermolecular disulfide bridges in a teleost fish (25), but has six Cys residues per dimer (at ␣18, ␣19, ␣104, ␣130, ␤23, and ␤100). Available data (36) show that apart from the highly conserved ␣104Cys, all crocodilian Hbs (as with chicken Hb) have Cys at ␣130 and ␤23 (Table 5).…”