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

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 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, and 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 the 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. Taken together, our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where build-up of blood 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.