Effects of hemoglobin symmetry in a statistical equilibrium model for oxygen binding

The effect of hemoglobin symmetry on the statistical mechanics of its motions is considered. Hemoglobin binding equilibrium constants are presented in which symmetry factors appear that differ from one binding step to another. Inclusion of the symmetry factors improves the fit of a symmetry-modified Koshland, Némethy, Filmer expression (1966, Biochemistry, 5:365-385) with tetrahedral oxy-oxyhemoglobin subunit interactions to the high-ionic-strength binding curve of Rossi-Fanelli, Antonini and Caputo (1961, J. Biol. Chem., 236:397-400).     

Reaction rates of oxygen with hemoglobin measured by non-equilibrium facilitated oxygen diffusion through hemoglobin solutions

The purpose of this study was to verify the concept of non-equilibrium facilitated oxygen diffusion. This work succeeds our previous study, where facilitated oxygen diffusion by hemoglobin was measured at conditions of chemical equilibrium, and which yielded diffusion coefficients of hemoglobin and of oxygen. In the present work chemical non-equilibrium was induced using very thin diffusion layers. As a result, facilitation was decreased as predicted by theory. Thus, this work presents the first experimental demonstration of non-equilibrium facilitated oxygen diffusion. In addition, association and dissociation rate parameters of the reaction between oxygen and bovine and human hemoglobin were calculated and the effect of the homotropic and heterotropic interactions on each rate parameter was demonstrated. The results indicate that the homotropic interaction--which leads to increasing oxygen affinity with increasing oxygenation--is predominantly due to an increase in the association rate. The heterotropic interaction--which leads to decreasing oxygen affinity by anionic ligands--appears to be effected in two ways. Cl- increases the dissociation rate. In contrast, 2,3-diphosphoglycerate decreases the association rate.     

Determination of hemoglobin affinity for oxygen by a polarographic method

The method of determination of hemoglobin affinity for oxygen based on polarographic analysis and the method of half-saturation values P50 calculation are described. A special design of a polarographic cell is proposed for measurements in a wide temperature range. The results of temperature and pH dependences of the hemoglobin affinity for oxygen and erythrocytes of the White-Sea cod are presented.     

Kinetics of oxygen binding to hemoglobin A

The two-state model [Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118] postulates a single conformational change which, in the case of hemoglobin, has been related to the structural differences between deoxy and ligated hemoglobins [Perutz, M. F. (1979) Nature (London) 228, 726-739]. In its simplest form, the model does not represent satisfactorily either the equilibrium or the kinetics of the hemoglobin-oxygen reaction. The kinetic difficulty is with the rate of dissociation from the T-state, and may be met by assuming a wide difference in behavior between alpha- and beta-subunits. Experiments with Ni-Fe hybrids, however, show almost identical rates of combination with, and dissociation from, the two types of subunit, both of which develop R-like reactions as the pH is raised, the alpha-Fe-subunits at lower pH than the beta-Fe-subunits [Shibayama, N., Yonetani, T., Regan, R. M., and Gibson, Q. H. (1995) Biochemistry 34, 14658-14667]. The reactions of oxygen with hemoglobin A and the effect of pH upon them may be represented by assuming behavior of its subunits similar to that of the Ni-Fe hybrids. In such a scheme, alpha-alpha and beta-beta interactions become important elements in cooperativity, and more than two allosteric states are required, for reconsideration of the structural basis of cooperativity.     

A thermodynamic model for gelation of sickle-cell hemoglobin

The two-step concept of gelation of sickle-cell hemoglobin (Minton, 1973) provides the basis for a thermodynamic model to account for the macroscopic solution properties of sickle-cell hemoglobin in terms of microscopic structure and interactions. Step 1, the formation of a rod-like microtubular array, is treated as thermodynamically equivalent to a precipitation. Step 2, the alignment of microtubules to form a nematic phase, is treated as an isotropic-nematic transition in a suspension of interacting rod-like particles. Upon combination of these two steps a qualitative temperature-composition phase diagram is obtained.The results of several experimental studies on sickle-cell hemoglobin solutions, including measurements of solubility, sedimentation equilibrium and viscosity, are reviewed and it is shown that the proposed model provides a unified interpretation of many of the observed physical properties of these solutions.     

A thin-layer optical cell for spectroscopic studies of oxygen binding to hemoglobin

A thin-layer sample cell for examination of the spectra of hemoglobin at various degrees of gaseous-ligand saturation has been constructed to fit into a Cary 17 spectrophotometer. Small sample volumes of 50 μl are used. Equilibration times of only a few minutes are required between taking spectra at different degrees of saturation. Apparatus stability is high as revealed by reproducibility and isosbestic point behavior. Incorporation of an oxygen electrode enables simultaneous measurement of oxygen partial pressure for intermediate spectra.     

Acute oxygen supply by infusion of hemoglobin solutions.

Preparations of 6% human hemoglobin have been investigated for 4 years. While the oxygen dissociation curve is shifted to the left, the hemoglobin solution has been shown to deliver oxygen adequately to the tissues in animal studies. Intracorporeal fate has been investigated by 131-I tagging, and the hemoglobin has not been found to accumulate in the tissues. Intravascular half-life has varied between 1.5 and 3.5 hours.     

The facilitated diffusion of oxygen by hemoglobin and myoglobin.

We have clarified the use of Wyman's differential equation for the facilitated oxygen flux through a slab of solution of myoglobin or hemoglobin by showing that there is a unique choice of boundary condition on the carrier concentration to be employed in conjunction with it. The singular perturbation solution of Wyman's equation, due to Murrayand Mitchell and Murray, has been extended. By means of it, the paradox of Wittenberg, that the facilitated oxygen flux per mole of heme is apparently independent of the protein carrier, has been resolved.