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 methemoglobin using a dual wavelength stopped-flow apparatus. The cellular reaction was considerably slower than the reaction of SCN- with methemoglobin in solution, indicating that SCN- diffusion and not chemical reaction was rate limiting. This view was confirmed by showing that the uptake rate followed saturation kinetics (K(m) approximately 9 mM), thus indicating that SCN- transport involves a facilitated diffusion process. Addition of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) totally inhibited SCN- uptake, thus identifying band 3 as the sole facilitator. Substitution of iodide or sulfate for trans chloride, slowed SCN- uptake by 4-fold and 35-fold respectively. Reducing the trans chloride concentration from 150 to 2 mM decreased the extent of the reaction, and slowed the observed rate by about 2-fold. These results define a new approach for the continuous monitoring of monovalent anion exchange by human erythrocyte band 3.
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, Nebraska, USA.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D000838:Anions / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008706:Methemoglobin / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013861:Thiocyanates / Q000097:blood
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D013861:Thiocyanates; D008706:Methemoglobin; C031760:thiocyanate