Synthesis of tritiated 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells.

The potent and specific inhibitor of anion permeability, 4,4'-diisothicyanostilbene-2,2'-disulfonic acid (DIDS) was synthesized in tritiated form ([3H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([3H]H2DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [3H]DIDS was substantially faster than that of [3H]H2DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [3H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2 X 10(6) molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein.