Denatured hemoglobin in sickle erythrocytes.

Asakura, Toshio; Minakata, Kumagusu; Adachi, K.; Russell, Marie O.; Schwartz, Elias

doi:10.1172/jci108681

Cited by 174 publications

(61 citation statements)

References 13 publications

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“…After washing the cell fraction of whole blood sample from the patient (collected in EDTA tubes) three times with isotonic saline by centrifugation at 15,000g at 4 C for 2 min and removal of the buffy coat, hemolysate was prepared by lysing the red blood cells (RBCs) in a hypotonic hemolysing solution [13]. To remove the stroma (RBC ghosts) efficiently, 1/10 volume of 10% NaCl was added followed by centrifugation at 15,000g at 4 C for 10 min.…”

Section: Materials and Methods Preparation Of The Hemolysatementioning

confidence: 99%

Characterization of hemoglobin bassett (α94Asp→Ala), a variant with very low oxygen affinity

et al. 2004

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Hemoglobin (Hb) Bassett, an abnormal Hb variant with a markedly reduced oxygen affinity, was discovered in a Caucasian (Anglo-Saxon) male child who experienced episodes of cyanosis. Cation-exchange and reversed-phase (RP) high-performance liquid chromatography (HPLC) showed that the patient has an abnormal Hb, with a mutation in the a-globin. Tryptic peptide digest of the abnormal a-globin with subsequent HPLC analysis revealed abnormal elution of the a-T11 peptide. Further studies with Edman sequencing and electrospray mass spectrometry of tryptic peptide a-T11, as well as structural analysis by X-ray crystallography revealed an AspfiAla substitution at the a94 (G1) position, a match for Hb Bassett. Detailed functional studies showed that this Hb variant had a markedly reduced oxygen affinity (P 50 at pH 7.0 = 22 mmHg; Hb A P 50 = 10.5 mmHg), reduced Bohr effect (-0.26 compared to -0.54 in Hb A), and low subunit cooperativity (n = 1.4, compared to 2.6 in Hb A). X-ray crystallography results explain the probable effects of the structural modification on the oxygen-binding properties of this Hb variant. Am.

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Section: Materials and Methods Preparation Of The Hemolysatementioning

confidence: 99%

Characterization of hemoglobin bassett (α94Asp→Ala), a variant with very low oxygen affinity

et al. 2004

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“…The incubations were terminated by centrifugation, and the undimerized FP was washed out of the pellet with 2.5% SDS dissolved in the standard medium. ␤-Hematin in the preparations before and after incubation was hydrolyzed to FP under alkaline conditions, and the FP was measured spectrophotometrically (12). This method has been used previously in quantitative studies of synthetic ␤-hematin and found to be satisfactory (8).…”

Section: Methodsmentioning

confidence: 99%

Chloroquine-induced Masking of a Lipid That Promotes Ferriprotoporphyrin IX Dimerization in Malaria

Fitch

Chen

Cai

2003

Journal of Biological Chemistry

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Mice infected with the NYU-2 strain of Plasmodium berghei were used to study the effect of chloroquine on masking of a lipid that promotes ferriprotoporphyrin IX dimerization. More than 40% of this lipid was masked and unable to promote dimerization in membrane ghosts from erythrocytes of untreated, infected mice. Thus, preparations of membrane ghosts dimerized 57 ؎ 6 nmol of ferriprotoporphyrin IX during a 2-h incubation, whereas the lipids extracted from these preparations dimerized 101 ؎ 11 nmol of ferriprotoporphyrin IX (means ؎ S.D. for four experiments). Exposure of membrane ghosts to sonication or cold significantly increased the extent of masking. In addition, chloroquine treatment of infected mice increased the extent of masking to ϳ90%. The lipid could be unmasked by extracting it into acetone or by aging erythrocyte membrane ghosts from untreated or chloroquine-treated, infected mice for 24 h at pH 7.4 and 25°C. These findings indicate that masking and unmasking of a lipid is central to the regulation of ferriprotoporphyrin IX dimerization in malaria parasites. They also indicate that chloroquine impairs the function of this regulatory process.When mice infected with Plasmodium berghei are treated with chloroquine, the parasites lose 80% or more of their ability to produce ␤-hematin (1), a dimer of ferriprotoporphyrin IX (FP) 1 (2). Consequently, undimerized, toxic FP accumulates (3) and, presumably, kills the parasites (4, 5). The biochemical basis for the effect of chloroquine on the production of betahematin in vivo presently is unknown. It is true that chloroquine and other quinoline derivatives, including quinine and mefloquine, bind to FP and inhibit dimerization in vitro (3, 6), but something else occurs in vivo. In fact, inhibition of FP dimerization by chloroquine is antagonized by quinine and mefloquine in vivo (3). These and other observations indicate that chloroquine acts by affecting a regulatory process rather than by directly inhibiting FP dimerization (7). To understand the biochemical basis for the antimalarial action of chloroquine, therefore, it is desirable to know more about the process that regulates FP dimerization.Current knowledge of FP dimerization in malaria parasites can be briefly summarized as follows. The process is promoted only by the lipid fraction of parasitized erythrocytes (8). Apparently, the lipid serves to concentrate monomeric FP and keep it in a state favorable for dimerization (8). Once formed, FP dimers spontaneously aggregate in an acidic milieu (2) such as occurs in the digestive vacuoles of malaria parasites. These aggregates are insoluble at physiologic pH or lower (5), and, in contrast to undimerized FP, they appear to be nontoxic for malaria parasites.Since malaria parasites are enriched with linoleic acid, it is likely that linoleate or a linoleate derivative is the lipid that promotes FP dimerization (9). The linoleate probably is released from the inner membranes of hemoglobin-laden endocytic vesicles as they are processed into digestive vacuoles (...

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“…There is already morphological evidence of "pitting" or "culling" of HMC by spleen macrophages. 48,49 On the other hand, elimination of HMC in the bone marrow has been previously hypothesized in β-thalassemia 6 and the loss of HMC through the vesiculation of specific membrane areas may play an important role in this phenomenon.…”

Section: N Protein Mw Score Coveragementioning

confidence: 96%

Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase

Ferru

Pantaleo

Carta³

et al. 2013

Haematologica

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© F e r r a t a S t o r t i F o u n d a t i o ndifferent genetic and clinical status provided new insights into the pathogenic role of circulating MP and possible interventions to control their amount in thalassemia. MethodsUnless otherwise stated, all materials were obtained from Sigma-Aldrich, St. Louis, MO, USA. Additional information about the methods are provided in the Online Supplementary Data. Treatment of red blood cellsVenous blood was drawn from 12 healthy individuals, eight non-splenectomized TI patients and six splenectomized TI patients. All subjects provided written, informed consent before entering the study. The study was approved by the local Ethics Committee and conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki.Clinical analyses were performed by routine laboratory tests at the Policlinico Hospital (Milan, Italy). Blood anti-coagulated with heparin was stored in citrate-phosphate-dextrose with adenine (CPDA-1) prior to its use. RBC were washed three times with phosphate-buffered saline (PBS: 137mM NaCl, 2.7mM KCl, 8.1mM K 2 HPO 4 , 1.5mM KH 2 PO 4 , pH 7.4) containing glucose 5 mM to obtain packed RBC.To stimulate HMC formation, RBC from healthy donors were suspended at a hematocrit of 30% and incubated with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 mM) of phenylhydrazine at 37 °C for 4 h. To test the antioxidant effect in MP release we incubated 200 μM of 2-mercaptoethanol in the presence of 1 mM phenylhydrazine. When necessary, RBC were pretreated with Syk kinase inhibitors (10 μM Syk inhibitors II and 10 μM Syk inhibitors IV, Calbiochem, Darmstadt, Germany), for 1 h at 37 °C in the dark. Each reaction was terminated by three washes with PBS-glucose. For all protocols described, untreated controls were processed identically except that the stimulant/inducer was omitted from the incubation. Red blood cell membrane preparationStandard hypotonic membranes were prepared, as previously described, 20 and stored frozen at -80°C until use. Membrane protein content was quantified using the DC Protein assay (Biorad, Hercules, CA, USA). Analysis of microparticlesThe MP in plasma were analyzed by flow cytometry using a modification of a previously described method:22 25 μL of plasma diluted 1:1 with PBS-glucose 5mM were analyzed using anti-CD41 (BD, Franklin Lakes, NJ, USA) and anti-glycophorin-A (Dako, Denmark), both diluted 1:10. Assay of hemichromesHMC were quantified by measuring heme absorbance (Abs) using the following equation:HMC= -133xAbs577 -114xAbs630 + 233xAbs560, and expressed as nMoles/mL of solubilized membranes. 23 Microparticle isolationTo induce MP release in vitro, RBC from each volunteer and phenylhydrazine-treated RBC in PBS (30% hematocrit) were incubated as previously described. 24 Electrophoresis and immunoblottingMembrane and MP proteins were solubilized in Laemmli buffer 25 under reducing [2% (w/v) dithiothreitol] or non-reducing conditions at a volume ratio of 1:1. Sodium dodecylsulfate polyacrylamide gel electropho...

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Denatured hemoglobin in sickle erythrocytes.

Cited by 174 publications

References 13 publications

Characterization of hemoglobin bassett (α94Asp→Ala), a variant with very low oxygen affinity

Characterization of hemoglobin bassett (α94Asp→Ala), a variant with very low oxygen affinity

Chloroquine-induced Masking of a Lipid That Promotes Ferriprotoporphyrin IX Dimerization in Malaria

Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase

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