Oxyhemoglobin In Solution Research Articles

Mössbauer and positron life-time spectroscopy of γ- and electron irradiated oxyhemoglobin solution

The primary processes of the oxyhemoglobin irradiation in solution were analyzed by Mössbauer and positron life-time spectroscopies. We studied the effect of γ-irradiation with an average energy of 15.5 MeV with doses from 100 to 600 kGy on the erythrocytes solution and that of γ-irradiation with average energy of 9 MeV with doses from 16.5 to 33 kGy on the oxyhemoglobin solution and the effect of electron irradiation with average energy of 10 MeV with doses from 1.7 to 5 kGy on oxyhemoglobin in solution. Mössbauer spectra showed the changes of oxyhemoglobin content in the samples after irradiation and deoxygenation of oxyhemoglobin as well as new iron-containing compounds formation. Positron life-time spectra reflected structural modifications of irradiated solutions.

Inhibition of endothelium-dependent vasorelaxation by sickle erythrocytes

Interactions between erythrocytes and vascular endothelium have been implicated in the pathogenesis of vaso-occlusion in sickle cell anemia. Sickle erythrocytes adhere to endothelial cells and facilitate trapping of rigid sickle cells in microvessels. Compensatory dilation of precapillary arterioles may mitigate the occlusion. The endothelium regulates vasoreactivity by elaborating endothelium-derived relaxing factor (EDRF), a small molecule that passes freely into vascular smooth muscle where it initiates vasorelaxation by activating soluble guanylate cyclase in the smooth muscle cell cytoplasm. Endothelial release of EDRF can be stimulated by agonists such as acetylcholine. It is highly sensitive to decomposition by superoxide anions and is rapidly bound and inactivated by oxyhemoglobin in solution. The purpose of this study was to determine whether sickle cell interaction with endothelium disrupts this mechanism of endothelial regulation of vasomotor tone. Transverse strips of rabbit aorta, mounted isometrically in organ baths, were contracted with norepinephrine, and relaxation responses to acetylcholine or other agonists were determined. Responses were measured under control conditions and again in the presence of oxyhemoglobin A or S, or erythrocytes or ghosts from normal control subjects or patients with homozygous sickle cell anemia. Sickle erythrocytes inhibited vasorelaxation to acetylcholine by 83%. Approximately half of the inhibition was attributable to a small amount of oxyhemoglobin S that was leaked into the buffer from the erythrocytes. Consistent with this, sickle erythrocyte ghosts inhibited vasorelaxation to acetylcholine by up to 45%. Ghosts from normal erythrocytes did not inhibit vasorelaxation to acetylcholine, and the inhibition seen with normal erythrocytes was entirely attributable to leakage of oxyhemoglobin A into the bath buffer. There was little or no inhibition of vasorelaxation to nitroglycerin, isoproterenol, or 8-bromocyclic guanosine monophosphate by sickle erythrocytes or ghosts, demonstrating that sickle erythrocytes predominantly inhibit endothelium-dependent vasorelaxation. In addition, superoxide dismutase significantly diminished the inhibition of endothelium-dependent vasorelaxation as a result of sickle erythrocytes and ghosts. These findings indicate that sickle erythrocytes and their membranes interfere with endothelium-dependent vasorelaxation by inhibiting EDRF. Disruption of endothelial regulation of vasoreactivity may contribute to the development of vaso-occlusive phenomena in patients with sickle cell anemia.

Relationship between protein/solvent proton exchange and progressive conformation and fluctuation changes in hemoglobin

The pH dependence of the tertiary structural changes and the quaternary reorganization of the alpha beta interface of oxyhemoglobin in solution has been previously observed in our laboratory. The present work aims to establish whether or not these progressive structural changes with pH result from proton exchange between the protein and the solvent. We therefore have used infrared spectroscopy, acid/base titration and 1H/2H exchange to assess the effect of external proton concentration on the structural and dynamic properties of the hemoglobin molecule in solution. This study is also performed on the carbonmonoxy form since the affinity of hemoglobin for CO is much higher than for oxygen. The present findings demonstrate that affinity changes are closely related to protein fluctuations, as well as structural modifications. An increased affinity, induced either by ligand replacement or pH variation, is associated with the constrained chains exhibiting a lower degree of fluctuation, together with a looser alpha beta interface association.

Spectral and oxygen-release kinetic properties of human hemoglobin bound to the cytoplasmic fragment of band 3 protein in solution

Binding of the cytoplasmic fragment of band 3 protein to oxyhemoglobin in solution caused a spectral change in the absorbance of the hemoglobin beta chain at a ratio of one monomer of band 3 protein per αβ dimer of hemoglobin. This spectral change was reversed at higher ratios of cytoplasmic fragment to hemoglobin. The unusual dependence on protein concentration was interpreted as indicating the formation of higher aggregates of the complex between hemoglobin and the cytoplasmic fragment of band 3 protein. Oxygen-release kinetic measurements also showed marked changes as a function of the concentration of the cytoplasmic fragment of band 3 protein. The higher ratio mixture had significantly different kinetic properties as compared with the lower ratio one, which in turn was different from oxyhemoglobin in solution. The significance of the formation of aggregates of band 3 protein containing oxyhemoglobin dimers is discussed in context with evidence suggesting that band 3 protein may exist as an equilibrium mixture of tetramers and dimers in the membrane.

A column method for deoxygenating human hemoglobin

The mixed-bed resin column charged with dithionite is shown to be an efficient deoxygenator. Deoxyhemoglobin prepared on this column from stock oxyhemoglobin in solution has normal spectral characteristics, and upon reoxygenation the oxyhemoglobin spectrum is identical with that of the original stock oxyhemoglobin solution. The deoxyhemoglobin solution has a pO 2 of less than 1 mm Hg and an ionic strength of less than 5 × 10 −5, and exhibits a Bohr effect and deoxygenation kinetics consistent with literature values. Deoxygenated water is also prepared by this method. Dithionite, oxidation products of dithionite, and ionic contaminants in the elluent remain bound to the resin.

Étude de l'action du chlorure de sodium sur l'oxyhémoglobine: I. Mesures de diffusion de la lumière

The method of light scattering has been used to measure the molecular weight of human oxyhemoglobin in solution, in the presence or absence of sodium chloride, near the pH corresponding to its isoelectric point. The sodium chloride causes the dissociation of the oxyhemoglobin molecule, probably into two halves. Neither by increasing the time of contact with the salt, nor by increasing the salt concentration, does this action ever go to completion in solution. After elimination of the sodium chloride, the molecules are formed again. Urea does not seem to have the same effect as sodium chloride.