Interaction of hemoglobin with red blood cell membranes as shown by a fluorescent chromophore

Shaklai, Nurith; Yguerabide, Juan; Ranney, Helen M.

doi:10.1021/bi00644a031

Cited by 231 publications

(152 citation statements)

References 17 publications

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“…When Hb A, C, or S was added to AS-labeled 10 Fig. 3 show that the binding constant of Hb C at pH 6.8 (35 X 106 M-') was slightly lower than the binding constant for Hb A at pH 6.0, calculated previously to be 80 X 106 M- (6).…”

Section: Resultsmentioning

confidence: 62%

“…In earlier studies we and others (6)(7)(8) have demonstrated binding of hemoglobin A to erythrocyte membranes. The method used in those and in the present studies utilized a fluorescent chromophore embedded in erythrocyte membranes prepared by hypotonic lysis: when hemoglobin approaches the chromophore, 12-(9-anthroyloxy)stearic acid, the fluorescence is quenched by an energy transfer mechanism.…”

Section: Introductionmentioning

confidence: 75%

See 1 more Smart Citation

Association of hemoglobin C with erythrocyte ghosts.

Reiss¹,

Ranney²,

Shaklai³

1982

J. Clin. Invest.

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Section: Resultsmentioning

confidence: 62%

Section: Introductionmentioning

confidence: 75%

Association of hemoglobin C with erythrocyte ghosts.

Reiss¹,

Ranney²,

Shaklai³

1982

J. Clin. Invest.

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“…If one makes the parsimonious and plausible assumption that the point mutation in globin is the only inherited genetic abnormality affecting CC red cells, it is likely that the abnormal globin interacts directly with the cation transport system or its regulators in such a way as to produce the observed transport abnormalities or with a nmembrane component not normally involved in cation transport. It has been shown that hemoglobin binds to the erythrocyte membrane (25,26) and that hemoglobin C binds with a higher affinity than hemoglobin A at neutral pH (27,28). An important site of interaction with the membrane is the N-terminal segment of band 3, which contains a sequence of 23 residues of which 14 are either glutamate or aspartate (29).…”

Section: Discussionmentioning

confidence: 99%

Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease.

Brugnara¹,

Kopin²,

Bunn³

et al. 1985

J. Clin. Invest.

168

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IntroductionErythrocytes from patients with homozygous hemoglobin C disease (CC cells) contain less K, Na, and water than do erythrocytes from normal subjects that contain only hemoglobin A (AA cells). In this paper, we provide evidence that the reduced K content and volume of CC cells are due to the activity in these but not in AA cells of a K transport system that is: (a) In this paper we examine the transport pathways for Na and K in CC erythrocytes. We show that CC cells regulate their volume through a ouabain-and bumetanide-insensitive pathway for K. This pathway seems to be responsible for the reduction in K and water content observed in CC cells. The pathway is dependent on cell volume and internal pH, and does not share any of the properties of the Ca-activated K transport system first described by Gardos (for a review see reference 5).A preliminary report of this work has previously appeared in another publication (6). Freedman and Hoffman (7).When the experiments were not performed on the same day of collection, the erythrocytes were stored at 4VC (20% hematocrit) in a preservation solution containing 140 mM KCI, 10 mM NaCI, 1 mM MgCl2, 2.5 mM NaH2PO4 buffer, pH 7.4, and 10 mM glucose. All experiments were performed within 2 d of sampling.Measurement of K efflux from fresh cells. K efflux from CC cells and controls was measured into Na or choline medium, as a function of external pH (pH.), at constant osmolarity (295-300 mosM), and as a function of osmolarity, at constant pHo (7.4). When the pH. was varied, the medium contained 140 mM NaCl, (or 140 mM choline chloride), I mM MgCI2, 10 mM gluc6oe, 0.1 mM ouabain, 0.01 mM bumetanide, and 10 mM Tris-MOPS, pH 6.2-8.0, at 37°C. When the osmolarity was varied, the medium contained 100 mM NaCl (or 100 mM choline chloride), I mM MgCl2, 10 mM Tris-MOPS, pH 7.4, at 37°C, 10 mM glucose, 0.1 mM ouabain, and 0.01 mM bumetanide. The osmolarity was varied from 220 to 400 mosM by adding choline chloride. For the flux assay, 0.4 ml of cell suspension in choline washing solution (hematocrit -30%) was added to 9 ml of flux medium, and this flux suspension was then distributed into six previously chilled 5-ml tubes. After capping, three tubes were incubated for 5 min and three for 25 min in a shaking water bath at 37°C. The time course of K efflux into hypotonic, isotonic, and hypertonic media was determined in preliminary experiments. A 25-min incubation was chosen in order to meet conditions of linear initial rate in the flux measurement. At the end of the incubation, the tubes were transferred to an ice bath and, after 2 min, they were spun at 3,000 g for 5 min. The supernatant was removed and the K concentration was measured by atomic absorption using standards for K with the same composition of the flux medium.Measurement of unidirectional radioactive influx in fresh cells. 9 ml of medium was chilled and mixed with 10 ACi of 22Na, or 30 Ci of 86Rb (86Rb was used as a tracer for K fluxes). The radioactivity in five aliquots of 20 Ml of medium was measured f...

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“…Hb is known to bind to membrane Band 3 protein especially under hypoxic conditions [28]. HbS and HbC have a higher affinity for band 3 than HbA [24,25].…”

Section: Membrane Damage Associated With In Vivo Heme Degradationmentioning

confidence: 99%

Heme degradation and oxidative stress in murine models for hemoglobinopathies: Thalassemia, sickle cell disease and hemoglobin C disease

Nagababu

Fabry

Nagel

et al. 2008

Blood Cells, Molecules, and Diseases

102

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Red blood cells with abnormal hemoglobins (Hb) are frequently associated with increased hemoglobin autoxidation, accumulation of iron in membranes, increased membrane damage and a shorter red cell life span. The mechanisms for many of these changes have not been elucidated. We have shown in our previous studies that hydrogen peroxide formed in association with hemoglobin autoxidation reacts with hemoglobin and initiates a cascade of reactions that results in heme degradation with the formation of two fluorescent emission bands and the release of iron. Heme degradation was assessed by measuring the fluorescent band at ex 321 nm. A 5.6 fold increase in fluorescence was found in red cells from sickle transgenic mice that expressed exclusively human globins when compared to red cells from control mice. When sickle transgenic mice co-express the γM transgene, that expresses HbF and inhibits polymerization, heme degradation is decreased. Mice expressing exclusively hemoglobin C had a 6.9 fold increase in fluorescence compared to control. Heme degradation was also increased 3.5 fold in β-thalassemic mice generated by deletion of murine β major . Membrane bound IgG and red cell metHb were highly correlated with the intensity of the fluorescent heme degradation band. These results suggest that degradation of the heme moiety in intact hemoglobin and/or degradation of free heme by peroxides are higher in pathological RBCs. Concomitant release of iron appears to be responsible for the membrane damage that leads to IgG binding and the removal of red cells from circulation.

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Interaction of hemoglobin with red blood cell membranes as shown by a fluorescent chromophore

Cited by 231 publications

References 17 publications

Association of hemoglobin C with erythrocyte ghosts.

Association of hemoglobin C with erythrocyte ghosts.

Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease.

Heme degradation and oxidative stress in murine models for hemoglobinopathies: Thalassemia, sickle cell disease and hemoglobin C disease

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