Transient-state kinetic evidence for intersubunit allosteric hysteresis during band 3 anion exchange

On-line, dual-wavelength stopped-flow spectroscopy was used to follow continuously, the uptake of dithionite (S2O4(2-)) into human erythrocyte resealed ghosts. We describe the general characteristics of a pre-steady-state transient phase measured both with chloride and sulfate as co-anions. We then quantitatively characterize the dithionite concentration dependence of the amplitude factor and relaxation constant (kR) for the transient phase measured in sulfate medium. We also study the dithionite dependence of the steady-state velocity. Our results suggest that dithionite induces a slow conformational change in band 3 leading to hysteresis in the transport velocity. As many as 25 turnovers of the transport cycle per monomer can occur prior to attainment of steady state. Both kR and the amplitude factor for the transient phase were dithionite concentration dependent. In addition, the steady-state velocity showed apparent negative cooperativity. To discriminate between monomeric and intersubunit allosteric hysteresis, we performed a series of critical kinetic tests with cells labeled partially with 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Coverage of 85% of the band 3 monomer population with DIDS caused kR to decrease about 10-fold, the dithionite concentration dependence of kR to change significantly, and the apparent negative cooperativity for the steady-state velocity to be eliminated. These results suggest that intersubunit allosteric hysteresis makes a significant contribution to dithionite transport by band 3.