Abstract
Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.
Highlights
The human adult hemoglobin (Hb A) is a tetrameric protein containing two a- and two b-chains, each associated with a heme molecule
It is admitted that normally a-Hb first binds Alpha hemoglobin stabilizing protein (AHSP), this chaperone maintaining the a-Hb in a soluble state until association with the b-hemoglobin chains (b-Hb) chain partner to form a2b2 tetramer [1,2,3]
The amino acid changes, deletions or insertions of some of these unstable Hbs are located in the G or H helices which are involved in the interaction with protein partners, AHSP or b-Hb
Summary
The human adult hemoglobin (Hb A) is a tetrameric protein containing two a- and two b-chains, each associated with a heme molecule. AHSP is a 102 amino-acid protein, synthesized at a high concentration in the erythroid precursors, which forms a stable soluble heterodimer with a-Hb [1]. This a chaperone recognizes the G and H helices of a-Hb, a region which binds to the b-Hb subunit [5,6]. The presence of AHSP facilitates reduction of the oxidized aHb chain which could trigger its release from AHSP toward its final Hb b-chain partner producing functional ferrous Hbtetramers [8]. These different data emphasize the important role of AHSP in Hb biosynthesis
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