Bircarb_CO2_crocodilian_hemoglobin

Crocodilians are reportedly unique among vertebrates in that their hemoglobin (Hb)-O2 affinity is allosterically regulated by bicarbonate ions. This unusual mode of allosteric control was originally inferred from indirect evidence and has not been verified by direct verification of bicarbonate binding tocrocodilian Hb. Here, we report results of equilibrium and kinetic experiments designed to distinguishand quantify the allosteric effects of CO2 and bicarbonate on Hb oxygenation in crocodilians. Studies onHb from spectacled caiman (Caiman crocodilus) revealed that both CO2 and bicarbonate stronglydecrease Hb-O2 affinity by binding with high affinity to the Hb, likely to the same positively charged site in the deoxy (T) protein conformation. We experimentally confirmed the same mode of allosteric regulation in the Hbs of a phylogenetically diverse set of other caiman, alligator, and crocodile species, suggesting that this novel allosteric property evolved >80-100 million years ago in the common ancestor of modern crocodilians. We found that the spectacled caiman hemolysate resolves into two functionally identical Hb fractions, a tetramer and as a higher-order aggregate, both containing the enzyme carbonicanhydrase. We propose that crocodilian Hb and carbonic anhydrase act in concert, whereby Hb converts bound CO2 to bicarbonate in the systemic circulation, followed by conversion of bicarbonate back to CO2 by carbonic anhydrase during pulmonary transit. This tight linkage between O2 and CO2 transport allows considerable O2 release to tissues when CO2 and bicarbonate levels increase in the blood, and it facilitates transport of Hb-bound CO2 and bicarbonate at low blood O2 levels.