Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, Alligator mississippiensis

Abstract

In the developing embryos of egg-laying vertebrates, O₂ flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O₂ flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O₂ transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O₂). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O₂ affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O₂ affinity and high CO₂ sensitivity. These results indicate that whole blood O₂ affinity is mainly regulated by ATP in the early embryo and by CO₂ and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

INTRODUCTION

In the developing embryos of egg-laying vertebrates, such as archosaurs, O₂ flux occurs across a fixed surface area of the eggshell and the chorioallantoic membrane (1–3). Egg shell properties may vary among oviparous vertebrates, but once the egg is deposited, shell composition is fixed and immutable. Under this constraint, changes in the gas composition of the nest microenvironment or the gas transport capacity of the developing embryo can influence the phenotype of the hatchling animal. The microenvironment of crocodilian nests can become hypoxic due to nest water saturation that accompanies rainfall or microbial metabolism, resulting in a decrease in the diffusion gradient for O₂ flux across the egg shell membrane to the developing embryo (2, 4, 5). Levels of hypoxia corresponding to 10% O₂ have been documented in the nests of crocodilians and other oviparous reptiles (6, 7). Nest hypoxia can therefore elicit compensatory changes in the cardiovascular system of prehatchling animals to increase O₂ transport capacity and O₂ uptake, or suppression of aerobic metabolism to decrease O₂ consumption (8–10). Specifically related to the cardiovascular system, regulatory changes in the expression of functionally distinct hemoglobin isoforms (isoHbs) during embryonic development could provide a mechanism for adjusting O₂ transport capacity if hypoxic nest conditions are experienced.

Plasticity of the embryonic cardiovascular system in crocodilians has been the focus of several recent investigations. The American alligator (Alligator mississippiensis; Duadin, 1801) undergoes both morphological and physiological changes in response to fluctuations in O₂ availability during development (8, 9, 11–16). Specifically, egg incubation in 10% O₂ induces changes in protein composition of cardiac tissue, cardiac growth, blood pressure, and cardiovascular regulatory mechanisms of embryonic alligators (14, 15, 17–20). Other factors, such as hypoxia-induced changes in gas exchanger morphology or capillary density, may also be involved. A critical component of convective O₂ transport in vertebrates is cardiac output, which is the product of the heart rate and the stroke volume, the volume of blood moved with each contraction. Therefore, the enlargement of the embryonic alligator heart relative to body mass has been suggested as a response to hypoxic development to increase O₂ transport (10, 21). It is possible that this change is combined with increased blood hematocrit (Hct) or changes in the relative abundance of isoHbs with different O₂ affinities and modes of allosteric regulation to increase the O₂-carrying capacity of the blood.

Crocodilians possess multiple copies of α- and β-type globin genes (22) and can therefore potentially express structurally distinct isoHbs at different stages of embryonic or postembryonic life, as is the case in birds (the closest living relatives of crocodilians) and other amniotes (23–26). All vertebrates examined to date express structurally and functionally distinct isoHbs during prenatal development (27–31). Among viviparous taxa, the isoHbs expressed during the earliest stages of embryonic development typically have especially high O₂ affinities, low sensitivities to allosteric cofactors, and low cooperativity (32–35). Oxygenation properties of isoHbs expressed during the earliest stages of embryogenesis may relate to O₂ scavenging/transport functions before the development of a circulatory system (30, 36). Much less is known about the functional properties of embryonic isoHbs in oviparous vertebrates. A previous study on the saltwater crocodile Crocodylus porosus (37) reported low O₂ affinity of whole blood at early stages of embryonic development compared with adults due to higher levels of intraerythrocytic ATP, which is a major allosteric effector of crocodilian Hbs together with chloride ions, CO₂, and bicarbonate (38–41). However, the expression of functionally different isoHbs during development, although reported, was not investigated in detail (37).

In this study, we examined the expression and functional properties of isoHbs during embryonic development of the American alligator, and we tested whether the expression of alternative isoHbs is modulated by exposure to hypoxia during development.

MATERIALS AND METHODS

Ethical Statement

This study was approved and performed in strict following of the Institutional Animal Care and Use Committee (IACUC; no. 11007) from the University of North Texas.

Animals and Blood Sampling

American alligator (Alligator mississippiensis, Daudin 1801) eggs were collected from six field nests in the Rockefeller Wildlife Refuge at Grand Chenier, LA. Embryonic age was established according to Ferguson (42). All eggs were weighed, numbered, and then randomly assigned to ziplock boxes (1 L) containing vermiculite water mixture at a ratio of 1:1 and the lidless box mass was recorded for each egg box. Water content of the egg boxes was maintained by adding water three times weekly to bring the box to the original egg containing mass without the box lid. Embryos were incubated at 30°C in a walk-in room (Percival Scientific, Perry, IA), ensuring that all embryos developed as females. At ∼20% of development (the total development period was 72 days at 30°C), all eggs were randomly assigned to either a 21% O₂ or 10% O₂ group. At this point in embryonic development, the heart is functioning to move blood through a rudimentary circulatory system (42). As eggs were added to the boxes, they were candled to determine the location of the embryo on top of the yolk. The O₂ percentages corresponding to normoxia and hypoxia incubation were used based on extensive studies on embryonic alligator development (10, 15, 43, 44) and the close proximity to a previous measure from a crocodilian nest (7). To maintain the O₂ level for each treatment group, the boxes were placed in 76-L ziplock bags that were connected to gas supply of either normoxia (21% O₂) or hypoxia (10% O₂), and eggs were incubated following a previously established protocol (9, 10, 21). The normoxic gas was supplied using an air pump (LT 11 Whitewater) passed through a rotameter flow controller. The hypoxic gas was generated using rotameters (Sho Rate; Brooks Instruments Division, Hatfield, PA) supplied with compressed N₂ and air supplied by an air pump (Whisper AP 300 tetra products). Normoxic and hypoxic air was humidified using bubbling chambers and delivered to the bags at a rate of 2–4 L/min. Gas mixtures were passed through a H₂O-bubbler to ensure adequate water saturation of ≥80–95% relative humidity. Gas composition was monitored continuously with an O₂ analyzer (S-3AI, Applied Electrochemistry, IL) connected to a PowerLab 16/35 data recording system connected to a computer running LabChart Pro software (v 7.2 AD Instruments), and data were recorded at 10 Hz (45).

At 30%, 40%, 50%, 70%, and 90% of developmental stage (42), we selected eggs from both incubation conditions candled to locate a major branch of the chorioallantoic membrane artery. Eggs were placed in a temperature-controlled surgical chamber that was maintained at 30°C, and a portion of the eggshell removed under a dissection microscope (Leica MZ6; Leica Microsystems, Waukegan, IL). A heat-pulled, heparinized saline-filled PE 50 tubing was then inserted into the vessel using methods previously described (21, 43) and forwarded into the major artery leaving the embryo. On completion of the catheter placement, 100- to 500-µL blood was collected into separate 50-µL heparinized microhematocrit tubes from each embryo (Scientific Products, MaGaw Park, IL) for blood analysis. The volume of blood collected was dependent on incubation percentage. Blood samples were then centrifuged at 20,854 g for 5 min (Micro-Hematocrit Damon/IEC Division) to separate the cellular components from the plasma. Hct was measured at 70% and 90% of incubation, where sufficient blood was obtained. The separate erythrocytes were flash frozen in liquid nitrogen and the samples were stored at −80°C until they were analyzed.

Hemolysate and Analysis of isoHb Multiplicity and ATP

Individual hemolysates were prepared from thawed blood or erythrocyte samples (20–50 µL) by adding an approximately fourfold volume of ice-cold 10 mM Hepes, 0.5 mM EDTA, pH 7.4, incubation on ice for 30 min, and centrifugation at 4°C (12,000 g, 15 min). Hemolysates were then frozen at −80°C and thawed on ice before use. For each individual hemolysate, we assessed expression of Hb isoforms by thin-layer polyacrylamide gel isoelectric focusing (IEF; pH range 3–9) on a PhastSystem (GE Healthcare). The position of red bands containing Hbs was recorded before staining gels with Coomassie blue. ATP was measured in freshly thawed individual hemolysates using a bioluminescence assay kit CLS II (Roche), as described (46). Hemolysate samples were kept on ice during performance of laboratory procedures.

Oxygen Equilibrium Curves

We measured O₂ equilibrium curves of individual hemolysates of alligators at developmental stages from 30% to 90% in both normoxia and hypoxia treatments (N = 4 and 3, respectively) and of purified isoHbs at 40% and 90% development (see materials and methods, Purification of IsoHbs). Curves were measured at 25°C using a thin-layer modified diffusion chamber (41, 46–49) connected to 1) a Cary 60 UV-Vis spectrophotometer with fiber optic probes (Agilent Technologies) for measuring changes in absorbance (415 nm, Soret peak of oxy Hb) at discrete O₂ partial pressure (Po₂) values and 2) a programmable Gas Mixing System (Loligo Systems, Viborg, Denmark) for mixing ultrapure N₂, O₂, and CO₂ to generate discrete Po₂ values and %CO₂. Hemolysate samples (4 µL) were analyzed in 0.1 M HEPES, 0.1 M KCl, 0.5 mM EDTA, and pH 7.2 in the absence and presence of 1% CO₂. Purified isoHbs were analyzed in 0.1 M HEPES, 0.5 mM EDTA, and pH 7.2 in the absence and presence of 0.1 KCl, 0.45 mM ATP and 1% CO₂. The pH of samples was measured using an InLab Micro pH electrode (Mettler Toledo) and it remained constant (within 0.05 units) after equilibration with 1% CO₂. Heme concentration was 0.3 mM in all experiments. Buffer conditions refer to final concentrations in samples used for measurements. Since CO₂ and bicarbonate affect crocodilian Hbs the same way and bind to the same protein site (40), we examined the CO₂ effect alone to save limited material as a proxy for the combined CO₂/bicarbonate allosteric effect. For each O₂ equilibrium curve, the O₂ affinity was expressed as P₅₀ (Po₂ at half saturation) and the degree of cooperativity by the Hill’s coefficient (n). Both parameters were obtained by fitting the sigmoidal Hill equation (Eq. 1) to the data (5–9 data points per curve), expressed as O₂ saturation (Y) as function of Po₂.

$$Y= \frac{{\mathrm{P}}_{{\mathrm{O}}_2}^n}{({\mathrm{P}}_{{\mathrm{O}}_2}^n+\mathrm{ }{\mathrm{P}}_{50}^n)}$$ (1)

Purification of isoHbs

To determine oxygenation properties and subunit composition of purified isoHbs expressed at early and late stages of development, we purified isoHb components using hemolysates of normoxic alligators at 40% and 90% developmental stage. We separated structurally distinct isoHbs by subjecting hemolysates to anion-exchange chromatography on a Mono Q 5/50 GL column (1 mL) connected to an Äkta Pure Chromatography System (GE Healthcare). The column was equilibrated with 20 mM HEPES, 0.5 mM EDTA, pH 7.65, and isoHbs were eluted with a 0–0.1 M NaCl linear gradient, at a flow rate of 1 mL/min, at room temperature. Absorbance was monitored at 415 and 280 nm, to identify heme-containing proteins. Hemolysates were first desalted on a PD-10 column (GE Healthcare) and were equilibrated with the same buffer as the Mono Q column before anion-exchange chromatography. Eluted peaks containing isoHbs were dialyzed against 10 mM HEPES, 0.5 mM EDTA, pH 7.4 to remove NaCl, concentrated by ultrafiltration (Amicon Ultra Centrifugal Devices, Millipore) at 4°C to a final heme concentration of >0.5 mM and stored in aliquots at −80°C. Peaks were analyzed by IEF as described above (see Hemolysate and Analysis of isoHb Multiplicity and ATP).

Structural Analysis of isoHbs

To identify the α- and β-chains of the purified isoHbs, we conducted tandem mass spectrometry (MS/MS) analyses on the fast-performance liquid chromatography (FPLC)-purified fractions of hemolysates from the 40% and 90% developmental stages. The purified isoHbs were separated in a mini-protean precast 4–20% SDS PAGE gel (Bio-Rad, Hercules, CA) and were subsequently stained with Coomassie brilliant blue-G. The stained bands were excised and processed for in-gel tryptic digestion (50), and the eluted peptides were then analyzed using a Thermo Orbitrap Fusion Lumos Tribrid (Thermo Scientific) mass spectrometer in data-dependent acquisition mode. Peptides were identified by searching MS/MS data against a customized reference database that contained the adult-expressed globin genes of 16 crocodilian species (41, 51) and the complete α- and β-globin gene repertoires of C. porosus, G. gangeticus, and A. mississippiensis (22), species that represent the three main lineages of extant crocodilians. For completeness, we included a representative avian α^D-globin sequence in the reference database because this gene encodes α-chain subunits of embryonic isoHbs in the limited number of squamate reptiles and birds that have been examined to date (52–54). Detection of α^D-globin in crocodilian isoHbs from any developmental stage would be a surprising finding since comparative genomic analyses indicate that this gene was deleted from its normal position in the α-globin gene cluster in the common ancestor of modern crocodilians (22). The database search was set up for full tryptic peptides with a maximum of two missed cleavage sites. The precursor mass tolerance threshold was set as 10 ppm and 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% (41, 50). For each identified isoHb, we used the MS/MS data to quantify the relative fraction of acetylated NH₂-termini of the α- and β-subunits (41, 55).

Statistical Analysis

Egg mass and Hct values were statistically compared between developmental stages and O₂ incubation conditions (i.e., normoxia and hypoxia) with two-way ANOVA. P₅₀ values of embryonic hemolysates were compared with three-way ANOVA using developmental stage, O₂ incubation conditions, and the absence and presence of CO₂ as factors. Multiple comparisons were performed by Holm–Sidak procedure, and statistical significance was considered with an α = 0.05. Statistical analysis and curve fitting were performed using SigmaPlot V.12.

RESULTS

We examined changes in isoHb expression and Hb-O₂ affinity in alligator embryos at different development stages in both normoxic and hypoxic conditions. Egg masses were similar (80 ± 2 g), when eggs were incubated in normoxia and hypoxia and across embryonic developmental stages (Table 1; P = 0.929). Thus, egg mass was not affected by age or incubation conditions, in accordance with previous results (9). Blood Hct was similar in normoxia and hypoxia treatment groups and decreased significantly from 70% development to 90% development (Table 1; P = 0.011), where it approached the low Hct values (∼25%) typical of adult alligator blood (∼22%) (41, 56). Hypoxia did not affect Hct significantly in either developmental stage (Table 1; P = 0.56).

Table 1.

Egg mass and hematocrit (Hct) during embryonic development in the American alligator

	Egg Mass, g		Hct, %
Embryonic Development	Normoxia	Hypoxia	Normoxia	Hypoxia
30% (8,7)	80.37 ± 6.11	81.39 ± 4.39	n.d.	n.d.
40% (4,6)	80.67 ± 7.73	81.48 ± 4.65	n.d.	n.d.
50% (6,6)	80.42 ± 5.66	79.08 ± 4.45	n.d.	n.d.
70% (6,6)	79.15 ± 4.71	80.53 ± 5.74	30.34 ± 2.83a	29.37 ± 5.14a
90% (6,6)	81.50 ± 4.29	79.90 ± 5.33	26.47 ± 1.46b	25.87 ± 1.62b

Data are expressed as means ± SD. Oxygen conditions of development are indicated (normoxia, 21% O_2; hypoxia, 10% O₂). Hct was measured in duplicate or quadruplicate for each sample. Statistical significance was considered with α = 0.05. Different superscript letters indicate statistically significant differences between 70% and 90% embryonic development. The number of animals for each developmental stage is reported in parenthesis (normoxia, hypoxia, respectively). n.d., not determined.

We examined isoHb multiplicity by IEF in all individual hemolysates. At each developmental stage, all individuals showed identical patterns of isoHb multiplicity (representative patterns for 30% development are shown in Fig. 1, A and B). Patterns of protein expression were identical in normoxic and hypoxic groups compared at the same developmental stage (Fig. 1, A and B), indicating that the level of hypoxia used did not induce changes in isoHb expression. In contrast, the pattern of isoHb expression underwent marked changes during development, as IEF revealed the presence of multiple Hb components at early developmental stages, and the progressive disappearance of Hb components with high isoelectric points at later developmental stages (Fig. 1, C and D). At 90% development, alligator embryos expressed a single major isoHb that is then retained throughout postnatal life (Fig. 1C).

Figure 1.

Analysis of IsoHb multiplicity by IEF on polyacrylamide gels. Representative IEF gels of individual hemolysates of embryonic alligators at 30% development during normoxia (N30, n = 7; A) and hypoxia (H30, n = 7; B), showing identical patterns of isoHbs expression. C: representative IEF gel of hemolysates of embryonic alligators at different stages of development during normoxia (N), showing ontogenetic changes in the pattern of expression of isoHb. D: the same gel as in C before staining with Coomassie blue. Similar results were obtained for hypoxia (not shown). The corresponding isoelectric points are reported on the left side. IEF, isoelectric focusing; isoHb, hemoglobin isoform; Std, standard marker proteins.

To investigate the functional consequences of the changes in isoHb expression during embryonic development, we measured the in vitro oxygenation properties of hemolysates from normoxic and hypoxic groups. These experiments showed a progressive decrease in O₂ affinity (i.e., increase in P₅₀) during development (Fig. 2, A and B; P = 0.001). The two earliest stages of development (30% and 40%) exhibited higher Hb-O₂ affinity than the later stages. To check if these differences in affinity could be due to variations in ATP content, we measured ATP of individual hemolysate, but no ATP was detected, presumably because of ATP degradation before hemolysate preparation or during storage. Thus, the progressive decrease in O₂ affinity of the hemolysate observed in vitro during development derived from a different composition of isoHbs. The presence of 1% CO₂ caused a significant increase in hemolysate P₅₀ (P = 0.001), which was particularly pronounced at the latest developmental stages (Fig. 2B). Exposure to hypoxia did not alter the P₅₀ of the hemolysate at any developmental stage (Fig. 2B; P = 0.230), consistent with the identical pattern of isoHb expression (Fig. 1, C and D). All hemolysate samples exhibited a low cooperativity of O₂ binding, with Hill coefficients slightly above unity (n ∼ 1.3 ± 0.4), reflecting largely hyperbolic O₂ equilibrium curves.

Figure 2.

Oxygenation properties of hemolysates of embryonic alligators at different developmental stages. A: representative O₂ binding curves of normoxic samples (from left to right, 30%, 40%, 50%, 70% and 90% development). B: P₅₀ values (means ± SE) of hemolysates of embryonic alligators during normoxia (circles) and hypoxia (squares), and in the absence (white symbols) or presence (black symbols) of 1% CO₂. P₅₀ values gradually increase in course of development in both normoxic and hypoxic groups. Different letters above bars indicate statistically significant differences between developmental stages (P ≤ 0.027). Hypoxia exposure during development did not affect P₅₀ values (P = 0.230). Addition of CO₂ increased P₅₀ values in later developmental stages (P = 0.001). *Significant differences in P₅₀ values between samples measured with or without CO₂ within each developmental stage (n = 3 biological replicates for each % development).

To examine the O₂ affinity of each Hb component in isolation and determine the origin of the P₅₀ shift during embryonic development, we purified isoHb components from normoxic hemolysates at 90% and 40% development. Since hemolysates at 30% and 40% development had identical IEF patterns of isoHb multiplicity (Fig. 1C) and identical functional properties (Fig. 2B), we used samples at 40% development as representative of the two earliest developmental stages. Hemolysates corresponding to each of the two developmental stages resolved into the same three elution peaks (Fig. 3, A and B), which MS/MS analyses identified as two structurally distinct isoHbs: HbI (peak 1) and HbII (peaks 2 and 3; Fig. 4). The HbII isoform may have been present in two separate elution peaks due posttranslational modifications. MS/MS analysis revealed that the two isoHbs contain identical α-type subunits but different β-type subunits: HbI incorporates the β-chain product of the HBB-T1 gene and HbII incorporates the β-chain product of the HBB-T4 gene (Fig. 4A). We recovered no homolog of the avian α^D chain protein. The HbII protein isoform represents the single Hb isolated from the adult RBCs of American alligator (41). In samples from both developmental stages, HbI and HbII exhibited high levels of NH₂-terminal acetylation (Fig. 4B) and the Hb subunits perfectly matched sequences of previously annotated α- and β-globin genes of American alligator (Fig. 4C). At both developmental stages, the α- and β-chains of the early expressed HbI exhibited lower rates of NH₂-terminal acetylation relative to those of the late-expressed HbII. The β-chain NH₂-termini of HbI (product of the HBB-T1 gene) were ∼80% acetylated, whereas those of HbII (product of HBB-T4 gene) were 100% acetylated (Fig. 4B).

Figure 3.

Purification and characterization of hemolysates of embryonic alligators at 40% and 90% developmental stages during normoxia. Representative elution profile of isoHb components purified by anion-exchange chromatography of hemolysates at 40% (A) and 90% (B) of development, showing elution of three peaks, with corresponding identification as HbI or HbII by subsequent MS/MS. Absorption at 280 nm (black) and 415 nm (gray) are indicated, along with the % buffer B (dashed line). isoHb, hemoglobin isoform.

Figure 4.

IsoHbs composition and NH₂-terminal acetylation profile of normoxic 40% and 90% developed alligators. A: isoHb subunit composition of the two major protein isoforms found in normoxic 40% and 90% developed alligators hemolysates. B: NH₂-terminal acetylation of the α- and β-chain subunits of expressed isoHbs of 40% and 90% developed alligators. C: amino acid sequences of adult α- and β-chain subunits and the corresponding genes (31). Accession numbers: HBA-α^A (XP_006261904), HBB-T4 (QHX99535), and HBB-T1 (AKHWO1000000). isoHbs, hemoglobin isoforms.

The ratio of the two isoHbs changed during development, as alligator embryos at the 40% stage had a HbI:HbII ratio of 0.7:0.3, whereas those at the 90% stage had a ratio of 0.26:0.74. Thus, HbII (which represents the sole-expressed isoform in adult RBCs) transitions to become the major isoHb sometime between the 40% and 90% developmental stages, whereas (embryonic) HbI is the predominant isoform in early developmental stages. In summary, relative expression levels of the two main isoHbs changes during the development but the same two isoHbs are present at both the 40% and 90% stages.

O₂ equilibrium curves of purified isoHbs at 40% and 90% development measured with and without CO₂ (Fig. 5) indicated that HbI had a lower P₅₀ than HbII under stripped conditions and with KCl added, but not in the presence of ATP, due to a large allosteric effect of ATP. Thus, HbI was sensitive to ATP but not to chloride ions. HbII showed highly similar P₅₀ values under all conditions investigated and was sensitive to chloride ions but not to ATP. The effect of CO₂ was more pronounced in HbII than in HbI under all conditions examined, i.e., with or without anionic cofactors. Overall, there were no detectable differences in P₅₀ values of the same isoHb between 40% and 90% development (Fig. 5). Taken together, these results show that HbI, the major isoHb expressed during early embryonic development, has a high O₂ affinity and high allosteric sensitivity to ATP, whereas HbII, which persists as the sole-expressed isoHb in adult RBCs, has a relatively low O₂ affinity and high allosteric sensitivity to chloride and CO₂/bicarbonate. Both isoHbs showed low cooperativity (n coefficient in the range 1.2–1.5), indicating nearly hyperbolic O₂ equilibrium curves, similar to the hemolysate.

Figure 5.

Oxygen affinity (P₅₀) of purified isohemoglobin fractions from alligator embryos at two different developmental stages (40% and 90%) in normoxic conditions. Measurements were performed in the absence (stripped) and presence of KCl and ATP as indicated, as well as in the absence (white bars) or presence (black bars) of 1% CO₂. A and B: P₅₀ of purified HbI from alligator embryos at 40% and 90% developmental stages, respectively. C and D: P₅₀ of purified HbII from alligator embryos at 40% and 90% developmental stages, respectively. Data are means ± SE with n = 3 technical replicates per treatment.

DISCUSSION

This study provides the first detailed experimental analysis of the developmental switch of Hb synthesis in crocodilians. Developmental changes in the expression of the HbI and HbII isoHbs were highly consistent across individuals, but expression patterns were not altered by hypoxia exposure.

During embryonic development, Hb expression in alligator erythrocytes changes progressively from HbI (the predominant isoHb at 30% and 40% development) with a high intrinsic O₂ affinity and high sensitivity to ATP to HbII (the predominant isoform at 90% development, matching the adult protein) with low intrinsic O₂ affinity and high sensitivity to CO₂/bicarbonate (Fig. 5). The P₅₀ values of purified HbII (Fig. 5, C and D) were similar to those of the purified adult Hb (41) measured under comparable conditions (e.g., 7.4–8.1 and 6.2 Torr in the presence of KCl and ATP, respectively). Thus, the progressive right-shift of the O₂ equilibrium curves of the hemolysate observed in vitro during development (Fig. 2A) is caused by the increase in the relative abundance of the low-affinity HbII isoform at the expense of the higher affinity HbI isoform. It is important to note that ATP was not present in these samples. We measured O₂ equilibrium curves in vitro under standard experimental conditions (i.e., at low Hb concentration and in purified samples) to reveal genetically based differences in the oxygenation properties of structurally distinct isoHbs. By contrast, studies of embryonic saltwater crocodiles (Crocodylus porosus) (37) reported a progressive left-shift of the O₂ equilibrium curves of whole blood during development. Assuming that crocodiles and alligators are comparable in their embryonic development, the apparent discrepancy between our results (obtained with ATP-free hemolysate) and those of Grigg et al. (37) may be explained by developmental changes in erythrocytic ATP concentration. High ATP at early development would shift whole blood O₂ equilibrium curves to the right, especially since ATP-sensitive HbI (Fig. 5, A and B) is highly abundant at these stages (Fig. 3A), whereas the use of CO₂ in whole blood experiments (37) mostly right shifted the O₂ equilibrium curves at later development. We note that the progressive shift of purified Hb isoforms from high- to low-O₂ affinity during alligator development (Fig. 5) is similar to that of humans and other jawed vertebrates (30, 33). However, in contrast to human embryonic Hbs, which are insensitive to organic phosphate 2,3-diphosphoglycerate (DPG) but sensitive to chloride ions (57, 58), the early-expressed alligator HbI is insensitive to chloride ions and is sensitive to ATP. Thus, alligator HbI may be able to regulate whole blood oxygenation during early development via its sensitivity to changes in ATP, whereby decreased ATP due to low available O₂ would shift the blood O₂ equilibrium to the left and enhance O₂ uptake, as it occurs in fish (46, 59). However, exposure of embryos to hypoxia did not alter RBC ATP levels and blood O₂ affinity in crocodiles (O₂ tension ∼80 Torr) (37) or isoHb expression in alligators (10% O₂; Fig. 1, A and B), but one cannot exclude that more severe hypoxia may elicit shifts in the blood O₂ equilibrium curve. In contrast to alligator HbI, a strong CO₂ (and by inference, bicarbonate) sensitivity appears as a hallmark of the adult protein HbII (Fig. 5, C and D). The progressive increase in the Hb-CO₂ sensitivity during development was also observed in crocodile embryos (37).

Differences in intrinsic O₂ affinity and cofactor sensitivity (CO₂, anions) between HbI and HbII (Fig. 5) must be attributable to amino acid substitutions in the β-chain subunits, as both isoHbs share the same α-chains (Fig. 4A). The β-chains of HbI and HbII (encoded by the HBB-T1 and HBB-T4 genes, respectively) differ at 59 of 146 amino acid sites, including several that are predicted to play a key role in anion-binding. Results of molecular dynamics simulations identified six β-chain residues (sites β1, β2, β82, β135, β139, and β143) that appear to play direct roles in allosteric phosphate-binding in avian and crocodilian Hbs (25, 41). Of those six sites, five differ between the HbI and HbII isoHbs of alligators (Fig. 4C). The higher ATP sensitivity of HbI relative to HbII may be largely explained by the fact that the former isoHb possesses positively charged β139-His and β143-Arg (both of which are predicted to bind ATP via charge-charge interactions (41), whereas the latter isoHb harbors uncharged Ala at both sites (Fig. 4C). The same amino acid differences at β139 and β143 that distinguish alligator HbI and HbII also distinguish alligator HbII from the HbA and HbD isoHbs of birds (25, 26). The fact that alligator HbI and the avian HbA and HbD isoHbs all exhibit significantly higher sensitivities to organic phosphates than alligator HbII (25, 41, 47, 60, 61) provides additional evidence that replacements of positively charged residues by Ala at β139 and β143 may be largely responsible for the reduced ATP sensitivity of alligator HbII. In addition to differences at the above-mentioned sites that have been directly implicated in oxygenation-linked phosphate-binding, differences between HbI and HbII in sensitivity to ATP and chloride ions may also be explained by the effects of amino acid substitutions that alter the orientation of the β-chain NH₂ termini (the isoHbs differ at 8 of the first 10 β-chain residues; Fig. 4C) or that otherwise alter the charge density of the central cavity. Differences in NH₂-terminal acetylation of the β-chains are likely attributable to differences in the amino acid state of the NH₂-terminal residue (following posttranslational removal of the initiator methionine), as the rate of acetylation is expected to be higher for β1-Ala (HbII) than for β1-Val (HbI) (62). However, it appears unlikely that isoHb differences in responsiveness to allosteric effectors could be explained by differences in NH₂-terminal acetylation, as experiments on recombinant alligator Hbs have revealed that accessibility of the –NH₂-terminal groups has little effect on sensitivity to ATP, chloride ions, or CO₂ (55). Interestingly, CO₂ has a modest effect on HbI and a strong and anion-independent effect on HbII (Fig. 5), suggesting that the different amino acid residues between the β chains of the two Hb are important for the CO₂ and bicarbonate-dependent allosteric regulation (40). Site-directed mutagenesis studies are ongoing in our laboratory to identify the site for CO₂ and bicarbonate allosteric binding.

Our findings illustrate that chronic exposure to 10% O₂ is not sufficient to elicit changes in isoHb expression in alligator embryos. Interestingly 10% O₂ is similar to the Po₂ experienced by alligator eggs under natural conditions and is therefore ecologically relevant. Exposing alligator eggs to the same level of hypoxia using an identical protocol (21) causes a significant increase in the heart-to-body mass ratio (10, 21) and is associated with more efficient (i.e., less uncoupled) heart mitochondria after hatching in juveniles, reflecting a better utilization of O₂ to generate ATP (8, 10). Thus, alligator embryos exhibit changes in response to environmental hypoxia such as increase heart mass, with consequent larger stroke volumes that may augment cardiac output, but hematological properties (Hct and blood-O₂ affinity) remain largely unchanged. At hatching, however, Hct decreases in crocodiles (37) and in alligators, when comparing values for embryos (Table 1) and adults (41, 56), indicating that some changes in the blood O₂-carrying capacity during embryonic development and in thetransition to juveniles may take place, also due to hypoxia (63).

Perspectives and Significance

We characterized the developmental regulation of Hb synthesis in American alligator and the structural and functional differences between early- and late-expressed (adult) isoHbs. We found developmental changes in isoHb composition in the erythrocytes of alligator embryos, resulting in a corresponding switch in the mode of allosteric regulation. Although the physiological consequences of our findings remain to be investigated further, our structural data on isoHb composition provide important clues for understanding the evolution of allosteric regulation in the Hb of vertebrates. During embryonic development of alligators, exposure to ecologically relevant levels of hypoxia induces changes in some cardiocirculatory traits (10, 21), but results of our experiments demonstrate that the ontogenetic switch in isoHb expression is unaffected.

GRANTS

This research was supported by a grant to D.A.C. II from the National Science Foundation (IOS-1755187) and by grants to J.F.S. from the National Institutes of Health (HL087216), the National Science Foundation (OIA-1736249 and IOS-1927675), and the Fulbright Foreign Scholar Program in Argentina.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.F.S., D.A.C., and A.F. conceived and designed research; N.M.B., E.E.P., R.J.J., and C.N. performed experiments; N.M.B., E.E.P., R.J.J., and C.N. analyzed data; N.M.B., C.N., J.F.S., D.A.C., and A.F. interpreted results of experiments; N.M.B. and C.N. prepared figures; N.M.B., J.F.S., D.A.C., and A.F. drafted manuscript; N.M.B., C.N., J.F.S., D.A.C., and A.F. edited and revised manuscript; N.M.B., E.E.P., R.J.J., C.N., J.F.S., D.A.C., and A.F. approved final version of manuscript.