Spectrophotometry of Hemoglobin: Absorption Spectra of Bovine Oxyhemoglobin, Deoxyhemoglobin, Carboxyhemoglobin, and Methemoglobin

The absorptivity at 540 nm of bovine hemiglobincyanide (cyanmethemoglobin) was determined on the basis of the iron content and found to be equal to the established value for human hemiglobincyanide (11.0 L · mmol⁻¹ · cm⁻¹). On this basis the absorption spectra of the common derivatives were determined for bovine hemoglobin. There proved to be only slight differences in the oxyhemoglobin, deoxyhemoglobin, and carboxyhemoglobin spectra between bovine and human hemoglobin. For comparison of the methemoglobin spectra a new series of measurements was made for human hemoglobin. As also found in the rat, the methemoglobin spectrum of bovine blood differed considerably from that in the human. These differences should be taken into account in multicomponent analysis.     

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.     

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.     

Electrostatic effects in hemoglobin: Bohr effect and ionic strength dependence of individual groups.

The electrostatic treatment applied in the preceding paper in this issue [Matthew, J. B., Hanania, G.I.H., & Gurd, F.R.N. (1979) Biochemistry (preceding paper in this issue)] to the titration behavior of individual groups in human deoxyhemoglobin and oxyhemoglobin was applied to the computation of the alkaline Bohr effect at various values of ionic strength. The enhanced proton binding of deoxyhemoglobin in the pH range of 6--9 was accounted for at ionic strength 0.01 M by the effects of the unique charge distributions of ionizable groups in the two quaternary states. At ionic strength 0.10 M the effects of 2--4 bound anions had to be considered in addition in the deoxyhemoglobin charge configuration. At the higher ionic strength 10 groups per tetramer contributed to the Bohr effect, whereas 28 groups were contributory at the lower ionic strength. The ionic strength dependence of individual groups in the two tetrameric structures as well as in the alpha-chain monomer was explained in terms of the electrostatic treatment. This examination showed that the differences in electrostatic behavior of deoxy- and oxyhemoglobin follow from particular dissymmetries in their configurations with respect to charge and static solvent accessibility.     

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.     

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.     

Molecular insights into the irreversible mechanical behavior of sickle hemoglobin

Sickle cell disease is caused by the amino acid substitution of glutamic acid to valine, which leads to the polymerization of deoxygenated sickle hemoglobin (HbS) into long strands. These strands are responsible for the sickling of red blood cells (RBCs), making blood hyper-coagulable leading to an increased chance of vaso-occlusive crisis. The conformational changes in sickled RBCs traveling through narrow blood vessels in a highly viscous fluid are critical in understanding; however, there are few studies that investigate the origins of the molecular mechanical behavior of sickled RBCs. In this work, we investigate the molecular mechanical properties of HbS molecules. A mechanical model was used to estimate the directional stiffness of an HbS molecule and the results were compared to adult human hemoglobin (HbA). The comparison shows a significant difference in strength between HbS and HbA, as well as anisotropic behavior of the hemoglobin molecules. The results also indicated that the HbS molecule experienced more irreversible mechanical behavior than HbA under compression. Further, we have characterized the elastic and compressive properties of a double stranded sickle fiber using six HbS molecules, and it shows that the HbS molecules are bound to each other through strong inter-molecular forces.     

Hemoglobin oxygen fractional saturation regulates nitrite-dependent vasodilation of aortic ring bioassays

Nitrite reacts with deoxyhemoglobin to generate nitric oxide (NO). This reaction has been proposed to contribute to nitrite-dependent vasodilation in vivo and potentially regulate physiological hypoxic vasodilation. Paradoxically, while deoxyhemoglobin can generate NO via nitrite reduction, both oxyhemoglobin and deoxyhemoglobin potently scavenge NO. Furthermore, at the very low O(2) tensions required to deoxygenate cell-free hemoglobin solutions in aortic ring bioassays, surprisingly low doses of nitrite can be reduced to NO directly by the blood vessel, independent of the presence of hemoglobin; this makes assessments of the role of hemoglobin in the bioactivation of nitrite difficult to characterize in these systems. Therefore, to study the O(2) dependence and ability of deoxhemoglobin to generate vasodilatory NO from nitrite, we performed full factorial experiments of oxyhemoglobin, deoxyhemoglobin, and nitrite and found a highly significant interaction between hemoglobin deoxygenation and nitrite-dependent vasodilation (P < or = 0.0002). Furthermore, we compared the effect of hemoglobin oxygenation on authentic NO-dependent vasodilation using a NONOate NO donor and found that there was no such interaction, i.e., both oxyhemoglobin and deoxyhemoglobin inhibited NO-mediated vasodilation. Finally, we showed that another NO scavenger, 2-carboxyphenyl-4,4-5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibits nitrite-dependent vasodilation under normoxia and hypoxia, illustrating the uniqueness of the interaction of nitrite with deoxyhemoglobin. While both oxyhemoglobin and deoxyhemoglobin potently inhibit NO, deoxyhemoglobin exhibits unique functional duality as an NO scavenger and nitrite-dependent NO generator, suggesting a model in which intravascular NO homeostasis is regulated by a balance between NO scavenging and NO generation that is dynamically regulated by hemoglobin's O(2) fractional saturation and allosteric nitrite reductase activity.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Nitric oxide formation from the reaction of nitrite with carp and rabbit hemoglobin at intermediate oxygen saturations

The nitrite reductase activity of deoxyhemoglobin has received much recent interest because the nitric oxide produced in this reaction may participate in blood flow regulation during hypoxia. The present study used spectral deconvolution to characterize the reaction of nitrite with carp and rabbit hemoglobin at different constant oxygen tensions that generate the full range of physiological relevant oxygen saturations. Carp is a hypoxia-tolerant species with very high hemoglobin oxygen affinity, and the high R-state character and low redox potential of the hemoglobin is hypothesized to promote NO generation from nitrite. The reaction of nitrite with deoxyhemoglobin leads to a 1 : 1 formation of nitrosylhemoglobin and methemoglobin in both species. At intermediate oxygen saturations, the reaction with deoxyhemoglobin is clearly favored over that with oxyhemoglobin, and the oxyhemoglobin reaction and its autocatalysis are inhibited by nitrosylhemoglobin from the deoxyhemoglobin reaction. The production of NO and nitrosylhemoglobin is faster and higher in carp hemoglobin with high O(2) affinity than in rabbit hemoglobin with lower O(2) affinity, and it correlates inversely with oxygen saturation. In carp, NO formation remains substantial even at high oxygen saturations. When oxygen affinity is decreased by T-state stabilization of carp hemoglobin with ATP, the reaction rates decrease and NO production is lowered, but the deoxyhemoglobin reaction continues to dominate. The data show that the reaction of nitrite with hemoglobin is dynamically influenced by oxygen affinity and the allosteric equilibrium between the T and R states, and that a high O(2) affinity increases the nitrite reductase capability of hemoglobin.     

Transformation of hemoglobin a into methemoglobin by sesamol

Sesamol (3,4-methylenedioxyphenol), a monophenolic antioxidant in sesame iol, produced methemoglobin from hemoglobin A (oxyhemoglobin and deoxyhemoglobin) and from red cells. The activity of the compound was more extensive than the polyphenolic compounds. The profiles of the methemoglobin formation by the compound were compared with those by nitrite and hydroxylamine. The formation of methemoglobin from oxyhemoglobin by the compound was rather slowly progressed, but the amount of methemoglobin formed was proportional to the concentration of oxyhemoglobin even when the concentration of the compound was low. The sesamol-induced methemoglobin formation was influenced by inositol hexaphosphate, an allosteric effector of hemoglobin. Thus, the phosphate enhanced the transformation of oxyhemoglobin and inhibited the transformation of deoxyhemoglobin.     

The effect of potassium chloride on the Bohr effect of human hemoglobin.

The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.     

Structure of nitric oxide hemoglobin

We have compared the structure of horse nitric oxide hemoglobin (HbNO) and methemoglobin in the oxy quaternary structure by difference Fourier analysis at 2.8 Å resolution. Both nitric oxide and oxygen assume bent co-ordination geometry and form low-spin complexes in binding to heme; on the basis of preferred ligand and heme stereochemistry, HbNO is the closest analog of HbO₂ (oxyhemoglobin) examined to date. To the resolution of the X-ray data, the stereochemistry of the heme-NO complex in hemoglobin and the corresponding free heme complex appears similar. In contrast, the ligand pockets in hemoglobin hinder binding of cyanide and carbon monoxide in their preferred linear axial co-ordination modes and force them to assume a strained off-axis binding stereochemistry. The structural similarity between HbNO and HbO₂ is reflected in their kinetic behavior, which is similar, and distinct from that of carboxyhemoglobin.     

ESR correlation times of 2,2,6,6-tetramethyl piperidone-N-oxyl (Tempone) in solutions of hemoglobin A and hemoglobin S.

Correlation times for the tumbling motion of the spin probe 2,2,6,6,-tetramethyl piperidone-N-oxyl (Tempone) were obtained in the presence of different concentrations of oxyhemoglobin A, oxyhemoglobin S, and deoxyhemoglobin S and compared to the viscosity of non-gelling hemoglobin solutions. Reorientational motion (or tumbling) of Tempone in gelled solutions of deoxyhemoglobin S is as great as that in non-gelled hemoglobins of the same total concentration. It is concluded that the gel does not exclusively partition Tempone into an aqueous phase of lower solute concentration after gel formation. The gel at room temperature is a highly mobile and dynamic structure on the microscopic level.     

The pH dependence of the binding of D-glycerate 2,3-bisphosphate to deoxyhemoglobin and oxyhemoglobin. Determination of the number of binding sites in oxyhemoglobin.

The number of Bohr protons released upon oxygenation has been measured over a large range of human hemoglobin concentrations (0.02 to 4.5 mM) in the presence of equimolar amounts of D-glycerate 2,3-bisphosphate. From these data the association constants for the binding of this organic phosphate to deoxyhemoglobin and oxyhemoglobin were calculated at different pH values. The maximum number of protons absorbed upon binding to oxyhemoglobin was determined as well. The maximum number of protons bound to deoxyhemoglobin upon binding of D-glycerate 2,3-bisphosphate was measured independently. From the pH dependence of the association constants and the maximum number of protons absorbed it could be concluded that only one D-glycerate 2,3-bisphosphate can be bound to both deoxyhemoglobin and oxyhemoglobin.     

Demonstration of lag phase in the sol-gel transformation of deoxygenated S hemoglobin without temperature alteration.

1). During the sol to gel transformation of deoxygenated sickle hemoglobin, a time-dependent process preceding gel formation (lag phase) was demonstrated that was inversely proportional to a function of the hemoglobin concentration and that occurred without alteration in temperature, pH, or oxygen tension. 2). As determined by the Schachman modification of the capillary viscometer, preparations of oxyhemoglobin S and A and deoxyhemoglobin A were indistinguishable when compared over a wide range of concentrations. Up to the concentration at which gelling occurred, deoxyhemoglobin S exhibited the same viscosity behavior. The viscosity of deoxygenated hemoglobin S within the lower gelling concentration range was normal during the lag phase and became abnormally high only at the time of gelation.     

Comparative spectral absorption characteristics of hemoglobins in elasmobranches,cartilaginous ganoids,and Teleostei

The spectrophotometric parameters of the main hemoglobin forms, deoxy-, oxy-, and carboxyhemoglobin, were studied in representatives of cartilaginous ganoids (giant sturgeon), elasmobranches (piked dogfish, thresher), and bony fish (the Mediterranean horse mackerel). It was established that the hemoglobin absorption spectra in the studied fish species do not have distinct specific features with respect to positions of maxima of absorption bands, their number, and extinctions. There were differences revealed in the process of deoxygenation of oxyhemoglobin in cartilaginous fish, on one hand, and cartilaginous ganoids and bony fish, on the other hand. Incomplete deoxygenation of the piked dogfish oxyhemoglobin appears to be due to the presence of two hemoglobin fractions with different values of the Bohr effect.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 29–32.Original Russian Text Copyright © 2005 by Vasilev.     

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.     

Shear wave elastography can assess the in-vivo nonlinear mechanical behavior of heel-pad

This study combines non-invasive mechanical testing with finite element (FE) modelling to assess for the first time the reliability of shear wave (SW) elastography for the quantitative assessment of the in-vivo nonlinear mechanical behavior of heel-pad. The heel-pads of five volunteers were compressed using a custom-made ultrasound indentation device. Tissue deformation was assessed from B-mode ultrasound and force was measured using a load cell to calculate the force – deformation graph of the indentation test. These results were used to design subject specific FE models and to inverse engineer the tissue’s hyperelastic material coefficients and its stress – strain behavior. SW speed was measured for different levels of compression (from 0% to 50% compression). SW speed for 0% compression was used to assess the initial stiffness of heel-pad (i.e. initial shear modulus, initial Young’s modulus). Changes in SW speed with increasing compressive loading were used to quantify the tissue’s nonlinear mechanical behavior based on the theory of acoustoelasticity. Statistical analysis of results showed significant correlation between SW-based and FE-based estimations of initial stiffness, but SW underestimated initial shear modulus by 64%(±16). A linear relationship was found between the SW-based and FE-based estimations of nonlinear behavior. The results of this study indicate that SW elastography is capable of reliably assessing differences in stiffness, but the absolute values of stiffness should be used with caution. Measuring changes in SW speed for different magnitudes of compression enables the quantification of the tissue’s nonlinear behavior which can significantly enhance the diagnostic value of SW elastography.