A comparison of the conformational stabilities of homologous hemoproteins: Myoglobin from several species, human hemoglobin and subunits

Using the magnitude of the Soret absorbance as an index of conformation, the unfolding of bovine, human, and turtle ferrimyoglobin (Mb), and of human ferrihemoglobin (Hb) and its subunits in guanidinium chloride has been studied. By applying the two-state unfolding model to the various Mb's and accounting for the protein-denaturant interactions using solubility data of the peptide groups and sidechains, estimates were made of the apparent conformational free energy of unfolding in the absence of denaturant. These results were compared with the corresponding values for horse and sperm whale Mb (Puett, D. (1973) J. Biol. Chem. 248, 4623–4639) and, also, with the values for seal and porpoise Mb calculated from H+ titration and Soret absorbance-pH data (Hartzell, Bradshaw, Hapner, and Gurd, (1968) J. Biol. Chem. 243, 690–696). The unfolding free energy of horse, bovine, seal, and sperm whale Mb was found to be in the range of 11–14 kcal/mole, and that of human, porpoise, and turtle Mb was between 7 and 9 kcal/mole. Thus, whereas differences occur in Mb stability within this phylogenetic spectrum, there is no correlation of the unfolding free energy with the emergence of these species during the evolutionary time scale. This indicates that, for the four orders of the mammalian class and the single order of the reptilian class investigated, there is no natural selection to increase the conformational stability of this particular hemoprotein in spite of the low magnitude of the unfolding free energy. Moreover, there appears to be no selection to produce a protein with a higher or lower oxygen affinity, at least in the mammals for which accurate data are available. The inter-species differences in the unfolding free energies do not correspond with the total number of amino acid substitutions between the various proteins. Also, relative to the structure of crystalline sperm whale Mb, it is not possible to explain in all cases the measured conformational stability differences of the various Mbs based on the location, structural role, and chemical nature of the substituted amino acids. The simplest interpretation of these data is that the total number of protein intramolecular and solvent interactions probably differ in most of these Mbs, and this may result in minor conformation changes in restricted regions of the proteins. The guanidinium chloride unfolding of Hb, the cyano derivative, and the cyano derivatives of the subunits, is quite different from that of the Mbs in that the transition occurs over a much broader concentration of guanidinium chloride. Also, the kinetics of unfolding and refolding indicate a more complicated unfolding scheme than exists with sperm whale Mb. The guanidinium chloride concentration required for 50% unfolding was appreciably higher in intact Hb than in the subunits, thus suggesting that an appreciable stabilization of the Hb structure can be attributed to subunit interactions. Glucosamine and mannose were found in the Mb fraction from turtle heart, but there was no evidence of carbohydrate in the mammalian Mbs studied.