NMR study of relative oxygen binding to the α and β subunits of human adult hemoglobin

NMR spectra of the downfield region of normal adult hemoglobin are reported as a function of oxygenation and temperature. Spectra were run in D₂O at pD 7.4. A specially made NMR tube insert allowed precise measurement of the degree of oxygenation and of methemoglobin formation before and after taking the NMR spectrum. Plots of the estimated intensity of the most downfield prominent NMR peak, identified as arising from a deoxy β subunit by Davis et al. versus the average degree of oxygenation y, measured optically, yield a nearly straight line within experimental error, for samples stripped of organic phosphates and for samples containing 2,3 diphosphoglycerate or inositol hexaphosphate. Intensities of peaks further upfield than this peak, previously attributed to deoxy α subunits, are difficult to measure directly especially for samples containing inositol hexaphosphate. The latter samples show broadening in these α peaks as the degree of oxygenation increases. This extra broadening appears to increase with temperature. Linearity of the β peak intensity with oxygenation is expected if there is no large oxygen affinity difference between α and β subunits. However, the cooperativity of binding, and inaccuracy of the data, make it impossible to make accurate estimates of affinity differences.