Rate of CO2 diffusion in the human red blood cell measured with pH-sensitive fluorescence

The diffusion rate of CO2, into and out of the red cell, was measured by using a stopped flow method with pH-sensitive fluorescence of 4-methylumbelliferone. A red cell suspension of 15% hematocrit with the PCO2 of 8 Torr (or 65 Torr) was mixed with the same amount of saline solution having 65 Torr (or 8 Torr). Carbonic anhydrase was added to the external solution at a concentration of 20 mg/100 ml in order to accelerate the hydration and dehydration reactions, so that the PCO2 change in the fluid could be observed instantaneously through pH. In the inward diffusion PCO2 showed a large change, suggesting a lack of HCO3- shift across the red cell membrane. In the outward diffusion, however, the PCO2 change was smaller, suggesting that H+ ions produced in the external solution by CO2 hydration were rapidly buffered by the red cell. The half-times of the inward and outward diffusions were, on an average, 0.08 and 0.13 sec, respectively. The results of the simulation revealed that the above difference in half-times was attributed to the difference in slope between the two dissociation curves with and without the HCO3- shift. The diffusion rate was almost constant and remained independent of the direction of CO2 flux. That is, at a low pH range the permeation of H+ ions across the red cell membrane was much faster than the diffusion rate of CO2.