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Dihydro; Cell Membrane Protein; Total Binding; Activation Enthalpy; Disulfonic Acid
Abstract:
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 >60% of DIDS binding were rapid, independent of temperature. Slow H2DIDS 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 μm, hematocrit 10%, 5-37°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 1.9×106 or 1.2×106 molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3×106 and 1.1×106. 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
