[Time Course] [Anion Transport Inhibitors] [Title page]

Results and Discussion: Hydration and Dehydration of CO2

The model of anion exchange in Figure 4 supposes that the only transport pathway for bicarbonate is through band 3. It is possible, however, for bicarbonate to be transported across the membrane as CO2. Bicarbonate dehydration and CO2 transport could provide a shunt to dissipate bicarbonate gradients set up in the stopped-flow experiments. The CO2-bicarbonate reactions are summarized by the chemical equations:
equation 6a
Reactions 1 and 2 are very fast compared to the time scale of our experiments. At pH 7.4, bicarbonate is the predominant species. In the absence of carbonic anhydrase, reaction 3 occurs on a time scale comparable to that of our experiments (13, 14), while reaction 4 occurs on a time scale of hours in the absence of vigorous solution agitation (15).

To examine reactions 3 and 4, pH stat experiments were performed. Aliquots of HCl were added to a freshly-prepared solution of 150 mM NaHCO3 and 20 mM HEPES to maintain a constant pH of 7.5. The kinetics of HCl addition was biphasic characterized by time constants of 14 sec and 9 hours (data not shown). The two kinetic components correspond to reactions 3 and 4, respectively (13). The decrease in bicarbonate concentration, calculated from the amount of HCl added after completion of the first reaction, was 7%.

In the stopped-flow apparatus, the concentration gradient of bicarbonate was imposed within milliseconds in the stopped-flow apparatus, and the longest time constant for SPQ quenching was 1 sec. Since these times are smaller than the measured dehydration times, bicarbonate equilibrates via chloride-bicarbonate exchange across the membrane before appreciable dehydration takes place. The maximal change in bicarbonate concentration due to dehydration is 5-7%, which would be a small perturbation on the observed SPQ time course.

To confirm the insignificance of bicarbonate dehydration, kinetic experiments with resealed ghosts were performed in the presence of intracellular and extracellular carbonic anhydrase, and also in the presence of acetazolamide, an inhibitor of carbonic anhydrase. None of these treatments affected the exchange rates observed by SPQ fluorescence. Control experiments showed that carbonic anhydrase activity was not inhibited by SPQ. We conclude that dehydration of bicarbonate does not contribute significantly to the observed SPQ time course.

[Time Course] [Anion Transport Inhibitors] [Title page]