Electrostatic effects in hemoglobin: Bohr effect and ionic strength dependence of individual groups.

The electrostatic treatment applied in the preceding paper in this issue [Matthew, J. B., Hanania, G.I.H., & Gurd, F.R.N. (1979) Biochemistry (preceding paper in this issue)] to the titration behavior of individual groups in human deoxyhemoglobin and oxyhemoglobin was applied to the computation of the alkaline Bohr effect at various values of ionic strength. The enhanced proton binding of deoxyhemoglobin in the pH range of 6--9 was accounted for at ionic strength 0.01 M by the effects of the unique charge distributions of ionizable groups in the two quaternary states. At ionic strength 0.10 M the effects of 2--4 bound anions had to be considered in addition in the deoxyhemoglobin charge configuration. At the higher ionic strength 10 groups per tetramer contributed to the Bohr effect, whereas 28 groups were contributory at the lower ionic strength. The ionic strength dependence of individual groups in the two tetrameric structures as well as in the alpha-chain monomer was explained in terms of the electrostatic treatment. This examination showed that the differences in electrostatic behavior of deoxy- and oxyhemoglobin follow from particular dissymmetries in their configurations with respect to charge and static solvent accessibility.

Absorption spectra of sperm-whale ferrimyoglobin.

1. Sperm-whale ferrimyoglobin was found to contain 0.308% of Fe, on a dry weight basis, corresponding to a molecular weight of 18130. The solid takes up moisture to an equilibrium state, and was then assayed to contain 0.280% of Fe. 2. Absorption spectra are presented for acidic ferrimyoglobin, Fe(+)(H(2)O), and its conjugate base, Fe-OH, as well as for the fluoride and cyanide complexes, within the range 200-2500mmu. Data for ferromyoglobin-carbon monoxide, Fe(II)-CO, in the visible region are also included. 3. Minor spectral differences have been found between whale and horse myoglobins, particularly in the effect of temperature on the visible-absorption spectrum of Fe-OH.

Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion.

The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.

Electrostatic effects in hemoglobin: hydrogen ion equilibria in human deoxy- and oxyhemoglobin A.

The modified Tanford-Kirkwood theory of Shire et al. [Shire, S. J., Hanania, G.I.H., & Gurd, F.R.N. (1974) Biochemistry 13, 2967] for electrostatic interactions was applied to the hydrogen ion equilibria of human deoxyhemoglobin and oxyhemoglobin. Atomic coordinates for oxyhemoglobin were generated by the application of the appropriate rigid rotation function to alpha and beta chains of the deoxyhemoglobin structure [Fermi, G. (1975) J. Mol. Biol. 97, 237]. The model employs two sets of parameters derived from the crystalline protein structures, the atomic coordinates of charged amino acid residues and static solvent accessibility factors to reflect their individual degrees of exposure to solvent. Theoretical titration curves based on a consistent set of pKint values compared closely with experimental potentiometric curves. Theoretical pK values at half-titration for individual protein sites corresponded to available observed values for both quaternary states. The results bring out the cumulative effects of numerous electrostatic interactions in the tetrameric structures and the major effects of the quaternary transition that result from changes in static solvent accessibility of certain ionizable groups.

Characterization of the reaction of methyl acetimidate with sperm whale myoglobin.

The effects of pH, acetimidate concentration, temperature, and reaction time of methyl acetimidate with sperm whale myoglobulin have been assessed. Reaction at pH 9.8 and 15 degrees C for 30 min with a sixfold excess of methyl acetimidate relative to each amino group yielded six acetimidomyoglobin derivatives which were separated and purified. Reaction with tetrahydrophthalic anhydride revealed the number of amino groups that remained unreacted in each separated component and made possible further subractionation. Modification at the NH2 terminus was quantitated by automated stepwise Edman degradation. The acetimidyl and tetrahydrophthalyl groups, were readily removable. The potentiometric titration of three of the completely deprotected components showed identity with the parent untreated sperm whale myoglobin. The first of two major products was acetimidated at all 19 epsilon-amino groups but not at the NH2 terminus. The second major product bore a blocked NH2 terminus but retained one unmodified epsilon-amino group, identified after modification by trinitrobenzenesulfonate as lysine residue 77. Of the minor components, one was identified as completely acetimidated at all 20 amino groups. The other three minor components appeared to contain irreversible by-products.

Electrostatic contributions to the energetics of dimer-tetramer assembly in human hemoglobin: pH dependence and effect of specifically bound chloride ions.

The pH dependence and effects of specifically bound chloride ions on the electrostatic contribution to the energetics of human hemoglobin dimer-tetramer assembly were computed for deoxy- and liganded hemoglobin. In the absence of bound chloride, the electrostatic contribution models the observed contrasting pH dependence of dimer-tetramer assembly for deoxy- and oxyhemoglobin. The effect of specifically bound chloride on the computations depends on the number and placement of the anions. Deoxy assembly shows a greater sensitivity to anion binding, with effects propagating as far as 32 A from the binding site. This sensitivity suggests a mechanism for electronic communication with the heme. At pH 7.4, 24-34% of the experimental value for deoxy and 73-85% for oxy dimer-tetramer assembly stabilization are predicted. Together with the findings of Chu and Ackers [Chu, A. H., & Ackers, G. K. (1981) J. Biol. Chem. 256, 1199] and other recent work, these results suggest that salt bridge formation is not the dominant energetic factor favoring deoxyhemoglobin dimer-tetramer assembly. Results of this work suggest that the marked electrostatic stabilization favoring oxy dimer-tetramer assembly may be a significant contributor to the quaternary enhancement observed in assembly reactions whereas the nonelectrostatic factors favoring deoxy dimer-tetramer assembly may be largely responsible for quaternary constraint.