New insights into the allosteric effects of CO2and bicarbonate on crocodilian hemoglobin

Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O 2 binding is allosterically regulated by bicarbonate, which forms in the red blood cell upon hydration of CO 2 . 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 CO 2 -mediated effects. Here we provide the first quantitative analysis of the separate allosteric effects of CO 2 and bicarbonate on Hb of the spectacled caiman ( Caiman crocodilus ). Using thin-layer gas diffusion chamber and Tucker chamber techniques, we demonstrate that both CO 2 and bicarbonate bind to Hb with high affinity and strongly decrease Hb-O 2 saturation, and propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O 2 affinity T conformation of the Hb. These results provide the first experimental evidence that bicarbonate binds directly to crocodilian Hb and promotes O 2 delivery independently of CO 2 . 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 CO 2 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 O 2 and CO 2 transport in the blood of crocodilians, where build-up of blood CO 2 and bicarbonate ions during breath-hold diving or digestion facilitates O 2 delivery, while Hb desaturation facilitates CO 2 transport as protein-bound CO 2 and bicarbonate.