Oxygen-linked CO2 binding to isolated beta subunits of human hemoglobin.

It is known that most of the oxygen-linked carbamate which is formed in normal adult human hemoglobin (Hb A) is confined to the beta subunits rather than to the alpha subunits. In order to find out if similar differences exist in the isolated protomers of Hb A we have measured the effect of various pressures of carbon dioxide (pCO2) on the oxygen affinity in the following heme pigments: isolated alpha and beta subunits with free --SH groups (alphaSH, betaSH), mercurated beta subunits (betaPMB), myoglobin (Mb), and betaSH/PLP in which the terminal alpha-amino group of betaSH was irreversibly blocked with pyridoxal phosphate (PLP). Similar measurements were done on Hb A and the fraction of oxygen-linked carbamate calculated from the effect of pCO2 (at constant pH) on the oxygen half-saturation pressure (p50). A distinct influence of CO2 on p50 was observed in betaSH which was absent in betaSH/PLP and thus indicates that the terminal alpha-amino group mediates the oxygen-linked binding of CO2 in betaSH as it does in the beta subunits of Hb A. However, the fraction of oxygen-linked carbamate was much less dependent on pH and pCO2 in betaSH than in Hb A. Neither alphaSH, betaPMB, or Mb, all of which are known to exist largely or wholly as monomers but have free terminal alpha-amino groups, showed a shift of p50 upon addition of CO2. As both betaSH and betaSH/PLP were shown to be tetrameric molecules, we conclude from this study that homotetramers composed of isolated beta subunits do exhibit a reciprocal interaction between the binding of O2 and CO2.     

Antagonistic interaction between oxygenation-linked lactate and CO2 binding to human hemoglobin

Oxygen binding to hemoglobin (Hb) depends on allosteric effectors (CO(2), lactate and protons) that may increase drastically in concentration during exercise. The effectors share common binding sites on the Hb molecules, predicting mutual interaction in their effects on Hb (de)oxygenation. We analysed the effects of lactate and CO(2), separately and in combination, on O(2) binding of purified human Hb at 37 degrees C and physiological pH and chloride values. We demonstrate pH-dependent, inhibitory interactions between lactate binding and CO(2) binding (carbamate formation); at pH 7.4, physiological CO(2) tension ( approximately 43 mm Hg) reduced lactate binding more markedly ( approximately 75%), than lactate (50 mM) inhibited carbamate formation ( approximately 25%). In contrast to previous studies on blood and Hb solutions, we moreover find that added lactate neither 'reverses' oxylabile carbamate formation (resulting in lower carbamate levels in deoxyHb than in oxyHb) nor exerts greater allosteric effects on Hb-O(2) affinity than equal increases in chloride ion concentrations.     

Carbon-13 NMR studies of 13CO binding to human hemoglobin

In the ¹³C NMR spectrum of hemoglobin A carbonylated with ¹³CO, separate resonances can be distinguished at 207.04 ppm and 206.60 ppm (with respect to the ¹³C resonance of external tetramethyl-silane) for ¹³Co bound to the α and β chains of the hemoglobin tetramer. A study of the ¹³Co derivatives of the isolated α and β chains, and of the abnormal hemoglobin M_IWATE which contains α chains which are in the met [Fe(III)] form and do not bind CO, has permitted an assignment of the high field (206.60 ppm) resonance to the β chain ¹³CO and the low field one to the α chain ¹³CO. The identification of these ¹³Co resonances permits a study of the differences in the chemistry of the α and β heme units in intact hemoglobin. Some results on the differences in the redox behavior of these chains are included.     

The binding of n-butyl isocyanide to human hemoglobin

The binding of n-butyl isocyanide to hemoglobin has been investigated by ¹⁹F-nuclear magnetic resonance spectroscopy. The ¹⁹F-nmr spectrum of hemoglobin trifluoroacetonylated at cysteine β93 exhibits chemical shift changes on binding of ligands to the β chains. Comparison of these changes to the fractional change in the visible spectrum, shows that in the presence of diphosphoglyceric acid initial ligands bind preferentially to α chains. In the absence of DPG, ligation of β chains increases linearly with overall fractional ligation, indicating that binding to α and β chains is random under these conditions.     

Oxygen and CO binding to triply NO and asymmetric NO/CO hemoglobin hybrids.

The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays a high bimolecular quantum yield and a large fraction of slow (deoxy-like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably reflecting the differences in the alpha and beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selenium binding to human hemoglobin via selenotrisulfide

Selenotrisulfide (e.g., glutathione selenotrisulfide (GSSeSG)) is an important intermediate in the metabolism of selenite. However, its reactivity with biological substances such as peptides and proteins in the subsequent metabolism is still far from clearly understood, because of its chemical instability under physiological conditions. Penicillamine (Pen) is capable of generating a chemically stable and isolatable selenotrisulfide, PenSSeSPen. To explore the metabolic fate of selenite in red blood cells (RBC), we investigated the reaction of selenotrisulfide with human hemoglobin (Hb) using PenSSeSPen as a model. PenSSeSPen rapidly reacted with Hb under physiological conditions. From the analysis of selenium binding using the Langmuir type binding equation, the apparent binding number of selenium per Hb tetramer almost corresponded to the number of reactive thiol groups of Hb. The thiol group blockade of Hb by iodoacetamide treatment completely inhibited the reaction of PenSSeSPen with Hb. In addition, MALDI-TOF mass spectrometric analysis of the selenium-bound Hb revealed that PenSSe moiety binds to the beta subunits of Hb. Overall, the reaction of PenSSeSPen with Hb appears to involve the thiol exchange between Pen and the cysteine residues on the beta subunit of Hb.     

High pK value of the N-terminal amino group of the gamma-chain causes low CO2 binding of human fetal hemoglobin.

The N-terminal amino acid residue of the γ-chains of human fetal hemoglobin (Hb F_II) is glycine rather than valine like in many other hemoglobins including the human adult pigment (Hb A). In the course of an evaluation of functional implications associated with this replacement we have studied the CO₂ binding properties of Hb F_II in comparison with Hb F_Ic where the N-termini of the γ-chains are blocked. By comparing Hb F_II and Hb F_Ic it is possible to specifically estimate carbamate formation at the N-termini of the γ-chains in Hb F_II. These data were used to calculate the carbamate equilibrium and ionization constant of these groups. At 37 °C, ?log₁₀ of the ionization constant (pK_z) was found to be 8.1 and is thus significantly higher than pK_z of the N-terminal valines of the β-chains of Hb A which has been reported to be 6.6 at 37 °C. The high pK_z value of the γ-chain α-amino group explains the much lower carbamate formation in Hb F_II compared to Hb A.     

Carbon monoxide binding to human hemoglobin A0

The carbon monoxide binding curve to human hemoglobin A0 has been measured to high precision in experimental conditions of 600 microM heme, 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, 0.1 M NaCl, 10 mM inositol hexaphosphate, 1 mM disodium ethylenediaminetetraacetic acid, pH 6.94, and 25 degrees C. Comparison to the oxygen binding curve in the same experimental conditions demonstrates that the two curves are not parallel. This result invalidates Haldane's two laws for the partitioning between carbon monoxide and oxygen to human hemoglobin. The partition coefficient is found to be 263 +/- 27 at high saturation, in agreement with previous studies, but is lowered substantially at low saturation. Although the oxygen and carbon monoxide binding curves are not parallel, both show the population of the triply ligated species to be negligible. The molecular mechanism underlying carbon monoxide binding to hemoglobin is consistent with the allosteric model [Di Cera, E., Robert, C. H., & Gill, S. J. (1987) Biochemistry 26, 4003-4008], which accounts for the negligible contribution of the triply ligated species in the oxygen binding reaction to hemoglobin [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry 26, 3995-4002]. The nature of the different binding properties of carbon monoxide stems largely from the lower partition coefficient of the T state (123 +/- 34), relative to the R state (241 +/- 19).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen binding to sickle cell hemoglobin.

The extent of oxygen binding and light scattering of concentrated solutions of hemoglobin S have been determined as a function of oxygen partial pressure using a thin film optical cell. Nearly reversible oxygen binding is observed as witnessed by the small hysteresis found between slow deoxygenation and reoxygenation runs. High co-operativity is noted from unusually large concentration-dependent Hill coefficients when aggregated hemoglobin S is present. The application of linkage theory with the inclusion of non-ideal solution properties permits a test of various simple models for oxygen binding to both the monomer (α₂β₂^s) and polymer (aggregated) phase. It is concluded that oxygen binding to the polymer is either negligible or small under present experimental conditions. Phase diagrams of the solution concentration in equilibrium with polymer phase as a function of oxygen partial pressure are derived using best fit values of polymer parameters.     

CO binding to hemoglobin and myoglobin in equilibrium with a gas phase of low PO2 value

The aim of this paper was to measure the binding of CO to myoglobin and hemoglobin at various PO2 values. For this purpose we have studied an "in vitro" system made up of solutions of hemoglobin and myoglobin equilibrated in two connected tonometers with the same gas phase of various PO2 and PCO. The results indicate that a significant proportion of CO is released by hemoglobin and binds myoglobin at low PO2 values (approximately 2-3 Torr), in qualitative agreement with the predictions of a previous computer simulation of the "in vivo" system.     

Calorimetric studies of oxygen and carbon monoxide binding to human hemoglobin. Sequential binding heats for oxygen

Two high precision techniques, titration microcalorimetry and thin-layer optical binding measurements, have made possible the evaluation of enthalpy changes for the overall oxygenation reactions for human hemoglobin (HbAo). Although the heat of adding three oxygen molecules could not be evaluated due to the indeterminate contribution of this species to the oxygen binding curve of the protein (Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., and Robert, C. H. (1987) Biochemistry, 26, 3995-4002), the heats for binding two and four oxygen molecules were found to be simple multiples of the first binding heat. A direct consequence of equal stepwise heats is invariance of the shape of the binding curve with temperature, as pointed out by Wyman (Wyman, J. (1939) J. Biol. Chem. 127, 581-599). Titration microcalorimetry was also performed for the binding of carbon monoxide to hemoglobin. While the tight binding of CO precludes high-precision binding measurements, it does allow one to accurately determine the heat of ligation as a function of the CO bound. In these titrations a uniform heat of reaction is not observed, but the heat of binding increases markedly near the end point. This implies that the stepwise binding enthalpy for adding the third CO molecule is anomalously endothermic and for adding the fourth strongly exothermic. A similar phenomenon cannot be ruled out in the case of oxygen because of imprecision intrinsic in the analysis of the weaker ligand binding.