Mechanism of Human Apohemoglobin Unfolding

Abstract

Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the αβ interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the αγ interface is more resistant to dissociation than the αβ interface.

Novel manufacturing method for producing apohemoglobin and its biophysical properties

“…This may be due to the lower thermal stability of these species leading to their precipitation when heated to 22°C. It is noteworthy that TFF‐apoHb shows much higher stability at 22°C than apoHb produced via other methodologies in the literature, which are highly unstable at 22°C leading to large precipitate formation at temperatures above 10°C (Samuel et al, ). Furthermore, for both the concentrated and unconcentrated samples, a higher fraction of heme‐bound TFF‐apoHb species was lost compared with the loss of heme‐free TFF‐apoHb.…”

“…As shown in Figure a, there was a minimal amount of apoHb tetramers even for a sample stored at 4°C for more than a year. Previous studies identified these tetrameric species as disulfide‐bonded tetramers that were not in equilibrium given that there were composed of two distinct peaks and there was no effect of sample loading onto column (Samuel et al, ). However, the relative amount of TFF‐apoHb tetramers observed in the elution chromatogram was dependent on sample loading onto the column (Figure b).…”

“…Not only was there minimal amounts of tetrameric species in our TFF‐apoHb preparations, but there were no higher orders species detected under any of the tested storage conditions. Previous SEC‐HPLC of MEK produced apoHb showed a large percentage of tetrameric species and other higher order aggregates which required use of a reducing agent (such as DTT) during preparation (Samuel et al, ). Furthermore, the tetrameric species of TFF‐apoHb were found to be dissociated at low concentrations, indicating that these species were not formed via irreversible disulfide bonds.…”

“…Because of the higher order complexity of Hb, which is due to its tetrameric structure, myoglobin (Mb) has been used as the primary model to study the unfolding of holoproteins. The complete mechanism for Hb assembly from its’ globin and heme components has not yet been fully defined, but various insights indicate that the Hb unfolding occurs via partial unfolding of the holoprotein followed by heme extraction via protonation of the proximal histidine (HisF8) or water molecules competing with the Fe–HisF8 bond (Boys & Konermann, ; Das et al, ; Liong et al, ; Peterson et al, ; Samuel et al, ). More specifically, the current evidence suggests that the unfolding of the F‐helix is required to allow solvent accessibility into the proximal heme‐binding pocket (Peterson et al, ).…”

“…ApoHb can react with heme to form reconstituted Hb (rHb), which shows virtually no difference in biophysical properties compared to native Hb (Antonini et al, 1964;Ascoli, Rossi Fanelli, & Antonini, 1981a;Rossi-Fanelli, Antonini, & Caputo, 1959). The heme-binding ability and heme-induced structural changes of apoHb make it an interesting precursor for studies investigating in vivo Hb synthesis and recombinant Hb production (Graves, Henderson, Horstman, Solomon, & Olson, 2008;Rose, Thompson, Light, & Olson, 1985;Samuel, Ou, Phillips, & Olson, 2017;Varnado et al, 2013a;Weickert, Pagratis, Curry, & Blackmore, 1997).…”