Bicarbonate-Chloride Exchange in Erythrocyte Suspensions: Stopped-Flow pH Electrode Measurements

Abstract

A pH-sensitive glass electrode was used in a temperature-controlled stopped-flow rapid reaction apparatus to determine rates of pH equilibration in red cell suspensions. The apparatus requires less than 2 ml of reactants. The electrode is insensitive to pressure and flow variations, and has a response time of < 5 ms. A 20% suspension of washed fresh human erythrocytes in saline at pH 7.7 containing NaHCO(3) and extracellular carbonic anhydrase is mixed with an equal volume of 30 mM phosphate buffer at pH 6.7. Within a few milliseconds after mixing, extracellular HCO(3) (-) reacts with H(+) to form CO(2), which enters the red cells and rehydrates to form HCO(3) (-), producing an electrochemical potential gradient for HCO(3) (-) from inside to outside the cells. HCO(3) (-) then leaves the cells in exchange for Cl(-), and extracellular pH increases as the HCO(3) (-) flowing out of the cells reacts with H(+). Flux of HCO(3) (-) is calculated from the dpH/dt during HCO(3) (-)-Cl(-) exchange, and a velocity constant is computed from the flux and the calculated intracellular and extracellular [HCO(3) (-)]. The activation energy for the exchange process is 18.6 kcal/mol between 5 degrees C and 17 degrees C (transition temperature), and 11.4 kcal/mol from 17 degrees C to 40 degrees C. The activation energies and transition temperature are not significantly altered in the presence of a potent anion exchange inhibitor (SITS), although the fluxes are markedly decreased. These findings suggest that the rate-limiting step in red cell anion exchange changes at 17 degrees C, either because of an alteration in the nature of the transport site or because of a transition in the physical state of membrane lipids affecting protein-lipid interactions.