The effect of the immobilization of hemoglobin on its oxygen binding]

Hemoglobin (Hb) covalently fixed to CM-Sephadex was found to bind oxygen in weakly acidic medium with higher affinity than free Hb. The opposite relation is seen in the alkaline pH region. The alkaline Bohr effect was determined to be -0.2 only. Cooperativity is pH dependent. The sigmoid coefficient at pH 6 is 0.7; at pH 8.7 n was determined to be 1.3. As the reason of these altered binding properties a blockade of the primary amino groups, disturbance of the salt bridges, and restrained cooperative mobility of the Hb-subunits are discussed. The Hill coefficient is additionally lowered by the heterogeneity of the immobilized Hb.     

Analysis of oxygen binding by hemoglobin on the basis of mean intrinsic thermodynamic quantities

The binding data for oxygenation of human hemoglobin, Hb, at various temperatures and in the absence and presence of 2,3-diphosphoglycerate, DPG, and inositol hexakis phosphate, IHP, were analyzed for extraction of mean intrinsic Gibbs free energy, DeltaGo, enthalpy, DeltaHo, and entropy, DeltaSo, of binding at various partial oxygen pressures. This method of analysis considers all the protein species present such as dimer and tetramer forms which were not considered by Imai et al. (Imai K et al., 1970, Biochim Biophys Acta 200: 189-196), in their analysis which was based on Adair equation. In this regard, the values of Hill equation parameters were estimated with high precision at all points of the binding curve and used for calculation of DeltaGo, DeltaHo and DeltaSo were also calculated by analysis of DeltaGo values at various temperatures using van't Hoff equation. The results represent the enthalpic nature of the cooperativity in Hb oxygenation and the compensation effect of intrinsic entropy. The interpretation of results also to be, into account the decrease of the binding affinity of sites for oxygen in the presence of DPG and IHP without any considerable changes in the site-site interaction (extent of cooperativity). In other words, the interactions between bound ligands, organic phosphates and oxygen, are more due to a decreasing binding affinity and not to the reduction of the cooperative interaction between sites. The results also document the more heterotropic effect of IHP compared to DPG.     

The effect of formaldehyde on the oxygen equilibrium of hemoglobin

1. When formaldehyde (0.10 M) is added to solutions of human hemoglobin, the oxygen affinity of the hemoglobin increases considerably (more than tenfold near pH 7). The interaction between hemes of the same hemoglobin molecule decreases, as shown by a drop in the value of n in Hill's equation from 2.9 to 1.5 or less. 2. In the presence of formaldehyde, both n and the oxygen pressure for half-saturation fall gradually as the pH rises in the range from pH 6.2 to 7.2. 3. Some of the effect of formaldehyde on the oxygen equilibrium may be due to combination with sulfhydryl groups of the protein, but nitrogenous groups are probably also involved.     

Effect of some organic cosolvents on the reaction of hemoglobin with oxygen

We studied the effects of some organic cosolvents (monohydric alcohols and amides) on the reaction of hemoglobin with oxygen. We present evidence showing that our data can be analyzed within the framework of the Monod-Wyman-Changeux model and that the main effect of cosolvents is to alter the T ? R conformational equilibrium of hemoglobin, without significantly affecting the intrinsic oxygen dissociation constants. Following a previously described phenomenological approach, the overall effects have been separated into effects related to the variation of the bulk dielectric constant of the solvent and effects not related to the variation of this constant.     

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.

     

Effect of oxygen binding on the dielectric properties of hemoglobin

The dielectric properties of horse hemoglobin have been investigated in the frequency range for 100 kcps to 15 Mcps at varying degrees of oxygenation. A linear dependence of the specific increment on the degree of oxygenation was found under a variety of experimental conditions, the increment of oxygenated hemoglobin being about 10% larger than that of deoxygenated hemoglobin. A similar difference was obtained with human adult and fetal hemoglobin. No variation of the dielectric parameters as reported by Takashima and Lumry could be detected.     

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.     

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.     

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin was investigated by accurately measuring and analyzing the oxygen equilibrium curves of human adult hemoglobin in the presence and absence of various concentrations of one or two of the following materials: chloride (Cl-), 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). Each equilibrium curve was analyzed according to the Adair equation to evaluate the four-step oxygen equilibrium constants (Adair constants) and the median oxygen pressure. The binding constants of the anions for the molecular species of hemoglobin carrying j oxygen molecules, Hb(O2)j(j=0,1,...,4), were evaluated from the dependences of the Adair constants and the median oxygen pressure on the anion concentration by introducing a model which takes the competitive binding of Cl- and DPG or IHP into account. Assumptions made in the model are: (a) the hemoglobin molecule has two oxygen-linked binding sites for Cl- which are equivalent and independent and (b) no Cl- can be bound to hemoglobin to which DPG or IHP is already bound and vice versa. Thus, we could obtain values for the intrinsic binding constants of Cl- and DPG, i.e., the constants in the absence of other competitive anions. For IHP, only the binding constants and apparent binding constants for Hb and Hb(O2)2 were obtained. Values of the Cl- binding constants and apparent binding constants for DPG and IHP, i.e., the binding constants in the presence of Cl- for Hb and Hb(O2)4, were in reasonable agreement with literature values. From the binding constants we calculated anion binding curves for Hb(O2)j(J=0,1,...,4), the number of anions bound to Hb(O2)J, And the relationship between fractional anion saturation of hemoglobin and fractional oxygen saturation. The numbers of released anions are not uniform with respect to oxygenation step. This non-uniformity is the reason for the changes in the shape of the oxygen equilibrium curve with anion concentration changes and for the non-uniform dependences of the Adair constants on anion concentration, and also results in non-linear relations between anion saturation and oxygen saturation. The anion binding constants and various binding properties of the anions derived from those constants are consistent with those observed by other investigators using different techniques, indicating that the present model describes the oxygen-linked competitive anion binding well.     

Linkage between carbon dioxide binding and four-step oxygen binding to hemoglobin

In order to elucidate the molecular mechanism of the effect of carbon dioxide on the four-step oxygenation equilibria of hemoglobin, accurate oxygen equilibrium curves of human adult hemoglobin were determined at different concentrations of CO₂ and in the presence and absence of chloride (Cl^?), 2,3-diphosphoglycerate (P₂G), and/or inositol hexaphosphate (IHP) and were analyzed according to Adair's stepwise oxygenation scheme to evaluate the four Adair constants, k_i (i = 1 to 4). The effects of CO₂ on oxygen affinity and co-operativity are influenced by H⁺, Cl^?, P₂G and IHP. The shape of the oxygen equilibrium curve varies with changes of CO₂ concentration; the four Adair constants are affected by CO₂ non-uniformly. Hence, the number of CO₂ molecules released upon oxygenation is not the same in the individual oxygenation steps. In the absence of added Cl^?, CO₂ lowers the overall oxygen affinity expressed by median oxygen pressure (p_m) and increases the co-operativity expressed by Hill's coefficient (n_max) by reducing k₁, k₂ and k₃ without changing k₄. significantly. The effect of CO₂ on oxygen affinity becomes smaller with decrease in pH, disappearing below pH 6.5. The alkaline Bohr effect is reduced by CO₂. The first oxygenation step contributes to the reduction of the Bohr effect more than the fourth step. When log p_m is plotted against log [CO₂] at several constant Cl^? concentrations, the plots converge to a common point that is named “iso-effective point”. When log p_p is plotted against log [Cl^?] at several constant CO₂ concentrations, the plots also converge to an iso-effective point. This phenomenon can be explained in terms of linkage relations in oxygen-linked competitive binding of CO₂ and Cl^?. It was found to be useful to consider in this analysis that the bicarbonate ion introduced by added CO₂ exerts a heterotropic effect equivalent to that of Cl^?. The combined effects of Cl^?, CO₂ and IHP were not explained satisfactorily by the present analysis using linkage relations.     

19 F-NMR studies of oxygen binding to hemoglobin

The binding of oxygen to hemoglobin has been investigated by ¹⁹F-nuclear magnetic resonance spectroscopy. The ¹⁹F-nmr spectrum of hemoglobin trifluoroacetonylated at cysteine β 93 exhibits chemical shift changes on binding of ligands, which differ depending on which chains are undergoing complexation. Comparison of these changes to the fractional ligation of all chains, determined concurrently from the fractional change in the visible spectrum, shows that initial oxygen molecules bind preferentially to α-chains. The ¹⁹F-nmr spectrum of partially oxygenated hemoglobin contains resonances at the normal chemical shift positions of the oxygenated and deoxy species, in addition to two small resonances at intermediate positions. Analysis of the relativ magnitudes of these four peaks as functions of oxygen pressure permits identification of the intermediate species