New insights into the allosteric effects of CO2 and bicarbonate on crocodilian hemoglobin

Abstract

Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O2 binding is allosterically regulated by bicarbonate, which forms in the red blood cell upon hydration of CO2. Although known for decades, this remarkable mode of allosteric control has not yet been experimentally verified with direct evidence of bicarbonate binding to crocodilian Hb, probably because of confounding CO2-mediated effects. Here we provide the first quantitative analysis of the separate allosteric effects of CO2 and bicarbonate on purified Hb of the spectacled caiman (Caiman crocodilus). Using thin-layer gas diffusion chamber and Tucker chamber techniques, we demonstrate that both CO2 and bicarbonate bind to Hb with high affinity and strongly decrease Hb-O2 saturation. We propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O2 affinity T conformation of the Hb. These results provide the first experimental evidence that bicarbonate binds directly to crocodilian Hb and promotes O2 delivery independently of CO2. Using the gas-diffusion chamber, we observed similar effects in Hbs of a phylogenetically diverse set of other caiman, alligator, and crocodile species, suggesting that the unique mode of allosteric regulation by CO2 and bicarbonate evolved >80-100 million years ago in the common ancestor of crocodilians.Our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where build-up of erytrocytic CO2 and bicarbonate ions during breath-hold diving or digestion facilitates O2 delivery, while Hb desaturation facilitates CO2 transport as protein-bound CO2 and bicarbonate.

Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin

“…We expressed and purified each of the three recombinant Hbs (rHbs), and we then measured oxygenation properties in vitro. The reconstructed AncArchosaur and AncCroc rHbs exhibited intrinsic O2-affinities, cooperativities, and pH sensitivities (Bohr effect) that are typical of native Hbs from modern crocodilians and non-avian reptiles (4,6,(17)(18)(19)(20)(Table S1). We used an experimental approach to confirm that all functional inferences are robust to uncertainty in ancestral sequence estimates (21)(Supplementary Text and Fig.…”

“…Crocodilians are able to remain submerged underwater for extraordinarily long periods of time -a physiological capacity that allows some species, such as the Nile crocodile (Crocodylus niloticus), to kill large, mammalian prey like wildebeest and zebras by dragging them underwater and drowning them. This capacity for breath-hold diving is made possible by the fact that the O2-affinity of crocodilian Hb is regulated by the inhibitory effects of bicarbonate ions (1)(2)(3)(4)(5)(6). During submergence, a progressive increase in the red cell concentration of bicarbonate (derived from the hydration of CO2) reduces Hb-O2 affinity, thereby promoting O2 unloading to the cells of metabolizing tissues (5).…”

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

“…Interestingly, CO 2 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 b chains of the two Hb are important for the CO 2 and bicarbonate-dependent allosteric regulation (40). Site-directed mutagenesis studies are ongoing in our laboratory to identify the site for CO 2 and bicarbonate allosteric binding.…”

“…Buffer conditions refer to final concentrations in samples used for measurements. Since CO 2 and bicarbonate affect crocodilian Hbs the same way and bind to the same protein site (40), we examined the CO 2 effect alone to save limited material as a proxy for the combined CO 2 /bicarbonate allosteric effect. For each O 2 equilibrium curve, the O 2 affinity was expressed as P 50 (PO 2 at half saturation) and the degree of cooperativity by the Hill's coefficient (n).…”

“…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 O 2 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, CO 2 , and bicarbonate (38)(39)(40)(41). However, the expression of functionally different isoHbs during development, although reported, was not investigated in detail (37).…”