Quaternary Structure and Spin State of Human Fetal Methemoglobin

In an attempt to shed more light on the physiological function of fetal hemoglobin, we studied the alteration of quaternary structure and switch in spin state of the fetal methemoglobin and compared it to the corresponding adult protein. Using the pulse‐radiolysis technique, solutions of fetal human methemoglobin were irradiated in order to reduce a single heme‐iron within the protein tetramers. The valence‐hybrids thus formed were reacted with oxygen. Kinetics of the reactions were studied. The effects of pH and inositol‐hexaphosphate (IHP) were examined. The kinetics of the ligation of oxygen to stripped valence‐hybrids showed a single‐phase behaviour at the pH range 7–9. As the pH was lowered below 6.5, a second slower phase became apparent. This slow phase consisted of ∼ 50% at pH 5.8. In the presence of IHP above pH 7.4, the kinetics of oxygen‐binding was of a single‐phase. As the pH was lowered a transition to a second, slower phase was noticed. Below pH 7 the slower phase was the only detectable one. The analysis of the relative contribution of the faster phase to the total reaction, as a function of the pH, showed a typical sigmoidal transition curve characterized by a pK = 7.2 and a Hill parameter n = 3.06. On this basis it is concluded that stripped, fetal human methemoglobin resides in an R quaternary structure while the presence of IHP stabilizes the T structure.at pH below 7.2. Thus, this protein exhibits a higher degree of intrinsic low affinity state than that of methemoglobin A, while in the presence of IHP the stabilization of the low affinity state of the fetal protein by the organic phosphate is smaller in comparison to the adult protein. The switch between the high spin aquomet‐ and the low spin hydroxymet‐derivatives of adult and fetal human hemoglobins was studied optically in detail. These switches were found to be only slightly affected by IHP, and exhibited very low cooperativity (pK = 8.04; n = 1.1 and pK = 8.10; n = 1.3 for adult methemoglobin when stripped and in the presence of IHP, respectively; pK = 8.18; n = 1.11 and pK = 8.21; n = 1.28 for fetal methemoglobin when stripped and in the presence of IHP, respectively). These findings lead to the conclusion that the transition between quaternary structures in either human or fetal methemoglobins is not coupled to the switch of the spin state of the ferric heme.