The plane of cleavage in human ferrihemoglobin. I. Ultraviolet difference spectroscopy.

X-ray diffraction studies have shown that hemoglobin has two predominant interfaces in the tetramer at which dissociation to dimers could occur. These interfaces have been designed as alpha1-beta1 and alpha1-beta2. There are 2 tyrosyl residues and 1 tryptophanyl residue in the alpha1-beta2- interface but only 1 tyrosyl residue in the alpha1-beta1 interface exposed to the solvent are perturbed. The ultraviolet difference spectrum between ferrihemoglobin dissociated in 1 M NaClO4 and undissociated hemoglobin revealed two negative peaks, one at 292.5 nm and another at 285 nm. This difference spectrum is due to tyrosyl and tryptophanyl residues which reside on the plane of cleavage and were exposed to 1 M NaClO4 upon dissociation. Hence, dissociation must have occurred along the alpha1-beta2 interface to yield alpha1 beta1 dimers. The deltaF degrees value extrapolated to zero salt concentration calculated on the basis of difference spectroscopy and sedimentation velocity experiments is 8.6 plus or minus 0.7 kcal per mol at pH 7.1 (K equals 4.5 times 10-7.     

pH-dependent Soret difference spectra of the deoxy and carbonmonoxy forms of human hemoglobin and its derivatives.

The transition from deoxy to oxystructure of hemoglobin A (Hb) is accompanied by the breaking of the salt bridges formed by C-terminal residues in deoxy-Hb. This, in turn, changes the state of the heme. The switch between these different allosteric forms can be followed by changes in the optical absorbance spectra (Perutz, M. F., Ladner, J. E., Simon, S. R., and Ho, C. (1974), Biochemistry 13, 2163). Using difference spectroscopy in the soret region, pH-dependent spectral changes of Hb and its derivatives (carbamylated at both the alpha-NH2 groups, alpha2cbeta2c; N-ethylsuccinimide hemoglobin, NES-Hb) in their deoxy and carbonmonoxy forms were measured. From these measurements, the pK values of histidine-146beta and valine-1alpha in deoxy-Hb were determined to be 8.6 +/- 0.2 and 7.7 +/- 0.1, respectively. In carbonmonoxy-Hb a pK value of 6.3 +/- 0.1 was found.     

EPR spectral changes of nitrosyl hemes and their relation to the hemoglobin T-R transition

EPR spectra of nitrosyl hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25 degrees C. After the equilibration of NO among the alpha and beta subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO-beta-hemes and the other two arising for NO-alpha-hemes of molecules in the high- and low-affinity conformations. The fractional amounts of alpha subunits exhibiting the high-affinity spectrum fitted the two-state model (Edelstein, S.J. (1974) Biochemistry 13, 4998-5002) with the allosteric constant L = 7.10(6) and relative affinities cNO alpha and cNO beta approx. 0.01. Hemoglobin has been marked with nitric oxide one chain using low-saturation amounts of nitric oxide. The EPR spectra was studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high-affinity spectrum fitted the two-state model with L = 7 . 10(6), c)2 = 0.0033 and cNO alpha = 0.08, instead of cNO alpha = 0.01. Thus, the two-state model is not adequate to describe the conformational transition of these hybrids. The results present evidence of the non-equivalence between oxygen and nitric oxide as ligands.