Human erythrocyte anion exchange site characterised using a fluorescent probe.

Abstract

A plasma membrane anion exchange system allows the red blood cell to replace intracellular bicarbonate rapidly with extracellular chloride to facilitate carbon dioxide release into the lungs. The protein responsible for this exchange is a glycoprotein of molecular weight about 93,000, called band 3, which comprises approximately 25% of the total red cell membrane protein and is present in the membrane as a non-covalent dimer1. The molecular mechanism of anion transport remains obscure, in spite of the characterisation of the extracellular anion binding site by a number of methods including interactions with disulphonic stilbene probes which act as specific inhibitors of anion exchange2. One of these compounds (4,4′-diisothiocyano-2,2′-disulphonic stilbene; DIDS) causes complete inhibition of anion exchange by reacting at approximately one site per band 3 monomer (∼2.5 nmol per mg protein)3. A fluorescent DIDS analogue, DBDS (4,4′-dibenzoamide-2,2′-disulphonic stilbene), binds reversibly and specifically to the DIDS reactive site where it also inhibits anion exchange6 . When bound, the fluorescence intensity of DBDS increases by two orders of magnitude and this has enabled us to investigate the binding kinetics of DBDS to red blood cell ghost membranes by fluorescence equilibrium and temperature-jump methods. Our equilibrium binding data indicate that there are two classes of DBDS binding sites on red cell ghosts. The reaction scheme that we present here suggests that the apparent high affinity (15–60 nM) for binding the first DBDS molecule is the result of a two step process: an initial binding of lower affinity (K1∼0.5 µM) to band 3 followed by a slow conformational change (∼5 s−1) in the binding site which acts to lock the DBDS molecule in place. A second DBDS molecule binds with low affinity (∼3 µM) to a site which is more difficult to characterise.