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.

The pH dependence of the binding of D-glycerate 2,3-bisphosphate to deoxyhemoglobin and oxyhemoglobin. Determination of the number of binding sites in oxyhemoglobin.

The number of Bohr protons released upon oxygenation has been measured over a large range of human hemoglobin concentrations (0.02 to 4.5 mM) in the presence of equimolar amounts of D-glycerate 2,3-bisphosphate. From these data the association constants for the binding of this organic phosphate to deoxyhemoglobin and oxyhemoglobin were calculated at different pH values. The maximum number of protons absorbed upon binding to oxyhemoglobin was determined as well. The maximum number of protons bound to deoxyhemoglobin upon binding of D-glycerate 2,3-bisphosphate was measured independently. From the pH dependence of the association constants and the maximum number of protons absorbed it could be concluded that only one D-glycerate 2,3-bisphosphate can be bound to both deoxyhemoglobin and oxyhemoglobin.

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.

Properties of protease-treated cytochrome c oxidase from beef heart.

About 45% of the protein can be removed from oxidized cytochrome c oxidase by treatment with proteolytic enzymes under a variety of conditions, leading to an increased heme to protein ratio. The principal spectroscopic parameters of cytochrome c oxidase are retained in the protease-treated enzyme. Of the overall catalytic activity 20% remained after digestion; the electron-transfer reactions were impaired but the affinity for cytochrome c appeared unchanged. Proteolysis resulted in removal of the hydrophobic subunit III and most of the smaller hydrophilic subunits, leaving a core, which basically consists of the two largest subunits I and II. The subunits I and/or II carry the prosthetic groups of the enzyme and at least one of the cytochrome c binding sites. The smaller subunits, however, are essential for optimal electron transfer and possibly have other functions as well.

The CO and NO Bohr effect of human hemoglobin with and without inositolhexaphosphate.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.