TRANSMEMBRANE HELIX-HELIX INTERACTIONS AND ACCESSIBILITY OF H2DIDS ONLABELED BAND-3, THE ERYTHROCYTE ANION-EXCHANGE PROTEIN

,4'Diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS), a bifunct ional inhibitor of anion exchange in erythrocytes, reacts with Lys-539 in band 3 at neutral pH and crosslinks to Lys-851 at alkaline pH. The accessibility of H2DIDS-labelled band 3 was determined using an anti- H2DIDS antibody and proteolysis. Competitive enzyme-linked immunosorbe nt assays (ELISAs) showed that a polyclonal antibody raised against H2 DIDS-labelled keyhole limpet hemocyanin bound a variety of stilbene di sulphonates in the following order of affinities, H2DIDS having the hi ghest affinity: H2DIDS>4,4'-diisothiocyanostilbene-2,2'- disulphonate (DIDS)> 4 cetamido-4'-isothiocyanostilbene-2,2'-disulphonate (SITS)>4, 4'-dinitrostilbene 2,2'-disulphonate (DNDS)>4,4'-diaminostilbene-2,2'- disulphonate (DADS). The antibody readily detected mono- or bifunction ally H2DIDS-labelled band 3 and proteolytic fragments on immunoblots. H2DIDS attached to Lys-539 is retained in a 7.5 kDa membrane associate d peptide after papain treatment of ghost membranes while the sequence around Lys-851 is more accessible. The band 3 proteolytic fragments p rotected by the membrane from proteolysis remained associated as a spe cific complex with a Stokes radius slightly smaller than the dimeric m embrane domain after solubilization in detergent solution and retained 82% of the amino acid content of the membrane domain. Circular dichro ism (CD) measurements of this H2DIDS-labelled complex showed that it h ad a very high helical content (86%). The loops connecting the transme mbrane segments in H2DIDS-labelled band 3 are therefore not required t o maintain transmembrane helix-helix interactions. Denatured band 3 pr elabelled with H,DIDS was more readily immunoprecipitated with the ant i-H2DIDS antibody than was native band 3 in detergent solution. Deglyc osylation of band 3 or proteolytic cleavage of the extramembranous loo ps did not enhance immunoprecipitation of H2DIDS-labelled band 3. The stilbene disulphonate inhibitor site is therefore relatively inaccessi ble and is bound by a bundle of helices in the native band 3 protein.