Molecular dynamics simulations indicate that deoxyhemoglobin,oxyhemoglobin, carboxyhemoglobin,and glycated hemoglobin under compression and shear exhibit an anisotropic mechanical behavior

We developed a new mechanical model for determining the compression and shear mechanical behavior of four different hemoglobin structures. Previous studies on hemoglobin structures have focused primarily on overall mechanical behavior; however, this study investigates the mechanical behavior of hemoglobin, a major constituent of red blood cells, using steered molecular dynamics (SMD) simulations to obtain anisotropic mechanical behavior under compression and shear loading conditions. Four different configurations of hemoglobin molecules were considered: deoxyhemoglobin (deoxyHb), oxyhemoglobin (HbO₂), carboxyhemoglobin (HbCO), and glycated hemoglobin (HbA_1C). The SMD simulations were performed on the hemoglobin variants to estimate their unidirectional stiffness and shear stiffness. Although hemoglobin is structurally denoted as a globular protein due to its spherical shape and secondary structure, our simulation results show a significant variation in the mechanical strength in different directions (anisotropy) and also a strength variation among the four different hemoglobin configurations studied. The glycated hemoglobin molecule possesses an overall higher compressive mechanical stiffness and shear stiffness when compared to deoxyhemoglobin, oxyhemoglobin, and carboxyhemoglobin molecules. Further results from the models indicate that the hemoglobin structures studied possess a soft outer shell and a stiff core based on stiffness.     

Effect of oxygen concentration on trasverse water proton relaxation times in erythrocytes homozygous and heterozygous for hemoglonin S.

The transverse water proton relaxation times (T₂) of erythrocytes homozygous and heterozygous for hemoglobin S have been measured as a function of oxyhemoglobin concentration at 37 °C. An immediate decrease in T₂ is observed in S/S erythrocytes as the amount of oxyhemoglobin is decreased and the maximum change is observed at 50% deoxyhemoglobin S. In heterozygous erythrocytes, the T₂ remains unchanged until a critical level of deoxyhemoglobin is attained. The critical level of deoxyhemoglobin is a function of the percentage of hemoglobin S in the heterozygous erythrocytes. A Hill plot of the data obtained from S/S erythrocytes gives an n value of around 2.4. These results suggest that the measurement of T₂ is sensitive to the very early stages of the polymerization process. This suggestion is supported by calculations; our T₂ measurements are sensitive to a range of correlation times expected for hemoglobin monomers at one extreme and linear polymers of seven hemoglobin molecules at the other extreme.     

Biphasic nature in the autoxidation reaction of human oxyhemoglobin

In comparison with myoglobin molecule as a reference, we have studied the autoxidation rate of human oxyhemoglobin (HbO₂) as a function of its concentration in 0.1 M buffer at 35°C and in the presence of 1 mM EDTA. At pH 6.5, HbA showed a biphasic autoxidation reaction that can be described completely by a first-order rate equation containing two rate constants — k_f, for fast autoxidation of the α-chain, and k_s, for slow autoxidation of the β-chain, respectively. When tetrameric HbO₂ was dissociated into αβ-dimers by dilution, the value of k_f increased markedly to an extent comparable with the autoxidation rate of horse heart oxymyoglobin (MbO₂). The rate constant k_s, on the other hand, was found to remain at an almost constant value over the whole concentration range from 1.0 × 10⁻³ M to 3.2 × 10⁻⁶ M in heme. At pH 8.5 and pH 10.0, however, the autoxidation of HbO₂ was monophasic, and no enhancement in the rate was observed by diluting hemoglobin solutions. Taking into consideration the effects of 2,3-diphosphoglyceric acid and chloride anion on the autoxidation rate of HbO₂, we have characterized the differential susceptibility of the α- and β-chains to the autoxidation reaction in aqueous solution.     

Observation of allosteric transition in hemoglobin

Two conclusions have been drawn from NMR studies of mixed state hemoglobins. First the α and β subunits in hemoglobin are not equivalent in their conformational properties. Second the mixed state hemoglobin (α^IIICN β^II)₂ can take two different quaternary structures without changing the degree of ligation. One of the two structures is similar to that of deoxyhemoglobin and the other to that of oxyhemoglobin.     

Oxygenation of hemoglobin: Correspondence of crystal and solution properties using diffusion coefficient measurements

Quasi-elastic light scattering has been used to measure the change in the translational diffusion coefficient of hemoglobin upon oxygenation and the difference in the diffusion coefficients of oxy- and methemoglobin. The diffusion coefficients of oxy- and methemoglobin were found to be the same within the experimental accuracy of 0.2%, while the diffusion coefficient of oxyhemoglobin tetramers in solution at 13 mg/ml was found to be 0.8% smaller than that of deoxyhemoglobin at the same concentration, when the reversible dissociation of oxyhemoglobin tetramers into dimers was taken into account. In the limit of zero concentration, the oxyhemoglobin diffusion coefficient was found to be 1.5% ± 1.0% smaller than that of deoxyhemoglobin. This result is in very good agreement with what we predict using atomic coordinates to model the liganded and unliganded hemoglobin molecules as ellipsoids of revolution.     

Red cell lysis induced by microorganisms as a case of superoxide- and hydrogen peroxide-dependent hemolysis mediated by oxyhemoglobin

Some bacteria, isolated from the blood of hospitalized patients, have been shown to hemolyze red blood cells through a mechanism which was dependent on the oxygenated state of intracellular hemoglobin, since transformation of hemoglobin into the CO-derivative inhibited the lysis. Hemolysis was also inhibited by superoxide dismutase and catalase, while only catalase prevented the formation of methemoglobin in experiments where isolated oxyhemoglobin was exposed to metabolizing bacteria. Production by bacteria of extracellular superoxide was demonstrated. It is suggested that hemolysis is due to interaction of O₂⁻ and/or H₂O₂ with intracellular hemoglobin and that some product of such interaction is the lytic agent.     

Oxygen dissociation of whole blood studied polarographically

The polarographic current of whole blood is in excess of that given by plasma at the same oxygen tension. The magnitude of this difference depends on (a) the oxygen content of the sample and thus is determined by the red blood cell content and by the state of oxygen saturation of hemoglobin, and (b) on the rate of dissociation of oxyhemoglobin and therefore is influenced by changes in pH, pCO₂, and temperature. The total current at 37°C. is proportional to the oxygen content of the sample and can be used to determine the latter. The theoretical basis of the studied phenomena is discussed in detail.     

Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.     

Calorimetric studies of oxyhemoglobin dissociation. I. Reaction of sodium dithionite with oxygen

Calorimetric studies of the reduction of free oxygen in solution by sodium dithionite are in agreement with a stoichiometry of 2 moles Na₂S₂O₄ per mole of oxygen. The reaction is biphasic with ΔH_t - 118±7 kcal mol^?1 (?494 ± 29 kJ mol^?1). The initial phase of the reaction proceeds with an enthalpy change of ca ?20 kcal (?84 kJ) and occurs when 0.5 moles of dithionite have been added per mole dioxygen present. This could be interpreted as the enthalpy change for the addition of a single electron to form the superoxide anion. Further reduction of the oxygen to water by one or more additional steps is accompanied by an enthalpy change of ca ?100 kcal (?418. 5 kJ). Neither of these reductive phases is consistent with the formation of hydrogen peroxide as an intermediate. The reduction of hydrogen peroxide by dithionite in 0.1 M phosphate buffer, pH 7.15, is a much slower process and with an enthalpy change of ca ? 74 kcal mol^?1 (?314 kJ mol^?1). Dissociation of oxyhemoglobin induced by the reduction of free oxygen tension with dithionite also shows a stoichiometry of 2 moles dithionite per mole oxygen present and an enthalpy change of ca. ?101 ±9 kcal mol^?1 (?423± 38 kJ mol^?1). The difference in the observed enthalpies (reduction of dioxygen vs. oxyhemoglobin) has been attributed to the dissociation of oxyhemoglobin, which is 17 kcal mol^?1 (71 kJ mol^?1).     

Laser-induced time-resolved photoacoustic calorimetry study on photo-dissociation of human and bovine oxyhemoglobin

The dynamics of the enthalpy and volume changes related to the photo-dissociation of oxygen from human and bovine oxyhemoglobin are investigated by nanosecond time-resolved photoacoustic calorimetry (PAC). The values of enthalpy and volume change associated with the above process are deltaH = 37.8 +/- 3 kcal/mol, deltaV = 5.0 +/- 1 ml/mol for human HbO(2); and deltaH = 35.7 +/- 3.5 kcal/mol, deltaV = 4.8 +/- 1 ml/mol for bovine HbO(2), respectively. A possible explanation for the similar values between both human and bovine oxyhemoglobin is proposed. In addition, the PAC results for human HbO(2) and HbCO are compared and discussed.     

Reaction of Oxyhemoglobin with Carbon Monoxide

The reaction of oxyhemoglobin and carbon monoxide was studied kinetically at pH 7.8 in a variety of suspending media. The dielectric constant of the suspending media, as well as the viscosity (and hence the Fick diffusion coefficients), was varied with the use of glycine, glycerol, and sucrose. The results showed that the reaction was unaltered by the various additions to the media, provided that the pO₂ and the concentration of carbon monoxide were held constant. Since the concentration of oxygen varies from medium to medium at constant pO₂ while the pCO varies at constant concentration of carbon monoxide, the differences in the reactions with oxygen and carbon monoxide were emphasized. The lack of variation of the rate constants with changes in dielectric constant can be interpreted as indicating that electrostatic effects are unimportant in this reaction.     

Stabilizing effect of organic solvents on oxyhemoglobin

The role of hemoglobin solutions as oxygen carriers in biotechnology are numerous, such as in the oxygen supply to biocatalysts or in the preparation of blood substitutes. However, the major barrier to the successful use of hemoglobin in biological and medical engineering is the autoxidation of heme iron during preparation, storage, and utilization. Fifty-six solvents, chosen among the group of Parker's classification, were studied with regard to the autoxidation kinetics of oxyhemoglobin under nondenaturant conditions. Among these solvents 27 present a concentration range in which the autoxidation rates were reduced compared to autoxidation in water. Three groups of solvent have been observed: one exhibiting only a destabilizing effect regardless of the solvent proportion, a second showing a strong stabilizing effect (k(H2O)/k(solvent) greater than 20) and a third showing a low stabilization (k(H2O)/k(solvent) less than 20). The most effective stabilizing solvents were glycerol, glycols, and alcohols. The effect of hydroorganic solvents could be explained by taking into account the globin solvation by water molecules. The solvents that enhance the structure of the water and form few hydrophobic interactions with globin prevent oxyhemoglobin autoxidation.     

The effect of diphenols upon the autoxiation of oxyhemoglobin and oxymyoglobin.

P-Hydroquinone and catechol catalytically promote the oxidation of oxyhemoglobin and Oxymyoglobin to the ferriform. Kinetic data for oxyhemoglobin oxidation indicates a first-order dependence upon the hemeprotein concentration and half-order dependence upon diphenol; however at high catalyst concentration, saturation is observed with similar V values for both diphenols despite the difference in reactivity.It is proposed that initially formed quinone oxidizes the hemeprotein with oxygen release; in turn, the semiquinone oxidizes a second molecule of hemeprotein and regenerates the quinone, with the bound oxygen acquiring two electrons. Except for the more reactive oxymyoglobin, the reduced form of the catalyst must be present to oppose semiquinone disappearance by dismutation.Since the expected release of O₂ for water formation is observed, the system may be considered a model for terminal oxidase, the couple $\text{Q}\text{S}$ replacing a $\text{Fe}{}^{\mathrm{2+}}\text{Fe}{}^{\mathrm{2+}}$ or a $\text{Cu}{}^{\mathrm{+}}\text{Cu}{}^{\mathrm{2+}}$ system.It is tentatively inferred that oxyhemoglobin has the structure HbFe²⁺---O₂ and that the rate of the catalyzed oxidation is limited by the rate of generation of the true reacting form, the superoxide ferri structure, HbFe³⁺---O₂^?.     

Self-diffusion of water-soluble fullerene derivatives in mouse erythrocytes

Self-diffusion of water-soluble fullerene derivative (WSFD) C₆₀[S(CH₂)₃SO₃Na]₅H in mouse red blood cells (RBC) was characterized by ¹H pulsed field gradient NMR technique. It was found that a fraction of fullerene molecules (~13% of the fullerene derivative added in aqueous RBC suspension) shows a self-diffusion coefficient of (5.5 ± 0.8)·10⁻¹² m²/s, which is matching the coefficient of the lateral diffusion of lipids in the erythrocyte membrane (D_L = (5.4 ± 0.8)·10⁻¹² m²/s). This experimental finding evidences the absorption of the fullerene derivative by RBC. Fullerene derivative molecules are also absorbed by RBC ghosts and phosphatidylcholine liposomes as manifested in self-diffusion coefficients of (7.9 ± 1.2)·10⁻¹² m²/s and (7.7 ± 1.2)·10⁻¹² m²/s, which are also close to the lateral diffusion coefficients of (6.5 ± 1.0)·10⁻¹² m²/s and (8.5 ± 1.3)·10⁻¹² m²/s, respectively. The obtained results suggest that fullerene derivative molecules are, probably, fixed on the RBC surface. The average residence time of the fullerene derivative molecule on RBC was estimated as 440 ± 70 ms. Thus, the pulsed field gradient NMR was shown to be a versatile technique for investigation of the interactions of the fullerene derivatives with blood cells providing essential information, which can be projected on their behavior in-vivo after intravenous administration while screening as potential drug candidates.     

Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

The dissociation of the extracellular hemoglobin of Tubifex tubifex at alkaline and acid pH, and its reassociation upon return to neutral pH, was investigated using gel filtration, ultracentrifugation, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Tubifex hemoglobin dissociated at pH above 8 and below 6; both dissociations appeared to be equilibrium processes. The extent of dissociation increased as the pH moved away from neutrality; although dissociation was virtually complete at pH 11, its extent at acid pH did not exceed 50–60% at pH 4. Ca(II), Mg(II), and Sr(II) cations over the range 1–100 mm decreased the extent of the dissociation only at alkaline pH. The visible absorption spectrum of the oxyhemoglobin remained unaltered in the pH range 4–9. At more extreme pH, it changed with time, altering irreversibly to that of the aquo ferri form. Gel filtration of the hemoglobin at both extremes of pH showed that it dissociated into two heme-containing fragments; one consisting of subunit 1 (M_r ~ 17,000) and the other containing subunits 2, 3, and 4 of the hemoglobin (M_r ~ 60,000). Upon return to neutral pH, the dissociated fragment reassociated to the extent of 50 to 80% to whole hemoglobin molecules. The reassociation decreased with increase in alkaline pH, and with decrease in acid pH to which the hemoglobin had been exposed; it increased in the presence of Ca(II), Sr(II), and Mg(II) only subsequent to dissociation at alkaline pH. The SDS-PAGE patterns, gel-filtration elution volumes, and α-helical contents, determined from circular dichroism at 222 nm, of the reassociated whole molecules were identical to those of the native hemoglobin.     

Sulfide Oxidation by a Noncanonical Pathway in Red Blood Cells Generates Thiosulfate and Polysulfides

A cardioprotectant at low concentrations, H₂S is a toxin at high concentrations and inhibits cytochrome c oxidase. A conundrum in H₂S homeostasis is its fate in red blood cells (RBCs), which produce H₂S but lack the canonical mitochondrial sulfide oxidation pathway for its clearance. The sheer abundance of RBCs in circulation enhances the metabolic significance of their clearance strategy for H₂S, necessary to avoid systemic toxicity. In this study, we demonstrate that H₂S generation by RBCs is catalyzed by mercaptopyruvate sulfurtransferase. Furthermore, we have discovered the locus of sulfide oxidation in RBCs and describe a new role for an old protein, hemoglobin, which in the ferric or methemoglobin state binds H₂S and oxidizes it to a mixture of thiosulfate and hydropolysulfides. Our study reveals a previously undescribed route for the biogenesis of hydropolysulfides, which are increasingly considered important for H₂S-based signaling, but their origin in mammalian cells is unknown. An NADPH/flavoprotein oxidoreductase system restores polysulfide-carrying hemoglobin derivatives to ferrous hemoglobin, thus completing the methemoglobin-dependent sulfide oxidation cycle. Methemoglobin-dependent sulfide oxidation in mammals is complex and has similarities to chemistry reported for the dissolution of iron oxides in sulfidic waters and during bioleaching of metal sulfides. The catalytic oxidation of H₂S by hemoglobin explains how RBCs maintain low steady-state H₂S levels in circulation, and suggests that additional hemeproteins might be involved in sulfide homeostasis in other tissues.     

STUDIES ON INVERTEBRATE HEMOGLOBINS (ERYTHROCRUORINS)

1. Two high molecular invertebrate hemoglobins (the erythrocruorins of Lumbricus terrestris and of Nereis virens) as well as the low molecular erythrocruorin of Glycera dibranchiata Ehlers were studied. Their physical chemical properties were compared with those of vertebrate hemoglobin. 2. The hemin of the blood pigment of Glycera dibranchiata Ehlers was shown to be identical with that of vertebrate hemoglobin. 3. The dissociation rates of Glycera and human oxyhemoglobin were measured in the reaction meter of DuBois and t ₅₀ (half time of the reaction) was found to be identical (0.027 second) for the two pigments. The t ₅₀ value for the high molecular Lumbricus erythrocruorin was 0.070 second. 4. The chemical constitution and physical chemical properties of erythrocruorins were compared with those of vertebrate hemoglobin and of hemocyanin.     

Exercise-induced oxyhemoglobin desaturation and pulmonary diffusing capacity during high-intensity exercise

The purpose of this investigation was to examine if exercise-induced arterial oxyhemoglobin desaturation selectively observed in highly trained endurance athletes could be related to differences in the pulmonary diffusing capacity (D _L) measured during exercise. The D _L of 24 male endurance athletes was measured using a 3-s breath-hold carbon monoxide procedure (to give D _LCO) at rest as well as during cycling at 60% and 90% of these previously determined O_2max. Oxyhemoglobin saturation (S _aO₂%) was monitored throughout both exercise protocols using an Ohmeda Biox II oximeter. Exercise-induced oxyhemoglobin desaturation (DS) (S _aO₂% < 91% at O_2max) was observed in 13 subjects [88.2 (0.6)%] but not in the other 11 nondesaturation subjects [NDS: 92.9 (0.4)%] (P ≤ 0.05), although O_2max was not significantly different between the groups [DS: 4.34 (0.65) l / min vs NDS: 4.1 (0.49) l / min]. At rest, no differences in either D _LCO [m1 CO · mmHg⁻¹ · min⁻¹: 41.7 (1.7) (DS) vs 41.1 (1.8) (NDS)], D _LCO / _A [8.2 (0.4) (DS) vs 7.3 (0.9) (NDS)], MVV [l / min: 196.0 (10.4) (DS) vs 182.0 (9.9) (NDS)] or FEV₁/FVC [86.3 (2.2) (DS) vs 82.9 (4.7) (NDS)] were found between groups (P ≥ 0.05). However, _E /O₂ at O_2max was lower in the DS group [33.0 (1.1)] compared to the NDS group [36.8 (1.5)] (P ≤ 0.05). Exercise D _LCO (m1 CO · mmHg⁻¹ · min⁻¹) was not different between groups at either 60% O_2max [DS: 55.1 (1.4) vs NDS: 57.2 (2.1)] or at 90% O_2max [DS: 61.0 (1.8) vs NDS: 61.4 (2.9)]. A significant relationship (r = 0.698) was calculated to occur between S _aO₂% and _E /O₂ during maximal exercise. The present findings indicate that the exercise-induced oxyhemoglobin desaturation seen during submaximal and near-maximal exercise is not related to differences in D _L, although during maximal exercise S _aO₂ may be limited by a relatively lower exercise ventilation. Accepted: 25 September 1996