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.     

Altered Membrane Structure and Surface Potential in Homozygous Hemoglobin C Erythrocytes

Background

Hemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes.

Methodology and Findings

We found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was ≈2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO₂ to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes.

Conclusion

Our data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure.     

Maintenance and mobility of hemoglobin and water within the human erythrocyte after detergent disruption of the plasma membrane.

Is an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water-holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D₂O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D₂O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin-sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij-permeabilized erythrocytes washed in D₂O saline or in a D₂O K buffer maintained a higher heavy water-holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij-treated and washed erythrocyte “shells” revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water-holding capacity of the erythrocyte can be accounted for by the water-holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the submembrane lamina.     

Microspectrophotometric and scanning microphotometric studies of carp (Cyprinus carpio L.) erythrocytes

Summary Carp (Cyprinus carpio) hemoglobin readily autoxidizes in blood smears. Quantification of Soret-band absorbance in individual erythrocytes by means of scanning cytophotometry therefore requires more elaborate methods of preparation of blood samples. Of the fixatives that have been tested, suspension of whole blood in isotonic salt solutions containing glutaraldehyde was most suitable. Glutar-aldehyde-fixed red blood cells are totally resistant to hemolysis. In the course of fixation, hemoglobin is transformed to methermoglobin. Spectrophotometry indicated extensive similarities between glutaraldehyde-fixed carp methemoglobin and human methemoglobin. In aqueous solutions, the intensity of the Soret-peak was pH-dependent. The allosteric modifier organic polyphosphate caused anR↔T transition, resulting in increased molar extinctions. Dried preparations showed Soret-spectra that were not influenced from either pH or organic polyphosphate concentration of the aqueous suspensions in which the erythrocytes had been stored. The same was true for slide preparations of cyano-methemoglobin, easily derived from methemoglobin on addition of potassium cyanide. In the absence of oxygen fresh blood cells from carp slowly transform their hemoglobin into deoxyhemoglobin. Spectra of the intermediate stages of deoxygenation, Hb₄(O₂)₃, Hb₄(O₂)₂ and Hb₄(O₂), as well as mixtures of these intermediates, could be monitored.     

Upregulation and Mitochondrial Sequestration of Hemoglobin Occur in Circulating Leukocytes during Critical Illness,Conferring a Cytoprotective Phenotype

The classical role of hemoglobin in the erythrocytes is to carry oxygen from the lungs to the tissues via the circulation. However, hemoglobin also acts as a redox regulator and as a scavenger of the gaseous mediators nitric oxide (NO) and hydrogen sulfide (H₂S). Here we show that upregulation of hemoglobin (α, β and δ variants of globin proteins) occurs in human peripheral blood mononuclear cells (PBMCs) in critical illness (patients with severe third-degree burn injury and patients with sepsis). The increase in intracellular hemoglobin concentration is a result of a combination of enhanced protein expression and uptake from the extra-cellular space via a CD163-dependent mechanism. Intracellular hemoglobin preferentially localizes to the mitochondria, where it interacts with complex I and, on the one hand, increases mitochondrial respiratory rate and mitochondrial membrane potential, and on the other hand, protects from H₂O₂-induced cytotoxicity and mitochondrial DNA damage. Both burn injury and sepsis were associated with increased plasma levels of H₂S. Incubation of mononuclear cells with H₂S induced hemoglobin mRNA upregulation in PBMCs in vitro. Intracellular hemoglobin upregulation conferred a protective effect against cell dysfunction elicited by H₂S. Hemoglobin uptake also was associated with a protection from, and induced the upregulation of, HIF-1α and Nrf2 mRNA. In conclusion, PBMCs in critical illness upregulate their intracellular hemoglobin levels by a combination of active synthesis and uptake from the extracellular medium. We propose that this process serves as a defense mechanism protecting the cell against cytotoxic concentrations of H₂S and other gaseous transmitters, oxidants and free radicals produced in critically ill patients.     

A proton nuclear magnetic resonance study of the interaction of mercury with intact human erythrocytes

The binding of mercuric ion (Hg(II)) by small molecules in the intracellular region of intact human erythrocytes has been studied by ¹H-NMR spectroscopy. HgCl₂ added to intact erythrocytes in saline-glucose suspension is found to cross the membrane and reach an equilibrium distribution among the molecules of the erythrocyte within 4 min. In the intracellular region Hg(II) reacts with GSH and hemoglobin to form the ternary mixed-ligand complex GSH-Hg(II)-hemoglobin. The analogous complex with ergothioneine is formed after all the GSH is complexed. ¹H-NMR spectra show that the GSH-Hg(II)-hemoglobin complex also forms in simpler solutions containing HgCl₂, GSH and hemoglobin, whereas the complex Hg(GSH)₂ predominates in solutions of GSH and HgCl₂. The lifetime of the GSH in the GSH-Hg(II)-hemoglobin complex is shown to be less than 30 s, which provides direct evidence for the first time that Hg(II) complexes in biological systems are quite labile, even though their thermodynamic stability is large. The effectiveness of eight sulfhydryl-containing ligands, some of which have been used as antidotes for Hg(II) poisoning, for releasing GSH from its Hg(II) complex in hemolyzed erythrocytes was also studied. Dithiol ligands were found to be more effective than monothiols, with dithioerythritol the most effective of the dithiols.     

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H₂O₂, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H₂O₂ or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.     

New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy

The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb_sm and Hb_c). We show that the conformation of Hb_sm differs from the conformation of Hb_c. This finding has an important application, as the comparative study of Hb_sm and Hb_c could be successfully used in biomedical research and diagnostic tests.     

Prostaglandin E2 effects on oxygen affinity by sickle erythrocytes

Prostaglandin E₂ slightly increases the oxygen affinity of normal and sickle cell trait erythrocytes. Sickle cell erythrocytes display decreased oxygen affinity in the presence of PGE₂, and more complex dissociation behavior, which is interpreted in terms of membrane mediated effects on hemoglobin oxygen carriage.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Effect of Glycation of Hemoglobin on its Interaction with Trifluoperazine

Trifluoperazine (TFZ), a phenothiazine drug, penetrates into human erythrocytes and releases oxygen by interaction with hemoglobin. TFZ-induced oxygen release from hyperglycemic erythrocytes isolated from diabetic patients is considerably less compared to that from the cells of normoglycemic individuals. In diabetes mellitus, hemoglobin is significantly glycated by glucose. Non-glycated hemoglobin, HbA₀ and its major glycated analog, HbA_1c have been separated from the blood samples of diabetic patients. TFZ releases considerable amount of oxygen from HbA₀, but very little from HbA_1c. Spectrofluorimetric studies reveal that TFZ forms excited state complexes with both HbA₀ and HbA_1c. Titration of HbA₀ with TFZ in a spectrophotometric study exhibits two isosbestic points. Similar experiment with HbA_1c causes gradual loss of the Soret peak without appearance of any isosbestic point indicating a possibility of heme loss during interaction, which is also supported by gel filtration experiment and SDS-PAGE experiment followed by heme staining. The results suggest that drug action on hemoglobin is influenced by glycation-induced structural modification of the protein.     

On the kinetics of erythroid cell differentiation in fetal mice. II. DNA and hemoglobin measurements of individual erythroblasts during gestation

Microspectrophotometric absorption measurements were used to determine the hemoglobin content of erythroid cells derived from the yolk sac during gestation of fetal C3H mice, from day 9 to day 15. Using the DNA content as a marker for the mitotic state between 2C and 4C phase, five successive cell generations and their mean hemoglobin contents were distinguished: 12 pg (pg, picogram = 10^?12 gm). 22.2 pg, 37 pg, 50 pg and 56 pg. In the final state, nucleated erythrocytes contained 98 ± 22 pg hemoglobin. Erythroid cells derived from the liver were measured on day 15 of fetal gestation. The hemoglobin content of proerythroblasts was below 0.3 pg. The two cell generations in the basophilic state had 0.6 pg and 1.7 pg respectively. Polychromatic erythroblasts yielded a hemoglobin content of 5.1 pg in the first cell generation and 7.5 pg in the second one. Orthochromatic erythroblasts contained 8 pg, reticulocytes 12 pg and mature erythrocytes 28 ± 7 pg hemoglobin. Calculations based on these data suggest that the rate of total hemoglobin synthesis is similar in both yolk sac and liver erythropoiesis. The difference between the final hemoglobin content in nucleated erythrocytes of yolk sac origin and that in hepatic erythrocytes can be explained by the different cell generation times.     

Post-translational modification of hemoglobin in alcoholism

Hemolysates from a majority of alcoholic individuals contain a hemoglobin fraction having chromatographic properties resembling those of hemoglobin A_Ic, a normally occurring glycosylated hemoglobin, but differing from the latter in not being bound by boronated agarose. A hemoglobin fraction having similar properties was also isolated from human erythrocytes exposed to acetaldehyde. These observations and other evidence suggest that the abnormal hemoglobin fraction present in men and women addicted to alcohol is produced by addition to hemoglobin A_o of an aldehyde or ketone formed metabolically from ethanol and thus may serve as a diagnostic indicator of alcoholism.     

Interaction between bacteria and erythrocytes and their role in the development of infectious anaemia

The concentration of hemoglobin in blood of patients with pyoinflammatory diseases was shown to depend on the ability of bacteria to interact with erythrocytes. Bacteria with high hemolytic activity (> or = 70% hemolysis) and anti-hemoglobin activity (>3 g/1) were isolated from pyoinflammatory foci of patients suffering from anaemia. Structural changes were investigated in vitro and in vivo erythrocytes in terms of their interaction with microorganisms by electron microscopy. For the first time, the phenomenon of coccal location within erythrocytes was established. Staphylococci with a high level of hemolytic and anti-hemoglobin activity appear to survive and multiply within erythrocytes that results in destroying hemoglobin. Staphylococci with a low level of mentioned activities are subjected to destructive changes within erythrocytes and subsequent killing.     

Bufferless lysis of erythrocytes for isolation of hemoglobin using modified cellulose acetate membranes

This paper presents a modified cellulose acetate membrane prepared using a dry casting technique that can be used to perform lysis of erythrocytes and isolation of hemoglobin. Isolation of hemoglobin is thus achieved without the use of lysis buffers. Cellulose acetate (CA) membranes are embedded with ammonium chloride (NH₄Cl) and potassium bicarbonate (KHCO₃), which act as lysing agents. The presence of embedded salts is confirmed using EDS analysis. The pores in the CA membrane act as filters. The average pore size in these membranes is designed to be 1.5 μM, as characterized by SEM analysis, so that they allow hemoglobin to pass through and block all other cells and unlysed erythrocytes present in blood. When a drop of blood is added to the membrane, the NH₄Cl and KHCO₃ embedded in the membrane dissolve in plasma and lyse the erythrocytes. The filtered hemoglobin is characterized using UV-Vis Spectroscopy. The results indicate extraction of higher concentration of hemoglobin compared with conventional methods.     

The fatty acid composition of phospholipids and absorption spectra of lipid extracts from lamprey,frog, and rat erythrocytes

The work studies the content and fatty acid composition of phospholipids as well as the absorption spectra of lipid extracts from red blood cells of poikilothermal and homoiothermal animals at different evolutionary levels. The objects of study include two poikilothermal species, the river lamprey (Lampetra fluviatilis) that uses oxygen dissolved in water, and the common frog (Rana temporaria) that consumes oxygen both from water and from air. A homoithermal animal is the white rat (Rattus rattus) that inhabits the terrestrial-aerial environment. The animals are studied in winter and spring. The phospholipid content in lamprey blood plasma is found to be twice higher than that in its erythrocytes. In the frog and the rat, the ratio is reverse. Determination of the fatty acid lipid composition of red blood cell phospholipids suggests that membranes in the lamprey are denser than in the frog. As for the fatty acids in the erythrocyte fraction of rat blood, they appear to be less diverse, with a double prevalence of saturated acids over unsaturated ones and devoid of long chain (C22) ω3 fatty acids. All of this results in a lower degree of unsaturation and a denser packing of fatty acids in the membrane structures of rat erythrocytes. The mechanism of reversible binding of O₂ molecules to hemoglobin in erythrocytes is discussed. Presumably, the mechanism of interaction between molecules of O₂ and molecules of water prevents the exchange interaction of electrons of the hemoglobin iron atoms with an oxygen molecule. This is confirmed by our obtained absorption spectra, which show that in the lipid extract almost totally devoid of water the heme isolated from erythrocytes is converted to hemin.     

Effect of vanadate on phosphoryl transfer enzymes involved in glucose metabolism

Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate. Phosph$\text{o}$ glucomutase and phosphoglycerate mutase are inhibited at the μM range. 2,3-Bisphosphoglycerate phosphatase is completely inhibited by 0.5 mM vanadate. 2,3-Bisphosphoglycerate synthase, hexokinase, phosphoglycerate kinase and fructose-1,6-P₂ phosphatase are partially inhibited by mM vanadate. Phosphofructokinase and pyruvate kinase are not affected. The glycolytic enzymes which mechanism does not involves phosphoryl transfer step are not affected by vanadate.     

Phosphorus-31 relaxation times of 2,3-diphosphoglycerate in intact human erythrocytes

The reciprocals of the spin-lattice relaxation times (T₁s) of the 2-P and 3-P nuclei of 2,3-diphosphoglycerate (DPG) increased linearly as percent DPG bound was raised in model hemoglobin solutions. The 2-P T₁ was slightly greater in intact erythrocytes than in model solutions under similar experimental conditions. The change in the 3-P T₁ with cellular deoxygenation was anomalous indicating that this nucleus should not be used to estimate DPG binding inside intact erythrocytes.     

Plasmodium falciparum: Activity of artemisinin against Plasmodium falciparum cultured in sickle trait hemoglobin AS and normal hemoglobin AA red blood cells

The presence of sickle hemoglobin causes accumulation of hemoglobin degradative products that favor oxidative reaction in erythrocytes. Artemisinin derivatives exert antiparasite effects through oxidative reactions within infected erythrocytes. Using [³H]-hypoxanthine incorporation, we therefore did an in vitro comparison of IC₅₀ values for artemisinin in Plasmodium falciparum-infected erythrocytes from sickle cell trait (AS) and normal (AA) individuals. IC₅₀ values for chloroquine served as control. Without drugs, parasite growth was similar in AA and AS erythrocytes. Gender, age and blood group of donors had no significant effects on parasite growth. IC₅₀ value for artemisinin was 27 ± 14 nM in AS (N = 22) compared to 24 ± 9 nM (N = 27) in AA erythrocytes (P = 0.4). IC₅₀ values for chloroquine were also similar in AA (22 ± 8 nM) and AS (20 ± 11 nM) erythrocytes. These results show no evidence of elevated artemisinin activity on P. falciparum in AS erythrocytes in vitro.     

Oxygen equilibria and structural characteristics of the tetrameric and polymeric intracellular hemoglobins from the bivalve molluscBarbatia reeveana

Summary The arcid bivalveBarbatia reeveana contains within its erythrocytes two hemoglobins with remarkably different structures and oxygen equilibrium properties. A tetrameric hemoglobin (M _r about 60,000) with non-identical subunits (₂₂) constitutes about 60% of the erythrocytic heme protein. This hemoglobin has a relatively low oxygen affinity (P ₅₀=19 Torr at 20°C, pH 7.2), shows cooperativityn _H=2.2, shows no Bohr effect between pH 6.8 and 7.6 and a heat of oxygenation (H) of –5.4 kcal/mole between 15 and 35°C. Its oxygen affinity appears to be insensitive to ATP.B. reeveana erythrocytes also contain another hemoglobin withM _r=430,000, the largest intracellular hemoglobin known in any organism. The subunit of this hemoglobin is unusual, having aM _r of 32–34,000 and two heme oxygen binding sites per polypeptide chain. The large hemoglobin has a very low oxygen affinity (P ₅₀=33 Torr at 20°C, pH 7.2), shows slight cooperativity,n _H=1.8, and no Bohr effect (Grinich and Terwilliger 1980). The H at pH 7.2 equals –2.9 kcal/mole, a low value for most hemoglobins, and its O₂ affinity appears to be insensitive to ATP. The two hemoglobins ofB. reeveana, so different in their structure, are also different in their functional properties.