Red blood cell carbonic anhydrase mediates oxygen delivery via the Root effect in red drum

Oxygen (O₂) and carbon dioxide (CO₂) transport are tightly coupled in many fishes as a result of the presence of Root effect hemoglobins (Hb), whereby reduced pH reduces O₂ binding even at high O₂ tensions. Red blood cell carbonic anhydrase (RBC CA) activity limits the rate of intracellular acidification, yet its role in O₂ delivery has been downplayed. We developed an in vitro assay to manipulate RBC CA activity while measuring Hb-O₂ offloading following a physiologically relevant CO₂-induced acidification. RBC CA activity in red drum (Sciaenops ocellatus) was inhibited with ethoxzolamide by 53.7±0.5%, which prompted a significant reduction in O₂ offloading rate by 54.3±5.4% (P=0.0206, two-tailed paired t-test; n=7). Conversely, a 2.03-fold increase in RBC CA activity prompted a 2.14-fold increase in O₂ offloading rate (P<0.001, two-tailed paired t-test; n=8). This approximately 1:1 relationship between RBC CA activity and Hb-O₂ offloading rate coincided with a similar allometric scaling exponent for RBC CA activity and maximum metabolic rate. Together, our data suggest that RBC CA is rate limiting for O₂ delivery in red drum.