Identification of the residues involved in the oxygen-linked chloride-ion binding sites in human deoxyhemoglobin and oxyhemoglobin.

The number of Bohr protons released upon oxygenation of human hemoglobin was measured at 25 degrees C and 37 degrees C as a function of the concentration of chloride ions. From the results obtained association constants could be evaluated for the binding of chloride ions to both deoxy and oxyhemoglobin at these two temperatures. Furthermore, pK values could be determined for those protonic groups involved in chloride ion binding to deoxy and oxyhemoglobin. From these data it was inferred that in oxyhemoglobin only imidazole groups are participating in chloride binding, whereas in deoxyhemoglobin the chloride binding site contained the alpha NH2 group of valine-1 alpha. The same conclusions were reached by measuring the pK values of the aminogroups of valine 1 alpha and valine-1 beta at different temperatures and ionic strengths. The pK values were measured by following the rate of reaction of 1-fluoro-2,4-dinitrobenzene with the alpha-NH2 group by spectrophotometric means. We further showed that binding of Cl-, Br- and I- to oxyhemoglobin follows the lyotropic or Hofmeister series, while this effect is much less for deoxyhemoglobin. This result indicates that for the binding of anions to oxyhemoglobin interactions with non-polar groups contribute to the free-energy change of binding.