Kinetics of conformational changes associated with inhibitor binding to the purified band 3 transporter. Direct observation of allosteric subunit interactions.

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 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) x 10(7) 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.(ABSTRACT TRUNCATED AT 250 WORDS)