Hydroxyl ion movements across the human erythrocyte membrane: Measurement of rapid pH changes in red cell suspensions

A stopped flow rapid reaction apparatus capable of following changes of ±0.02 pH unit in 0.1 ml of solution in less than 0.005 sec has been developed, utilizing a commercially available pH-sensitive glass electrode. Using this instrument, extracellular pH at 37°C was followed from less than 0.025 sec to 300 sec after mixing equal volumes of the following C0₂ -free solutions: (A) normal human red cells, washed three times and resuspended in 150 mM NaCI at pH 7.2 with a hematocrit of 18 %; and, (B) 150 mm NaCl adjusted with HC1 or NaOH to pH 2.1 to pH 10.3. A minimum of 2 ml of mixture had to flow through the electrode chamber to ensure complete washout. The mixing process produced a step change in the pH of the extracellular fluid, after which exchanges across the red cell membrane and buffering by intracellular hemoglobin caused it to return toward pH 7.2 with an approximately exponential time course. Under the assumption that pH changes after mixing represent exchanges of hydroxyl for chloride ions across the cell membrane, hydroxyl ion permeabilities (Pol-H in cm/sec) were calculated and found to vary from 2× 10^-4 at pH 9 to 4 × 10^-1 at p_H 4 according to the empirical relationship P_OH- = 170 exp (-1.51 pH). The form of the dependence of P_OE⁻ on extracellular pH does not appear compatible with a simple fixed charge theory of membrane permselectivity.