Molecular Dissection of an Allosteric Protein by Using Ionic and Non-Ionic Co-Solutes and Their Impact on the Protein Function

We have studied the impact of co-solutes not considered allostetic effectors, both ionic and non-ionic, on the oxygenation function of human hemoglobin (Hb). In the presence of NaI, the oxygenation characteristics of Hb showed a complex behavior depending on the concentration of the halide salt. At concentrations of 0.1 M NaI or below, the oxygenation affinity for the first oxygen, K1, decreased as the concentration of the halide salt was increased. At concentrations between 0.1 M and 0.5 M NaI, the oxygenation affinity for the last oxygen, K4, decreased slightly, while K1 was unaffected. At concentrations between 0.5 M and 1 M NaI, K1 values increased gradually with increasing concentrations of the salt, while K4 values remained practically constant. Compared to Hb under stripped solution conditions, the oxygenation curve for Hb in the presence of 2 M NaI showed a decreased affinity for oxygen and a reduced cooperativity, being its symmetric shape the most striking feature, i.e., the curve resembled that of an allosterically linked two-oxygen binding site derivative. Size exclusion chromatography revealed that Hb was mostly dimerized under the above-mentioned solution condition. The retention of cooperativity contrast greatly with the widely accepted knowledge that dimers: (1) do not show cooperativity, and (2) exhibit high oxygen affinity for oxygen. Oxygenation experiments carried out using non-ionic amphiphatic solutes of low molecular weight showed characteristics that were similar to those exhibited in the presence of NaI, especially in the presence of co-solutes that show moieties with high hydrophobicity. Since these features are not observed in the presence of NaCl, we conclude that the dimerization is caused by the iodide ion altering the characteristics of water.