Reconstitution of native human hemoglobin from separated globin chains and alloplex intermediates.

Abstract

A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free a-and -globin chains (a', ft') and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the a losteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at hig dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For a-globin, the yield is about 50% because of incomplete combination with the heme-containing P chain. For -globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.In two previous papers (1, 2), we described the preparation and properties of isolated heme-free human globin chains (a", a°) and of two kinds of reassembly intermediates in which both kinds of chains are present but only half of the heme sites are filled (HFa and HFft molecules). The first paper demonstrated that the separated globin chains near neutral pH have different conformations, a" being substantially more disordered than a", in contrast to the separated heme-containing subunits, which are folded similarly, and in contrast also to the heme-free chains when bound together in dimeric apohemoglobin (a°,B°). Moreover, it was shown that, once separated, the globin chains do not readily recombine at neutral pH and accessible temperatures to form apohemoglobin. The second paper demonstrated that if either chain contains a heme, then the complementary chains do specifically recombine. During such a recombination, the disordered a-globin is refolded to the conformation it possesses in apohemoglobin. The coupled binding and refolding was termed an alloplex interactiont, to emphasizethe extensive refolding induced by a neighboring subunit.In future papers on this subject, the kinetics and mechanism of the alloplex interaction and the equilibrium of the HFa and HFB intermediates will be reported. The present paper shows that human hemoglobin can be reconstituted from separated globin chains and hemin by two different routes and that the reconstituted hemoglobin is similar or identical to native hemoglobin with respect to cooperativity in oxygen binding, the Bohr effect, and response to the allosteric effector 2,3-diphosphoglycerate (P-glycerate).In the course of our work on this problem, we found it necessary to modify several of our previous...

Biophysical Characterization of the α-Globin Binding Protein α-Hemoglobin Stabilizing Protein

“…Analysis by CD spectropolarimetry revealed that the resulting apo-␣-globin retains considerable, but much reduced, ␣-helical structure (ϳ30% ␣-helix) in the absence of the heme group. This result is in agreement with previous studies of apo-hemoglobin and its subunits, which demonstrated that removal of heme causes a greater destabilization of ␣-globin secondary structure than of ␤-globin (24,25). A UV-visible wavelength absorption spectrum indicated that contamination of the apo-␣-globin preparation with heme-containing protein was at levels below 1% (ratio of absorption, A 420 /A 280 ϭ 0.04 (26)).…”

“…In the absence of AHSP, apo-␣-globin cannot be detected by gel filtration chromatography; it appears to interact with the chromatography system in such a way that no discrete protein peak is observed. Notably, apo-␣-globin has previously been shown to form high sedimentation coefficient aggregates resulting in exclusion from gel filtration systems (24). In addition, higher yields of soluble apo-␣-globin were obtained when lyophilized protein was taken up in buffer already containing AHSP.…”

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

“…As noted above, however, likely the exact sequence of events during Hb folding and assembly in vitro can be modulated by external conditions such as solvent, pH, presence of chaperones, etc. [45]. …”

“…is still a matter of debate [28,[42][43][44], and apparently there is more than one viable assembly pathway leading from the isolated subunits to native Hb [45].…”

“…Previous attempts to carry out related experiments were frustrated by very low heterotetramers yields due to extensive aggregation and precipitation [55]. In fact, aggregation is a common problem even for reconstitution schemes employing folded ␣-and ␤-subunits [45].°This°could°imply that Hb folding and self-assembly necessarily requires a chaperone system, and possibly other components of the cellular machinery. However, the experiments described in the current study demonstrate that this is not the case.…”