Inhibition of anion transport in human red blood cells by 5,5'-dithiobis(2-nitrobenzoic acid)

The uptake of [32P]phosphate into human red blood cells was inhibited (Ki = 0.6 mM) by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor (Ki = 7 mM). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[14C]-benzamido-4'-aminostilbene-2,2'-disulfonate, a potent inhibitor of anion transport (Ki = 1-2 microM), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.