Subunit interaction effects were identified for isolated human erythrocyte band 3, the anion exchanger, by observing both static and stopped-flow kinetic protein fluorescence changes associated with inhibitor binding to the intramonomeric stilbenedisulfonate site. We measured the the rate of conformational changes associated with reversible binding of H2DIDS (4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate). The rate of H2DIDS release was also measured. As a test for subunit interactions, we studied the effect of partial labeling of the band 3 monomer population with H2DIDS on the equilibrium and kinetics of H2DIDS reversible binding to the remaining monomers. The results showed biphasic kinetics for control band 3, with a pseudo-first-order ligand dependence for the fast phase followed by a slow ligand-independent relaxation. A second-order “on” rate constant for the fast phase was determined to be (1.2 ± 0.1) × 107 M−1 s−1, while the associated “off” rate constant was found to be 1.1 ± 0.5 s−1. From these kinetic constants, we calculated a Kd value of 95 ± 50 nM, which is in excellent agreement with the Kd value determined at thermodynamic equilibrium (110 ± 9 nM). Covalent labeling of 75% of the band 3 monomer population with H2DIDS changed the kinetics of the fast phase, slowing the apparent rate by changing the order of the reaction from pseudo-first-order to zero-order. Partial labeling did not affect the ligand-independent relaxation. Separate measurements of the H2DIDS “off” rate also showed a biphasic time course, with a 20-fold difference in apparent rate constants. Covalent labeling of 75% of the band 3 monomer population increased the ratio of fast to slow phases for H2DIDS release from the remaining monomers. This change in the “off” reaction occurred after about 50% of the band 3 monomer population was labeled. A similar nonlinear effect was observed for the apparent value for H2DIDS binding to band 3. In this case, partial labeling caused a 5-fold increase in Kd. On the basis of these findings, we conclude that covalent labeling of one band 3 monomer with H2DIDS significantly alters H2DIDS reversible binding to the second monomer in the dimer. These inhibitor binding results, when taken together with our transport kinetic findings [Salhany, J. M., & Cordes, K. A. (1992) Biochemistry 31, 7301–7310], offer strong support for an allosteric subunit interaction hypothesis for band 3 structure-function.
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