Assembly of Human Hemoglobin (Hb) β- and γ-Globin Chains Expressed in a Cell-free System with α-Globin Chains to Form Hb A and Hb F

Abstract

Rates of in vitro synthesis of radiolabeled ␥ and ␤ chains made in a cell-free transcription/translation system were similar, but expressed globin chains were unstable. The addition of unlabeled ␤ or ␥ chains at the start of chain synthesis generated radiolabeled ␤ 4 or ␥ 2 and ␥ 4 chains, respectively. If unlabeled ␣-globin chains were added at the start of chain synthesis, then approximately equal amounts of radiolabeled ␣␤ or ␣␥ bands were generated. If unlabeled Hb A or Hb F was added to reactions containing radiolabeled ␣␤ or ␣␥ prior to electrophoresis, then radiolabeled Hb A or Hb F tetramers, respectively, were generated. If ␣ chains were added after synthesis of radiolabeled ␥ chains made in the presence of unlabeled ␥ chains, then little radiolabeled ␣␥ formed. In contrast, if ␣ chains were added after synthesis of radiolabeled ␤ chains made in the presence of unlabeled ␤ chains, then radiolabeled ␣ 2 ␤ 2 formed. These findings suggest that ␤ and ␥ chains associate with ␣ chains during or soon after translation. This would prevent the formation of unstable monomers as well as stable ␥ 2 dimers and suggests that ␣ chains may bind to nascent non-␣ chains, acting as folding catalysts to promote functional tetrameric hemoglobin formation in vivo.Assembly in vitro of human Hb 1 subunits (␣ and non-␣ chains) into stable Hb heterotetramers (e.g. ␣ 2 ␤ 2 or ␣ 2 ␥ 2 ) using purified globin chains has been explored and a 3-step mechanism proposed (1-8). The ␣ chains are in monomer/dimer equilibrium-favoring monomers, whereas non-␣ chains are in monomer/tetramer equilibrium-favoring tetramers (9, 10). It is generally assumed that dissociation of these oligomeric subunits into monomers must occur before these two different chains can combine to form ␣␤ or ␣␥ dimers, which then associate to form tetrameric Hb (␣ 2 ␤ 2 or ␣ 2 ␥ 2 ) (11, 12). In addition, the assembly of ␣␤ or ␣␥ dimer was postulated to be the rate-limiting step for assembly in vivo and has been theorized to be governed by electrostatic attractions between ␣-and non-␣ partner subunits (8, 12). Furthermore, from in vitro studies it is known that Hb F formation using purified ␥-and ␣-globin chains is very slow compared with Hb A using purified ␤-and ␣-globin chains (11). In fact, our previous studies showed approximately a 10 5 -fold slower rate of assembly in vitro for Hb F compared with Hb A (11). The slow rate of Hb F formation in vitro is caused by stable ␥ 2 dimer formation and is unlikely to occur in erythroid precursors (11). We also found that even at low concentrations (Ͻ5 M), ␥ chain dimers do not dissociate readily into monomers, resulting in decreased assembly with ␣ chains. Furthermore, our results showed that the assembly of [Ile 116 3 His]␥ chains with ␣ chains was similar to that of ␤ chains, whereas the assembly of [Thr 112 3 Cys]␥ with ␣ chains was similar to wild type ␥ chains (11). These findings indicate that amino acid differences between [Ile 116 ]␥ and [His 116 ]␤ at ␣ 1 ␥ 1 and ␣ 1 ␤ 1 interaction sites, respectively, a...

Insights into Hemoglobin Assembly through in Vivo Mutagenesis of α-Hemoglobin Stabilizing Protein

“…This role is supported by kinetic studies showing that both ferrous and ferric holo-␣ bind ϳ20 times more rapidly to AHSP than to ferrous ␤ subunits (14,25,42). However, ␤ subunits have a 10,000-fold higher affinity for ␣-globin than AHSP due to an extremely small ␣1␤1 dissociation rate constant, which is on the order of 10 Ϫ5 s Ϫ1 (25,65). In comparison, the rate of ␣ dissociation from AHSP is 10 2 -10 4 larger for all the AHSP variants listed in Table 1.…”

Analysis of human α globin gene mutations that impair binding to the α hemoglobin stabilizing protein

“…Approximately 3 ng of 35 S-labeled ␣ globin containing bound CN-hemin was generated in each TNT reaction (ϳ7.5 nM in a 25-L reaction). 18,19 The reaction was incubated with 20 nM unlabeled purified human oxy-␤ globin for 10 minutes at room temperature and then loaded onto precast agarose gels (FR9120 Hemoglobin Test Kit; pH range, 6-8; PerkinElmer Life and Analytical Sciences). Electrophoresis was performed on an LKB 2117 Multiphor at 1500 V for 65 minutes.…”

An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis

“…In vitro TNT was performed using the wheat germ TNT kit (Promega) according to published protocols (68,69). Plasmids containing human α-or β-globin cDNAs (pcDNA3-α, T7 promoter or pcDNA3-β, Sp6 promoter) were added to the TNT mixture with CN-hemin.…”

“…After the reaction, purified human αHb or βHb were added to complex with the newly synthesized radiolabeled globin chain. Additionally, purified unlabeled HbA was added to facilitate the formation and subsequent analysis of radiolabeled HbA tetramers (68,69). HbA assembly was monitored by electrophoresis on Titan IIIH cellulose acetate membranes (Helena Laboratories) according to the manufacturer's instructions.…”