Separation of asymmetrical hybrid containing hemoglobin F by anaerobic anion-exchange high-performance liquid chromatography

Ip, C Y; Asakura, Toshio

doi:10.1016/0003-2697(86)90264-2

Cited by 7 publications

(5 citation statements)

References 16 publications

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“…HemoglobinsInitial analysis of hemoglobin tetramers was performed by anion exchange high performance liquid chromatography (HPLC) utilizing a Synchropak AN 300 (4.6 ϫ 25 mm) column (MICRA Scientific, Northbrook, IL) (17). Preparative isoelectric focusing was performed on 4% acrylamide gels with 2% Pharmalyte (Amersham Biosciences AB, Uppsala, Sweden) (pH 6.7-7.7).…”

Section: Analysis and Purification Of Recombinant Humanmentioning

confidence: 99%

“…Analysis of Heterotetramer Formation-Equimolar amounts of purified oxygenated HbS and HbAS3 or HbA were mixed and allowed to equilibrate overnight at 0°C as described previously (17). The mixtures were analyzed by cation exchange chromatography as described above with the addition of 3 mM sodium dithionite to the elution buffers.…”

Section: Analysis and Purification Of Recombinant Humanmentioning

confidence: 99%

“…These constructs were injected into fertilized mouse eggs, and transgenic lines were established. Hemolysates obtained from several animals were analyzed by HPLC to quantitate the amounts of human, mouse, and hybrid hemoglobins (17). The purity of the human hemoglobins was assessed by denaturing reverse-phase HPLC, which separated the ␣-and ␤-globin subunits (19,20).…”

Section: Analysis and Purification Of Recombinant Humanmentioning

confidence: 99%

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A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

Levasseur

Ryan

Reilly

et al. 2004

Journal of Biological Chemistry

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A new recombinant, human anti-sickling ␤-globin polypeptide designated ␤ AS3 (␤Gly 16 3 Asp/␤Glu 22 3 Ala/␤Thr 87 3 Gln) was designed to increase affinity for ␣-globin. The amino acid substitutions at ␤22 and ␤87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxyHbS polymerization. The ␤16 substitution confers the recombinant ␤-globin subunit (␤ AS3 ) with a competitive advantage over ␤ S for interaction with the ␣-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (␣ 2 ␤ AS3 2 ) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that ␤ AS3 has a distinct advantage over ␤ S for dimerization with ␣-globin. When equal amounts of ␤ S -and ␤ AS3 -globin monomers compete for limiting ␣-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the ␤ AS3 polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of ␤ AS3 -globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit ␣-thalassemia. Therefore, constructs expressing the ␤ AS3 -globin gene may be suitable for future clinical trials for sickle cell disease.Sickle cell disease (SCD) 1 results from an A to T transversion at the sixth codon of the human ␤-globin gene on chromosome 11 (1, 2). The mutation of a single DNA base leads to the substitution of a valine for a glutamic acid in the ␤-globin polypeptide of sickle hemoglobin (HbS). The positioning of a hydrophobic residue at this position permits an interaction with a hydrophobic pocket on another hemoglobin tetramer (see Fig. 1). This interaction allows deoxy-HbS to polymerize in an entropy-driven process (3-5). The polymerization of deoxyHbS leads to erythrocyte deformation from a biconcave morphology into the sickle shapes for which SCD is named.Polymerization of deoxy-HbS is effectively inhibited by fetal hemoglobin (HbF), and individuals who are homozygous for the sickle mutation but also express high levels of HbF are typically asymptomatic (6 -8). The level of HbF necessary to significantly reduce the symptoms of SCD ranges from about 20 to 25%; however, data showing an enhanced red cell survival with as little as 9% HbF have been reported (9). The efficacy of HbF in inhibiting HbS polymerization suggests that transduction of fetal globin genes into hematopoietic stem cells might be an effective strategy for sickle cell disease gene therapy. However, high levels of ␥-globin gene expression are difficult to achieve in adult erythroid cells even...

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Section: Analysis and Purification Of Recombinant Humanmentioning

confidence: 99%

Section: Analysis and Purification Of Recombinant Humanmentioning

confidence: 99%

Section: Analysis and Purification Of Recombinant Humanmentioning

confidence: 99%

See 1 more Smart Citation

A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

Levasseur

Ryan

Reilly

et al. 2004

Journal of Biological Chemistry

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“…In effect separation leads to loss of asymmetric species through conversion to symmetric forms. Attempts to prevent loss of asymmetric tetramers by chemical cross-linking (9 -11), retarding dissociation rate by analysis of Hb under anaerobic conditions in the more stable deoxy form (1,2,(12)(13)(14)(15), or performing separation at cryogenic temperatures (16) have been used successfully; however, these approaches are laborious or resolve the hybrid poorly, and are technically difficult.…”

mentioning

confidence: 99%

Mass Spectral Analysis of Asymmetric Hemoglobin Hybrids: Demonstration of Hb FS (α2γβS) in Sickle Cell Disease

Ofori-Acquah

Green²,

Davies

et al. 2001

Analytical Biochemistry

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“…These constructs were injected into fertilized mouse eggs, and blood from mice that developed was analyzed by IEF to identify transgenic animals that synthesize high levels of human hemoglobin (26,28). Transgenic lines were established and hemolysates obtained from several animals were analyzed by anion-exchange HPLC to quantitate the amounts of human, mouse, and hybrid hemoglobins (27). Fig.…”

mentioning

confidence: 99%

Recombinant human hemoglobins designed for gene therapy of sickle cell disease.

McCune

Reilly

Chomo

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Two human hemoglobins designed to inhibit the polymerization of sickle hemoglobin (Hb S; a2os2) have been produced. Mutations that disrupt the ability of Hb S to form polymers were introduced into the normal human 3-globin gene by site-specific mutagenesis. These mutations affect the axial and lateral contacts in the sickle fiber. The recombinant hemoglobin designated anti-sickling hemoglobin 1 (Hb AS1) contains the mutations 1822 glutamic acid to alanine and 1380 asparagine to lysine. Hb AS2 has the same 1822 glutamic acid to alanine mutation combined with p87 threonine to glutamine. Human a-and 13s-globin genes were separately fused downstream of 3-globin locus control region sequences and these constructs were coinjected into fertilized mouse eggs.Transgenic mouse lines that synthesize high levels of each anti-sickling hemoglobin were established and anti-sickling hemoglobins were purified from hemolysates and characterized. Both AS hemoglobins bind oxygen cooperatively and the oxygen afnities of these molecules are in the normal range. Delay time experiments demonstrate that Hb AS2 is a potent inhibitor of Hb S polymerization; therefore, locus control

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Separation of asymmetrical hybrid containing hemoglobin F by anaerobic anion-exchange high-performance liquid chromatography

Cited by 7 publications

References 16 publications

A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

Mass Spectral Analysis of Asymmetric Hemoglobin Hybrids: Demonstration of Hb FS (α2γβS) in Sickle Cell Disease

Recombinant human hemoglobins designed for gene therapy of sickle cell disease.

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