The anion-transport inhibitor H 2DIDS cross-links hemoglobin interdimerically and enhances oxygen unloading

Abstract

Human hemoglobin treated with equal concentrations of the anion-transport inhibitor H 2DIDS produces a right shift in the oxygen dissociation curve. Concomitantly, the Hill coefficient is reduced from n = 2.7 to 2.1. When higher concentrations of H 2DIDS are applied (H 2DIDS: hemoglobin= 5:0.5 mM), the Hill coefficient decreases further to 1.5 and the oxygen dissociation curve of hemoglobin is shifted slightly to the left of the control. Similar results were also obtained with DIDS instead of H 2DIDS. SDS-PAGE shows that H 2DIDS cross-links hemoglobin monomers mainly into dimers. Cross-linking is more effective under anaerobic conditions. With tritiated H 2DIDS the larger part of the radioactivity is found in the dimer position of hemoglobin. Separation of the α and β units of hemoglobin reacted with tritiated H 2DIDS demonstrated a stoichiometry of 2.2 and 2.4 molecules H 2DIDS per molecule α and β unit hemoglobin, leading to about 8–9 H 2DIDS molecules per native hemoglobin. The right shift produced in the hemoglobin oxygen dissociation curve and the cross-linking of monomers into dimers, especially under anaerobic condition, suggest that H 2DIDS can also react with those amino groups of hemoglobin which are involved in 2,3-DPG binding. A comparison of H 2DIDS, DIDS and 2,3-DPG at three different concentrations close to the hemoglobin concentration revealed a concentration dependent right shift in the oxygen dissociation curve with the order of potency 2,3-DPG > H 2DIDS > DIDS. The Hill coefficients ( n) at the three concentrations of 2,3-DPG demonstrated no changes, but H 2DIDS and DIDS reduced in a concentration-dependent manner the cooperativity of hemoglobin. Again, H 2DIDS is more potent than DIDS, especially at the low concentration. These anion-transport inhibitors provide novel approaches to the exploration of hemoglobin function.