Enhancement of Anion Equilibrium Exchange by Dansylation of the Red Blood Cell Membrane

Abstract

Dansylation of resealed red cell ghosts enhances the band 3 protein-mediated equilibrium exchange of sulfate ions. After dansylation the specific anion transport inhibitor 4,4''-diisothiocyanato-dihydrostilbene-2,2''-disulfonate (H2DIDS) is still capable of combining with its original binding site on the band 3 protein and of producing the same high degree of inhibition of sulfate exchange as in the untreated red cell ghost. Dansylation causes allosteric effects at the H2DIDS-binding site that exhibit themselves by an increased susceptibility to dinitrophenylation of 1 of the amino acid residues that is involved in the covalent bond formation with H2-DIDS and a decrease of the apparent KI values for 2 reversibly acting inhibitors that are known to produce their effects at the H2DIDS-binding site of the band 3 protein. The degree of enhancement of divalent anion exchange depends on both the pH that existed during dansylation and the pH at which the anion equilibrium exchange across the dansylated membrane is measured. The effect of dansylation reaches a broad maximum around pH 6.6. In untreated ghosts, divalent anion equilibrium exchange passes through a maximum around pH 6.3. After dansylation under optimal conditions at pH 6.6, anion equilibrium exchange as measured below the maximum of pH 6.3 is much less enhanced than above the maximum. Under suitable experimental conditions the maximum may be replaced by a plateau that extends up to at least pH 8.5. At this pH, the enhancement is about 100-fold. The pH dependence of divalent anion exchange becomes more similar to that of monovalent anion exchange. The apparent activation enthalpies for sulfate-equilibrium exchange across the modified membrane, as measured at pH 6.3 and 7.9, are indistinguishable, independent of temperature between 0 and 37.degree. C and amount to 146 kJ/mol. This is similar to the activation enthalpies measured in the untreated membrane. The mode of action of dansyl chloride is discussed on the basis of currently considered mechanisms of divalent anion transport, for which the pertinent equations are presented.