A NOVEL RAPID-REACTION SPECTROPHOTOMETRIC METHOD FOR MONITORING MONOVALENT ANION-EXCHANGE BY HUMAN ERYTHROCYTE BAND-3

Thiocyanate (SCN-) uptake into human erythrocytes and resealed ghosts was measured by monitoring the intracellular reaction of SCN- with met hemoglobin using a dual wavelength stopped-flow apparatus. The cellula r reaction was considerably slower than the reaction of SCN- with meth emoglobin in solution, indicating that SCN- diffusion and not chemical reaction was rate limiting. This view was confirmed by showing that t he uptake rate followed saturation kinetics (K-m similar to 9 mM), thu s indicating that SCN- transport involves a facilitated diffusion proc ess. Addition of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) totally inhibited SCN- uptake, thus identifying band 3 as the sole fa cilitator. Substitution of iodide or sulfate for trans chloride, slowe d SCN- uptake by 4-fold and 35-fold respectively. Reducing the trans c hloride concentration from 150 to 2 mM decreased the extent of the rea ction, and slowed the observed rate by about 2-fold. These results def ine a new approach for the continuous monitoring of monovalent anion e xchange by human erythrocyte band 3.