Effect of NH₂-terminal acetylation on the oxygenation properties of vertebrate haemoglobin

Abstract

In vertebrate haemoglobin (Hb), the NH₂-terminal residues of the α- and β-chain subunits are thought to play an important role in the allosteric binding of protons (Bohr effect), CO₂ (as carbamino derivatives), chloride ions, and organic phosphates. Accordingly, acetylation of the α- and/or β-chain NH₂-termini may have significant effects on the oxygenation properties of Hb. Here we investigate the effect of NH₂-terminal acetylation by using a newly developed expression plasmid system that enables us to compare recombinantly expressed Hbs that are structurally identical except for the presence or absence of NH₂-terminal acetyl groups. Experiments with native and recombinant Hbs of representative vertebrates reveal that NH₂-terminal acetylation does not impair the Bohr effect, nor does it significantly diminish responsiveness to allosteric cofactors, such as chloride ions or organic phosphates. These results suggest that observed variation in the oxygenation properties of vertebrate Hbs is principally explained by amino acid divergence in the constituent globin chains rather than post-translational modifications of the globin chain NH₂-termini.

Introduction

Haemoglobin (Hb) is a heterotetramer comprising two identical α- and β-chain subunits, each of which contains a heme group that reversibly binds a single O₂ molecule. Hb is one of the most extensively studied proteins with regard to structure-function relationships, and has been used as a model system to investigate mechanisms of allosteric regulation [1-5]. Although the experimental study of native and recombinant Hb mutants has provided a wealth of information about how particular amino acid replacements affect O₂-binding properties [6-9] the functional consequences of post-translational modifications such as NH₂-terminal acetylation are less well characterized. Many vertebrate taxa express Hb isoforms with NH₂-terminal acetylation [10-13], suggesting that such post-translational modifications could contribute to the wide variation in structural and functional properties among species.

NH₂-terminal acetylation is a common post-translation modification of Hb and other eukaryotic proteins. In mammals, at least six isoforms of Nt-acetyltransferases (NATs) have been identified and characterized [14]. The NatA complex has a catalytic subunit Naa10 that acetylates the nascent polypeptide, and Naa15 is an auxiliary ribosomal subunit that helps anchor the complex [15]. The NatA complex binds to the ribosome and acetylates the smaller, polar amino acids (alanine, serine, threonine, cysteine, valine, and glycine) at the NH₂-terminus of the nascent polypeptide, and this process is performed simultaneously with the cleavage of the initiator methionine [16-18].

The NH₂-termini of the α- and β-type subunits of vertebrate Hb are thought to play an important role in the allosteric binding of protons, CO₂, Cl⁻ ions, and organic phosphates [19-22]. The NH₂-termini have pK_a values within the physiological range, and can therefore contribute to the allosteric regulation of Hb-O₂ affinity via oxygenation-linked binding of protons and other effector molecules. A number of missense mutations at the NH₂-termini of human α- and β-chain have been reported, some of which affect post-translational modifications such as cleavage of the initiator methionine and NH₂-terminal acetylation. Some amino acid replacements of both α1(NA1) and β1(NA1) permit normal cleavage of the initiator methionine, followed by acetylation of the new NA1 residue if it provides a suitable substrate for N-acetyltransferases, as in the case with human Hb mutant Antananarivo (α1(NA1)Val → ac-Gly) [23], Hb Lyon-Bron (α1(NA1)Val → ac-Ala) [24], and Hb Raleigh (β1(NA1)Val → ac-Ala) [25]. In several prenatally expressed human Hb isoforms, the NH₂-termini of the ζ- and γ chains (Ser ζ1(NA1) and Gly γ1(NA1), respectively) are partly acetylated, which affects the function of embryonic Hbs Gower I (ζ₂ε₂) and Portland (ζ₂γ₂) and fetal Hb, HbF (α₂γ₂) [26]. Similar to the Hb Raleigh mutant and the prenatally expressed Hb Portland and HbF isoforms, the adult Hbs of a number of mammals (e.g. feloid carnivores such as cats, hyenas, and civets) and crocodilians also have an acetylated alanine at β1(NA1) [3,10,13,27]. Likewise, the α-chain NH₂-termini are acetylated in the adult Hbs of numerous cartilaginous fishes, teleost fishes, and amphibians [28-35]. Other replacements of α1(NA1) and β1(NA1) result in the retention and complete acetylation of the initiator methionine, such as the human Hb mutant Thionville (α1(NA1)Val → Glu) [36]. Since some of these rare NA1 mutations in adult human Hb are wild-type in prenatally expressed Hb isoforms and adult isoforms of other vertebrates, information about the functional consequences of NH₂-terminal modifications can provide insight into possible sources of variation in O₂-binding properties.

The functional effects of NH₂-terminal acetylation in Hb have not been experimentally characterized due to the difficulty of isolating the effect of post-translational modifications in comparisons between otherwise identical proteins. Perhaps reflecting these technical difficulties, there are conflicting reports about how acetylation and other chemical modifications of NH₂-terminal groups influence the Bohr effect [22,37-45] and the allosteric binding of anions in the central cavity [25,46]. Here we investigate the functional consequences of NH₂-terminal acetylation by using a newly developed expression plasmid system that enables us to compare recombinantly expressed Hbs that are structurally identical except for the presence or absence of NH₂-terminal acetyl groups.

We had previously designed a plasmid system for expressing recombinant Hb (rHb) in E. coli that cleaves the NH₂-terminal methionine, a post-translational modification that does not occur in prokaryotic cells [47,48]. In the present study, we co-expressed globin genes with a pNatA plasmid that contains a cassette for NatA complex genes from yeast [49]. This additional plasmid enables us to produce acetylated NH₂-termini for the α- and β-chains [50], and therefore enables us to isolate the effect of this specific post-translational modification. For these experiments, we used native and recombinant Hbs of two representative vertebrates: human and American alligator (Alligator mississippiensis). The motivation for investigating human Hb is that structure-function relationships are extremely well-characterized [1,3,8,9], so we have a solid framework for interpreting experimental results. The motivation for investigating alligator Hb is that the NH₂-termini of α- and β-globin chains of crocodilians are naturally acetylated [13,27,51,52]. Moreover, adult Hbs of alligator and other crocodilians exhibit unusual allosteric properties relative to those of other vertebrates [13,27,51], so the naturally occurring variation in NH₂-terminal acetylation could have distinct functional effects on Hb-O₂ affinity that are not shared with other vertebrates.

Materials and methods

Expression plasmid construction

We synthesized the cassette for human α- and β-globin genes (P69905, P68871) and for the American alligator α- and β-globin genes (MN905608, MN905621) (GeneArt Gene Synthesis, Thermo Fishers scientific). Both globin cassettes were optimized according to E. coli codon preferences. We subcloned the globin cassette into the pGM vector using NcoI and SacI restriction enzyme [47,50] and we experimentally confirmed the proper coding sequences. We expressed and purified acetylated and non-acetylated versions of rHbs of both American alligator (AAHb) and human (HHb). We used following combinations of subcloning the plasmids: (1) pGMAAHb + pMAP, (2) pGMAAHb + pMAP + pNat, (3) pGMHHb + pMAP and (4) pGMHHb + pMAP + pNat (Figure 1A-C). Our current protocol allows us to co-express all three pGMHHb/pGMAAHb, pMAP, and pNatA plasmids together in the JM109(DE3) cells (Figure 1D,E). The pNatA plasmid (pACYCduet-naa10-naa15) was kindly provided by Dan Mulvihill (Addgene plasmid # 72928; http://n2t.net/addgene:72928; RRID: Addgene _72928) [49].

Figure 1. Vector maps for the expression plasmids and their co-expression in E. coli host cells.

(A) The pGM expression plasmid contains the genes for ampicillin resistance and α- and β-globin cloned tandemly with a copy of the MAP gene. (B) The pMAP expression plasmid contains the kanamycin resistance gene and an additional copy of the MAP gene [47]. (C) The pNatA plasmid contains the gene that confers chloramphenicol resistance and the naa10 and naa15 genes of the NatA complex [49,50]. (D) Schematic diagram showing the co-expression of pGM + pMAP plasmids in E. coli host cells. (E) Co-expression of pGM + pMAP + pNat plasmids, with a depiction of MAP and pNatA enzyme complex during translation. The pNatA complex facilitates acetylation of the NH₂-termini of alanine, valine, serine, glycine, cystine, and threonine.

Large-scale rHb expression

The pGMHHb/pGMAAHb, pMAP, and pNatA plasmids contained genes for ampicillin, kanamycin, and chloramphenicol resistance, respectively (Figure 1A-C). We can select the co-transformed JM109(DE3) colonies through double and triple antibiotics on an LB agar plate. A single colony was picked and inoculated in 100 ml of LB medium in an orbital shaker at 37°C overnight at 200 rpm. A 10 ml inoculum from the overnight startup culture was added to 300 ml of TB medium of the final volume of 50 μg/ml ampicillin, kanamycin, and chloramphenicol (if needed) in a 1000 ml Baffled Erlenmeyer Flask. A batch of six flasks containing a total of 2 L of TB medium was used, and the culture was grown at 37°C in an orbital shaker at 200 rpm until the absorbance reached 0.6–0.8 at 600 nm. The Hb expression was induced with 0.5 mM IPTG and also supplemented with hemin (50 μg/ml), sodium hydrosulfite (50 mg/L), and glucose (20 g/L). Following induction, the cells were grown at 28°C for 16 h in an orbital shaker at 200 rpm.

Purification of rHb

The overnight culture was saturated with CO for 15 min, and the cells were pelleted and stored at −80°C. The pellets were resuspended with Tris lysis buffer (50 mM Tris, 0.5 mM DDT, 1 mM EDTA). Lysozyme enzyme (1 mg/g of cells) was added for proper lysis before sonication. Polyethyleneimine solution was added to the crude lysate to a final concentration of 0.5 to 1% to precipitate the bacterial nucleic acids. The crude lysate was centrifuged for 15,000g for 45 min at 4°C and the clarified supernatants were dialyzed overnight against the anion-exchange buffer for chromatography. We performed a two-step ion-exchange chromatography using ÄKTA start protein purification system (GE Healthcare Life sciences). We used the Q-Sepharose column (HiTrap QHP, 5 ml, 17-5159-01; GE Healthcare) pre-equilibrated with Tris buffer (20 mM Tris with 0.5 mM EDTA, pH 8.8) and CAPS buffer (20 mM CAPS with 0.5 mM EDTA, pH 9.3) to purify human and alligator rHbs, respectively. The rHb was eluted using a linear gradient of 0–1.0 M NaCl with an appropriate buffer and pH. The eluted sample was desalted and dialyzed overnight against the second column buffer. We used prepackaged SP-Sepharose columns (HiTrap SPHP, 5 ml, 17-516101; GE Healthcare) equilibrated with HEPES buffer (20 mM HEPES with 0.5 mM EDTA, pH 7.0) and phosphate buffer (50 mM Phosphate with 0.5 mM EDTA, pH 6.8) for human and alligator rHbs respectively. The rHb sample was eluted with a linear gradient of 0–1.0 M NaCl with the respective buffer and pH. The purified rHb samples were analyzed using 4–20% SDS–polyacrylamide gel electrophoresis. We carried out in vitro measurements of O₂-binding properties of the native Hb and rHb samples in the absence and presence of allosteric cofactors and of 1% CO₂.

O₂ equilibrium curves

O₂-binding properties of purified native Hbs and recombinantly expressed Hbs were measured using the same protocol. For the analysis of native Hbs, we added 100 μl of whole blood to a 5-fold greater volume of HEPES buffer (10 mM HEPES, 0.5 mM EDTA, pH 7.4) and incubated the sample on ice for 30 min to lyse the red blood cells. Human blood was obtained from a volunteer and alligator blood was obtained from an adult specimen at the Crocodile Zoo (Eskilstrup, Falster, Denmark) as described previously [13]. Alligators express a single Hb isoform during adult life [13,27], and it is this adult isoform that we examined in experiments on both native and recombinantly expressed Hbs. After adding NaCl to a final concentration of 0.2 M, samples were centrifuged at 20,000g for 10 min to remove cell debris. Native and recombinant Hb samples were desalted by passing samples through a PD-10 desalting column (GE Healthcare) equilibrated with 0.01 M HEPES, 0.5 mM EDTA, pH 7.4, and concentrated using Amicon Ultra-4 Centrifugal Filter Units (Millipore).

O₂-equilibrium curves for Hb solutions (0.1 mM Hb tetramer in 100 mM HEPES, 0.5 mM EDTA buffer) were measured at 37°C (human) or 25°C (alligator) using a Blood Oxygen Binding System (Loligo Systems). The Blood Oxygen Binding System is a gas diffusion chamber with built-in spectrophotometer coupled to a precision gas mixer which measures O₂-affinity of whole-blood or Hb solutions by monitoring spectral absorbance in response to changes in the partial pressure of O₂ (PO₂). Each Hb solution was sequentially equilibrated with 3–5 different PO₂s at saturation levels between 30 to 70%, while absorbance was continually monitored at 430 nm (deoxy peak) and 421 nm (oxy/deoxy isosbestic point). Hb-O₂ saturation was estimated at each equilibrium step by comparing the absorbance at 430 nm to fully oxygenated and deoxygenated baselines. Estimates of the PO₂ at half-saturation (P₅₀) and the cooperativity coefficient (n₅₀) were then estimated from linear Hill plots (log[fractional saturation/[1 – fractional saturation]] vs. logPO₂). O₂-equilibrium curves were measured in the absence (stripped) and presence of Cl⁻ ions (0.1 M KCl) and organic phosphates (0.2 mM 2,3-diphosphoglycerate [DPG] for human Hb and 0.2 mM adenosine triphosphate [ATP] for alligator Hb). O₂-equilibrium curves were also measured at three different pH levels, where the pH of working solutions was adjusted with NaOH to 7.2, 7.4 or 7.6 and were measured with an Orion Star A211 pH meter and an Orion™ PerpHecT™ ROSS™ Combination pH Micro Electrode. Linear regressions were fit to plots of logP₅₀ vs. pH, and the resulting equation was used to estimate P₅₀ values at pH 7.40 (±SE of the regression coefficient). The n₅₀ values are presented as mean ± SE of three measurements.

CO₂ effect

The CO₂ sensitivity for the individual rHbs was tested in the HEPES buffer (0.1 M HEPES, and 0.5 mM EDTA, pH 7.2 at 25°C). The samples were dialyzed and concentrated to 0.3 mM heme concentration. The rHb samples were measured for CO₂ sensitivity using a modified diffusion chamber technique described elsewhere [13,53-55] at a constant PO₂ corresponding to the P₅₀. After equilibrating the samples to the PO₂ corresponding to 50% O₂ saturation (P₅₀) in the chamber, 1% CO₂ was added to the gas mixture while maintaining the PO₂ constant, and the decrease in O₂ saturation was recorded, as described earlier [13]. We measured the CO₂ effect in the acetylated and unacetylated versions for alligator and human rHbs. We also used human HbA (Sigma–Aldrich) as control.

Mass spectrometry

NH₂-terminal acetylation of the α- and β-chain subunits of alligator and human rHbs was assessed with tandem mass spectrometry (MS/MS). Purified rHbs were separated in a mini-protean precast 4–20% SDS PAGE gel (Bio-Rad, Hercules, CA) and then stained with Coomassie brilliant blue-G overnight. The stained bands were excised and processed for in-gel digestion [56]. The eluted peptides were then analyzed using a Thermo Orbitrap Fusion Lumos Tribrid (Thermo Scientific™) mass spectrometer in data-dependent acquisition mode. The peptides were identified by searching MS/MS data against a customized reference database containing adult American alligator and human globin sequences. Acetylation of α- and β-chain NH₂-termini and the oxidation of methionines were included as variable modifications and the carbamidomethylation of cysteines was set as a fixed modification. The precursor mass tolerance threshold was set as 10 ppm, and the maximum fragment mass error was set at 0.02 Da. Qualitative analysis was performed using PEAKS X software. The significance threshold of the ion score was calculated based on a false discovery rate of ≤1%. We measured the relative percentages of acetylated and unacetylated NH₂-termini for each globin subunits.

Results

NH₂-terminal acetylation

By co-transforming the pNatA plasmid along with our Hb expression plasmids, we successfully expressed and purified human and alligator rHbs with and without NH₂-terminal acetylation in E. coli. In human Hb, valine is the NH₂-terminal residue of both the α- and β-chains (Supplementary Figure S1). In adult-expressed alligator Hb, valine and alanine are the NH₂-terminal residues of the α- and β-chains, respectively [13,27]. Previous MS/MS results for native Hb of alligator revealed that the percentage of NH₂-terminal acetylation was ~35% for the α-chain and ~90% for the β-chain [13]. In the case of the alligator rHb expressed in conjunction with the pNat plasmid, MS/MS results revealed NH₂-terminal acetylation of 80% and 100% for the α- and 6β-chains, respectively. In the case of human rHb expressed in the same system, MS/MS results revealed NH₂-terminal acetylation of 97% and 84% for the α- and β-chains, respectively (Figure 2).

Figure 2. NH₂-terminal acetylation profile for alligator and human rHbs that were synthesized using different plasmid systems.

Histograms depict the percentage of NH₂-terminal acetylation for the α- and β-chain subunits and reveal that rHbs synthesized with the pGM + pMAP + pNatA expression plasmid resulted in successful addition of acetyl groups to the amino termini.

Oxygenation properties

Native and recombinant Hbs of both alligator and human exhibited cooperative O₂-binding, as indicated by n₅₀ values >1.0, and measured O₂-affinities were consistent with results of previous studies (Table 1). The relatively low cooperativity of alligator Hb is consistent with previous experimental studies of native adult-expressed Hbs of alligator and other crocodilians [13,27]. NH₂-terminal acetylation did not produce a detectable change in the intrinsic O₂-affinity of either alligator or human rHb, as revealed by comparisons of stripped P₅₀ values of acetylated and unacetylated rHbs from the same species (Table 1 and Figure 3). Likewise, structurally identical rHbs that only differed with respect to NH₂-terminal acetylation exhibited highly similar sensitivities to Cl⁻ ions and organic phosphates (Figures 3 and 4) and similar sensitivities to changes in pH (Bohr effect), both in the presence and absence of anionic cofactors (Figure 4 and Supplementary Figure S2). The responsiveness of alligator and human rHbs to the addition of CO₂ was measured at a constant pH and PO₂. The alligator rHbs exhibited a pronounced CO₂ effect compared with human rHbs, consistent with the results of previous studies [13]. However, the CO₂ sensitivity of alligator rHb was not significantly affected by NH₂-terminal acetylation (Figure 5).

Table 1.

O₂-affinities and allosteric properties of native and recombinant Hbs of alligator and human

	Native alligator Hb		Unacetylated alligator rHb		Acetylated alligator rHb		Native human Hb		Unacetylated human rHb		Acetylated human rHb
Treatments	P50	n50	P50	n50	P50	n50	P50	n50	P50	n50	P50	n50
Stripped	3.80 ± 0.06	1.70 ± 0.1	3.77 ± 0.27	1.10 ± 0.01	3.54 ± 0.38	1.41 ± 0.17	8.43 ± 0.09	1.69 ± 0.10	9.05 ± 0.74	2.11 ± 0.10	8.95 ± 1.27	1.83 ± 0.04
+KCl	11.82 ± 0.10	1.40 ± 0.01	6.95 ± 0.08	1.27 ± 0.01	6.03 ± 0.20	1.35 ± 0.05	16.90 ± 0.70	2.15 ± 0.09	17.74 ± 0.46	2.15 ± 0.05	16.00 ± 0.06	2.13 ± 0.01
+OP	5.12 ± 0.38	1.55 ± 0.06	3.97 ± 0.22	1.22 ± 0.04	3.52 ± 0.09	1.19 ± 0.01	14.42 ± 0.72	2.21 ± 0.09	10.25 ± 0.58	2.14 ± 0.05	11.20 ± 0.38	2.79 ± 0.26
+KCl +OP	11.24 ± 0.26	1.40 ± 0.01	7.13 ± 0.14	1.25 ± 0.01	5.61 ± 0.09	1.23 ± 0.02	17.25 ± 0.69	2.20 ± 0.22	17.40 ± 0.18	2.02 ± 0.06	15.69 ± 0.12	2.30 ± 0.08
ΔlogP50
KCl – stripped	0.493		0.266		0.231		0.302		0.292		0.252
OP* – stripped	0.129		0.022		−0.002		0.233		0.054		0.097
(KCl + OP*) – stripped	0.471		0.277		0.200		0.311		0.284		0.244

Sensitivities to allosteric effectors are measured as the difference in the log-transformed P₅₀ values in the presence and absence of anions (Cl⁻ and organic phosphates), individually and in combination.

OP* organic phosphate (ATP in experiments on alligator Hb, DPG in experiments on human Hb).

Figure 3. O₂-binding properties of the native and recombinant Hbs of alligator and human.

O₂-affinities (P₅₀) in the absence (stripped) and presence of anionic effectors (Cl⁻ ions and organic phosphates) are shown for native Hbs, unacetylated rHbs (produced using the pGM + pMAP plasmid), and acetylated rHbs (produced using the pGM + pMAP + pNatA plasmid). ATP and DPG were used as physiologically relevant organic phosphates in experiments using alligator and human Hbs, respectively.

Figure 4. Effect of NH₂-terminal acetylation on pH sensitivity (Bohr effect) of alligator and human Hbs in the presence and absence of anions.

(A and B) Effect of Cl⁻ on O₂-affinities of alligator and human Hbs as a function of pH. (C) Effect of ATP on the O₂-affinity of alligator Hb as a function of pH. (D) Effect of DPG on the O₂-affinity of human Hb as a function of pH.

Figure 5. Effect of NH₂-terminal acetylation on CO₂ sensitivity of alligator and human Hbs.

Responsiveness to CO₂ is measured as the change in SO₂ in response to the addition of 1% CO₂ (see Materials and Methods for details).

Discussion

Results of structural studies provide some clues as to why NH₂-terminal acetylation of the α- and β-chains does not produce a detectable effect on Hb-O₂ affinity in the presence of Cl⁻ and organic phosphates. When human Hb is in the low-affinity ‘T’ state, a single Cl⁻ ion binds to the free NH₂-group of Val α1(NA1), jointly coordinated with the β-hydroxyl group of Ser α131(H14) on the same chain, and the guanidinium ion of the C-terminal Arg α141(HC3) on the other α-chain. Experiments with bovine Hb have demonstrated that the binding of a single Cl⁻ ion to the triad of α-chain sites (Val α1(NA1)/Ser α131(H14)/Arg α141(HC3)) helps stabilize the T-state of the Hb tetramer, thereby reducing O₂-affinity [57]. Likewise, Cl⁻ binds to β1(NA1), jointly coordinated with β82(EF6). However, Cl⁻ binding to these β-chain sites does not appear to be oxygenation-linked and therefore does not produce a direct allosteric effect on Hb-O₂ affinity [42,58-61]. The fact that we observe no substantial impairment of Cl⁻ sensitivity in human Hb with NH₂-terminal acetylation suggests the possibility that Ser α131(H14) and Arg α141(HC3) alone are sufficient for the allosteric binding of Cl⁻ in the T-state. The same explanation could apply to our results for alligator rHbs, as Ser α131(H14) and Arg α141(HC3) are also present (Supplementary Figure S1).

A previous study of embryonic Hbs suggested that naturally occurring NH₂-terminal acetylation of Gly γ1 (NA1) in Hb Portland impairs Cl⁻ binding and therefore accounts for its reduced Cl⁻ sensitivity relative to adult HbA [62]. However, Hb Portland is distinguished from HbA by many other substitutions, including two (α138Ser → ζ138Glu, and β143His → γ143Ser) that reduce the net positive charge in the central cavity. Contrary to the hypothesis of Hofmann et al. (1995), our direct comparison of structurally identical adult rHbs with and without NH₂-terminal acetylation revealed no detectable effect on Cl⁻ sensitivity (Table 1 and Figure 3), suggesting that the lower Cl⁻ sensitivity of Hb Portland relative to adult HbA is mainly attributable to the substitutions at ζ138, γ143, or other sites.

In the case of phosphate-binding to human Hb in the T-state, DPG carries four negative charges which allow it to bind between the opposing β1 and β2 chains via charge-charge interactions with the Val β1(NA1) residue of one chain, and with His β2(NA2), Lys82(EF6) and His β43(H21) of both chains. Val β(NA1) is the only charged residue without a symmetry-related interaction with the DPG molecule, suggesting that it may play a less important role than the other charged residues in the central cavity. Consistent with this hypothesis, in vitro experiments with Hb Raleigh demonstrate that blocking the α-amino group of the βNA1 residue does not significantly impair oxygenation-linked DPG-binding [25]. In the case of alligator Hb, molecular dynamics simulations suggest that NH₂-terminal acetylation of the β-chains may alter the stereochemistry of ATP-binding in the central cavity of deoxyHb [13]. However, any such alteration in binding-site configuration is apparently not sufficient to produce a significant change in Hb-O₂ affinity under the experimental conditions that we investigated (Table 1 and Figure 3).

Based on X-ray crystallographic results [63], the alkaline Bohr effect was thought to be partly attributable to an oxygenation-linked increase in the ionization constant of Val α1(NA1). However, subsequent experiments cast doubt on this hypothesis [64], and chemical manipulations revealed no significant shift in the pK value of either Val α1(NA1) or Val β1(NA1) upon deoxygenation [38]. Our results indicate that blocking the NH₂-terminal valines with an acetyl group does not produce any detectable change in the Bohr effect, either in the presence or absence of anions (Supplementary Figure S2) which is consistent with the conclusions of Kaplan et al. [38].

Our conclusions about the effects of NH₂-terminal acetylation are based on comparisons between structurally identical rHbs that only differ with respect to the presence or absence of the NH₂-terminal acetyl groups. We included data for the native Hbs of American alligator and human as controls, and it is worth noting some minor discrepancies between the native and recombinant proteins of the same species. In particular, native alligator Hb exhibited slightly higher sensitivities to ATP and Cl⁻ and human Hb exhibited slightly higher sensitivity to DPG relative to recombinantly expressed versions of the same proteins (Table 1 and Figure 3). The source of these minor differences in anion-sensitivity is unclear, especially since intrinsic O₂-affinities of the native and recombinant Hbs from the same species are essentially identical. Regardless, these minor discrepancies do not affect our main conclusions as the comparisons between rHbs of the same species provided decisive results regarding the functional consequences of NH₂-terminal acetylation.

Conclusion

Comparative studies of native and recombinant Hbs from diverse vertebrate species have identified specific amino acid substitutions that are responsible for evolved changes in the oxygenation properties of Hb [3,54,65-81]. In principle, post-translational modifications such as NH₂-terminal acetylation could represent an additional source of variation in such properties. However, our experiments indicate that this is not the case and suggest that among-species variation in Hb-O₂ affinity, Bohr effect, and responsiveness to anionic cofactors is primarily attributable to genetically based variation in the primary structure of the constituent α- and β-type globin subunits.

Abbreviations

AAHb

American Alligator recombinant haemoglobin

ATP

adenosine triphosphate

DPG

2,3-diphosphoglycerate

EDTA

ethylenediaminetetraacetic acid

Hb

haemoglobin

HHb

human recombinant haemoglobin

MAP

methionine aminopeptidas

NAT

N-terminal acetyltransferase

NH₂-terminal

amino-terminal

PO₂

partial pressure of oxygen

rHb

recombinant haemoglobin

SO₂

oxygen saturation