Diffusion of carbon dioxide through lipid bilayer membranes: effects of carbonic anhydrase, bicarbonate, and unstirred layers.

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Abstract
Diffusion of 14C-labeled CO2 was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in n-decane. The results indicate that CO2, but not HCO3-, crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments one-way fluxes between identical solutions at constant pCO2 [CO2 tension] but differing [HCO3-] and pH were studied. In the absence of carbonic anhydrase (CA) the diffusion of CO2 through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of CO2 is too slow to permit the high [HCO3-] to facilitate tracer diffusion through the unstirred layers. Addition of CA (1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the CO2 flux, which is proportional to [HCO3-] over the pH range 7-8. In the presence of CA the hydration-dehydration reaction is so fast that CO2 transport across the entire system is rate limited by diffusion of HCO3- through the unstirred layers. In the presence of CA when the ratio [HCO3- + CO3=]:[CO2] > 1000 (pH 9-10), the CO2 flux reaches a maximum value. Under these conditions the diffusion of CO2 through the membrane becomes rate limiting, which allows an estimate of the permeability coefficient of the membrane to CO2 of 0.35 cm s-1. In a 2nd series of experiments the effects of CA and buffer concentration on the net flux of CO2 were studied. CA stimulates the net CO2 flux in well buffered, but not in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCO3- to facilitate the net transport of CO2 through the unstirred layers.