Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO 2 . Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and β-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO 2 , which would permit effective O 2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O 2 -affinities, there is considerable variation in sensitivity to Cl − ions and ATP, which appears to be at least partly attributable to variation in the extent of NH 2 -terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O 2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.
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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.
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- Award ID(s):
- 1736249
- NSF-PAR ID:
- 10356591
- Date Published:
- Journal Name:
- Journal of Experimental Biology
- Volume:
- 224
- Issue:
- 15
- ISSN:
- 0022-0949
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1242/jeb.242615
