PROTON NUCLEAR MAGNETIC-RESONANCE INVESTIGATION OF STRUCTURAL-CHANGES ASSOCIATED WITH COOPERATIVE OXYGENATION OF HUMAN ADULT HEMOGLOBIN

The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-27°C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H2O) and two exchangeable proton resonances (at about -6.4 and -9.4 ppm from H2O) as a function of oxygenation, the amount of oxygen bound to the α and β chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: in the absence of organic phosphates, there is no preferential O2 binding to the α or β chains; in the presence of organic phosphates, the α hemes have a higher affinity for O2 as compared to the β hemes; the ligand-induced structural changes in the hemoglobin molecule are not concerted; and 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 one or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric hemoglobin molecule. Thus, the present results show that two-state allosteric models are not adequate to describe the cooperative oxygenation of hemoglobin. In addition, the present results provide 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 hemoglobin molecules and in the solution state.