Phenylhydrazine-induced changes in erythrocyte membrane surface lipid packing

Phenylhydrazine-induced oxidative damage in red cells results in increased binding of merocyanine 540, a fluorescence probe sensitive to changes in lipid packing. Fluorescence polarization studies with diphenylhexatriene did not reveal major changes in order parameters both in intact red cells and lysates treated with phenylhydrazine. These fluorescence studies indicate that major changes are observed in membrane lipids. Analytical studies of membrane phospholipids revealed a significant decrease in phosphatidylethanolamine. The results of the fluorescence and lipid studies, taken in association with our previously reported findings on spectrin and other cytoskeletal protein degradation in red cells exposed to phenylhydrazine, suggests that degradation of cytoskeleton membrane proteins is also responsible for changes in the lipid bilayer surface of the red cell membrane.     

Membrane alterations in G6PD- and PK-deficient erythrocytes exposed to oxidizing agents

After in vitro treatment of normal, glucose-6-phosphate dehydrogenase-deficient or pyruvate kinase-deficient human erythrocytes with three different oxidizing agents, the extent of lipid peroxidative degradation and the alterations of membrane proteins were evaluated. Exposure to tert-butylhydroperoxide induced, most markedly in G6PD- and PK-deficient erythrocytes, a reduction of protein bands 1, 2, 2.1, 3, 4.1, 4.2, and 5, with the appearance of high-molecular-weight aggregates and of "new" polypeptide components in the 29- to 23-kDa region and with a marked increase of membrane-bound globin. Malonyldialdehyde production was highest in G6PD-deficient cells and relatively low in PK-deficient ones. Methylene blue, which had similar but less relevant effects on lipid peroxidation, in G6PD-deficient erythrocytes caused a conspicuous appearance of high-molecular-weight aggregates and a simultaneous relevant decrease of bands 1 and 2 and of membrane-bound globin; it brought about an almost opposite effect in PK-deficient red cells. Acetylphenylhydrazine, which under our conditions appeared the mildest agent, failed, in normal and PK-deficient erythrocytes, to increase malonyldialdehyde production or to alter membrane proteins, whereas it caused, in G6PD-deficient cells, a slight decrease of bands 1 and 2, a more pronounced decrease of band 3, and a marked increase of bands 4.5 and 4.9.     

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.     

Member-associated changes during erythropoiesis. On the mechanism of maturation of reticulocytes to erythrocytes

The mature mammalian erythrocyte has a unique membranoskeleton, the spectrin-actin complex, which is responsible for many of the unusual membrane properties of the erythrocyte. Previous studies have shown that in successive stages of differentiation of the erythropoietic series leading to the mature erythrocyte there is a progressive increase in the density of spectrin associated with the membranes of these cells. An important stage of this progression occurs during the enucleation of the late erythroblast to produce the incipient reticulocyte, when all of the spectrin of the former cell is sequestered to the membrane of the reticulocyte. The reticulocyte itself, however, does not exhibit a fully formed membranoskeleton. In particular, the in vitro binding of multivalent ligands to specific membrane receptors on the reticulocyte was shown to cause a clustering of some fractions of these ligand-receptor complexes into special mobile domains on the cell surface. These domains of clustered ligand-receptor complexes became invaginated and endocytosed as small vesicles. By immunoelectron microscopic experiments, these invaginations and endocytosed vesicles were found to be specifically free of spectrin on their cytoplasmic surfaces. These earlier findings then raised the possibility that the maturation of reticulocytes to mature erythrocytes in vivo might involve a progressive loss of reticulocyte membrane free of spectrin, thereby producing a still more concentrated spectrin-actin membranoskeleton in the erythrocyte than in the reticulocyte. This proposal is tested experimentally in this paper. In vivo reticulocytes were observed in ultrathin frozen sections of spleens from rabbits rendered anemic by phenylhydrazine treatment. These sections were indirectly immunolabeled with ferritin-antibody reagents directed to rabbit spectrin. Most reticulocytes in a section had one or more surface invaginations and one or more intra-cellular vesicles that were devoid of spectrin labeling. The erythrocytes in the same sections did not exhibit these features, and their membranes were everywhere uniformly labeled for spectrin. Spectrin-free surface invaginations and intracellular vesicle were also observed with reticulocytes within normal rabbit spleens. Based on these results, a scheme for membrane remodeling during reticulocyte maturation in vivo is proposed.     

Membrane alterations in phenylhydrazine-induced reticulocytes

It is known that reticulocytes formed in animals in response to phenylhydrazine treatment have a shorter life span than those formed as a consequence of bleeding. The experiments presented illustrate that the reticulocytes formed consequent to the action of this drug exhibit membrane alterations, in the absence of intracellular oxidative changes (e.g., to hemoglobin), which might be expected to contribute to their decreased survival. These membrane alterations include the formation of flourescent chromolipids, a decrease in spectrin polypeptides, and an increase in high molecular weight membrane protein polymers. It is suggested that these effects are unique to the reticulocytes formed as a response to phenylhydrazine since they are a consequence of the peroxidation of membrane phospholipids initiated by this agent.     

Formation of disulfide bonds between glutathione and membrane sh groups in human erythrocytes

In erythrocytes treated with the SH-oxidizing agent, diamide, mixed disulfide bonds between membrane proteins and GSH are formed involving 20% of the membrane SH groups. To study the distribution of these mixed disulfides over the membrane protein fractions, intracellular GSH was labelled biosynthetically with [2-³H]glycine prior to diamide treatment of the cells and the radioactivity of defined membrane peptide fractions determined. Mixed disulfides preferentially occur in the extrinsic protein, spectrin (six SH groups), in addition to the formation of peptide disulfides. Intrinsic proteins are much less reactive: only one SH group of the major intrinsic protein (band 3) reacts with GSH, which accounts for previously observed impossibility to dimerize band 3 via disulfide bonds in intact cells. The labelling method described offers a promising strategy to label and map exposed endofacial SH groups of membrane proteins with a physiological, impermeable marker, GSH.In ghosts treated with diamide and GSH the number of mixed disulfides formed is greater than in erythrocytes. Polymerization of spectrin via intermolecular disulfide bridges is suppressed, while intramolecular disulfides are still formed, providing a means for the analysis of spectrin structure.The diamide-induced mixed membrane-GSH disulfides are readily reduced by GSH. This suggests, that GSH may also be able to reduce mixed disulfides formed in the erythrocyte membrane under oxidative stress in vivo. The reversible formation of mixed disulfides may serve to protect sensitive membrane structures against irreversible oxidative damage.     

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT^?) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT^? erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT^? erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT^? erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.     

Polarization-optical investigation (topo-optical analysis) of the structure of the human erythrocyte glycocalyx. Influence of pH, ionic strength and diamide-induced spectrin cross linking

The value of membrane anisotropy after fixation and topo-optical analysis of erythrocytes stained with toluidine blue is a measure for the degree of spatial order of the dyestuff-binding acidic residues of the glycocalyx and thus a parameter for the characterization of the glycocalyx structure. Lowering the pH value and/or the ionic strength of the staining medium results in a decrease of membrane anisotropy indicating a lower order of the anionic residues. In agreement with the findings of other authors this phenomenon seems to be connected with an expansion of the glycocalyx. Diamide-induced oxidative crosslinking of spectrin before fixation and staining with toluidine blue at physiological pH and ionic strength also results in a decreased anisotropy. This indirect influence on the glycocalyx structure may be caused by an increase of the charge density within the glycocalyx due to a diamide-induced rearrangement of the membrane skeleton and of the transmembrane proteins bound to it.     

Hyperthermia, unlike ionizing radiation and chemical oxidative stress, does not stimulate proteolysis in erythrocytes

1. Oxidative stress by phenazine methosulfate stimulated proteolysis in erythrocytes. 2. Gamma-irradiation of erythrocytes in the range of 50-1000 Gy also resulted in the induction of proteolysis. 3. Though it has been suggested that hyperthermia imposes an oxidative stress on a cell, hyperthermic exposure of erythrocytes (30 min, 39-49 degrees C) did not stimulate proteolysis during subsequent incubation of whole cells or hemolysates. 4. Proteolytic degradation of spectrin was accelerated during incubation of membranes isolated from cells heated above 45 degrees C but this effect seems to be due rather to thermal denaturation of spectrin than to oxidative modification of cellular proteins by hyperthermia.     

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.     

Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging

The addition of malondialdehyde to erythrocytes in vitro causes a decrease in bands 1 and 2 of spectrin and an increase in high molecular weight protein polymers. Additionally, this agent causes the formation of fluorscent chromolipids characteristic of those produced during the peroxidation of endogenous membrane phospholipids. These same alterations in proteins and lipids are observed in the membranes of older cells fractionated from freshly drawn blood and in the membranes of reticulocytes induced by treatment of animals with phenylhydrazine, but not in reticulocytes induced by bleeding. The former reticulocytes have a much shorter half-life in the circulation than do either normal erythrocytes or reticulocytes produced consequent to bleeding. These experiments and the apparent paradox of "young" reticulocytes with short half-lives suggest that the in vivo polymerization of membrane proteins consequent to radical-induced peroxidation of membrane lipids may contribute to the altered rheological behavior and hence to the splenic sequestration of cells. They also suggest that increases in intrinsic membrane rigidity due to lipid peroxidation, malondialdehyde, and protein polymerization may be a common feature of both aging in normal erythrocytes and in the accelerated aging that accompanies the administration of radical-generating, hemolytic agents. However, it is cautioned that other polymerization reactions involving disulfides, calcium, or direct radical attack on protein monomers may also be important determinants of the visco-elastic properties of erythrocyte membranes.     

Erythrocyte membrane defects in hemolytic anemias found through derivative thermal analysis of electric impedance

Hereditary hemolytic anemias originate mainly from defects in hemoglobin and plasma membrane proteins. Here, we propose a new method, thermal analysis of impedance, sensitive to membrane defects. It detects three processes in erythrocyte membrane; fall in membrane capacity at 49.5 degrees C and activation of passive PO(4)(2+) permeability at 37 degrees C and inorganic ions at 61.5 degrees C. The denaturation of spectrin is involved in the first process whilst the anion channel is involved in latter processes. Using this method three persons with xerocytosis were found whereby the fall in membrane capacity and spherization of erythrocytes were both postponed (53 degrees C) compared to control (49.5 degrees C). In contrast to control cells, strong activation of passive permeability for Cl(-) at 37 degrees C and sucrose at 61 degrees C were detected that were both eliminated by pre-inhibition of the anion channel with 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). In addition, erythrocytes from 15 patients with various forms of anemia were studied in intact state and after refreshment. The results were compared with the data of clinical laboratory and osmotic fragility test. The final conclusion is that this method detects membrane defects with altered spectrin and anion channel syndrome (hereditary xerocytosis, spherocytosis, poikilocytosis and pyropoikilocytosis, elliptocytosis and stomatocytosis) and, after refreshment, helps differentiate them from the anemia with hemoglobinopathy.     

Hemin-mediated dissociation of erythrocyte membrane skeletal proteins

Spectrin tetramers and oligomers in normal erythrocytes are cross-linked by actin and protein 4.1 to form a two-dimensional membrane skeletal network. In the present study, we find that hemin, a breakdown product of hemoglobin, progressively (a) alters the conformation of spectrin as revealed by electron microscope studies and by the decreased resistance of spectrin to proteolytic degradation, (b) alters the conformation of protein 4.1 as revealed by the increased mobility of protein 4.1 on nondenaturing gel electrophoresis, (c) weakens spectrin dimer alpha beta-dimer alpha beta, spectrin alpha-spectrin beta, as well as spectrin-protein 4.1 associations as analyzed by nondenaturing gel electrophoresis, and (d) diminishes the structural stability of erythrocyte membrane skeletons (i.e. Triton-insoluble ghost residues) subjected to mechanical shearing. Since hemin may be liberated from oxidized or unstable mutant hemoglobin under pathological conditions, these hemin-induced effects on spectrin, protein 4.1, and membrane skeletal stability may play a role in the membrane lesion of these erythrocytes.     

Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy

Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-¹³C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13±0.02 μmol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21±0.02 μmol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59±0.10 μmol/h per ml erythrocytes, compared to normal erythrocytes, 1.64±0.10 μmol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16±0.16 μmol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to simulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.     

Alterations of red cell membrane proteins and hemoglobin under natural and experimental oxidant stress

We compared on red cell membrane proteins and hemoglobin (Hb) the effects of (i) natural oxidant stress that has been suggested to occur in a variety of oxidative hemolytic anemias, and (ii) experimental stress induced by hydrogen peroxide. SDS-polyacrylamide gel electrophoresis was used for protein analysis. Under natural conditions (thalassemias, hemoglobinopathies with Hb unstability), a high molecular weight polymer (HMWP) and variable amounts of globin mono- and dimers became apparent. Furthermore, a major 12 kDa polypeptide, its dimer, and conspicuous spectrin degradation products in the band 2.2–2.6 region occurred in a patient carrying the highly unstable Hb Hammersmith. Under experimental conditions, incubation of erythrocyte ghosts with H₂O₂ in the presence of minimal concentration (25 μM) of Hb generated a HMWP at the expense of membrane proteins, mainly spectrin. Incubation of a diluted (200 μM) membrane-free hemolysate with H₂O₂ induced a HMWP, an array of globin oligomers and a 12 kDa polypeptide similar to that mentionned above. Therefore, the damage to the red cell membrane present in various oxidative hemolytic anemias, including polypeptide polymerisation and breakdown, can be produced by experimental oxidant stress. These observations support the view that the alterations described in the patients result directly from oxidative reactions. However, we did not observe in the patient the sharp breakdown of polyunsaturated fatty acids that was triggered in vitro by H₂O₂ in the presence of Hb acting as a catalyst. In most cases, oligo- and polymers were resistant to β-mercaptoethanol, and the chemical nature of the underlying cross-links is discussed. To our knowledge, the 12 kDa polypeptide, that we consider as arising from globin proteolysis, has never been reported under pathological conditions.     

Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association

Human erythrocytes vesiculate upon exposure to temperatures of 49 degrees C and above. Pretreatment of the cells with the thiol-alkylating agent N-ethylmaleimide (NEM) lowers the temperature needed to produce the same effect. Concomitant with the cells' heat susceptibility, skeletal mechanical instability and an increase in spectrin dissociation have been reported (Smith and Palek (1983) Blood 62, 1190). In the present study, similar results were achieved by preincubation of the cells with diamide, which could be reversed by reduction with dithiothreitol. Another oxidative agent, sodium tetrathionate, could only induce the temperature susceptibility, with little effect on spectrin dissociation. Incubation of spectrin solutions with NEM or diamide caused decreased association of spectrin dimers and increased dissociation of spectrin tetramers. Estimation of membrane and spectrin thiols in the treated cells showed that NEM was effective while blocking less than 20% of the thiols. Diamide and tetrathionate blocked more than 50% of the thiols, but were less effective than NEM. It is suggested that some very defined population of thiols is essential for spectrin self-association and for membrane thermal stability. They are more available to NEM than to diamide and less so to tetrathionate. Other thiols participate in maintaining the membrane thermal stability only.     

Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for alpha-globin over that for beta-globin and intact HbA in an adenosine 5'-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the alpha-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in alpha-globin compared with intact haemoglobin, HbA and the beta-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the alpha-globin chain over that of the beta-globin in the haemoglobin disorder beta-thalassaemia.     

Structural modification of erythrocyte membranes during oxidative stress and activity of membrane bound NADH-methemoglobin reductase

The activity of NADH-methemoglobin reductase (metHb-reductase) in membranes isolated from human erythrocytes treated with phenylhydrazine at its sublytic concentration was studied. A decrease in the activity of membrane-bound metHb-reductase was shown to depend on the concentration of phenylhydrazine. Simultaneously, an increase in the level of membrane-bound methemoglobin and a change in the fluorescence parameters of membrane-bound 4,4'-diisothiocy-anatostilbene-2,2'-disulfonic acid were registered. In the case when Hb-free erythrocyte ghosts were treated with 0.2-2.0 mM phenylhydrazine, the activity of metHb-reductase did not change. The obtained results indicate that the inhibition of the activity of membrane-bound metHb-reductase by phenylhydrazine-induced oxidative stress in human erythrocytes is not caused by the direct action of the oxidant on the enzyme. The reason for this is the interaction of the products of hemoglobin oxidation with erythrocyte membrane (protein band 3) and structural changes in membrane proteins.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for α-globin over that for β-globin and intact HbA in an adenosine 5′-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the α-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in α-globin compared with intact haemoglobin, HbA and the β-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the α-globin chain over that of the β-globin in the haemoglobin disorder β-thalassaemia.