Fusion of erythrolysosomes in epithelial cells of the sheep placenta

Summary In trophoblastic epithelial cells of the sheep placenta the breakdown of erythrocytes within complex erythrolysosomes was studied at the ultrastructural level.It was found that the formation of complex erythrolysosomes containing from two to several erythrocytes as a result of fusion of erythrolysosomes within the epithelial cells was a common occurrence when the epithelial cells engulfed a large number of erythrocytes. The erythrocytes enclosed in complex erythrolysosomes appear to be either in the same or in different stages of hemolysis.In the process of breakdown of erythrocytes within complex erythrolysosomes five successive stages of hemolysis could be distinguished. Acid phosphatase activity was demonstrated in the complex erythrolysosomes and appeared to be located in the angular interspaces between the erythrocytes and the lysosomal membrane. The fragmentation of complex erythrolysosomes with formation of small hemoglobin-containing lysosomes also occurred.The fusion of erythrolysosomes with formation of complex erythrolysosomes can be considered as an additional mechanism in the process of erythrocyte breakdown in the epithelial cells of the sheep placenta.     

Final stages of erythrophagocytosis in the sheep placenta

Summary In trophoblastic epithelial cells of the sheep placenta the final stages of erythrocyte breakdown within the lysosomal apparatus were studied at the ultrastructural level.As a result of hemoglobin digestion lysosomes containing hemoglobin-derived pigments (HDP) were formed. The HDP-lysosomes were acid phosphatase-positive, highly electron-dense bodies of round to irregular shape containing whorled membranous formations. The accumulation of these lysosomes in epithelial cells led to fusion resulting in the formation of conglomerates. At the end of the gestation period the amount of HD Plysosomes and their conglomerates markedly increased.In addition to erythrocytes the trophoblastic epithelial cells in the erythrophagocytic regions phagocytosed maternal leukocytes and neighbouring epithelial cells and giant cells.By gradual accumulation of HDP-lysosomes and remnants of phagocytosed cells, highly electron-dense acid phosphatase-positive residual bodies of variable appearance were formed within the epithelial cells.At the end of pregnancy the spaces between juxtaposed villi of the trophoblastic epithelium in the erythrophagocytic zones were occluded by apposition of the epithelial cells. In these occluded regions an increase in highly electron-dense large-sized residual bodies (15–22 m of dimension) occurred as a result of multiple cell phagocytosis in combination with fusion. In these residual bodies the numerous incorporated HDP-lysosomes and the remnants of phagocytosed cells could still be recognized.     

Investigations into combined dietary deficiencies of copper,selenium, and vitamin E in the rat

Interactions between dietary Cu, Se, and vitamin E in ascorbate-induced hemolysis of erythrocytes obtained from rats fed diets deficient or adequate in these elements were investigated. Hemolysis was affected by all three dietary factors, through closely interrelated but distinct mechanisms. In vitamin E-deficient cells, hemolysis was increased and the amount of hemolysis was directly related to the amount of hemoglobin breakdown. Deficiency of Cu or Se decreased hemolysis, but only in vitamin E-deficient cells. Vitamin E did not affect the breakdown of hemoglobin, but Cu and Se did. Hemolysis and hemoglobin breakdown were decreased by the addition of glucose, through mechanisms independent of that involving reduced glutathione metabolism. These results suggest that vitamin E acts within erythrocyte membranes to prevent products of hemoglobin breakdown from initiating peroxidation and subsequent hemolysis. Effects of Cu and Se are linked with that of vitamin E by the involvement of glutathione peroxidase and Cu superoxide dismutase in the cytoplasmic breakdown of hemoglobin, rather than by a direct effect of these enzymes on lipid peroxidation. It is concluded that the erythrocyte, because of its high heme content, probably represents a special system in terms of peroxidative pathways, and these findings may not be directly applicable to other tissues.     

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.     

The red cell revisited--matters of life and death.

An erythrocyte-fractionating method combining volume and subsequent density separation is described. Iron isotope (59Fe)-validation proved this combination of methods to be complementary. By deploying HbA1c as cell age marker, obtained fractions demonstrated that circulating erythrocytes lose 20% of hemoglobin and membrane by shedding vesicles. Vesiculation from older cells proved to be facilitated by the spleen. Animal studies revealed that such vesicles are rapidly removed from the circulation by scavenger receptors on Kupffer cells with phosphatidylserine acting as the principal ligand. These studies reveal the existence of an alternative pathway of erythrocyte breakdown. This means that the premortal substrate of 20% of any erythrocyte is at our disposal. As this kind of vesiculation takes place during the entire erythrocyte lifespan, loss and sometimes reutilisation of marker substances limits the usefulness of isotope studies to the first half of the erythrocyte lifespan, thereby putting the dogmatic lifespan of 120 days into question. Furthermore, these studies add to the understanding of hemoglobin A1c (HbA1c) metabolism and the origin of the wide variation of erythrocyte parameters in peripheral blood. Removal of old erythrocytes from the circulation and from donor blood may open new ways into the treatment of both bilirubin and secondary iron overload.     

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.     

Association of glyceraldehyde-3-phosphate dehydrogenase with the plasma membrane of the intact human red blood cell

Glycolytic enzymes have been observed to associate in vitro with membranes and cytoplasmic filaments in a variety of systems, but their distribution in vivo is contested. We have therefore examined the distribution of glyceraldehyde-3-phosphate dehydrogenase (G3PD) in the intact human erythrocyte using indirect immunofluorescence and affinity-purified rabbit antibodies to G3PD. Antibody specificity was demonstrated by immunoblotting as well as immunofluorescence experiments with ghosts specifically depleted of and reconstituted with G3PD. Anti-G3PD immunolabeling experiments utilized both fixed whole cells and fixed cell suspensions infused with 2.3 M sucrose, frozen and thick-sectioned. In all experiments a two-step fixation protocol was employed which ensured that cytoplasmic hemoglobin was retained when cells were subjected to Triton X-100 permeabilization, the anti-genicity of G3PD was preserved, and antibody penetration was complete. We used mixtures of biotinylated affinity-purified antibodies to G3PD and dichlorotriazinylaminofluorescein-labeled, affinity-purified antibodies to hemoglobin, followed by rhodamine-streptavidin, in double-label experiments. In both whole and sectioned human erythrocytes, G3PD staining was predominantly membrane associated while hemoglobin staining was diffusely distributed throughout the cytoplasm. In isolated ghosts, some G3PD was tightly bound to the membrane and was resistant to elution with phosphate-buffered saline and NAD+/arsenate. However, in immunolabeled rat reticulocytes and erythrocytes G3PD was cytoplasmic. Nucleated human blood cells and platelets also exhibited cytoplasmic G3PD. In approximately 10% of the human erythrocyte population G3PD was also cytoplasmic. These cells were flatter in shape and exhibited strong cytoplasmic immunolabeling for hemoglobin which was sometimes concentrated along the cell membrane; possibly, these cells were late reticulocytes or early erythrocytes. We conclude that G3PD is preferentially associated with the plasma membrane of human erythrocytes in a specific fashion.     

Immunoelectron microscopy localization of immunoglobulin G in human placenta

Immunoelectron microscopy of IgG molecules in human mature placenta has shown that IgG bound to microvillar surfaces and the inner wall of endocytotic vesicles of syncytiotrophoblasts. The endocytotic vesicles, containing both bound and unbound IgG molecules, tended to fuse with each other or with other cellular organelles, particularly with lysosomes. The phagolysosomes were more abundant in the basal regions of the cells. Apparently some IgG molecules were not digested by lysosomal enzymes. Vesicles with residual IgG were found to fuse with the basal and basolateral cell membrane and to discharge their contents into the extracellular space by exocytosis. It is suggested that IgG molecules were transported through the trophoblastic basement membrane and the interstitial space by diffusion to the endothelial basement membrane. The IgG molecules then migrated into the fetal vascular lumen via endothelial gaps and interendothelial spaces.     

Selective radiolabeling of cell surface proteins to a high specific activity

A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125I-(hydroxyphenyl)propionyl groups via 125I-sulfosuccinimidyl (hydroxyphenyl)propionate (125I-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[125I]iodide labeling technique revealed that 125I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spatial orientations on erythrocytes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.     

Erythrocyte membrane antigen expression during friend cell differentiation: Analysis of two non-inducible variants

Erythrocyte membrane antigens have been detected on induced Friend erythroleukemic cells with a rabbit antiserum raised against mouse erythrocyte membranes. The antibody specificities of this antiserum have been quantitatively analyzed using a cellular radioimmunoassay. After absorption with thymocytes, the rabbit anti-erythrocyte membrane serum bound to dimethylsulfoxide (DMSO)-induced Friend erythroleukemic cells and to mouse erythrocytes but not to uninduced Friend cells or thymocytes. Reciprocal inhibition studies demonstrated that, following complete thymocyte absorption, the antiserum detected similar antigenic specificities, termed erythrocyte membrane antigens (EMA), on both mature erythrocytes and induced Friend cells. The expression of these erythrocyte membrane antigens was also induced on Friend cells by other agents, such as ouabain and dimethylacetamide (DMA). In contrast, exogenous hematin, which did not induce hemoglobin synthesis in the Friend cell clones used in this study, also did not induce erythrocyte membrane antigen expression. Two independently derived variant clones which do not produce hemoglobin in reponse to DMSO were analyzed for their ability to produce erythrocyte membrane antigens in response to various inducers of Friend cell differentiation. Clone TG-13 is not inducible by DMSO or hematin but is weakly induced by DMA for both hemoglobin production and erythrocyte membrane antigen expression. Another variant clone, M18, was also analyzed. This clone does not synthesize detectable hemoglobin when grown in either DMSO or hematin alone, but undergoes extensive hemoglobin synthesis when grown in medium containing both DMSO and hematin. M18 does, however, express erythrocyte membrane antigens when grown in DMSO alone: the presence of hematin and DMSO together in the growth medium does not enhance expression of these antigens. Thus M18 appears to be defective for hemoglobin inducibility, and this defect can be overcome by exogenous hematin; however, the expression of erythrocyte membrane antigens is not affected by this block in hemoglobin synthesis. The results with the variant clones are discussed in terms of a program for Friend cell differentiation in which the induction of hemoglobin synthesis and erythrocyte membrane antigen expression are under both co-ordinate and separate controls.     

Susceptibility of erythrocytes from several animal species to Vibrio vulnificus hemolysin

The hemolytic activity of Vibrio vulnificus hemolysin (VVH) against erythrocytes from several animal species (sheep, horse, cow, rabbit, chicken) was investigated. VVH was active against erythrocytes from all species, but the amount of VVH causing 50% hemolysis under identical conditions (hemolytic susceptibility to VVH) differed. The degree of 125I-labeled VVH (125I-VVH) binding to each erythrocyte species correlated with the susceptibility of the cells to hemolysis. However, marked differences in the binding ability of 125I-VVH were not observed against liposomes constructed with lipids from each erythrocyte membrane. On the other hand, release of hemoglobin (Hb) differed for each of the erythrocyte species despite administration of approximately the same hemolytic VVH concentration to each species. Furthermore, under hypotonic conditions, the stability of each erythrocyte species varied markedly; the more susceptible the erythrocyte to VVH, the more unstable it was under such conditions. These results, therefore, suggest that the susceptibility of erythrocytes to VVH may be closely associated with the binding ability of VVH and erythrocyte membrane stability.     

Iron release, oxidative stress and erythrocyte ageing

Iron, to be redox cycling active, has to be released from its macromolecular complexes (ferritin, transferrin, hemoproteins, etc.). Iron is released from hemoglobin or its derivatives in a nonprotein-bound, desferrioxamine-chelatable form (DCI) in a number of conditions in which the erythrocytes are subjected to oxidative stress. Such conditions can be related to toxicological events (haemolytic drugs) or to physiological situations (erythrocyte ageing, reproduced in a model of prolonged aerobic incubation), but can also result from more subtle circumstances in which a state of ischemia-reperfusion is imposed on erythrocytes (e.g., childbirth). The released iron could play a central role in oxidation of membrane proteins and senescent cell antigen (SCA) formation, one of the major pathways for erythrocyte removal. Iron chelators able to enter cells (such as ferrozine, quercetin, and fluor-benzoil-pyridoxal hydrazone) prevent both membrane protein oxidation and SCA formation. The increased release of iron observed in beta-thalassemia patients and newborns (particularly premature babies) suggests that fetal hemoglobin is more prone to release iron than adult hemoglobin. In newborns the release of iron in erythrocytes is correlated with plasma nonprotein-bound iron and may contribute to its appearance.     

Membrane orientation of sheep spectrin

Based primarily on studies of human erythrocytes, current theories of the structure and organization of erythrocyte membrane localize spectrin to the membrane cytoplasmic surface. Affinity purified anti-sheep spectrin antibodies were used in indirect immunofluorescence studies of intact erythrocytes from various vertebrate species and inside-out and right-side-out impermeable sheep erythrocyte vesicles. This investigation detected immunologically reactive external and potentially transmembranal determinant(s) of the sheep erythrocyte spectrin "assembly." Parallel studies using anti-sheep and anti-human spectrin antibodies, as well as 125I surface-labelling studies of intact sheep and human erythrocytes, indicated that this particular membrane orientation of spectrin was evident in sheep but not in human erythrocytes. Antisera containing antibodies to the external portion of this spectrin "assembly" demonstrated external fluorescence to a variable degree on some, but not all, vertebrate erythrocytes surveyed, confirming that the sheep erythrocyte was not the only exception. It is suggested that there may be subtle species variability in the intermolecular associations of the spectrin "assembly" with(in) the erythrocyte membrane not requiring alterations of the spectrin molecule itself.     

Fine structure of developing hagfish erythrocytes with particular reference to the cytoplasmic organelles

Cytological changes accompanying the maturation of erythrocytes in the “Pacific hagfish” (Eptatretus stoutii) were studied. Great numbers of immature and mitotically dividing red blood cells in the peripheral circulation of the hagfish appear to indicate that extensive differentiation and proliferation occurs in the blood stream of this animal. The immature erythrocytes contained mitochondria, Golgi membranes, centrioles, microtubules and a high density of ribosomes in the cytoplasm. Intermediate stages revealed lysosomes in the cytoplasm. With progressive differentiation the hagfish erythrocytes accumulate hemoglobin and lose most of their cytoplasmic organelles. The various cytoplasmic organelles are apparently lost through a degradation process brought about by lysosomal autolysis. The undigested products of degradation such as mitochondrial and other intercellular membranes are apparently extruded by way of the plasma membrane. The plasma membrane of young as well as mature erythrocytes display evidence of intense pinocytotic activity. The nucleolus undergoes a reduction in size with progressive maturation. The cytoplasm of mature erythrocytes consists predominantly of hemoglobin. An equatorial microtubular marginal band is identifiable in differentiating erythrocytes.     

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.     

Differential time‐dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum‐infected hemoglobinopathic erythrocytes

During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid‐osmotic model on the grounds of time‐resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid‐osmotic model can predict time‐dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model‐predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid‐osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum‐infected erythrocytes. The contribution of vesiculation to the malaria‐protective function of hemoglobin S is discussed.     

Beneficial effect of succinic acid monoethyl ester on erythrocyte membrane bound enzymes and antioxidant status in streptozotocin-nicotinamide induced type 2 diabetes

Succinic acid monoethyl ester (EMS) was recently proposed as an insulinotropic agent for the treatment of non-insulin dependent diabetes mellitus. In the present study the effect of EMS and metformin on erythrocyte membrane bound enzymes and antioxidants activity in plasma and erythrocytes of streptozotocin-nicotinamide induced type 2 diabeteic model was investigated. Succinic acid monoethyl ester was administered intraperitonially for 30 days to control and diabetic rats. The effect of EMS on glucose, insulin, hemoglobin, glycosylated hemoglobin, TBARS, hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (Gpx), glutathione-S-transferase (GST), vitamins C and E, reduced glutathione (GSH) and membrane bound enzymes were studied. The effect of EMS was compared with metformin, a reference drug. The levels of glucose, glycosylated hemoglobin, TBARS, hyderoperoxide, and vitamin E were increased significantly whereas the level of insulin and hemoglobin, as well as antioxidants (SOD, CAT, Gpx, GST, vitamin C and GSH) membrane bound total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase were decreased significantly in streptozotocin-nicotinamide diabetic rats. Administration of EMS to diabetic rats showed a decrease in the levels of glucose, glycosylated hemoglobin, lipid peroxidation markers and vitamin E. In addition the levels of insulin, hemoglobin, enzymic antioxidants, vitamin C, and GSH and the activities of membrane bound enzymes also were increased in EMS and metformin treated diabetic rats. The present study indicates that the EMS possesses a significant beneficial effect on erythrocyte membrane bound enzymes and antioxidants defense system in addition to its antidiabetic effect.     

Platelet plasma membrane lectin activity

The lectin activity of human platelet and erythrocyte membranes was evaluated using trypsinized, formalinized erythrocytes from eight species. Platelet membranes had the greatest lectin activity against cow erythrocytes, but also had significant activity against human, sheep, electric eel, and rabbit erythrocytes. In contrast, erythrocyte membranes only had low lectin activity against electric eel erythrocytes with no activity against the other types of erythrocytes tested. The platelet membrane lectin activity was found to reside in protein molecules on the external surface of the platelet plasma membrane. The lectin activity of platelet membranes was inhibited by amino sugars and some basic amino acids: N-acetylated amino sugars and other neutral sugars were without effect. These results demonstrate that the external surface of the platelet plasma membrane has a specific lectin activity.     

Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis)]

The activation energy of thermohemolysis of erythrocytes changes from 36 +/- 5 kcal/mol (35-45 degrees C) to 97 +/- 5 kcal/mol (45-55 degrees C) at the temperature about 45 degrees C in isotonic buffer. The break on Arhenius' plot is preserved also when erythrocytes are placed into plasma. The character of Arhenius' plot is the same when erythrocyte hemoglobin is totally oxidated into methemoglobin by chemical way, though thermal stability of such erythrocytes is decreased. The scheme is presented in which thermohemolysis of erythrocytes occurs by two independent ways: thermodenaturation of hemoglobin (limiting stage of the process when t greater than 45 degrees C) and modification of membrane proteins by hemin, the last being a product of hemoglobin oxidation (limiting stage of the process when t less than 45 degrees C).     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

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 H(2)O(2); 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.