A comparison of the resistance of human and horse ferrihemoglobin to acid denaturation.

Many of the stability characteristics of horse ferrihemoglobin (Hb+) in acid solutions, such as pH dependence and susceptibility to stabilization by iron ligands, are shared by human ferrihemoglobin, but striking differences between the two proteins exist. The most noticeable is the much greater rate of denaturation of the human protein at all pH values. Other differences include a shift to higher pH in the equilibrium between native and acid-denatured forms, differences in the temperature at which the temperature effect on the equilibrium-pH curve reverses, a complete absen.ce in human Hb+ of the dependence of regeneration rate on extent of denaturation that is found with horse Hb+, and a related failure of the regeneration kinetics of human Hb+ to follow the inhibited regeneration model developed for horse Hb+. pH-stat experiments, and rapid flow titration curves of both native and denatured forms, indicate that human Hbf has about four more masked groups (presumably imidazoles) than horse Hb+, although both proteins contain the same number of histidine residues. Although regeneration as measured by reappearance of the Soret band occurs readily, there is very little remasking of the liberated groups unless a stabilizing ligand (cyanide) is present; even then recovery is far from complete. It has been observed with both proteins that the band at 370 rnp, characteristic of denatured oxidized heme-protein complexes, disappears much more rapidly during regeneration than the Soret band, characteristic of native protein, reappears. The discrepancy is more marked with human than with horse Hb+.