The pH dependence of red cell membrane transport of titratable anions studied by NMR spectroscopy

The effects of varying extracellular pH on the rates of uptake of titratable anions by human erythrocytes under conditions of constant intracellular pH have been determined for a series of highly related anions, the phosphate "analogs." These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratable protons: phosphate (HPO4(2-), pKa 6.8); phosphite (HPO3(2-), pKa 6.4); hypophosphite (H2PO2-); methylphosphonate ((CH3)PO3(2-), pKa 7.4); dimethylphosphinate ((CH3)2PO2-); fluorophosphate PO3F2-, pKa 4.7); and thiophosphate (HSPO3(2-), pKa 5.5). Suspensions of intact, Cl(-)-loaded erythrocytes (intracellular pH, 7.2) were incubated at 37 degrees C in isotonic buffers (pH 4-8) containing 60 mM phosphate analog for specified time intervals, whereupon influx was halted by the addition of 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), an inhibitor of anion exchange. The intracellular anion concentrations were determined from 31P or 19F nuclear magnetic resonance spectra from the erythrocyte suspensions. The influx rates for the titratable phosphate analogs exhibited bimodal pH dependence, reaching maximal levels at pH values that increased with increasing anion pK. This pH-dependent behavior is consistent with a transport channel that contains a titratable regulatory site which interacts with the translocated anion. Based upon the Henderson-Hasselbalch equation, the probability that a titratable anion will have an electric charge of equal magnitude to that of the titratable carrier is highest at a pH value exactly midway between the pK of the regulatory site and that of the anion. The pH maxima observed for the phosphate analogs indicate a pK for this site of 5.5 at 37 degrees C. Intracellular pH changes associated with influx indicated that transport of the "fast" anion phosphite is largely in monoionized form. Intracellular pH changes associated with transport of slow anions were predominantly determined by partial ionic equilibrium effects and did not indicate the ionization state of the transported anion.