Allosteric Control of Oxygen Binding by Haemoglobin During Embryonic Development in the Crocodile Crocodylus Porosus: The Role of Red Cell Organic Phosphates and Carbon Dioxide

Crocodiles and alligators form an ancient group of vertebrates surviving relatively unchanged from the Mesozoic age and, from evidence based on haemoglobin sequencing and on immuno-electrophoresis, show little variation between species (Schneck et al. 1984; Densmore and Owen, 1989). The functional properties of their haemoglobin and mechanisms of allosteric control have therefore attracted interest. Juvenile and adult crocodilians have very low levels of red-cell organic phosphates (Bauer and Jelkmann, 1977; Grigg and Gruca, 1979) and appear to be unique because bicarbonate undertakes the allosteric role that organic phosphate compounds play in regulating haemoglobin oxygen-affinity in most vertebrates (Bauer et al. 1978; Brittain and Wells, 1991). This unusual mechanism for regulating haemoglobin function has been interpreted in terms of adaptations to diving (Grigg and Gruca, 1979; Grigg and Cairncross, 1980; Weber and White, 1986) and the unique cardiovascular responses associated with this behaviour (Grigg and Johansen, 1987; Grigg, 1991). Crocodiles, like a small number of mammalian species (ungulates and felids), have low or zero red-cell ATP and 2,3-DPG concentrations, a contrast with most other vertebrates, including fish, amphibians, other reptiles and birds, which employ red-cell organic phosphates (RCOP) as modulators of oxygen affinity.

Grigg and Gruca (1979) have pointed out that, in general, low RCOP concentrations are associated with a reduction of the fixed-acid Bohr effect and an enhanced CO₂ Bohr effect. Accordingly, they have interpreted the low levels of RCOP in crocodilians and other low-RCOP vertebrates in functional terms, as conferring an advantage which offsets the effects of low pH experienced at the end of a prolonged dive or active period.

Regardless of what the functional significance may be, if any, the occurrence of only low levels of RCOP in crocodilians poses the question of whether the bicarbonate-mediated control of oxygen delivery offsets the loss of organic phosphate sensitivity. Alternatively, the phosphate cofactors may also be absent from the embryo. A second question is whether the crocodilian embryo regulates oxygen transport during the course of development and has the potential to improve oxygen supply to tissues in the face of altered environmental circumstances.

We report here the patterns of changes in blood oxygen-affinity and concentrations of red cell organic phosphates, ATP and 2,3-DPG in the estuarine crocodile Crocodylus porosus (Schneider) from early in embryonic development through to hatching and juvenility. We report also the effects of ATP, 2,3-DPG and CO₂ on the oxygen affinity of haemolysates and the apparent unresponsiveness of embryonic whole-blood oxygen affinity to low ambient oxygen levels. Further, in order to understand better the evolutionary origins of allosteric regulation, we examine whether the oxygen-binding traits of the red cells in early embryonic development appear to have been subject to selection pressures.

We studied blood from embryonic (15–86 days), hatchling and juvenile crocodiles in a makeshift laboratory at the Edward River Crocodile Farm, Pormpuraaw, Queensland, Australia (14°54′ S, 141°36′ E) in March 1990 and April 1991, late in the nesting season. Additional eggs were transported from Pormpuraaw to Brisbane by light aircraft, the eggs travelling for the day-long journey in moist vermiculite in a polystyrene box while being handled with great care in order to avoid bumps which may have ruptured fragile attachment membranes. Some of these were sampled as embryos and some were allowed to hatch to provide known-age hatchlings. The work was conducted under a permit from the Queensland National Parks and Wildlife Service and followed guidelines approved by The University of Queensland’s Animal Ethics Committee.

To sample embryonic blood, an emery cutting wheel (Dremel Tool Co.) was used to remove the upper third of the egg shell, exposing the embryo. Blood was then drawn into a heparinized syringe from either a chorio-allantoic vein or the heart, using a 30-gauge needle. Blood from the smallest embryos was collected from the heart into a heparinized glass tube drawn in a match flame to a fine point. In this way, we were able to obtain samples from embryos as early as 15 days through to hatching (approximately 86 days). Samples from hatchlings were drawn by cardiac puncture using a 26-gauge needle. Blood from juveniles was collected at the farm from individuals shot in the medulla at close range (0–1cm) with a.22Z-calibre bullet, the standard method of farm slaughter. A deep incision was made immediately, behind the skull platform, severing the cervical artery. A large sample of clean blood could easily be taken into a heparinized syringe from the profuse flow welling from the cut.

In a number of full-term embryos we were able to draw samples anaerobically from the chorio-allantoic artery and vein, for measurement of ‘arterial’ and ‘venous’ O₂ and CO₂ partial pressures using Radiometer BMS3 and PHM71 or PHM73 gas analysis equipment stabilized at 30°C. In these cases, removal of the scribed shell section was accomplished rapidly, and sampling was completed with the assumption that blood gases were not significantly contaminated.

Blood was subdivided immediately after sampling for determinations of the oxygen equilibrium curve (2 μl), haematocrit (10 μl) and haemoglobin content (5 μl) by the method of Dacie and Lewis (1984). Haematocrit measurements using microcap capillaries were validated against conventional 75mm tubes. 2,3-DPG (50μl) and ATP (100 μl) were assayed using Sigma enzymatic test chemicals and an LKB Ultraspec spectrophotometer. It must be noted that the enzymatic method cannot distinguish ATP from other nucleotide triphosphates. In some of the youngest embryos, not all determinations could be made because there was insufficient blood.

Nucleotide phosphate fractions were characterized further by high-performance liquid chromatography (HPLC) (Ryder, 1985). Samples of whole blood from 40-and 70-day embryos and from juvenile animals were diluted sixfold with 0.6mol 1⁻1 perchloric acid and the extracts neutralized with 5mol l⁻1 K₂CO₃. Filtered extracts were chromatographed on a Waters Associates reverse-phase 250mm 5μ C-18 Lichrosorb column (Merck) eluted with 0.1mol 1⁻1 phosphate buffer at pH7.2. Peaks were identified from the column retention times of a series of standard nucleotide solutions and their areas were integrated.

Oxygen equilibrium curves were determined at 30°C, the temperature of incubation at the farm, using an Aminco Hem-O-Scan (American Instrument Co.) taking particular care to ensure the chamber was kept fully moist and using other precautions described by Wells and Weber (1989). Gases for equilibration were pre-mixed and pre-analysed; 2 % CO₂ in air, 2% CO₂ in N₂, 6% CO₂ in air and 6% CO ₂ in N₂ (Commonwealth Industrial Gases, Australia). This afforded determination of whole blood or haemolysate oxygen equilibria at 2.08kPa (15.6mmHg) and 5.91kPa (44.3mmHg) CO₂, respectively. pH was calculated from pH– diagrams for C. porosus (Seymour et al. 1985). This has not been confirmed for early embryos, because insufficient volumes of blood were collected. Full saturation was confirmed with brief exposure to 100% oxygen and the absence of haem oxidation was checked spectrophotometrically (see Dacie and Lewis, 1984).

Lysates were prepared by freeze-thawing erythrocytes in 5mmol l⁻1 Tris–HCl (pH8.2) buffer containing 100mmol l⁻1 NaCl and 10mmol l⁻1 dithiothreitol, protected from oxidation by carbon monoxide. Polyacrylamide gel isoelectric focusing (IEF) was performed according to the basic procedures described by Riggs (1981) in order to establish the pattern of isohaemoglobins during development. Tube gels containing 5 % ampholines mixed in the ranges pH5–8 and pH6–9 (1:1) were prefocused for 30min prior to sample application. Anodal and cathodal electrolyte solutions were 0.01moll⁻1 phosphoric acid and 0.01mol l⁻1 NaOH respectively. Ten 1 μl samples of 60 μmol l⁻1 Hb-lysate were applied and IEF was initiated at 3mA per tube, maximum power 0.25 W per tube, for approximately 5h, until the current dropped to 0.6mA per tube. After focusing was complete the gels were scanned in a densitometer, then removed from the rods. Bands were circumsected and the haemoglobins eluted overnight in cold 10mmol l⁻1 KCl. Concentrations were estimated spectrophotometrically and the proportions calculated.

A lysate of embryonic Hb was prepared from pooled blood taken from embryos less than 40 days old and the absence of HbA was confirmed by IEF. Red cells were washed in 1 mmol l⁻1 Tris–HCl buffer (pH8.2) and brought to 100mmol l⁻1 with NaCl. Stroma were removed by centrifugation and the clear lysate was ‘stripped’ of organic phosphates by Sephadex gel-filtration as described by Jelkmann and Bauer (1976). Lysates were adjusted to 0.6mmol l⁻1 (Hb-tetramer) with distilled water and combined with equal volumes of 100mmol l⁻1 Tris–HCl or Bis–Tris–HCl with a final concentration of 0.3mmol l⁻1 Hb and 100mmol l⁻1 chloride. Buffers were also prepared containing ATP (disodium salt) or 2,3,-DPG (pentacyclohexammonium salt) in 10-fold molar excess to compensate for dissociation of the Hb–phosphate complex in dilute solution. Experiments were carried out in the presence and absence of CO₂.

Five control and five experimental eggs, 40 days old, were exposed at 30°C in separate containers to humidified gas mixtures for 7 days. The flow rate through each chamber was held at 400mlmin⁻1. The controls received humidified air while the experimental eggs received air supplemented with nitrogen to reduce to approximately 10.7kPa (80mmHg). This was achieved using precision flowmeters (GAP and Meter Rule, UK) and the mix was confirmed by periodic measurements of oxygen partial pressure in the excurrent gas.

Haematocrits were higher during embryonic life, with a conspicuous fall immediately after hatching (Fig. 1). Haemoglobin concentration, in contrast, rose during development, followed by a similarly conspicuous drop at hatching (Fig. 2). These results imply a steady increase in mean corpuscular haemoglobin concentration during embryonic life to the values seen in juveniles (262±72 gl⁻1, S.D., N=10) and a decrease in blood viscosity without compromising oxygen transport capacity (Wells et al. 1991).

The pattern of changing levels of organic phosphates during development is shown in Fig. 3. For the first half of development, ATP (Fig. 3A) is the dominant RCOP, with levels near 100 μmol g⁻1 Hb at 15 days (as early as measurement was practical). There is an exponential fall in ATP concentration to a plateau of 5–10 μmolg⁻-1 Hb at hatching, a level which persists past hatching into juvenile life. Assuming a relative molecular mass of 65000 for both embryonic and adult tetrameric Hb, these values correspond to a fall from 6.7 to 0.3molATPmol⁻1 Hb. Levels of 2,3-DPG (Fig. 3B) are initially undetectable but rise dramatically after day 40, so that late in embryonic life there is a marked pulse rising to about 18 μmol g⁻1 Hb (≈1.2molmol⁻1 Hb), a result which confirms the high levels of 2,3-DPG found in embryos of C. porosus close to hatching (Hinchliffe, 1980). This pulse decays within 10 days after hatching to the low levels (<1 μmol g⁻1 Hb) typical of posthatching crocodilians in general.

Identification of the components in the nucleotide phosphate pools in erythrocytes from early and late embryos and from juveniles was made using HPLC chromatograms. The percentage contribution of each is shown in Table 1. ATP is the principal erythrocyte nucleotide throughout development, but GTP makes up a significant fraction of the nucleotide triphosphate pool; approximately 27% in the juvenile and 33% in the early embryo, with the balance ATP. Since the enzymatic method for detecting ATP also detects GTP, the pattern in Fig. 3 must be interpreted accordingly. Small fractions of inosine monophosphate (IMP) occur in the erythrocytes from embryos and juveniles. Appreciable fractions of adenine nucleotides in the embryonic samples are consistent with metabolically active erythrocytes in the embryos.

Early in embryonic life the blood has a strikingly low affinity for oxygen [P₅₀ at 30°C and of 20.08kPa (15.6mmHg) is approximately 6.7kPa (50mmHg) at 15 days]. P₅₀ decreases as development proceeds (Fig. 4) to about half this value by about day 80, an affinity typical of hatchlings and juveniles. Fig. 5 shows the progressive change in the position of the oxygen equilibrium curve from early embryonic life through to that seen in juveniles. The increase in oxygen affinity parallels the fall in ATP levels (Fig. 3). P₅₀ tends to be slightly lower before hatching than after hatching, as in birds (Lomholt, 1975), but there is no suggestion of any effect from the pulse of 2,3-DPG at 60–80 days.

The CO₂ Bohr effect increases gradually throughout development (Fig. 4). Fig. 4 also shows that the oxygen affinity of crude haemolysates of whole blood is high and remains essentially unchanged during development, implying that the observed changes in whole-blood oxygen-affinity are a consequence of the allosteric effects of the changing levels of red-cell organic phosphates, rather than a consequence of a changing population of haemoglobin components.

The developmental profile of the Hb components was resolved by isoelectric focusing of lysates protected from autoxidation with carbon monoxide, in which a well-defined embryonic band (HbE), a poorly focused adult component (HbA) and several trace bands were revealed (Fig. 6). In the early developmental stages, HbE predominates and it is gradually replaced by HbA so that by hatching only traces of HbE persist (Fig. 7).

The molecular integrity of HbE and HbA was confirmed at 500–700nm and the pH of the eluted fractions indicated isoelectric points of 7.2 and 7.8 for HbE and HbA, respectively. The trace components were assumed to be derived artefacts as they were absent in fresh samples of oxyHb taken from eggs and from hatchlings returned to the University for analysis. Further, the fuzzy component observed previously in crocodiles (Bauer and Jelkmann, 1977) appeared to increase in stored lysates, but resolved into a sharp band when treated with 10mmol l⁻1 dithiothreitol. This result suggested that polymerization by disulphide linkage of native tetramers occurs very readily in HbA.

Pooled erythrocytes harvested from embryos younger than 40 days were shown by isoelectric focusing to be free of HbA and HbE was absent from erythrocytes of juvenile crocodiles. Preparative procedures did not promote significant formation of oxidized Hb derivatives as judged from spectral ratios (Benesch et al. 1973).

The oxygen-binding properties of HbE and HbA are compared in Fig. 8A,B. The intrinsic affinity of HbE for oxygen is higher than that of HbA throughout the pH range tested, and it is slightly less sensitive to pH as quantified by the fixed-acid Bohr factor, Ф=ΔlogP₅₀/ΔpH, of −0.21 vs −0.25 calculated linearly between pH7.1 and 7.7. Furthermore, HbE shows an almost hyperbolic equilibrium curve (Hill coefficient, n₅₀=1.1–1.3) compared with the significantly sigmoidal curve (n₅₀=2.2–2.8) seen in HbA. The most striking difference between HbE and HbA lies in their relative responsiveness to organic phosphates and CO₂ modulators. Whereas HbA shows a small, insignificant decrease in oxygen affinity in the presence of ATP or 2,3-DPG, the affinity is decreased dramatically by molecular CO₂ (Fig. 8). Conversely, HbE is most responsive to ATP and much less so to 2,3-DPG. The CO₂ effect is significant in HbE but decreases oxygen affinity less than in HbA.

Assuming a linear Bohr relationship between pH7.1 and 7.7, the fixed-acid value of Ф= −0.21 for HbE was essentially similar with either added 2,3-DPG (−0.20) or ATP −0.14) in the absence of CO₂. Both phosphates increased the cooperativity of HbE and the effect was more marked at alkaline pH. Molecular CO₂ superimposed on varying fixed acid loads (pH) appeared to reduce considerably the Bohr effect of both HbE and HbA but, whereas cooperativity was decreased in the former, it was enhanced in the latter.

The essential regulating effects of ATP and CO₂ on the shape and position of the oxygen equilibrium curve at pH7.7 are illustrated in Fig. 9.

Exposure of 40-day embryos to a low-oxygen environment [10.7kPa (80mmHg)] for 7 days did not result in either an increased oxygen-affinity of embryonic whole blood or any change in red-cell ATP levels, the dominant organic phosphate at 40 days. Mean oxygen affinities (± S.D.) of the whole blood in the hypoxic and control groups were 4.49kPa (33.7mmHg) (±0.69kPa, N=5) and 4.78kPa (35.9mmHg) (±0.75kPa, N=5) respectively. Mean ATP levels in the hypoxic and control groups were, respectively, 53.4±12.8 μmol g⁻1 Hb, N=4, and 54.6±8.2, μmol g⁻1 Hb, N=5.

Obtaining adequate blood volumes to make these measurements was technically difficult. The results are shown in Table 2 and, with reference to Fig. 5, imply an increase from approximately 30% to above 80% in the saturation of the blood as it traverses the chorio-allantois. A comparison of the ‘crocodile egg oxygen cascade’ with that of chickens is shown in Fig. 10.

The affinity of C. porosus blood for oxygen increased throughout the embryonic phase (Fig. 4). It was correlated with both the progressive replacement of embryonic haemoglobin by adult haemoglobin (HbA) (Fig. 6), which was essentially complete soon after hatching (Fig. 7), and with the progressive fall in red-cell ATP concentration (Fig. 3). Of particular note is the apparent lack of response of the whole-blood oxygen-affinity to the peak in 2,3-DPG prior to hatching.

The replacement of one Hb type by another during development parallels that in several species of precocial birds (Borgese and Nagel, 1977; Baumann et al. 1982) and in turtles (Isaacks et al. 1978). These events have been interpreted in terms of falling Po₂ at the chorio-allantoic surface immediately prior to hatching (Lomholt, 1975; Snyder et al. 1982; Lapennas and Reeves, 1983) and on the basis of measurements of metabolic rates in embryos (Whitehead and Seymour 1990). A similar argument may be advanced for C. porosus. Reptiles belonging to the Squamata, in contrast, do not appear to have similar ontogenetic changes in haemoglobin components, and oxygen affinity appears to be regulated allosterically (Korsgaard and Weber, 1989). The general pattern of change in blood oxygen-affinity during embryonic development is geared to the regulation of oxygen supply in response to metabolism. There is no evidence that oxygen becomes limiting in the nest environment of C. porosus (G. C. Grigg and L. A. Beard, unpublished observations).

The higher oxygen affinity of haemolysates compared to whole blood in embryos of C. porosus supports an allosteric role for ATP in regulating embryonic oxygen transport, but not apparently for 2,3-DPG (Figs 8, 9). This poses a riddle in seeking an adaptive explanation for the function of 2,3-DPG in early development. 2,3-DPG is an allosteric modulator that generally decreases blood oxygen-affinity, and yet we found no change in whole-blood oxygen-affinity during development when the 2,3-DPG level peaks and when embryonic Hb is present in significant proportion. Similar results have been obtained in other species: a flush of 2,3-DPG appears late in the embryonic development of several birds and turtles (Isaacks and Harkness, 1975; Bartlett, 1976, 1978, 1982; Snyder et al. 1982) and, prior to hatching, the 2,3-DPG peak in embryonic red cells of the green sea turtle has little effect on the oxygen-transporting properties of the embryonic haemoglobin (Isaacks et al. 1978).

Of particular interest in relation to any adaptive interpretation of the role of organic phosphates is the lack of an increase in whole-blood oxygen-affinity in response to an exposure to low ambient oxygen levels for 7 days. This contrasts with results in many fish such as Pagothenia borchgrevinki and in other vertebrates (Wells et al. 1989a). Ingermann et al. (1983) observed that hypoxia in the chick embryo led to an increase in 2,3-DPG and a fall in ATP levels, suggesting a regulatory role for 2,3-DPG near the end of the incubation period when oxygen is limited. 2,3-DPG is, however, a feeble effector in the allosteric regulation of oxygen affinity in chick and goose embryos (Baumann et al. 1982; Snyder et al. 1982), despite the presence of the requisite erythrocyte metabolites and phosphoglyceromutase enzymes providing for a potential regulatory role (Harkness et al. 1980).

The observation of fluxes in 2,3-DPG levels in the early development of birds and reptiles may be extended to mammals: 2,3-DPG appears in high concentrations in the early development of the marsupial wallaby (Baudinette et al. 1988), in rabbits (Jelkmann and Bauer, 1977) and in mice (Wells, 1979). In mammals, however, a correlation of falling 2,3-DPG levels with increased oxygen affinity during intrauterine development has been established (Brittain and Wells, 1983). More puzzling is the peak of 2,3-DPG appearing during sheep development (Aufderheide et al. 1980) because, as in crocodiles, adult sheep lack 2,3-DPG and there is no direct allosteric effect of 2,3-DPG on either foetal or adult sheep haemoglobin (Baumann et al. 1972). Similar patterns of development occur in goats (Blunt, 1972) and in cattle, although the latter retain traces of 2,3-DPG in the adult erythrocytes (Zinkl and Kaneko, 1973).

The embryonic erythrocytes of C. porosus contained equivalent amounts of ATP and DPG at 70 days. In late alligator and turtle embryos, ATP is about 5–7 times as abundant as 2,3-DPG, and in crocodiles inositol polyphosphate (IPP) is absent (Bartlett, 1978). An allosteric role for ATP in C. porosus during development therefore seems likely. Nucleotide triphosphates also regulate the foetal–maternal shift in an ovoviviparous snake (Birchard et al. 1984) and are implicated in the development of viviparous lizards (Grigg and Harlow, 1981).

The progressive shift from organic phosphate to CO₂ modulation of oxygen affinity parallels the replacement of the embryonic component by adult haemoglobin, and the process is fully completed soon after hatching.

Physiologists often view traits in haemoglobin function as the optimized result of natural selection, when a different kind of explanation may be warranted (Wells, 1990). Both reptiles and birds have developed separate and distinct strategies in allosteric regulation of haemoglobin function in adults which may reasonably be interpreted in adaptationist terms (see Weber and Wells, 1989, for a review). Embryonic development, in contrast, shows remarkable uniformity in that blood oxygen affinity is increased near to the time of hatching, and appreciable quantities of 2,3-DPG are accumulated. This occurs despite the fact that 2,3-DPG has less effect on chicken HbA than does either ATP or IPP, little effect on turtle HbA (Lutz and Lapennas, 1982) and no effect on HbA from C. porosus (Bauer et al. 1978). Moreover, the embryonic haemoglobins of green sea turtles and loggerhead turtles appear to be functionally indistinguishable, despite marked differences in the oxygen affinity of the adult blood (Isaacks et al. 1978).

These observations have alerted some authors to the dangers of pan-selectionism. Bartlett (1982, p. 134) stated that 2,3-DPG in bird embryos ‘… has not been shown to have any clear functional purpose’ and, of turtles, Bartlett (1978, p. 197) wrote ‘… the pool of DPG in the late embryos is a non-functional vestige…’. In a similar vein, Isaacks et al. (1978), writing about marine turtles, stated, ‘… phosphate modulators have little effect upon the mechanisms regulating haemoglobin function…’. These comments invite us to consider the possibility that Hb function in embryonic development has not been scrutinized by natural selection and support the contention of Von Baer (1828) that early development is a highly conserved process.

2,3-DPG accumulates when glycolysis predominates and ATP accumulates with oxidative phosphorylation. Viewed in this way, the period of ATP depletion and 2,3-DPG accumulation in the erythrocytes of the crocodile egg just prior to hatching represents a fully discharged energy unit and need not be seen in adaptationist terms. We may therefore envisage embryonic development as an orderly metabolic integration in which ATP is the ‘currency’ of metabolic economy, its accumulation in early embryos being an index of the extent of phosphorylation, or adenylate energy charge (cf. Atkinson, 1968; Wells et al. 1989b). Although few people would seriously wish to resurrect Haekel’s Biogenetic Law (‘ontogeny recapitulates phylogeny’), this structuralist approach to embryonic development may be preferable to a functionalist one (Wells, 1990).

This project was supported by an Australian Research Council Small Grant, for which we are very grateful. We thank staff at the Edward River Crocodile Farm, Pormpuraaw, particularly Victor Onions, Don Morris and the Edward River Board for providing facilities, crocodile eggs and for allowing us to work there. We thank Lynne Pryor for assistance with the artwork and Jan Mehaffey and Helen Bouter for processing words.