pH equilibration in human erythrocyte suspensions

Summary

A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH⁻ or H⁺ was determined over a wide range of extracellular pH (4–11) in CO₂-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0>pH>11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH⁻ or H⁺ moves across the red cell membrane. Flux and velocity constants can be calculated from the initiald pH/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4–11), the greater the absolute value of total OH⁻ and/or H⁺ flux. Pretreatment with SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H⁺ and OH⁻ may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H⁺ and OH⁻ transport during CO₂-free pH equilibration.