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.     

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).     

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 reaction cell with short optical path and oxygen electrode compartment for convenient quantitative determination of oxygen binding by hemoglobin

• 1.1. A simple and compact apparatus, with which the oxygen equilibrium of hemoglobin can be determined easily, was constructed and its performance was examined.
• 2.2. The oxygen partial pressure and the degree of oxygen saturation in a hemoglobin solution were determined by combined use of a Clark oxygen electrode and a spectrophotometer.
• 3.3. This apparatus can be used in a usual 1 cm square cell holder of a commercial spectrophotometer without any modifications, and has the advantage that the light path length of the optical cell is variable and can be shortened to about 2 mm.

     

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.     

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.     

The modification of hemoglobin affinity for oxygen and tumor radiosensitivity by antilipidemic drugs

It has been recognized for some time that alterations in the affinity of Hb for oxygen could offer a means of improving oxygen delivery to tumors and achieving radiosensitization. Three antilipidemic drugs, clofibrate, bezafibrate, and gemfibrozil, two of which had previously been shown to reduce Hb/O2 affinity in vitro, were tested in mice for their ability to affect Hb/O2 affinity and to alter the radiosensitivity of the RIF-1 sarcoma. Each of the drugs produced a significant increase in the P50 of the blood, from a mean control value of 45 mm Hg to 55, 74, and 51 mm Hg after a dose of 1 g/kg of clofibrate, bezafibrate, and gemfibrozil, respectively. However, they had very different effects on the radiosensitivity of the RIF-1 tumor. When the mice breathed air at the time of irradiation, clofibrate produced a marked sensitization equivalent at the optimum time to a 20-fold reduction in hypoxic fraction; bezafibrate gave a lower sensitization equivalent to a 4-fold reduction, while gemfibrozil caused dramatic radioresistance equivalent to a 10-fold increase in hypoxic fraction. When the mice were given 95% O2/5% CO2 to breathe at the time of irradiation to ensure complete Hb saturation in the lungs, a large increase in the sensitization by bezafibrate was seen, but there was only a small change with clofibrate. We conclude that drugs which reduce Hb/O2 affinity could have a role in sensitizing tumors to radiation.