Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, alligator mississippiensis

In the developing embryos of egg-laying vertebrates, O₂ flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O₂ flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O₂ transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O₂). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O₂ affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O₂ affinity and high CO₂ sensitivity. These results indicate that whole blood O₂ affinity is mainly regulated by ATP in the early embryo and by CO₂ and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.