Interaction of haptoglobin with hemoglobin octamers based on the mutation <i>α</i>Asn78Cys or <i>β</i>Gly83Cys

Abstract

Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min; however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours; a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αβ dimers may be of importance for the binding to haptoglobin.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

“…We and others have previously generated B10Leu mutants and shown that replacement of the ␣B10Leu residue (␣Leu-29) with phenylalanine increases the oxygen affinity and decreases the autoxidation rate of the macromolecule (6,13,15,31,32). We have also produced an octameric hemoglobin with wild type biological activities by substituting the ␣-subunit Asn-78 residue with cysteine (33). Combination of these mutations produced octameric macromolecules with unique properties that were utilized as resuscitation solutions in mice suffering traumatic brain injury combined with hemorrhagic shock.…”

Interaction of recombinant octameric hemoglobin with endothelial cells

“…In parallel to our work, another recombinant octameric Hb has been obtained by introducing analogous substitution on the a-chains at aAsn78(EF7). This rHb aN78C also forms a stable octameric structure with a similar oxygen affinity as that of Hb A [18]. The mutation aN78C has been also associated with the aL29F or with aL29W mutation leading to an increase or decrease in oxygen affinity, respectively [38].…”

“…1) with a slightly increased oxygen affinity relative to a Hb A solution [15]. We have previously reported that this octameric rHbbG83C is resistant toward potential disulfide reducing agents present in fresh human plasma and does not interact rapidly with haptoglobin, a glycoprotein that binds Hb (ab) dimers as part of the elimination of Hb from blood circulation [15][16][17][18]. In contrast to other molecules produced by recombinant technology [19], rHbbG83 C exhibits a homogeneous size and does not dissociate into small molecular species at low concentration [15].…”