Carbonic anhydrase activity in intact red blood cells measured with 18O exchange

We have used a stirred, temperature-regulated, reaction vessel separated by a Teflon membrane from the ion source of a mass spectrometer to monitor continuously the time course of disappearance of C18O16O, mass 46, at chemical equilibrium as the 18O exchanges with 16O in water. This instrument is sensitive to less than 0.01 mm Hg of partial pressure of C18O16O with a response time of less than 3 s. The equation of Mills and Urey was used to calculate the hydration velocity constant for uncatalyzed or catalyzed homogenous solutions from the exponential disappearance of mass 46. Addition of red blood cells to the reaction mixture produces biphasic (double exponential) disappearance curve for mass 46. A theory of this process has been developed which describes the time course of [C18O16O] as a function of the catalytic factor for intracellular carbonic anhydrase (A) and the permeability of the cell membrane to HCO3- (P) in addition to the known values; water volume of the cells in the suspension, extracellular pH, the extracellular hydration reaction velocity constant, ku, and dehydration reaction velocity constant, ku. Using this theory, A and P were estimated from the disappearance curve for mass 46 at different values of hematocrit in the reaction mixture, both by a trial and error curve fitting procedure and by a more convenient graphical linearization method. The values of A and P obtained were very sensitive to small amounts of lysis (less than 1%), but the graphical method of analysis minimized this effect. For the blood cells of five normal subjects suspended in 24 mM bicarbonate in 145 mM NaCl at pH 7.4 and 37 degrees, using the graphical method we obtained an average value of 9,906 for A as compared to 19,900 for a comparable concentration of hemolysate. Correcting for a lower pH and chloride concentration inside the cell the latter figure would reduce to 17,500, still 80% higher than the intracellular value. The reason for this discrepancy is not clear. The average permeability of the red cell to bicarbonate ion was 3 X 10(-4) cm/s.