pH equilibration in human erythrocyte suspensions

Abstract

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 greater than pH greater than 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 initial dp H/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 valute 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.