Proton nuclear magnetic resonance investigation of structural changes associated with cooperative oxygenation of human adult hemoglobin

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Abstract
The structural changes associated with cooperative oxygenation of human adult Hb as a function of O2 saturation in aqueous media at neutral pH and at 25-27.degree. C were investigated by high-resolution NMR spectroscopy at 250 and 360 MHz. By monitoring the intensities of 2 hyperfine shifted proton resonances (at about -12 and -18 ppm from H2O) and 2 exchangeable proton resonances (at about -6.4 and -9.4 ppm from H2O) as a function of oxygenation, the amount of O2 bound to the .alpha. and .beta. chains of a Hb molecule could be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions could be investigated. In the absence of organic phosphates, there is apparently no preferential O2 binding to the .alpha. or .beta. chains. In the presence of organic phosphates, the .alpha. hemes apparently have a higher affinity for O2 as compared to the .beta. hemes. The ligand-induced structural changes in the Hb molecule are apparently not concerted. Some cooperativity must be present within the deoxy quaternary state during the oxygenation process. The variations of the exchangeable proton resonances as a function of oxygenation strongly suggest that the breaking of 1 or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric Hb molecule. Two-state allosteric models are not adequate to describe the cooperative oxygenation of Hb. Direct correlation to the ligand-induced structural changes (such as in the heme pockets and subunit interfaces) observed to occur in the crystals of deoxy- and oxy-like Hb molecules and in the solution state was provided.