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 red blood cells 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 O2 saturation of Hb. 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 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 the 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.

Keywords: Adaptation; Allosteric regulation; Blood; Carbon dioxide; Oxygen.

Fig. 1.

Reversibility of CO₂ effect on O₂ saturation in the stripped hemolysate of the spectacled caiman. Representative absorbance traces (absorbance at 415 nm; range ∼0.3–0.8) in the absence (A) and presence (B) of acetazolamide monitored by the diffusion chamber technique. After equilibration with 100% O₂ (100% O₂ saturation) and 100% N₂ (0% O₂ saturation), the hemolysate was equilibrated with 0.44% O₂ to achieve approximately 50% O₂ saturation before equilibration with 1% CO₂, 0.44% O₂, as indicated by the shaded area. Changes in the percentage gas in the mixture are indicated by arrows. Traces are representative of one sample out of N=3 biological replicates.

Fig. 2.

FPLC purification, SDS-PAGE and functional analysis of hemoglobin (Hb) fractions of spectacled caiman. (A) Elution profile of caiman hemolysate (absorbance at 280 nm; representative of one sample out of N=3 biological replicates), resolving in two peaks (Hb1 and Hb2) indicated by arrows, is shown superimposed on that of standard proteins with known molecular mass: human Hb (64.6 kDa), horse myoglobin (Mb, 17.6 kDa) and bovine carbonic anhydrase II (CA, 30 kDa). (B) Calibration curve by regression analysis for molecular weight (MW, kDa) against elution volume. (C) SDS PAGE under reducing conditions (10 mmol l⁻¹ DTT) of (left to right) bovine CA II, spectacled caiman hemolysate (H), purified Hb1, purified Hb2 and standard protein (Std) as indicated. The bands corresponding to α- and β-type chains in the hemolysate and in the two purified Hb fractions are indicated. (D) logP₅₀ measured at various P_CO2 values, showing an identical effect of CO₂ on the O₂ affinity of purified Hb1 and Hb2 fractions (N=3).

Fig. 3.

Effect of CO₂ and bicarbonate on the O₂ saturation of purified Hb1 and Hb2 from spectacled caiman and apparent CO₂ and bicarbonate binding curves. (A) Representative traces measured using the thin-layer diffusion chamber of the decrease in Hb saturation induced by changes in P_CO2 at a fixed P_O2 close to P₅₀ (0.44% O₂, P_O2 ∼3.3 Torr). (B,C) Apparent CO₂ saturation curves of (B) Hb1 and (C) Hb2 purified from N=3 individuals. Non-linear regression fitting of a single hyperbola equation is shown (details and fitted parameters are in Table S1). (D) Representative traces (one sample out of N=3) showing the increase in O₂ released from Hb1 and Hb2 upon injection of bicarbonate at a fixed P_O2 of ∼4.2 Torr. The gray line represents a control trace in which bicarbonate was added to buffer alone. (E,F) Apparent bicarbonate saturation curves of (E) Hb1 and (F) Hb2 obtained as shown in D. Non-linear regression fitting of a single hyperbola is shown (details and fitted parameters are in Table S2).

Fig. 4.

Proposed mechanistic model of the observed CO₂- and bicarbonate-dependent changes in Hb oxygenation in crocodilians. (A) General scheme of carbamino protein derivative formation (R-NH-COO⁻) at a non-protonated amino group (R-NH₂) from CO₂. (B) Identical putative binding site for CO₂ (top) and bicarbonate (bottom) on the T-state of crocodilian Hb, including a reactive uncharged amino group surrounded by positively charged residues (indicated by the half-circle). The negatively charged carbamino formed at the amino group (top) is stabilized by the positively charged amino acid residues, stabilizing the T-state and causing a decrease in O₂ saturation. The proton generated in carbamino formation is picked up by the protein molecule, likely contributing to further electrostatic stabilization of the carbamino. Note the very small structural difference between covalent CO₂ binding via carbamino formation (top) and non-covalent bicarbonate binding (bottom) to the protein.