A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS).
DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.
D001557:Benzenesulfonates / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D013267:Stilbenes / Q000378:metabolism* / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism*; D013696:Temperature; D013861:Thiocyanates / Q000378:metabolism
D001557:Benzenesulfonates; D008565:Membrane Proteins; D013267:Stilbenes; D013431:Sulfates; D013861:Thiocyanates